Rho-associated kinase 2 (ROCK2) regulates the secretion of proinflammatory cytokines and the development of autoimmunity in mice. Data from a phase 1 clinical trial demonstrate that oral administration of KD025, a selective ROCK2 inhibitor, to healthy human subjects down-regulates the ability of T cells to secrete IL-21 and IL-17 by 90% and 60%, respectively, but not IFN-? in response to T-cell receptor stimulation in vitro. Pharmacological inhibition with KD025 or siRNA-mediated inhibition of ROCK2, but not ROCK1, significantly diminished STAT3 phosphorylation and binding to IL-17 and IL-21 promoters and reduced IFN regulatory factor 4 and nuclear hormone RAR-related orphan receptor ?t protein levels in T cells derived from healthy subjects or rheumatoid arthritis patients. Simultaneously, treatment with KD025 also promotes the suppressive function of regulatory T cells through up-regulation of STAT5 phosphorylation and positive regulation of forkhead box p3 expression. The administration of KD025 in vivo down-regulates the progression of collagen-induced arthritis in mice via targeting of the Th17-mediated pathway. Thus, ROCK2 signaling appears to be instrumental in regulating the balance between proinflammatory and regulatory T-cell subsets. Targeting of ROCK2 in man may therefore restore disrupted immune homeostasis and have a role in the treatment of autoimmunity.
Aging is a contributing factor in cancer occurrence. We recently demonstrated that systemic immunotherapy (IT) administration in aged, but not young, mice resulted in induction of rapid and lethal cytokine storm. We found that aging was accompanied by increases in visceral fat similar to that seen in young obese (ob/ob or diet-induced obese [DIO]) mice. Yet, the effects of aging and obesity on inflammatory responses to immunotherapeutics are not well defined. We determine the effects of adiposity on systemic IT tolerance in aged compared with young obese mice. Both young ob/ob- and DIO-generated proinflammatory cytokine levels and organ pathologies are comparable to those in aged ad libitum mice after IT, culminating in lethality. Young obese mice exhibited greater ratios of M1/M2 macrophages within the peritoneal and visceral adipose tissues and higher percentages of TNF(+) macrophages in response to ?CD40/IL-2 as compared with young lean mice. Macrophage depletion or TNF blockade in conjunction with ?CD40/IL-2 prevented cytokine storms in young obese mice and protected from lethality. Calorie-restricted aged mice contain less visceral fat and displayed reduced cytokine levels, protection from organ pathology, and protection from lethality upon ?CD40/IL-2 administration. Our data demonstrate that adiposity is a critical factor in the age-associated pathological responses to systemic anti-cancer IT.
The common gamma chain (CD132) is a subunit of the interleukin (IL) receptors for IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21. Because levels of several of these cytokines were shown to be increased in the serum of patients developing acute and chronic graft-versus-host disease (GvHD), we reasoned that inhibition of CD132 could have a profound effect on GvHD. We observed that anti-CD132 monoclonal antibody (mAb) reduced acute GvHD potently with respect to survival, production of TNF, IFN-? and IL-6, and GvHD histopathology. Anti-CD132 mAb afforded protection from GvHD partly via inhibition of granzyme B production in CD8 T cells, while exposure of CD8 T cells to IL-2, IL-7, IL-15, and IL-21 increased granzyme B production. Also, T cells exposed to anti-CD132 mAb displayed a more naive phenotype in microarray based analyses and showed reduced JAK3 phosphorylation upon activation. Consistent with a role of JAK3 in GvHD, Jak3(-/-) T cells caused less severe GvHD. Additionally, anti-CD132 mAb treatment of established chronic GvHD reversed liver and lung fibrosis, and pulmonary dysfunction characteristic of bronchiolitis obliterans. We conclude that acute GvHD and chronic GvHD, caused by T cells activated by common gamma chain cytokines, each represent therapeutic targets for anti-CD132 mAb immunomodulation.
IL-33 is a recently characterized IL-1 family member that is proposed to function as an alarmin, or endogenous signal of cellular damage, as well as act as a pleiotropic cytokine. The ability of IL-33 to potentiate both Th1 and Th2 immunity supports its role in pathogen clearance and disease immunopathology. Yet, IL-33 restrains experimental colitis and transplant rejection by expanding regulatory T cells (Treg) via an undefined mechanism. We sought to determine the influence of IL-33 on hematopoietic cells that drives Treg expansion and underlies the therapeutic benefit of IL-33 administration. In this study, we identify a feedback loop in which conventional mouse CD11c(+) dendritic cells (DC) stimulated by IL-33 secrete IL-2 to selectively expand IL-33R(ST2(+))- suppressive CD4(+)Foxp3(+) Treg. Interestingly, this occurs in the absence of classical DC maturation, and DC-derived (innate) IL-2 increases ST2 expression on both DC and interacting Treg. ST2(+) Treg represent an activated subset of Foxp3(+) cells, demonstrated to be ICOS(high)CD44(high) compared with their ST2(-) counterparts. Furthermore, although studies have shown that IL-33-exposed DC promote Th2 responses, we reveal that ST2(+) DC are required for IL-33-mediated in vitro and in vivo Treg expansion. Thus, we have uncovered a relationship between IL-33 and innate IL-2 that promotes the selective expansion of ST2(+) Treg over non-Treg. These findings identify a novel regulatory pathway driven by IL-33 in immune cells that may be harnessed for therapeutic benefit or for robust expansion of Treg in vitro and in vivo.
The Notch signaling pathway plays a substantial role in human NK cell development. However, the role of Notch on killer Ig-like receptor (KIR) upregulation and acquisition of effector function has not been explored. To evaluate how Notch influences terminal differentiation, cord blood-derived NK cells or sorted peripheral blood NK cells were cultured with IL-15 for 7 d with inhibitory or activating Notch signals. Inhibition of Notch signaling significantly decreased KIR expression, whereas activation enhanced it. Overexpression of activated Notch on cord blood-derived NK cells resulted in a 2-fold increase in KIR expression, indicating that Notch signaling plays a direct, cell-intrinsic role in KIR regulation. Moreover, Notch-mediated KIR expression on NK cells is regulated through cis inhibition by delta-like ligand 1. Notch signaling also enhances CD16 upregulation that precedes KIR expression. Concomitant with the upregulation of KIR and CD16, Notch signaling induces increased cytolytic effector capacity and cytokine secretion, even in posttransplant samples in which NK cell function is inherently defective. Given these attributes of Notch signaling, we propose that Notch agonists may enhance NK cell maturation and tumor killing in a posttransplant setting.
Chronic GVHD (cGVHD) is the major cause of late, nonrelapse death following stem cell transplantation and characteristically develops in organs such as skin and lung. Here, we used multiple murine models of cGVHD to investigate the contribution of macrophage populations in the development of cGVHD. Using an established IL-17-dependent sclerodermatous cGVHD model, we confirmed that macrophages infiltrating the skin are derived from donor bone marrow (F4/80+CSF-1R+CD206+iNOS-). Cutaneous cGVHD developed in a CSF-1/CSF-1R-dependent manner, as treatment of recipients after transplantation with CSF-1 exacerbated macrophage infiltration and cutaneous pathology. Additionally, recipients of grafts from Csf1r-/- mice had substantially less macrophage infiltration and cutaneous pathology as compared with those receiving wild-type grafts. Neither CCL2/CCR2 nor GM-CSF/GM-CSFR signaling pathways were required for macrophage infiltration or development of cGVHD. In a different cGVHD model, in which bronchiolitis obliterans is a prominent manifestation, F4/80+ macrophage infiltration was similarly noted in the lungs of recipients after transplantation, and lung cGVHD was also IL-17 and CSF-1/CSF-1R dependent. Importantly, depletion of macrophages using an anti-CSF-1R mAb markedly reduced cutaneous and pulmonary cGVHD. Taken together, these data indicate that donor macrophages mediate the development of cGVHD and suggest that targeting CSF-1 signaling after transplantation may prevent and treat cGVHD.
Posttransplantation cyclophosphamide (PTCy) is an effective prophylaxis against graft-versus-host disease (GVHD). However, it is unknown whether PTCy works singularly by eliminating alloreactive T cells via DNA alkylation or also by restoring the conventional (Tcon)/regulatory (Treg) T-cell balance. We studied the role of Tregs in PTCy-mediated GVHD prophylaxis in murine models of allogeneic blood or marrow transplantation (alloBMT). In 2 distinct MHC-matched alloBMT models, infusing Treg-depleted allografts abrogated the GVHD-prophylactic activity of PTCy. Using allografts in which Foxp3(+) Tregs could be selectively depleted in vivo, either pre- or post-PTCy ablation of donor thymus-derived Tregs (tTregs) abolished PTCy protection against GVHD. PTCy treatment was associated with relative preservation of donor Tregs. Experiments using combinations of Foxp3(-) Tcons and Foxp3(+) Tregs sorted from different Foxp3 reporter mice indicated that donor Treg persistence after PTCy treatment was predominantly caused by survival of functional tTregs that retained Treg-specific demethylation and also induction of peripherally derived Tregs. Finally, adoptive transfer of tTregs retrieved from PTCy-treated chimeras rescued PTCy-treated, Treg-depleted recipients from lethal GVHD. Our findings indicate that PTCy-mediated protection against GVHD is not singularly dependent on depletion of donor alloreactive T cells but also requires rapidly recovering donor Tregs to initiate and maintain alloimmune regulation.
We have previously demonstrated that immunotherapy combining agonistic anti-CD40 and IL-2 (IT) results in synergistic anti-tumor effects. IT induces expansion of highly cytolytic, antigen-independent "bystander-activated" (CD8(+)CD44high) T cells displaying a CD25(-)NKG2D(+) phenotype in a cytokine dependent manner, which were responsible for the anti-tumor effects. While much attention has focused on CD4(+) T cell help for antigen-specific CD8(+) T cell expansion, little is known regarding the role of CD4(+) T cells in antigen-nonspecific bystander-memory CD8(+) T cell expansion. Utilizing CD4 deficient mouse models, we observed a significant expansion of bystander-memory T cells following IT which was similar to the non-CD4 depleted mice. Expanded bystander-memory CD8(+) T cells upregulated PD-1 in the absence of CD4(+) T cells which has been published as a hallmark of exhaustion and dysfunction in helpless CD8(+) T cells. Interestingly, compared to CD8(+) T cells from CD4 replete hosts, these bystander expanded cells displayed comparable (or enhanced) cytokine production, lytic ability, and in vivo anti-tumor effects suggesting no functional impairment or exhaustion and were enriched in an effector phenotype. There was no acceleration of the post-IT contraction phase of the bystander memory CD8(+) response in CD4-depleted mice. The response was independent of IL-21 signaling. These results suggest that, in contrast to antigen-specific CD8(+) T cell expansion, CD4(+) T cell help is not necessary for expansion and activation of antigen-nonspecific bystander-memory CD8(+) T cells following IT, but may play a role in regulating conversion of these cells from a central memory to effector phenotype. Additionally, the expression of PD-1 in this model appears to be a marker of effector function and not exhaustion.
Chronic graft-versus-host disease (cGVHD) following allogeneic hematopoietic stem cell transplantation (HSCT) has emerged as a predominant complication following HSCT and has a distinct etiology. We and others have previously demonstrated that bortezomib, a proteasome inhibitor, can prevent but not treat acute GVHD in mice. To assess the effects of bortezomib on cGVHD, a mouse minor histocompatibility antigen-mismatched strain combination was used to mimic clinical cGVHD sclerodermatous pathogenesis and phenotype. Treatment of ongoing cGVHD with bortezomib ameliorated cutaneous lesions, which were also associated with a reduction in total numbers of germinal center B cells and lower B-cell activating factor gene expression levels in cutaneous tissues. Importantly, lymphoma-bearing mice receiving allogeneic HSCT with bortezomib preserved graft-versus-tumor (GVT) effects. Based on these animal studies, we initiated an intrapatient dose escalation clinical trial in patients with extensive steroid-intolerant, dependent, or resistant cGVHD. Marked clinical improvement was observed in patients, which was also associated with reductions of peripheral B cells and minimal toxicity. These results indicate that bortezomib can be of significant use in the treatment of cGVHD and may also allow for maintenance of GVT. This trial was registered at www.clinicaltrials.gov as #NCT01672229.
Development of T cells in the thymus requires continuous importation of T-lineage progenitors from the bone marrow via the circulation. Following bone marrow transplant, recovery of a normal peripheral T-cell pool depends on production of naïve T cells in the thymus; however, delivery of progenitors to the thymus limits T-lineage reconstitution. Here, we examine homing of intravenously delivered progenitors to the thymus following irradiation and bone marrow reconstitution. Surprisingly, following host conditioning by irradiation, we find that homing of lymphoid-primed multipotent progenitors and common lymphoid progenitors to the thymus decreases more than 10-fold relative to unirradiated mice. The reduction in thymic homing in irradiated mice is accompanied by a significant reduction in CCL25, an important chemokine ligand for thymic homing. We show that pretreatment of bone marrow progenitors with CCL25 and CCL21 corrects the defect in thymic homing after irradiation and promotes thymic reconstitution. These data suggest new therapeutic approaches to promote T-cell regeneration.
CD4 regulatory T cells play a critical role in establishment of immune tolerance and prevention of graft-versus-host disease after allogeneic hematopoietic stem cell transplantation. The recovery and maintenance of regulatory T cells is dependent on homeostatic factors including the generation of naïve regulatory T cells from hematopoietic precursor cells, the proliferation and expansion of mature regulatory T cells, and the survival of regulatory T cells in vivo. In this study, quantitation of mitochondrial apoptotic priming was used to compare susceptibility of regulatory T cells, conventional CD4 T cells and CD8 T cells to intrinsic pathway apoptosis in 57 patients after allogeneic hematopoietic stem cell transplantation and 25 healthy donors. In healthy donors, regulatory T cells are more susceptible to mitochondrial priming than conventional T cells. Mitochondrial priming is increased after hematopoietic stem cell transplantation in all T-cell subsets and particularly in patients with chronic graft-versus-host disease. Regulatory T cells express high levels of CD95 and are also more susceptible than conventional T cells to apoptosis through the extrinsic pathway. However, CD95 expression and extrinsic pathway apoptosis is not increased after hematopoietic stem cell transplantation. Decreased expression of BCL2 and increased expression of BIM, a mitochondrial cell death activator protein, in regulatory T cells contributes to increased mitochondrial priming in this T-cell subset but additional factors likely contribute to increased mitochondrial priming following hematopoietic stem cell transplantation.
Chronic graft-versus-host disease (cGVHD) is a leading cause of morbidity and mortality after allogeneic hematopoietic stem cell transplantation. Having shown that germinal center (GC) formation and immunoglobulin deposition are required for multiorgan system cGVHD and associated bronchiolitis obliterans syndrome (BOS) in a murine model, we hypothesized that T follicular helper (Tfh) cells are necessary for cGVHD by supporting GC formation and maintenance. We show that increased frequency of Tfh cells correlated with increased GC B cells, cGVHD, and BOS. Although administering a highly depletionary anti-CD20 monoclonal antibody (mAb) to mice with established cGVHD resulted in peripheral B-cell depletion, B cells remained in the lung, and BOS was not reversed. BOS could be treated by eliminating production of interleukin-21 (IL-21) by donor T cells or IL-21 receptor (IL-21R) signaling of donor B cells. Development of BOS was dependent upon T cells expressing the chemokine receptor CXCR5 to facilitate T-cell trafficking to secondary lymphoid organ follicles. Blocking mAbs for IL-21/IL-21R, inducible T-cell costimulator (ICOS)/ICOS ligand, and CD40L/CD40 hindered GC formation and cGVHD. These data provide novel insights into cGVHD pathogenesis, indicate a role for Tfh cells in these processes, and suggest a new line of therapy using mAbs targeting Tfh cells to reverse cGVHD.
Chronic graft-versus-host disease (GVHD) induces significant morbidity and mortality after allogeneic hematopoietic stem cell transplantation. Corticosteroids are standard initial therapy, despite limited efficacy and long-term toxicity. Based on our experience using bortezomib as effective acute GVHD prophylaxis, we hypothesized that proteasome-inhibition would complement the immunomodulatory effects of corticosteroids to improve outcomes in chronic GVHD (cGVHD). We undertook a single-arm phase II trial of bortezomib plus prednisone for initial therapy of cGVHD. Bortezomib was administered at 1.3 mg/m(2) i.v. on days 1, 8, 15, and 22 of each 35-day cycle for 3 cycles (15 weeks). Prednisone was dosed at .5 to 1 mg/kg/day, with a suggested taper after cycle 1. All 22 enrolled participants were evaluable for toxicity; 20 were evaluable for response. Bortezomib plus prednisone therapy was well tolerated, with 1 occurrence of grade 3 sensory peripheral neuropathy possibly related to bortezomib. The overall response rate at week 15 in evaluable participants was 80%, including 2 (10%) complete and 14 (70%) partial responses. The organ-specific complete response rate was 73% for skin, 53% for liver, 75% for gastrointestinal tract, and 33% for joint, muscle, or fascia involvement. The median prednisone dose decreased from 50 mg/day to 20 mg/day at week 15 (P < .001). The combination of bortezomib and prednisone for initial treatment of cGVHD is feasible and well tolerated. We observed a high response rate to combined bortezomib and prednisone therapy; however, in this single-arm study, we could not directly measure the impact of bortezomib. Proteasome inhibition may offer benefit in the treatment of cGVHD and should be further evaluated.
Natural killer (NK) cell efficacy correlates with in vivo proliferation, and we hypothesize that NK cell product manipulations may optimize this endpoint. Xenotransplantation was used to compare good manufacturing practice (GMP) grade freshly activated NK cells (FA-NK) and ex vivo expanded NK cells (Ex-NK). Cells were infused into NOD scid IL2 receptor gamma chain knockout (NSG) mice followed by IL-2, IL-15, or no cytokines. Evaluation of blood, spleen, and marrow showed that persistence and expansion was cytokine dependent, IL-15 being superior to IL-2. Cryopreservation and immediate infusion resulted in less cytotoxicity and fewer NK cells in vivo, and this could be rescued in FA-NK by overnight culture and testing the next day. Marked differences in the kinetics and homing of FA-NK versus Ex-NK were apparent: FA-NK cells preferentially homed to spleen and persisted longer after cytokine withdrawal. These data suggest that cryopreservation of FA-NK and Ex-NK is detrimental and that culture conditions profoundly affect homing, persistence, and expansion of NK cells in vivo. The NSG mouse model is an adjuvant to in vitro assays before clinical testing.
In recent years, immune-based therapies have become an increasingly attractive treatment option for patients with cancer. Cancer immunotherapy is often used in combination with conventional chemotherapy for synergistic effects. The alkylating agent cyclophosphamide (CTX) has been included in various chemoimmunotherapy regimens because of its well-known immunostimulatory effects. Paradoxically, cyclophosphamide can also induce suppressor cells that inhibit immune responses. However, the identity and biologic relevance of these suppressor cells are poorly defined. Here we report that cyclophosphamide treatment drives the expansion of inflammatory monocytic myeloid cells (CD11b(+)Ly6C(hi)CCR2(hi)) that possess immunosuppressive activities. In mice with advanced lymphoma, adoptive transfer (AT) of tumor-specific CD4(+) T cells following cyclophosphamide treatment (CTX+CD4 AT) provoked a robust initial antitumor immune response, but also resulted in enhanced expansion of monocytic myeloid cells. These therapy-induced monocytes inhibited long-term tumor control and allowed subsequent relapse by mediating functional tolerization of antitumor CD4(+) effector cells through the PD-1-PD-L1 axis. PD-1/PD-L1 blockade after CTX+CD4 AT therapy led to persistence of CD4(+) effector cells and durable antitumor effects. Depleting proliferative monocytes by administering low-dose gemcitabine effectively prevented tumor recurrence after CTX+CD4 AT therapy. Similarly, targeting inflammatory monocytes by disrupting the CCR2 signaling pathway markedly potentiated the efficacy of cyclophosphamide-based therapy. Besides cyclophosphamide, we found that melphalan and doxorubicin can also induce monocytic myeloid suppressor cells. These findings reveal a counter-regulation mechanism elicited by certain chemotherapeutic agents and highlight the importance of overcoming this barrier to prevent late tumor relapse after chemoimmunotherapy.
Haploidentical natural killer (NK) cell infusions can induce remissions in some patients with acute myeloid leukemia (AML) but regulatory T-cell (Treg) suppression may reduce efficacy. We treated 57 refractory AML patients with lymphodepleting cyclophosphamide and fludarabine followed by NK cell infusion and interleukin (IL)-2 administration. In 42 patients, donor NK cell expansion was detected in 10%, whereas in 15 patients receiving host Treg depletion with the IL-2-diphtheria fusion protein (IL2DT), the rate was 27%, with a median absolute count of 1000 NK cells/?L blood. IL2DT was associated with improved complete remission rates at day 28 (53% vs 21%; P = .02) and disease-free survival at 6 months (33% vs 5%; P < .01). In the IL2DT cohort, NK cell expansion correlated with higher postchemotherapy serum IL-15 levels (P = .002), effective peripheral blood Treg depletion (<5%) at day 7 (P < .01), and decreased IL-35 levels at day 14 (P = .02). In vitro assays demonstrated that Tregs cocultured with NK cells inhibit their proliferation by competition for IL-2 but not for IL-15. Together with our clinical observations, this supports the need to optimize the in vivo cytokine milieu where adoptively transferred NK cells compete with other lymphocytes to improve clinical efficacy in patients with refractory AML. This study is registered at clinicaltrials.gov, identifiers: NCT00274846 and NCT01106950.
Myelodysplastic syndromes (MDS) are stem cell disorders that can progress to acute myeloid leukemia. Although hematopoietic cell transplantation can be curative, additional therapies are needed for a disease that disproportionally afflicts the elderly. We tested the ability of a CD16xCD33 BiKE to induce natural killer (NK) cell function in 67 MDS patients. Compared with age-matched normal controls, CD7(+) lymphocytes, NK cells, and CD16 expression were markedly decreased in MDS patients. Despite this, reverse antibody-dependent cell-mediated cytotoxicity assays showed potent degranulation and cytokine production when resting MDS-NK cells were triggered with an agonistic CD16 monoclonal antibody. Blood and marrow MDS-NK cells treated with bispecific killer cell engager (BiKE) significantly enhanced degranulation and tumor necrosis factor-? and interferon-? production against HL-60 and endogenous CD33(+) MDS targets. MDS patients had a significantly increased proportion of immunosuppressive CD33(+) myeloid-derived suppressor cells (MDSCs) that negatively correlated with MDS lymphocyte populations and CD16 loss on NK cells. Treatment with the CD16xCD33 BiKE successfully reversed MDSC immunosuppression of NK cells and induced MDSC target cell lysis. Lastly, the BiKE induced optimal MDS-NK cell function irrespective of disease stage. Our data suggest that the CD16xCD33 BiKE functions against both CD33(+) MDS and MDSC targets and may be therapeutically beneficial for MDS patients.
Bortezomib, a proteasome inhibitor capable of direct antitumor effects, has been shown to prevent acute graft-versus-host disease (GVHD) when administered in a short course immediately after bone marrow transplantation (BMT) in mice. However, when bortezomib is given continuously, CD4(+) T cell-mediated gastrointestinal tract damage increases GVHD mortality. To investigate the protective effects of bortezomib on other organs, we used a CD8-dependent acute GVHD (aGVHD) model of C3H.SW donor T cells engrafted into irradiated C57BL/6 recipients (minor MHC mismatch), which lack significant gut GVHD. Our data in this model show that bortezomib can be given continuously to prevent and treat aGVHD mediated by CD8(+) T cells, but this effect is organ specific, such that only skin, and not liver, protection was observed. Despite the lack of hepatic protection, bortezomib still significantly improved survival, primarily because of its skin protection. Reduced skin GVHD by bortezomib was correlated with reduced serum and skin IL-6 levels. Administration of a blocking IL-6 antibody in this model also resulted in similar cutaneous GVHD protection. These results indicate that bortezomib or blockade of IL-6 may prevent CD8(+) T cell-mediated cutaneous acute GVHD.
Acquisition of a functional NK cell repertoire, known as education or licensing, is a complex process mediated through inhibitory receptors that recognize self. We found that NK cells containing self-killer Ig-like receptors for cognate HLA ligand in vivo were less susceptible to apoptosis. In vitro IL-15 withdrawal showed that uneducated NK cells upregulated Bim and Fas. Conversely, educated NK cells upregulated Fas ligand (FasL) under these conditions. Induction of cell death and Bim expression on uneducated cells correlated with increased IL-2R? expression. Overexpression and knockdown studies showed that higher IL-2R? limits NK cell survival in a novel manner that is independent from the role of IL-2 in activation-induced cell death. To study the role of FasL in induction of IL-2R?(hi) NK cell death, a coculture assay with FasL-blocking Abs was used. IL-15 withdrawal led to FasL-dependent killing of IL-2R?(hi) NK cells by more educated IL-2R?(lo) NK cells. Finally, CMV reactivation induces a potent long-lasting population of licensed NK cells with enhanced survival. These findings show that education-dependent NK cell survival advantages and killing of uneducated NK cells result in the maintenance of a functional repertoire, which may be manipulated to exploit NK cells for cancer immunotherapy.
The majority of allogeneic stem cell transplants are currently undertaken using G-CSF mobilized peripheral blood stem cells. G-CSF has diverse biological effects on a broad range of cells and IL-10 is a key regulator of many of these effects. Using mixed radiation chimeras in which the hematopoietic or nonhematopoietic compartments were wild-type, IL-10(-/-), G-CSFR(-/-), or combinations thereof we demonstrated that the attenuation of alloreactive T cell responses after G-CSF mobilization required direct signaling of the T cell by both G-CSF and IL-10. IL-10 was generated principally by radio-resistant tissue, and was not required to be produced by T cells. G-CSF mobilization significantly modulated the transcription profile of CD4(+)CD25(+) regulatory T cells, promoted their expansion in the donor and recipient and their depletion significantly increased graft-versus-host disease (GVHD). In contrast, stem cell mobilization with the CXCR4 antagonist AMD3100 did not alter the donor T cell's ability to induce acute GVHD. These studies provide an explanation for the effects of G-CSF on T cell function and demonstrate that IL-10 is required to license regulatory function but T cell production of IL-10 is not itself required for the attenuation GVHD. Although administration of CXCR4 antagonists is an efficient means of stem cell mobilization, this fails to evoke the immunomodulatory effects seen during G-CSF mobilization. These data provide a compelling rationale for considering the immunological benefits of G-CSF in selecting mobilization protocols for allogeneic stem cell transplantation.
Primary viral infections induce activation of CD8(+) T cells responsible for effective resistance. We sought to characterize the nature of the CD8(+) T cell expansion observed after primary viral infection with influenza. Infection of naive mice with different strains of influenza resulted in the rapid expansion of memory CD8(+) T cells exhibiting a unique bystander phenotype with significant up-regulation of natural killer group 2D (NKG2D), but not CD25, on the CD44(high) CD8(+) T cells, suggesting an antigen non-specific phenotype. We further confirmed the non-specificity of this phenotype on ovalbumin-specific (OT-I) CD8(+) T cells, which are not specific to influenza. These non-specific CD8(+) T cells also displayed increased lytic capabilities and were observed primarily in the lung. Thus, influenza infection was shown to induce a rapid, antigen non-specific memory T cell expansion which is restricted to the specific site of inflammation. In contrast, CD8(+) T cells of a similar phenotype could be observed in other organs following administration of systemic agonistic anti-CD40 and interleukin-2 immunotherapy, demonstrating that bystander expansion in multiple sites is possible depending on whether the nature of activation is either acute or systemic. Finally, intranasal blockade of NKG2D resulted in a significant increase in viral replication early during the course of infection, suggesting that NKG2D is a critical mediator of anti-influenza responses prior to the initiation of adaptive immunity. These results characterize further the local bystander expansion of tissue-resident, memory CD8(+) T cells which, due to their early induction, may play an important NKG2D-mediated, antigen non-specific role during the early stages of viral infection.
There is accumulating evidence that mesenchymal stem cells (MSCs) have their origin as perivascular cells (PVCs) in vivo, but precisely identifying them has been a challenge, as they have no single definitive marker and are rare. We have developed a fluorescent transgenic vertebrate model in which PVC can be visualized in vivo based upon sdf1 expression in the zebrafish. Prospective isolation and culture of sdf1(DsRed) PVC demonstrated properties consistent with MSC including prototypical cell surface marker expression; mesodermal differentiation into adipogenic, osteogenic, and chondrogenic lineages; and the ability to support hematopoietic cells. Global proteomic studies performed by two-dimensional liquid chromatography and tandem mass spectrometry revealed a high degree of similarity to human MSC (hMSC) and discovery of novel markers (CD99, CD151, and MYOF) that were previously unknown to be expressed by hMSC. Dynamic in vivo imaging during fin regeneration showed that PVC may arise from undifferentiated mesenchyme providing evidence of a PVC-MSC relationship. This is the first model, established in zebrafish, in which MSC can be visualized in vivo and will allow us to better understand their function in a native environment.
Chronic graft-versus-host disease (cGVHD) is a life-threatening impediment to allogeneic hematopoietic stem cell transplantation, and current therapies do not completely prevent and/or treat cGVHD. CD4+ T cells and B cells mediate cGVHD; therefore, targeting these populations may inhibit cGVHD pathogenesis. Ibrutinib is an FDA-approved irreversible inhibitor of Bruton's tyrosine kinase (BTK) and IL-2 inducible T cell kinase (ITK) that targets Th2 cells and B cells and produces durable remissions in B cell malignancies with minimal toxicity. Here, we evaluated whether ibrutinib could reverse established cGVHD in 2 complementary murine models, a model interrogating T cell-driven sclerodermatous cGVHD and an alloantibody-driven multiorgan system cGVHD model that induces bronchiolar obliterans (BO). In the T cell-mediated sclerodermatous cGVHD model, ibrutinib treatment delayed progression, improved survival, and ameliorated clinical and pathological manifestations. In the alloantibody-driven cGVHD model, ibrutinib treatment restored pulmonary function and reduced germinal center reactions and tissue immunoglobulin deposition. Animals lacking BTK and ITK did not develop cGVHD, indicating that these molecules are critical to cGVHD development. Furthermore, ibrutinib treatment reduced activation of T and B cells from patients with active cGVHD. Our data demonstrate that B cells and T cells drive cGVHD and suggest that ibrutinib has potential as a therapeutic agent, warranting consideration for cGVHD clinical trials.
Excessive levels of B cell activating factor (BAFF) are found in patients with active chronic graft-versus-host disease (cGVHD). In mice, BAFF has been shown to be essential for B cell recovery after myeloablation. To assess how BAFF levels relate to transplantation factors and subsequent development of cGVHD, we prospectively monitored 412 patients in the first year after allogeneic peripheral blood or bone marrow hematopoietic stem cell transplantation (HSCT) and censored data at time of cGVHD onset. In patients who did not develop cGVHD, we affirmed a temporal pattern of gradually decreasing BAFF levels as B cell numbers increase after myeloablative conditioning. In contrast, after reduced-intensity conditioning, BAFF levels remained high throughout the first post-HSCT year, suggesting that the degree of myeloablation resulted in delayed B cell recovery associated with persistence of higher BAFF levels. Given that high BAFF/B cell ratios have been associated with active cGVHD, we examined differences in early BAFF/B cell ratios and found significantly different BAFF/B cell ratios at 3 months post-HSCT only after myeloablative conditioning in patients who subsequently developed cGVHD. In addition to HSCT conditioning type, the use of sirolimus was significantly associated with higher BAFF levels after HSCT, and this also was potentially related to lower B cell numbers. Taken together, our results are important for interpreting BAFF measurements in cGVHD biomarker studies.
Graft-versus-host disease (GVHD) is a systemic inflammatory response due to the recognition of major histocompatibility complex disparity between donor and recipient after hematopoietic stem cell transplantation (HSCT). T-cell activation is critical to the induction of GVHD, and data from our group and others have shown that regulatory T cells (Tregs) prevent GVHD when given at the time of HSCT. Using multiphoton laser scanning microscopy, we examined the single cell dynamics of donor T cells and dendritic cells (DCs) with or without Tregs postallogeneic transplantation. We found that donor conventional T cells (Tcons) spent very little time screening host DCs. Tcons formed stable contacts with DCs very early after transplantation and only increased velocity in the lymph node at 20 hours after transplant. We also observed that Tregs reduced the interaction time between Tcons and DCs, which was dependent on the generation of interleukin 10 by Tregs. Imaging using inducible Tregs showed similar disruption of Tcon-DC contact. Additionally, we found that donor Tregs induce host DC death and down-regulate surface proteins required for donor T-cell activation. These data indicate that Tregs use multiple mechanisms that affect host DC numbers and function to mitigate acute GVHD.
Nfatc2 and Tob1 are intrinsic negative regulators of T cell activation. Nfatc2-deficient and Tob1-deficient T cells show reduced thresholds of activation; however, whether these factors have independent or overlapping roles in negative regulation of T cell responses has not been previously examined. Here, we show that Nfatc2 knockout (KO) but not Tob1 KO mice have age-associated accumulation of persistently activated T cells in vivo and expansion of the CD44+ memory cell compartment and age-associated lymphocytic infiltrates in visceral organs, without significant changes in numbers of CD4+CD25+Foxp3+ regulatory T cells (Treg). In vitro, CD4+CD25- "conventional" T cells (Tconvs) from both KO strains showed greater proliferation than wild type (WT) Tconvs. However, while Tregs from Nfatc2 KO mice retained normal suppressive function, Tregs from Tob1 KOs had enhanced suppressive activity. Nfatc2 KO Tconvs expanded somewhat more rapidly than WT Tconvs under conditions of homeostatic proliferation, but their accelerated growth capacity was negated, at least acutely, in a lymphoreplete environment. Finally, Nfatc2 KO mice developed a previously uncharacterized increase in B-cell malignancies, which was not accelerated by the absence of Tob1. The data thus support the prevailing hypothesis that Nfatc2 and Tob1 are non-redundant regulators of lymphocyte homeostasis.
We tested the hypothesis that a novel vaccine developed from autologous dendritic cells (DC) loaded with cells from a unique allogeneic brain tumor cell line (GBM6-AD) would be well-tolerated and would generate an immune response.
Cancer commonly occurs in the elderly and immunotherapy (IT) is being increasingly applied to this population. However, the majority of preclinical mouse tumor models assessing potential efficacy and toxicities of therapeutics use young mice. We assessed the impact of age on responses to systemic immune stimulation. In contrast to young mice, systemic cancer IT regimens or LPS given to aged mice resulted in rapid and lethal toxicities affecting multiple organs correlating with heightened proinflammatory cytokines systemically and within the parenchymal tissues. This inflammatory response and increased morbidity with age was independent of T cells or NK cells. However, prior in vivo depletion of macrophages in aged mice resulted in lesser cytokine levels, increased survival, and decreased liver histopathology. Furthermore, macrophages from aged mice and normal human elderly volunteers displayed heightened TNF and IL-6 production upon in vitro stimulation. Treatment of both TNF knockout mice and in vivo TNF blockade in aged mice resulted in significant increases in survival and lessened pathology. Importantly, TNF blockade in tumor-bearing, aged mice receiving IT displayed significant anti-tumor effects. These data demonstrate the critical role of macrophages in the age-associated hyper-inflammatory cytokine responses to systemic immunostimulation and underscore the importance of performing preclinical assessments in aged mice.
Programmed death 1 (PD-1) and its ligands, PD-L1 and PD-L2, play an important role in the maintenance of peripheral tolerance. We explored the role of PD-1 ligands in regulating graft-versus-host disease (GVHD). Both PD-L1 and PD-L2 expression were upregulated in the spleen, liver, colon, and ileum of GVHD mice. Whereas PD-L2 expression was limited to hematopoietic cells, hematopoietic and endothelial cells expressed PD-L1. PD-1/PD-L1, but not PD-1/PD-L2, blockade markedly accelerated GVHD-induced lethality. Chimera studies suggest that PD-L1 expression on host parenchymal cells is more critical than hematopoietic cells in regulating acute GVHD. Rapid mortality onset in PD-L1-deficient hosts was associated with increased gut T-cell homing and loss of intestinal epithelial integrity, along with increased donor T-cell proliferation, activation, Th1 cytokine production, and reduced apoptosis. Bioenergetics profile analysis of proliferating alloreactive donor T-cells demonstrated increased aerobic glycolysis and oxidative phosphorylation in PD-L1-deficient hosts. Donor T-cells exhibited a hyperpolarized mitochondrial membrane potential, increased superoxide production, and increased expression of a glucose transporter in PD-L1-deficient hosts. Taken together, these data provide new insight into the differential roles of host PD-L1 and PD-L2 and their associated cellular and metabolic mechanisms controlling acute GVHD.
The transcription factor Runx1 (AML1) is a central regulator of hematopoiesis and is required for the formation of definitive hematopoietic stem cells (HSCs). Runx1 is alternatively expressed from two promoters: the proximal (P2) prevails during primitive hematopoiesis, while the distal (P1) dominates in definitive HSCs. Although some transcription factor binding sites and cis-regulatory elements have been identified, a mechanistic explanation for the alternative promoter usage remains elusive. We investigated DNA methylation of known Runx1 cis-elements at stages of hematopoietic development in vivo and during differentiation of murine embryonic stem cells (ESCs) in vitro. In vivo, we find loss of methylation correlated with the primitive to definitive transition at the P1 promoter. In vitro, hypomethylation, acquisition of active chromatin modifications, and increased transcriptional activity at P1 are promoted by direct interaction with HOXB4, a transcription factor that confers definitive repopulation status on primitive hematopoietic progenitors. These data demonstrate a novel role for DNA methylation in the alternative promoter usage at the Runx1 locus and identify HOXB4 as a direct activator of the P1 promoter. This epigenetic signature should serve as a novel biomarker of HSC potential in vivo, and during ESC differentiation in vitro.
Allogeneic hematopoietic cell transplantation (HCT) is the most effective therapy for hematopoietic malignancies through T-cell-mediated graft-vs-leukemia (GVL) effects but often leads to severe graft-vs-host disease (GVHD). Given that protein kinase C? (PKC?), in cooperation with PKC?, is essential for T-cell signaling and function, we have evaluated PKC? and PKC? as potential therapeutic targets in allogeneic HCT using genetic and pharmacologic approaches. We found that the ability of PKC?(-/-)/?(-/-) donor T cells to induce GVHD was further reduced compared with PKC?(-/-) T cells in relation with the relevance of both isoforms to allogeneic donor T-cell proliferation, cytokine production, and migration to GVHD target organs. Treatment with a specific inhibitor for both PKC? and PKC? impaired donor T-cell proliferation, migration, and chemokine/cytokine production and significantly decreased GVHD in myeloablative preclinical murine models of allogeneic HCT. Moreover, pharmacologic inhibition of PKC? and PKC? spared T-cell cytotoxic function and GVL effects. Our findings indicate that PKC? and ? contribute to T-cell activation with overlapping functions essential for GVHD induction while less critical to the GVL effect. Thus, targeting PKC? and PKC? signaling with pharmacologic inhibitors presents a therapeutic option for GVHD prevention while largely preserving the GVL activity in patients receiving HCT.
B cells are implicated in the pathophysiology of chronic graft-vs-host disease (GVHD), and phase 2 trials suggest that B cell depletion can treat established chronic GVHD. We hypothesized that posttransplantation B cell depletion could prevent the occurrence of chronic GVHD. We performed a 65-patient phase 2 trial of rituximab (375 mg/m(2) IV), administered at 3, 6, 9, and 12 months after transplantation. Rituximab administration was safe without severe infusional adverse events. The cumulative incidences of chronic GVHD and systemic corticosteroid-requiring chronic GVHD at 2 years from transplantation were 48% and 31%, respectively, both lower than the corresponding rates in a concurrent control cohort (60%, P = .1, and 48.5%, P = .015). There was no difference in relapse incidence, but treatment-related mortality at 4 years from transplantation was significantly lower in treated subjects when compared with controls (5% vs 19%, P = .02), and overall survival was superior at 4 years (71% vs 56%, P = .05). At 2 years from transplantation, the B-cell activating factor/B-cell ratio was significantly higher in subjects who developed chronic GVHD in comparison with those without chronic GVHD (P = .039). Rituximab can prevent systemic corticosteroid-requiring chronic GVHD after peripheral blood stem cell transplantation and should be tested in a prospective randomized trial.
Graft-versus-host disease (GVHD) is a critical complication after allogeneic bone marrow transplantation. During GVHD, donor T cells are activated by host antigen-presenting cells and differentiate into T-effector cells (Teffs) that migrate to GVHD target organs. However, local environmental factors influencing Teff differentiation and migration are largely unknown. Vitamin A metabolism within the intestine produces retinoic acid, which contributes to intestinal homeostasis and tolerance induction. Here, we show that the expression and function of vitamin A-metabolizing enzymes were increased in the intestine and mesenteric lymph nodes in mice with active GVHD. Moreover, transgenic donor T cells expressing a retinoic acid receptor (RAR) response element luciferase reporter responded to increased vitamin A metabolites in GVHD-affected organs. Increasing RAR signaling accelerated GVHD lethality, whereas donor T cells expressing a dominant-negative RAR? (dnRAR?) showed markedly diminished lethality. The dnRAR? transgenic T cells showed reduced Th1 differentiation and ?4?7 and CCR9 expression associated with poor intestinal migration, low GVHD pathology, and reduced intestinal permeability, primarily via CD4(+) T cells. The inhibition of RAR signaling augmented donor-induced Treg generation and expansion in vivo, while preserving graft-versus-leukemia effects. Together, these results suggested that reagents blunting donor T-cell RAR signaling may possess therapeutic anti-GVHD properties.
The infusion of donor regulatory T cells (Tregs) has been used to prevent acute graft-versus-host disease (GVHD) in mice and has shown promise in phase 1 clinical trials. Previous work suggested that early Treg migration into lymphoid tissue was important for GVHD prevention. However, it is unclear how and where Tregs function longitudinally to affect GVHD. To better understand their mechanism of action, we studied 2 Treg-associated chemokine receptors in murine stem cell transplant models. CC chemokine receptor (CCR) 4 was dispensable for donor Treg function in the transplant setting. Donor Tregs lacking CCR8 (CCR8(-/-)), however, were severely impaired in their ability to prevent lethal GVHD because of increased cell death. By itself, CCR8 stimulation was unable to rescue Tregs from apoptosis. Instead, CCR8 potentiated Treg survival by promoting critical interactions with dendritic cells. In vivo, donor bone marrow-derived CD11c(+) antigen-presenting cells (APCs) were important for promoting donor Treg maintenance after transplant. In contrast, host CD11c(+) APCs appeared to be dispensable for early activation and expansion of donor Tregs. Collectively, our data indicate that a sustained donor Treg presence is critical for their beneficial properties, and that their survival depends on CCR8 and donor but not host CD11c(+) APCs.
Acute graft-versus-host disease (GVHD) occurs in 40% to 60% of recipients of partially matched umbilical cord blood transplantation (UCBT). In a phase I study, adoptive transfer of expanded CD4(+)CD25(+)Foxp3(+) natural regulatory T cells (nTregs) resulted in a reduced incidence of grade II-IV acute GVHD. To investigate potential mechanisms responsible for the reduced GVHD risk, we analyzed peripheral blood mononuclear cell mRNA expression of a tolerance gene set previously identified in operation- tolerant kidney transplant recipients, comparing healthy controls and patients who received nTregs and those who did not receive nTregs with and without experiencing GVHD. Samples from patients receiving nTregs regardless of GVHD status showed increased expression of Foxp3 expression, as well as B cell-related tolerance marker. This was correlated with early B cell recovery, predominately of naïve B cells, and nearly normal T cell reconstitution. CD8(+) T cells showed reduced signs of activation (HLA-DR(+) expression) compared with conventionally treated patients developing GVHD. In contrast, patients with GVHD had significantly increased TLR5 mRNA expression, whereas nTreg-treated patients without GVHD had reduced TLR5 mRNA expression. We identified Lin(-)HLADR(-)CD33(+)CD16(+) cells and CD14(++)CD16(-) monocytes as the main TLR5 producers, especially in samples of conventionally treated patients developing GVHD. Taken together, these data reveal interesting similarities and differences between tolerant organ and nTreg-treated hematopoietic stem cell transplantation recipients.
Clinical trials reveal that plasmid DNA (pDNA)-based gene delivery must be improved to realize its potential to treat human disease. Current pDNA platforms suffer from brief transgene expression, primarily due to the spread of transcriptionally repressive chromatin initially deposited on plasmid bacterial backbone sequences. Minicircle (MC) DNA lacks plasmid backbone sequences and correspondingly confers higher levels of sustained transgene expression upon delivery, accounting for its success in preclinical gene therapy models. In this study, we show for the first time that MC DNA also functions as a vaccine platform. We used a luciferase reporter transgene to demonstrate that intradermal delivery of MC DNA, relative to pDNA, resulted in significantly higher and persistent levels of luciferase expression in mouse skin. Next, we immunized mice intradermally with DNA encoding a peptide that, when presented by the appropriate major histocompatibility complex class I molecule, was recognized by endogenous CD8(+) T cells. Finally, immunization with peptide-encoding MC DNA, but not the corresponding full-length (FL) pDNA, conferred significant protection in mice challenged with Listeria monocytogenes expressing the model peptide. Together, our results suggest intradermal delivery of MC DNA may prove more efficacious for prophylaxis than traditional pDNA vaccines.
CD4(+)Foxp3(+) regulatory T cells (Tregs) play a central role in the maintenance of immune tolerance after allogeneic hematopoietic stem cell transplantation. We recently reported that daily administration of low-dose interleukin-2 (IL-2) induces selective expansion of functional Tregs and clinical improvement of chronic graft-versus-host disease (GVHD). To define the mechanisms of action of IL-2 therapy, we examined the immunologic effects of this treatment on homeostasis of CD4(+) T cell subsets after transplant. We first demonstrated that chronic GVHD is characterized by constitutive phosphorylation of signal transducer and activator of transcription 5 (Stat5) in conventional CD4(+) T cells (Tcons) associated with elevated amounts of IL-7 and IL-15 and relative functional deficiency of IL-2. IL-2 therapy resulted in the selective increase of Stat5 phosphorylation in Tregs and a decrease of phosphorylated Stat5 in Tcons. Over an 8-week period, IL-2 therapy induced a series of changes in Treg homeostasis, including increased proliferation, increased thymic export, and enhanced resistance to apoptosis. Low-dose IL-2 had minimal effects on Tcons. These findings define the mechanisms whereby low-dose IL-2 therapy restores the homeostasis of CD4(+) T cell subsets and promotes the reestablishment of immune tolerance.
Recessive dystrophic epidermolysis bullosa (RDEB) is characterized by a functional deficit of type VII collagen protein due to gene defects in the type VII collagen gene (COL7A1). Gene augmentation therapies are promising, but run the risk of insertional mutagenesis. To abrogate this risk, we explored the possibility of using engineered transcription activator-like effector nucleases (TALEN) for precise genome editing. We report the ability of TALEN to induce site-specific double-stranded DNA breaks (DSBs) leading to homology-directed repair (HDR) from an exogenous donor template. This process resulted in COL7A1 gene mutation correction in primary fibroblasts that were subsequently reprogrammed into inducible pluripotent stem cells and showed normal protein expression and deposition in a teratoma-based skin model in vivo. Deep sequencing-based genome-wide screening established a safety profile showing on-target activity and three off-target (OT) loci that, importantly, were at least 10?kb from a coding sequence. This study provides proof-of-concept for TALEN-mediated in situ correction of an endogenous patient-specific gene mutation and used an unbiased screen for comprehensive TALEN target mapping that will cooperatively facilitate translational application.
The ability of cells to detect changes in the microenvironment is important in cell signaling and responsiveness to environmental fluctuations. Our interest is in understanding how human bone marrow stromal-derived cells (MSC) and their relatives, vascular smooth muscle cells (VSMC), interact with their environment through novel receptors. We found, through a proteomics screen, that MSC express the bitter taste receptor, TAS2R46, a protein more typically localized to the taste bud. Expression was also confirmed in VSMCs. A prototypical bitter compound that binds to the bitter taste receptor class, denatonium, increased intracellular calcium release and decreased cAMP levels as well as increased the extracellular release of ATP in human MSC. Denatonium also bound and activated rodent VSMC with a change in morphology upon compound exposure. Finally, rodents given denatonium in vivo had a significant drop in blood pressure indicating a vasodilator response. This is the first description of chemosensory detection by MSC and VSMCs via a taste receptor. These data open a new avenue of research into discovering novel compounds that operate through taste receptors expressed by cells in the marrow and vascular microenvironments.
Steroid-refractory chronic graft-versus-host disease (cGVHD) carries a poor prognosis with no agreed upon algorithm for treatment. Because both B and T cells contribute to the pathophysiology of cGVHD, we conducted a phase I study in subjects with steroid-refractory cGVHD using the anti-CD52 antibody alemtuzumab to transiently deplete most mononuclear subsets. Three regimens were investigated in a 3+3 dose-escalation design: 3 mg × 6 (dose level 1), 3 mg × 1, then 10 mg × 5 (dose level 2) and 3 mg × 1, 10 mg × 1, then 30 mg × 4 (dose level 3) administered over 4 weeks. The maximum tolerated dose of alemtuzumab was dose level 2. Thirteen patients were assessable for toxicities, which were primarily infectious and hematologic. Rates of infectious complications in the first 12 weeks were 0% at dose level 1 (n = 3), 50% at dose level 2 (1 death, n = 6), and 75% at dose level 3 (2 deaths, n = 4). Of 10 patients assessable for response, 7 (70%) responded at 12 weeks, with a 30% complete response rate. Four subjects reduced steroid dose or discontinued an immunosuppressant at 12 weeks. The median decrease in steroid dose at 1 year was 61.6%. Infectious complications occurred predominantly in the first 3 months after therapy, but full B and T cell recovery took well over 12 months. Immunophenotypic profiling revealed early recovery by natural killer cells and relative sparing of CD4+ and CD8+ central memory T cell subsets. Our study indicates that therapy with alemtuzumab for steroid-refractory cGVHD is tolerable with close attention to dosing and may be active in subjects who have failed multiple therapies. The pattern of lymphocyte recovery after alemtuzumab will inform the biology and future therapy of cGVHD. The use of alemtuzumab in the context of therapy for cGVHD deserves study in larger phase II trials.
Spontaneous reversion of disease-causing mutations has been observed in some genetic disorders. In our clinical observations of severe generalized recessive dystrophic epidermolysis bullosa (RDEB), a currently incurable blistering genodermatosis caused by loss-of-function mutations in COL7A1 that results in a deficit of type VII collagen (C7), we have observed patches of healthy-appearing skin on some individuals. When biopsied, this skin revealed somatic mosaicism resulting from the self-correction of C7 deficiency. We believe this source of cells could represent an opportunity for translational "natural" gene therapy. We show that revertant RDEB keratinocytes expressing functional C7 can be reprogrammed into induced pluripotent stem cells (iPSCs) and that self-corrected RDEB iPSCs can be induced to differentiate into either epidermal or hematopoietic cell populations. Our results give proof in principle that an inexhaustible supply of functional patient-specific revertant cells can be obtained-potentially relevant to local wound therapy and systemic hematopoietic cell transplantation. This technology may also avoid some of the major limitations of other cell therapy strategies, eg, immune rejection and insertional mutagenesis, which are associated with viral- and non-viral-mediated gene therapy. We believe this approach should be the starting point for autologous cellular therapies using "natural" gene therapy in RDEB and other diseases.Journal of Investigative Dermatology accepted article preview online, 6 December 2013. doi:10.1038/jid.2013.523.
Human interleukin (IL)-22-producing ROR?t(+) innate lymphoid cells (ILC22) and conventional natural killer (cNK) cells are present in secondary lymphoid tissues. Both have an immunophenotype corresponding to stage III NK progenitors (CD56(+/-)CD117(high)CD94(-)). Using an in vitro differentiation and primary human tissues, we investigated their developmental relationships. cNK cells showed a CD56(+)CD117(+)CD7(+/-)LFA-1(high) phenotype and expressed surface receptors, cytokines, and transcription factors found on mature cNK cells. In contrast, ILC22 cells were contained within the CD56(+)CD117(high)CD94(-)CD7(-)LFA-1(-) fraction and produced IL-22, IL-8, and granulocyte macrophage colony stimulating factor. Although ILC22 cells expressed NKp44 and CD161, they lacked most other NK receptors and NK-associated transcription factors (T-bet and Eomes) and were incapable of interferon-? production or cytotoxic responses. Most purified CD56(+)CD117(+)CD7(+/-)LFA-1(-) remained as ILC22 cells and never became cNK cells. In the absence of IL-15, CD34(+) cells showed a complete block in cNK differentiation and instead gave rise to a CD56(+) population of ILC22 cells. Conversely, in the absence of IL-7 and stem cell factor, cNK cells were generated but ILC22 cells showed minimal differentiation. Although human ILC22 cells and cNK progenitors have a phenotype that overlaps with stage III NK progenitors, they have unique cytokine requirements and can be distinguished by LFA-1 expression.
At sites of inflammation, certain regulatory T cells (Treg cells) can undergo rapid reprogramming into helper-like cells without loss of the transcription factor Foxp3. We show that reprogramming is controlled by downregulation of the transcription factor Eos (Ikzf4), an obligate corepressor for Foxp3. Reprogramming was restricted to a specific subset of "Eos-labile" Treg cells that was present in the thymus and identifiable by characteristic surface markers and DNA methylation. Mice made deficient in this subset became impaired in their ability to provide help for presentation of new antigens to naive T cells. Downregulation of Eos required the proinflammatory cytokine interleukin-6 (IL-6), and mice lacking IL-6 had impaired development and function of the Eos-labile subset. Conversely, the immunoregulatory enzyme IDO blocked loss of Eos and prevented the Eos-labile Treg cells from reprogramming. Thus, the Foxp3(+) lineage contains a committed subset of Treg cells capable of rapid conversion into biologically important helper cells.
Although exceptionally high radiation dose-rates are currently attaining clinical feasibility, there have been relatively few studies reporting the biological consequences of these dose-rates in hematopoietic cell transplant (HCT). In zebrafish models of HCT, preconditioning before transplant is typically achieved through radiation alone. We report the comparison of outcomes in adult zebrafish irradiated with 20 Gy at either 25 or 800 cGy/min in the context of experimental HCT. In non-transplanted irradiated fish we observed no substantial differences between dose-rate groups as assessed by fish mortality, cell death in the kidney, endogenous hematopoietic reconstitution, or gene expression levels of p53 and ddb2 (damage-specific DNA binding protein 2) in the kidney. However, following HCT, recipients conditioned with the higher dose rate showed significantly improved donor-derived engraftment at 9 days post transplant (p ? 0.0001), and improved engraftment persisted at 31 days post transplant. Analysis for sdf-1a expression, as well as transplant of hematopoietic cells from cxcr4b -/- zebrafish, (odysseus), cumulatively suggest that the sdf-1a/cxcr4b axis is not required of donor-derived cells for the observed dose-rate effect on engraftment. Overall, the adult zebrafish model of HCT indicates that exceptionally high radiation dose-rates can impact HCT outcome, and offers a new system for radiobiological and mechanistic interrogation of this phenomenon. Key words: Radiation dose rate, Total Marrow Irradiation (TMI), Total body irradiation (TBI), SDF-1, Zebrafish, hematopoietic cell transplant.
In rodent graft-versus-host disease (GVHD) models, anti-IL-21 neutralizing mAb treatment ameliorates lethality and is associated with decreases in Th1 cytokine production and gastrointestinal tract injury. GVHD prevention was dependent on the in vivo generation of donor-inducible regulatory T cells (Tregs). To determine whether the IL-21 pathway might be targeted for GVHD prevention, skin and colon samples obtained from patients with no GVHD or grade 2 to 4 GVHD were analyzed for IL-21 protein expression. By immunohistochemistry staining, IL-21 protein-producing cells were present in all gastrointestinal tract samples and 54% of skin samples obtained from GVHD patients but not GVHD-free controls. In a human xenogeneic GVHD model, human IL-21-secreting cells were present in the colon of GVHD recipients and were associated with elevated serum IL-21 levels. A neutralizing anti-human IL-21 mAb given prophylactically significantly reduced GVHD-associated weight loss and mortality, resulting in a concomitant increase in Tregs and a decrease in T cells secreting IFN-? or granzyme B. Based on these findings, anti-IL-21 mAb could be considered for GVHD prevention in the clinic.
Chronic GVHD (cGVHD) poses a significant risk for HSCT patients. Preclinical development of new therapeutic modalities has been hindered by models with pathologic findings that may not simulate the development of human cGVHD. Previously, we have demonstrated that cGVHD induced by allogeneic HSCT after a conditioning regimen of cyclophosphamide and total-body radiation results in pulmonary dysfunction and airway obliteration, which leads to bronchiolitis obliterans (BO), which is pathognomonic for cGVHD of the lung. We now report cGVHD manifestations in a wide spectrum of target organs, including those with mucosal surfaces. Fibrosis was demonstrated in the lung and liver and was associated with CD4(+) T cells and B220(+) B-cell infiltration and alloantibody deposition. Donor bone marrow obtained from mice incapable of secreting IgG alloantibody resulted in less BO and cGVHD. Robust germinal center reactions were present at the time of cGVHD disease initiation. Blockade of germinal center formation with a lymphotoxin-receptor-immunoglobulin fusion protein suppressed cGVHD and BO. We conclude that cGVHD is caused in part by alloantibody secretion, which is associated with fibrosis and cGVHD manifestations including BO, and that treatment with a lymphotoxin-? receptor-immunoglobulin fusion protein could be beneficial for cGVHD prevention and therapy.
With recent approval of the first dendritic cell (DC) vaccine for patient use, many other DC vaccine approaches are now being tested in clinical trials. Many of these DC vaccines employ tumor cell lysates (TL) generated from cells cultured in atmospheric oxygen (?20% O?) that greatly exceeds levels found in tumors in situ. In this study, we tested the hypothesis that TLs generated from tumor cells cultured under physiologic oxygen (?5% O?) would be more effective as a source for DC antigens. Gene expression patterns in primary glioma cultures established at 5% O? more closely paralleled patient tumors in situ and known immunogenic antigens were more highly expressed. DCs treated with TLs generated from primary tumor cells maintained in 5% O? took up and presented antigens to CD8 T cells more efficiently. Moreover, CD8 T cells primed in this manner exhibited superior tumoricidal activity against target cells cultured in either atmospheric 20% O? or physiologic 5% O?. Together, these results establish a simple method to greatly improve the effectiveness of DC vaccines in stimulating the production of tumoricidal T cells, with broad implications for many of the DC-based cancer vaccines being developed for clinical application.
The microenviroment of acute myelogenous leukemia (AML) is suppressive for immune effector cells. Regulatory T cells (Tregs) have been recognized as a contributor factor and may be recruited and exploited by leukemic cells to evade immunesurveillance. Studies have shown that the frequencies of marrow and blood Tregs are greater in patients with AML than in control patients. Although increased Tregs have been associated with a decreased risk of GVHD after allogeneic HCT and hence may impede the graft-versus-tumor effect, recent findings indicate that that this may not be the case. Because there is a need to improve outcomes of standard treatment (chemotherapy with or without allogeneic HCT) in AML, targeting Tregs present an outstanding opportunity in AML because discoveries may apply throughout its treatment. Here, we review data on the roles of Tregs in mediating immune system-AML interactions. We focused on in vitro, animal, and observational human studies of Tregs in AML biology, development, prognosis, and therapy in different settings (eg, vaccination and HCT). Manipulation of Tregs or other types of immunomodulation may become a part of AML treatment in the future.
Few therapeutic strategies exist for hematologic malignancies relapsing post allogeneic hematopoietic cell transplantation. We present outcomes on 35 patients with nonchronic myelogenous leukemia (CML) hematologic malignancies, the majority having acute myelogenous leukemia (AML) or myelodysplastic syndromes/myeloproliferative disorders (MDS/MPD) (n = 22) receiving lymphodepleting chemotherapy followed by donor lymphocyte infusion (DLI) at 2 T cell dose levels (0.5 and 1.0 × 10(8) CD3/kg). Forty-nine percent of patients achieved complete remission (CR), with a median duration of remission of 6 months (range: 2-71+). CR rates were similar between the 2 groups. The incidence of acute graft-versus-host disease (aGVHD) of any grade was 49%. We saw a higher incidence of grade II-IV aGVHD, with a rate of 66% using the higher-dose DLI (grade III, 33% and grade 4, 20%) versus only 25% (10% grade III-IV) with the lower-dose DLI (P = .06). Overall survival at 1 and 2 years was 30% (95% confidence interval [CI], 16%-45%) and 19% (95% CI, 8%-34%); however, for those achieving CR, 1- and 2-year survival was improved at 44% (95% CI, 20%-66%) and 28% (95% CI, 8%-52%) (P = .03), respectively. These results demonstrate that DLI after lymphodepleting chemotherapy for relapsed hematologic malignancies results in frequent CRs. The lower DLI dose regimen improved the tolerability of this therapeutic approach, with modest rates of severe aGVHD.
Engineered zinc finger nucleases (ZFNs) are a tool for genome manipulation that are of great interest to scientists in many fields. To meet the needs of researchers wishing to employ ZFNs, an inexpensive, rapid assembly procedure would be beneficial to laboratories that do not have access to the proprietary reagents often required for ZFN production. Using freely available sequence data derived from the Zinc Finger Targeter database, we developed a protocol for synthesis and directed insertion of user-defined ZFNs into a versatile plasmid expression system. This oligonucleotide-based isothermal DNA assembly protocol was used to determine whether we could generate functional nucleases capable of endogenous gene editing. We targeted the human ?-l-iduronidase (IDUA) gene on chromosome 4, mutations of which result in the severe lysosomal storage disease mucopolysaccharidosis type I. In approximately 1 week we were able to design, assemble, and test six IDUA-specific ZFNs. In a single-stranded annealing assay five of the six candidates we tested performed at a level comparable to or surpassing previously reported ZFNs. One of the five subsequently showed nuclease activity at the endogenous genomic IDUA locus. To our knowledge, this is the first demonstration of in silico-designed, oligonucleotide-assembled, synthetic ZFNs, requiring no specialized templates or reagents that are capable of endogenous human gene target site activity. This method, termed CoDA-syn (context-dependent assembly-synthetic), should facilitate a more widespread use of ZFNs in the research community.
Dendritic cells (DCs) competent to express the regulatory enzyme IDO in mice are a small but distinctive subset of DCs. Previously, we reported that a high-dose systemic CpG treatment to ligate TLR9 in vivo induced functional IDO exclusively in splenic CD19(+) DCs, which stimulated resting Foxp3-lineage regulatory T cells (Tregs) to rapidly acquire potent suppressor activity. In this paper, we show that IDO was induced in spleen and peripheral lymph nodes after CpG treatment in a dose-dependent manner. Induced IDO suppressed local T cell responses to exogenous Ags and inhibited proinflammatory cytokine expression in response to TLR9 ligation. IDO induction did not occur in T cell-deficient mice or in mice with defective B7 or programmed death (PD)-1 costimulatory pathways. Consistent with these findings, CTLA4 or PD-1/PD-ligand costimulatory blockade abrogated IDO induction and prevented Treg activation via IDO following high-dose CpG treatment. Consequently, CD4(+)CD25(+) T cells uniformly expressed IL-17 shortly after TLR9 ligation. These data support the hypothesis that constitutive interactions from activated T cells or Tregs and IDO-competent DCs via concomitant CTLA4?B7 and PD-1?PD-ligand signals maintain the default potential to regulate T cell responsiveness via IDO. Acute disruption of these nonredundant interactions abrogated regulation via IDO, providing novel perspectives on the proinflammatory effects of costimulatory blockade therapies. Moreover, interactions between IDO-competent DCs and activated T cells in lymphoid tissues may attenuate proinflammatory responses to adjuvants such as TLR ligands.
Idiopathic pneumonia syndrome (IPS) is a significant cause of morbidity and mortality post-bone marrow transplantation (BMT) in humans. In our established murine IPS model in which lethally conditioned recipients are given allogeneic bone marrow and splenocytes, recruitment of host monocytes occurs early post-BMT, followed by donor T cells concomitant with development of severe lung dysfunction. Because matrix metalloproteinase 12 (MMP12) is important for macrophage infiltration and injury in other mouse models of lung disease such as emphysema, lethally conditioned MMP12(-/-) mice were used as allogeneic recipients to determine whether MMP12 plays a similar role in potentiating lung injury in IPS. Surprisingly, MMP12(-/-) mice developed IPS and exhibited an accelerated allogeneic T cell-dependent decrease in compliance compared with wild-type (WT) recipients. MMP12(-/-), but not WT, mice also had allogeneic T cell-dependent elevated lung resistance post-BMT. Recruitment of monocytes and T cells into the lungs was not altered on day 7 post-BMT, but the lungs of MMP12(-/-) recipients had increased collagen deposition, a feature normally not seen in our IPS model. MMP12(-/-) mice had a compensatory increase in MMP2 in the lungs post-BMT, as well as increased ?6-integrin compared with WT recipients, and only in the presence of allogeneic T cells. Levels of total transforming growth factor (TGF)-?1 protein in the lungs were elevated compared with WT recipients, consistent with the profibrotic function of ?6-integrin as an activator of TGF-?. These data indicate that host-derived MMP12 may be important in limiting development of IPS by allowing proper remodeling of extracellular matrix and effective repair of BMT-related injury.
The Sleeping Beauty (SB) transposon system can insert defined sequences into chromosomes to direct the extended expression of therapeutic genes. Our goal is to develop the SB system for nonviral complementation of Fanconi anemia (FA), a rare autosomal recessive disorder accompanied by progressive bone marrow failure.
Programmed death-1 ligand 1 (PD-L1) is a coinhibitory molecule that negatively regulates multiple tolerance checkpoints. In the NOD mouse model, PD-L1 regulates the development of diabetes. PD-L1 has two binding partners, programmed death-1 and B7-1, but the significance of the PD-L1:B7-1 interaction in regulating self-reactive T cell responses is not yet clear. To investigate this issue in NOD mice, we have compared the effects of two anti-PD-L1 Abs that have different blocking activities. Anti-PD-L1 mAb 10F.2H11 sterically and functionally blocks only PD-L1:B7-1 interactions, whereas anti-PD-L1 mAb 10F.9G2 blocks both PD-L1:B7-1 and PD-L1:programmed death-1 interactions. Both Abs had potent, yet distinct effects in accelerating diabetes in NOD mice: the single-blocker 10F.2H11 mAb was more effective at precipitating diabetes in older (13-wk-old) than in younger (6- to 7-wk-old) mice, whereas the dual-blocker 10F.9G2 mAb rapidly induced diabetes in NOD mice of both ages. Similarly, 10F.2H11 accelerated diabetes in recipients of T cells from diabetic, but not prediabetic mice, whereas 10F.9G2 was effective in both settings. Both anti-PD-L1 mAbs precipitated diabetes in adoptive transfer models of CD4(+) and CD8(+) T cell-driven diabetes. Taken together, these data demonstrate that the PD-L1:B7-1 pathway inhibits potentially pathogenic self-reactive effector CD4(+) and CD8(+) T cell responses in vivo, and suggest that the immunoinhibitory functions of this pathway may be particularly important during the later phases of diabetogenesis.
The programmed death ligand 1 (PDL1)/programmed death 1 (PD1) costimulatory pathway plays an important role in the inhibition of alloimmune responses as well as in the induction and maintenance of peripheral tolerance. It has been demonstrated recently that PDL1 also can bind B7.1 to inhibit T cell responses in vitro. Using the bm12 into B6 heart transplant model, we investigated the functional significance of this interaction in alloimmune responses in vivo. PD1 blockade unlike PDL1 blockade failed to accelerate bm12 allograft rejection, suggesting a role for an additional binding partner for PDL1 other than PD1 in transplant rejection. PDL1 blockade was able to accelerate allograft rejection in B7.2-deficient recipients but not B7.1-deficient recipients, indicating that PDL1 interaction with B7.1 was important in inhibiting rejection. Administration of the novel 2H11 anti-PDL1 mAb, which only blocks the PDL1-B7.1 interaction, aggravated chronic injury of bm12 allografts in B6 recipients. Aggravated chronic injury was associated with an increased frequency of alloreactive IFN-?-, IL-4-, and IL-6-producing splenocytes and a decreased percentage of regulatory T cells in the recipients. Using an in vitro cell culture assay, blockade of the interaction of PDL1 on dendritic cells with B7.1 on T cells increased IFN-? production from alloreactive CD4(+) T cells, whereas blockade of dendritic cell B7.1 interaction with T cell PDL1 did not. These data indicate that PDL1 interaction with B7.1 plays an important role in the inhibition of alloimmune responses in vivo and suggests a dominant direction for PDL1 and B7.1 interaction.
Three main types of CD4+ regulatory T cells can be distinguished based upon whether they express Foxp3 and differentiate naturally in the thymus (natural Tregs) or are induced in the periphery (inducible Tregs); or whether they are FoxP3 negative but secrete IL-10 in response to antigen (Tregulatory type 1, Tr1 cells). Adoptive transfer of each cell type has proven highly effective in mouse models at preventing graft vs. host disease (GVHD) and autoimmunity. Although clinical application was initially hampered by low Treg frequency and unfavorable ex vivo expansion properties, several phase I trials are now being conducted to assess their effect on GVHD following hematopoietic stem cell transplantation (HSCT) and in type I diabetes. Human Treg trials for HSCT recipients have preceded other indications because GVHD onset is precisely known, the time period needed for prevention relatively short, initial efficacy is likely to provide life-long protection, and complications of GVHD can be lethal. This review will summarize the clinical trials conducted to date that have employed Tregs to prevent GVHD following HSCT and discuss recent advances in Treg cellular therapy.
Although rodent graft-versus-host disease (GVHD) models have suggested that indoleamine 2,3-dioxygenase (IDO) is a critical regulator of gastrointestinal GVHD, parallel human studies on IDO expression have not been reported. IDO expression was assessed in 20 patients who underwent duodenal biopsy. IDO was upregulated in epithelial cells. In situ analyses reveal that macrophages and dendritic cells stain positive for IDO, but that most of the IDO(+) cells were a novel population of CD3(+)CD4(+)IDO(+) cells. The proportion of CD4(+)IDO(+) T cells was significantly higher in patients with moderate GVHD. In situ regulatory T cell and Th17 numbers correlated with overall severity. Although needing confirmatory results from larger sample sets, these data are consistent with the hypothesis that IDO is involved in regulating gastrointestinal GVHD.
In mammals, stromal cell-derived factor-1 (SDF-1) promotes hematopoietic cell mobilization and migration. Although the zebrafish, Danio rerio, is an emerging model for studying hematopoietic cell transplantation (HCT), the role of SDF-1 in the adult zebrafish has yet to be determined. We sought to characterize sdf-1 expression and function in the adult zebrafish in the context of HCT. In situ hybridization of adult zebrafish organs shows sdf-1 expression in kidney tubules, gills, and skin. Radiation up-regulates sdf-1 expression in kidney to nearly 4-fold after 40 Gy. Assays indicate that zebrafish hematopoietic cells migrate toward sdf-1, with a migration ratio approaching 1.5 in vitro. A sdf-1a:DsRed2 transgenic zebrafish allows in vivo detection of sdf-1a expression in the adult zebrafish. Matings with transgenic reporters localized sdf-1a expression to the putative hematopoietic cell niche in proximal and distal renal tubules and collecting ducts. Importantly, transplant of hematopoietic cells into myelosuppressed recipients indicated migration of hematopoietic cells to sdf-1a-expressing sites in the kidney and skin. We conclude that sdf-1 expression and function in the adult zebrafish have important similarities to mammals, and this sdf-1 transgenic vertebrate will be useful in characterizing the hematopoietic cell niche and its interactions with hematopoietic cells.
Regulatory T cells (T(reg) cells) are essential for self-tolerance and immune homeostasis. Lack of effector T cell (T(eff) cell) function and gain of suppressive activity by T(reg) cells are dependent on the transcriptional program induced by Foxp3. Here we report that repression of SATB1, a genome organizer that regulates chromatin structure and gene expression, was crucial for the phenotype and function of T(reg) cells. Foxp3, acting as a transcriptional repressor, directly suppressed the SATB1 locus and indirectly suppressed it through the induction of microRNAs that bound the SATB1 3 untranslated region. Release of SATB1 from the control of Foxp3 in T(reg) cells caused loss of suppressive function, establishment of transcriptional T(eff) cell programs and induction of T(eff) cell cytokines. Our data support the proposal that inhibition of SATB1-mediated modulation of global chromatin remodeling is pivotal for maintaining T(reg) cell functionality.
Graft-versus-host disease (GVHD) is a frequent and severe complication after hematopoietic cell transplantation. Natural CD4(+)CD25(+) regulatory T cells (nT(regs)) have proven highly effective in preventing GVHD and autoimmunity in murine models. Yet, clinical application of nT(regs) has been severely hampered by their low frequency and unfavorable ex vivo expansion properties. Previously, we demonstrated that umbilical cord blood (UCB) nT(regs) could be purified and expanded in vitro using good manufacturing practice (GMP) reagents; however, the initial number of nT(regs) in UCB units is limited, and average yield after expansion was only 1 × 10(9) nT(regs). Therefore, we asked whether yield could be increased by using peripheral blood (PB), which contains far larger quantities of nT(regs). PB nT(regs) were purified under GMP conditions and expanded 80-fold to yield 19 × 10(9) cells using anti-CD3 antibody-loaded, cell-based artificial antigen-presenting cells (aAPCs) that expressed the high-affinity Fc receptor and CD86. A single restimulation increased expansion to ~3000-fold and yield to >600 × 10(9) cells while maintaining Foxp3 expression and suppressor function. nT(reg) expansion was ~50 million-fold when flow sort-purified nT(regs) were restimulated four times with aAPCs. Indeed, cryopreserved donor nT(regs) restimulated four times significantly reduced GVHD lethality induced by the infusion of human T cells into immune-deficient mice. The capability to efficiently produce donor cell banks of functional nT(regs) could transform the treatment of GVHD and autoimmunity by providing an off-the-shelf, cost-effective, and proven cellular therapy.
The skin is constantly exposed to environmental insults and requires effective repair processes to maintain its protective function. Wound healing is severely compromised in people with congenital absence of structural proteins of the skin, such as in dystrophic epidermolysis bullosa, a severe congenital mechanobullous disorder caused by mutations in collagen type VII. Remarkably, stem cell transplantation can ameliorate deficiency of this skin-specific structural protein in both animal models and in children with the disorder. Healthy donor cells from the hematopoietic graft migrate to the injured skin; simultaneously, there is an increase in the production of collagen type VII, increased skin integrity, and reduced tendency to blister formation. How hematogenous stem cells from bone marrow and cord blood can alter skin architecture and wound healing in a robust, clinically meaningful way is unclear. We review the data and the resulting hypotheses that have a potential to illuminate the mechanisms for these effects. Further modifications in the use of stem cell transplantation as a durable source of extracellular matrix proteins may make this regenerative medicine approach effective in other cutaneous and extracutaneous conditions.
The goal of this study was to determine if we could establish a mesenchymal stromal line from zebrafish that would support hematopoietic cells. Such a coculture system would be a great benefit to study of the hematopoietic cell-stromal cell interaction in both in vitro and in vivo environments. Zebrafish stromal cells (ZStrC) were isolated from the "mesenchymal" tissue of the caudal tail and expanded in a specialized growth media. ZStrC were evaluated for phenotype, gene expression, and ability to maintain zebrafish marrow cells in coculture experiments. ZStrC showed mesenchymal and endothelial gene expression. Although ZStrC lacked the ability to differentiate into classic mesenchymal stromal cell lineages (i.e., osteocytes, adipocytes, chondrocytes), they did have the capacity for endotube formation on Matrigel and low-density lipoprotein uptake. ZStrC supported marrow cells for >2 weeks in vitro. Importantly, marrow cells were shown to retain homing ability in adoptive transfer experiments. ZStrC were also shown to improve hematopoietic recovery after sublethal irradiation after adoptive transfer. As the zebrafish model grows in popularity and importance in the study of hematopoiesis, new tools to aid in our understanding of the hematopoietic cell-stromal cell interaction are required. ZStrC represent an additional tool in the study of hematopoiesis and will be useful in understanding the factors that mediate the stromal cell-hematopoietic cell interactions that are important in hematopoietic cell maintenance.
Survival rates after allogeneic hematopoietic cell transplantation (HCT) for Fanconi anemia (FA) have increased dramatically since 2000. However, the use of autologous stem cell gene therapy, whereby the patients own blood stem cells are modified to express the wild-type gene product, could potentially avoid the early and late complications of allogeneic HCT. Over the last decades, gene therapy has experienced a high degree of optimism interrupted by periods of diminished expectation. Optimism stems from recent examples of successful gene correction in several congenital immunodeficiencies, whereas diminished expectations come from the realization that gene therapy will not be free of side effects. The goal of the 1st International Fanconi Anemia Gene Therapy Working Group Meeting was to determine the optimal strategy for moving stem cell gene therapy into clinical trials for individuals with FA. To this end, key investigators examined vector design, transduction method, criteria for large-scale clinical-grade vector manufacture, hematopoietic cell preparation, and eligibility criteria for FA patients most likely to benefit. The report summarizes the roadmap for the development of gene therapy for FA.
T cell costimulation is important for T cell activation. The CD27/CD70 pathway contributes to effector and memory T cell development and is involved in T cell and B cell activation. CD27/CD70 is known for having opposing roles during different models of antigenic challenges. During primary T cell responses to influenza virus infection or during tumor challenges, CD27/CD70 costimulation has a positive role on T cell responses. However, during some chronic infections, constitutive triggering of this signaling pathway has a negative role on T cell responses. It is currently unclear what specific characteristic of an antigen determines the outcome of CD27/CD70 costimulation. We investigated the effect of a transient CD70 blockade during an acute or a chronic lymphocytic choriomeningitis virus (LCMV) infection in mice. Blockade of this pathway during acute LCMV infection (Armstrong strain) resulted in delayed T cell responses and decreased CD127 (interleukin-7 receptor ? [IL-7R?] chain) conversion. Upregulation of CD127 is an important event in T cell differentiation that heralds the passage of an effector T cell to a long-lived memory T cell. In contrast to the reduced CD8 T cell responses after CD70 blockade during acute infection, CD70 blockade during chronic LCMV infection resulted in increased CD8 T cell responses. Our data show the dual roles of this costimulatory pathway in acute versus persistent antigen challenge. Our findings suggest that antigen persistence may determine the effect of CD27/CD70 signaling on CD8 T cell responses. Tailored triggering or blockade of this costimulatory pathway may be important in vaccination regimens against acute or chronic pathogens.
Natural killer (NK) cell subsets can be defined by the differential expression of inhibitory receptors for MHC class I molecules. Early after congenic HSCT, we found that Ly49G2(high) single-positive NK cells repopulated, displayed an activated phenotype, and were highly cytolytic. Over time, this subset was replaced with NK cells with a normal pattern of Ly49 expression. Treatment of mice with IL-2 also resulted in the rapid expansion of these Ly49G2(high) single-positive NK cells. Only the Ly49g (Klra7) Pro1 transcript was highly induced in both HSCT- and IL-2-treated recipients. MHC-independent expansion of the Ly49G2(+) subset was also observed after Listeria monocytogenes or mouse cytomegalovirus infection. Our data indicate that during reconstitution after HSCT and various activation stimuli, Ly49G2(+) NK cells represent the "first-responder" NK cells, which occur independently of NK-cell licensing via Ly49-MHC interactions. These data suggest that the inhibitory Ly49G2 receptor represents an activation marker on mouse NK cells under various conditions.
Cytoreductive conditioning regimens used in the context of allogeneic hematopoietic cell transplantation (HCT) elicit deficits in innate and adaptive immunity, which predispose patients to infections. As such, transplantation outcomes depend vitally on the successful reconstruction of immune competence. Restoration of a normal peripheral T-cell pool after HCT is a slow process that requires the de novo production of naive T cells in a functionally competent thymus. However, there are several challenges to this regenerative process. Most notably, advanced age, the cytotoxic pretransplantation conditioning, and posttransplantation alloreactivity are risk factors for T-cell immune deficiency as they independently interfere with normal thymus function. Here, we discuss preclinical allogeneic HCT models and clinical observations that have contributed to a better understanding of the transplant-related thymic dysfunction. The identification of the cellular and molecular mechanisms that control regular thymopoiesis but are altered in HCT patients is expected to provide the basis for new therapies that improve the regeneration of the adaptive immune system, especially with functionally competent, naive T cells.
Tumor-associated immune suppression can lead to defective T cell-mediated antitumor immunity. Here, we identified a unique phenotype of exhausted T cells in mice with advanced acute myelogenous leukemia (AML). This phenotype is characterized by the coexpression of Tim-3 and PD-1 on CD8(+) T cells in the liver, the major first site of AML metastases. PD-1 and Tim-3 coexpression increased during AML progression. PD-1(+)Tim-3(+) CD8(+) T cells were deficient in their ability to produce IFN-?, TNF-?, and IL-2 in response to PD-1 ligand (PDL1) and Tim-3 ligand (galectin-9) expressing AML cells. PD-1 knockout (KO), which were partially resistant to AML challenge, up-regulated Tim-3 during AML progression and such Tim-3(+)PD-1- KO CD8(+) T cells had reduced cytokine production. Galectin-9 KO mice were more resistant to AML, which was associated with reduced T-regulatory cell accumulation and a modest induction of PD-1 and Tim-3 expression on CD8(+) T cells. Whereas blocking the PD-1/PDL1 or Tim-3/galectin-9 pathway alone was insufficient to rescue mice from AML lethality, an additive effect was seen in reducing-albeit not eliminating-both tumor burden and lethality when both pathways were blocked. Therefore, combined PD-1/PDL1 and Tim-3/galectin-9 blockade may be beneficial in preventing CD8(+) T-cell exhaustion in patients with hematologic malignancies such as advanced AML.
Dendritic cells (DCs) are potent antigen-presenting cells derived from hematopoietic progenitor cells and circulating monocytes. To investigate the role of microRNAs (miRNAs) during DC differentiation, maturation, and function, we profiled miRNA expression in human monocytes, immature DCs (imDCs), and mature DCs (mDCs). Stage-specific, differential expression of 27 miRNAs was found during monocyte differentiation into imDCs and mDCs. Among them, decreased miR-221 and increased miR-155 expression correlated with p27(kip1) accumulation in DCs. Silencing of miR-221 or overexpressing of miR-155 in DCs resulted in p27(kip1) protein increase and DC apoptosis. Moreover, mDCs from miR-155(-/-) mice were less apoptotic than those from wild-type mice. Silencing of miR-155 expression had little effect on DC maturation but reduced IL-12p70 production, whereas miR-155 overexpression in mDCs enhanced IL-12p70 production. Kip1 ubiquitination-promoting complex 1, suppressor of cytokine signaling 1, and CD115 (M-CSFR) were functional targets of miR-155. Furthermore, we provide evidence that miR-155 indirectly regulated p27(kip1) protein level by targeting Kip1 ubiquitination-promoting complex 1. Thus, our study uncovered miRNA signatures during monocyte differentiation into DCs and the new regulatory role of miR-221 and miR-155 in DC apoptosis and IL-12p70 production.
Human secondary lymphoid tissues (SLTs) contain interleukin-22 (IL-22)-producing cells with an immature NK phenotype. Given their location, these cells are difficult to study. We have generated large numbers of NK22 cells from hematopoietic stem cells. HSC-derived NK22 cells show a CD56(+)CD117(high)CD94(-) phenotype, consistent with stage III NK progenitors. Like freshly isolated SLT stage III cells, HSC-derived NK22 cells express NKp44, CD161, CCR6, IL1 receptor, AHR, and ROR-??. IL-1? and IL-23 stimulation results in significant IL-22 but not interferon-? production. Supernatant from these cells increases CD54 expression on mesenchymal stem cells. Thus, IL-22-producing NK cells can be generated in the absence of SLT. HSC-derived NK22 cells will be valuable in understanding this rare NK subset and create the opportunity for human translational clinical trials.
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