quantification-neurovascular-protection-following-repetitive-hypoxic

Quantification of Neurovascular Protection Following Repetitive Hypoxic Preconditioning and Transient Middle Cerebral Artery Occlusion in Mice

Experimental animal models of stroke are invaluable tools for understanding stroke pathology and developing more effective treatment strategies. A 2 week ...

University of Texas Southwestern Medical Center

Multiple sclerosis (MS) is an inflammatory, demyelinating disease of the central nervous system (CNS) with features suggestive of T-cell-mediated pathology. Most prior reports have focused on CD4(+) T cells with the underlying assumption that MS is predominantly a CD4(+) T helper 1 (Th1)-mediated disease. In this report, we used a novel flow cytometric approach to evaluate autoreactive T-cell responses against a large variety of neuroantigenic targets. We found that both CD4(+) and CD8(+) T cells targeted against several CNS autoantigens were widely prevalent in patients with MS and healthy individuals. Whereas the distribution of CD4(+) responses was similar in different groups, patients with relapsing-remitting MS showed a higher proportion of CNS-specific CD8(+) responses. Autoreactive CD4(+) T cells from patients with MS exhibited a more differentiated Th1 phenotype compared with healthy subjects. Similarly, CNS-specific CD8(+) T-cell responses from patients with MS were functionally distinct from those in healthy individuals. Collectively, these studies reveal the high prevalence of class I-restricted autoreactive CD8(+) T-cell responses in MS that has been underappreciated thus far. The results emphasize the need to evaluate both CD4(+) and CD8(+) T-cell responses in MS and to make both subsets a consideration in the development of novel therapeutic strategies.

High prevalence of autoreactive, neuroantigen-specific CD8+ T cells in multiple sclerosis revealed by novel flow cytometric assay.

In the setting of autoimmunity, one of the goals of successful therapeutic immune modulation is the induction of peripheral tolerance, a large part of which is mediated by regulatory/suppressor T cells. In this report, we demonstrate a novel immunomodulatory mechanism by an FDA-approved, exogenous peptide-based therapy that incites an HLA class I-restricted, cytotoxic suppressor CD8+ T cell response. We have shown previously that treatment of multiple sclerosis (MS) with glatiramer acetate (GA; Copaxone) induces differential up-regulation of GA-reactive CD8+ T cell responses. We now show that these GA-induced CD8+ T cells are regulatory/suppressor in nature. Untreated patients show overall deficit in CD8+ T cell-mediated suppression, compared with healthy subjects. GA therapy significantly enhances this suppressive ability, which is mediated by cell contact-dependent mechanisms. CD8+ T cells from GA-treated patients and healthy subjects, but not those from untreated patients with MS, exhibit potent, HLA class I-restricted, GA-specific cytotoxicity. We further show that these GA-induced cytotoxic CD8+ T cells can directly kill CD4+ T cells in a GA-specific manner. Killing is enhanced by preactivation of target CD4+ T cells and may depend on presentation of GA through HLA-E. Thus, we demonstrate that GA therapy induces a suppressor/cytotoxic CD8+ T cell response, which is capable of modulating in vivo immune responses during ongoing therapy. These studies not only explain several prior observations relating to the mechanism of this drug but also provide important insights into the natural immune interplay underlying this human immune-mediated disease.

Therapeutic induction of regulatory, cytotoxic CD8+ T cells in multiple sclerosis.

We have demonstrated that GA therapy induces a differential upregulation of GA-specific, cytotoxic/suppressor CD8+ T-cell responses in MS patients. We utilized a novel combination of flow sorting and anchored PCR to analyze the evolving clonal composition of GA-specific CD4+ and CD8+ T-cells. TCRbeta chain analysis revealed the development of an oligoclonal GA-specific CD8+ repertoire with persistence of dominant clones over long periods. Interestingly, some sequences resembled published oligoclonal CD8+ TCR sequences from MS lesions. In contrast, GA-specific CD4+ responses were polyclonal and showed continual evolution of their repertoire. This clonotypic and functional analysis provides mechanistic insights into GA therapy.

Glatiramer acetate (GA) therapy induces a focused, oligoclonal CD8+ T-cell repertoire in multiple sclerosis.

CD4(+)CD25(+)FOXP3(+) regulatory T-cells (T(regs)) form an important arm of the immune system responsible for suppressing untoward immune responses. T(regs) can be thymically derived or peripherally induced, even from CD4(+)CD25(-)FOXP3(-) T-cells. FOXP3 expression and in vitro suppressive activity are considered unique hallmarks of this dedicated and stable lineage of regulatory cells. Here we show that virtually all human CD4(+)CD25(-)FOXP3(-) T-cells and CD8(+)CD25(-)FOXP3(-) T-cells attain a transient FOXP3(+)CD25(+) state during activation. In this state of activation, these cells possess the classic phenotype of T(regs), in that they express similar markers and inhibit in vitro proliferation of autologous CD4(+)CD25(-) T-cells. This state is characterized by suppressed IFN-gamma production and robust TNF-alpha and IL-10 production. Interestingly, the great majority of the activated cells eventually downregulate FOXP3 expression, with a concomitant drop in suppressive ability. Our results show that, in humans, FOXP3 expression and T(reg) functionality are not exclusive features of a stable or unique lineage of T-cells but may also be a transient state attained by almost all T-cells. These results warrant caution in interpreting human studies using FOXP3 and suppressive activity as readouts and suggest that attempts to induce "T(regs)" may paradoxically result in induction of effector T-cells, unless stability is confirmed.

Transient regulatory T-cells: a state attained by all activated human T-cells.

Graft-versus-host disease (GVHD) is a major cause of transplant-related morbidity and mortality in recipients of allogeneic hematopoietic stem cell transplantation. As GVHD is mediated predominantly by alloreactive donor T cells, selective allodepletion from the graft may alleviate GVHD, whereas potentially maintaining other advantages conferred by donor T cells, such as graft survival, antiviral immunity, and graft-versus-leukemia effect. In this study, we evaluated the ability of methotrexate, a clinically approved antimetabolite drug, to deplete alloreactive T cells in HLA-mismatched mixed lymphocyte reactions (MLR). We observed that methotrexate could inhibit the proliferation of alloreactive T cells in primary in vitro MLR. On reexposure of methotrexate-treated cells to the same allostimulus, a significant reduction in the alloreactive immune response was observed, whereas responses to third-party allostimuli and viral antigens were preserved. Thus, our results provide preclinical evidence that in vitro methotrexate treatment results in specific allodepletion and may be used as an effective agent for preventing GVHD.

In vitro methotrexate as a practical approach to selective allodepletion.

Immune-based self-recognition and failure to modulate this response are believed to contribute to the debilitating autoimmune pathology observed in multiple sclerosis (MS). Studies from its murine model, experimental autoimmune encephalomyelitis (EAE), have shown that neuroantigen-specific CD4+T cells are capable of inducing disease, while their immune sibling, the CD8+T cells, have largely been ignored. To understand their role in autoimmune demyelination, we first confirmed that, similar to our observations in human MS, there is robust induction of neuroantigen-reactive CD8+T cells in several models, including MOG(35-55)/CFA-induced EAE. However, MOG(35-55)-specific CD8+T-cells, when purified, were unable to adoptively transfer disease into naïve mice (in contrast to CD4+T-cells). In fact, we observed that the transfer of these neuroantigen-specific CD8+T cells was able to suppress the induction of EAE and to inhibit ongoing EAE. These regulatory CD8+T cells produced IFN-gamma and perforin and were able to kill MOG loaded CD4+T-cells as well as CD4-depleted APC, suggesting a cytotoxic/suppressor mechanism. Inhibition of EAE was associated with both the modulation of APC function as well as decreased MOG-specific CD4+T cell responses. Our studies reveal a novel and unexpected immune regulatory function for neuroantigen-specific CD8+T cells and have interesting biologic and therapeutic implications.

Immune regulatory CNS-reactive CD8+T cells in experimental autoimmune encephalomyelitis.

Multiple sclerosis (MS) is an inflammatory, demyelinating disease of the central nervous system (CNS). MS is thought to be T-cell-mediated, with prior research predominantly focusing on CD4+ T-cells. There is a high prevalence of CNS-specific CD8+ T-cell responses in MS patients and healthy subjects. However, the role of neuroantigen-specific CD8+ T-cells in MS is poorly understood, with the prevalent notion that these may represent pathogenic T-cells. We show here that healthy subjects and MS patients demonstrate similar magnitudes of CD8+ and CD4+ T-cell responses to various antigenic stimuli. Interestingly, CD8+ T-cells specific for CNS autoantigens, but not those specific for control foreign antigens, exhibit immune regulatory ability, suppressing proliferation of CD4+CD25- T-cells when stimulated by their cognate antigen. While CD8+ T-cell-mediated immune suppression is similar between healthy subjects and clinically quiescent treatment-naïve MS patients, it is significantly deficient during acute exacerbation of MS. Of note, the recovery of neuroantigen-specific CD8+ T-cell suppression correlates with disease recovery post-relapse. These studies reveal a novel immune suppressor function for neuroantigen-specific CD8+ T-cells that is clinically relevant in the maintenance of peripheral tolerance and the intrinsic regulation of MS immune pathology.

Neuroantigen-specific CD8+ regulatory T-cell function is deficient during acute exacerbation of multiple sclerosis.

Patients with multiple sclerosis (MS) show a high prevalence of myelin-reactive CD8+ and CD4+ T-cell responses, which are the putative effectors/modulators of CNS neuropathology. Utilizing a novel combination of short-term culture, CFSE-based sorting and anchored PCR, we evaluated clonal compositions of neuroantigen-targeting T-cells from RRMS patients and controls. CDR3 region analysis of TCRβ chains revealed biased use of specific TCRBV-bearing CD4+ clones. CD8+ clones showed homology to published TCR from CNS-infiltrating T-cells in MS lesions. These studies are the first description of TCR usage of CNS-specific CD8+ T-cells and provide insights into their potential regulatory role in disease.

Clonal composition of neuroantigen-specific CD8+ and CD4+ T-cells in multiple sclerosis.

The autoimmune disease systemic lupus erythematosus is characterized by loss of tolerance to nuclear Ags and a heightened inflammatory environment, which together result in end organ damage. Lyn-deficient mice, a model of systemic lupus erythematosus, lack an inhibitor of B-cell and myeloid cell activation. This results in B-cell hyper-responsiveness, plasma cell accumulation, autoantibodies, and glomerulonephritis (GN). IL-21 is associated with autoimmunity in mice and humans and promotes B-cell differentiation and class switching. Here, we explore the role of IL-21 in the autoimmune phenotypes of lyn(-/-) mice. We find that IL-21 mRNA is reduced in the spleens of lyn(-/-) IL-6(-/-) and lyn(-/-) Btk(lo) mice, neither of which produce pathogenic autoantibodies or develop significant GN. While IL-21 is dispensable for plasma cell accumulation and IgM autoantibodies in lyn(-/-) mice, it is required for anti-DNA IgG antibodies and some aspects of T-cell activation. Surprisingly, GN still develops in lyn(-/-) IL-21(-/-) mice. This likely results from the presence of IgG autoantibodies against a limited set of non-DNA Ags. These studies identify a specific role for IL-21 in the class switching of anti-DNA B cells and demonstrate that neither IL-21 nor anti-DNA IgG is required for kidney damage in lyn(-/-) mice.

IL-21 promotes the production of anti-DNA IgG but is dispensable for kidney damage in lyn-/- mice.

Multiple sclerosis (MS) is an immune-mediated demyelinating disease of the CNS, and CD8 T cells are the predominant T cell population in MS lesions. Given that transfer of CNS-specific CD8 T cells results in an attenuated clinical demyelinating disease in C57BL/6 mice with immunization-induced experimental autoimmune encephalomyelitis (EAE), we investigated the cellular targets and mechanisms of autoreactive regulatory CD8 T cells. In this study we report that myelin oligodendrocyte glycoprotein peptide (MOG35-55)-induced CD8 T cells could also attenuate adoptively transferred, CD4 T cell-mediated EAE. Whereas CD8(-/-) mice exhibited more severe EAE associated with increased autoreactivity and inflammatory cytokine production by myelin-specific CD4 T cells, this was reversed by adoptive transfer of MOG-specific CD8 T cells. These autoregulatory CD8 T cells required in vivo MHC class Ia (K(b)D(b)) presentation. Interestingly, MOG-specific CD8 T cells could also suppress adoptively induced disease using wild-type MOG35-55-specific CD4 T cells transferred into K(b)D(b-/-) recipient mice, suggesting direct targeting of encephalitogenic CD4 T cells. In vivo trafficking analysis revealed that autoregulatory CD8 T cells are dependent on neuroinflammation for CNS infiltration, and their suppression/cytotoxicity of MOG-specific CD4 T cells is observed both in the periphery and in the CNS. These studies provide important insights into the mechanism of disease suppression mediated by autoreactive CD8 T cells in EAE.

The disease-ameliorating function of autoregulatory CD8 T cells is mediated by targeting of encephalitogenic CD4 T cells in experimental autoimmune encephalomyelitis.

Repetitive hypoxic preconditioning (RHP) creates an anti-inflammatory phenotype that protects from stroke-induced injury for months after a 2-week treatment. The mechanisms underlying long-term tolerance are unknown, though one exposure to hypoxia significantly increased peripheral B cell representation. For this study, we sought to determine if RHP specifically recruited B cells into the protected ischemic hemisphere, and whether RHP could phenotypically alter B cells prior to stroke onset.

Repetitive hypoxic preconditioning induces an immunosuppressed B cell phenotype during endogenous protection from stroke.

Multiple sclerosis (MS) is an inflammatory, demyelinating disease of the central nervous system (CNS). Although its etiology remains unknown, pathogenic T cells are thought to underlie MS immune pathology. We recently showed that MS patients harbor CNS-specific CD8+ Tregs that are deficient during disease relapse. We now demonstrate that CNS-specific CD8+ Tregs were cytolytic and could eliminate pathogenic CD4+ T cells. These CD8+ Tregs were present primarily in terminally differentiated (CD27-, CD45RO-) subset and their suppression was IFNγ, perforin and granzyme B-dependent. Interestingly, MS patients with acute relapse displayed a significant loss in terminally differentiated CD8+ T cells, with a concurrent loss in expression of perforin and granzyme B. Pre-treatment of exacerbation-derived CD8+ T cells with IL-12 significantly restored suppressive capability of these cells through upregulation of granzyme B. Our studies uncover immune-suppressive mechanisms of CNS-specific CD8+ Tregs, and may contribute to design of novel immune therapies for MS.

Disease exacerbation of multiple sclerosis is characterized by loss of terminally differentiated autoregulatory CD8+ T cells.

Experimental autoimmune encephalomyelitis (EAE) is a well-established murine model of multiple sclerosis, an immune-mediated demyelinating disorder of the central nervous system (CNS). We have previously shown that CNS-specific CD8+ T cells (CNS-CD8+) ameliorate EAE, at least in part through modulation of CNS-specific CD4+ T cell responses. In this study, we show that CNS-CD8+ also modulate the function of CD11c+ dendritic cells (DC), but not other APCs such as CD11b+ monocytes or B220+ B cells. DC from mice receiving either myelin oligodendrocyte glycoprotein-specific CD8+ (MOG-CD8+) or proteolipid protein-specific CD8+ (PLP-CD8+) T cells were rendered inefficient in priming T cell responses from naïve CD4+ T cells (OT-II) or supporting recall responses from CNS-specific CD4+ T cells. CNS-CD8+ did not alter DC subset distribution or MHC class II and CD86 expression, suggesting that DC maturation was not affected. However, the cytokine profile of DC from CNS-CD8+ recipients showed lower IL-12 and higher IL-10 production. These functions were not modulated in the absence of immunization with CD8-cognate antigen, suggesting an antigen-specific mechanism likely requiring CNS-CD8-DC interaction. Interestingly, blockade of IL-10 in vitro rescued CD4+ proliferation and in vivo expression of IL-10 was necessary for the suppression of EAE by MOG-CD8+. These studies demonstrate a complex interplay between CNS-specific CD8+ T cells, DC and pathogenic CD4+ T cells, with important implications for therapeutic interventions in this disease.

Article	Year
High prevalence of autoreactive, neuroantigen-specific CD8+ T cells in multiple sclerosis revealed by novel flow cytometric assay. Blood\| PubMed ID: 14976054	2004
Therapeutic induction of regulatory, cytotoxic CD8+ T cells in multiple sclerosis. Journal of immunology (Baltimore, Md. : 1950)\| PubMed ID: 16709875	2006
Glatiramer acetate (GA) therapy induces a focused, oligoclonal CD8+ T-cell repertoire in multiple sclerosis. Journal of neuroimmunology\| PubMed ID: 16935352	2006
Transient regulatory T-cells: a state attained by all activated human T-cells. Clinical immunology (Orlando, Fla.)\| PubMed ID: 17185041	2007
In vitro methotrexate as a practical approach to selective allodepletion. Biology of blood and marrow transplantation : journal of the American Society for Blood and Marrow Transplantation\| PubMed ID: 17531774	2007
Immune regulatory CNS-reactive CD8+T cells in experimental autoimmune encephalomyelitis. Journal of autoimmunity\| PubMed ID: 20172692	2010
Neuroantigen-specific CD8+ regulatory T-cell function is deficient during acute exacerbation of multiple sclerosis. Journal of autoimmunity\| PubMed ID: 21257291	2011
Clonal composition of neuroantigen-specific CD8+ and CD4+ T-cells in multiple sclerosis. Journal of neuroimmunology\| PubMed ID: 21397340	2011
IL-21 promotes the production of anti-DNA IgG but is dispensable for kidney damage in lyn-/- mice. European journal of immunology\| PubMed ID: 23169140	2013
The disease-ameliorating function of autoregulatory CD8 T cells is mediated by targeting of encephalitogenic CD4 T cells in experimental autoimmune encephalomyelitis. Journal of immunology (Baltimore, Md. : 1950)\| PubMed ID: 23733879	2013
Repetitive hypoxic preconditioning induces an immunosuppressed B cell phenotype during endogenous protection from stroke. Journal of neuroinflammation\| PubMed ID: 24485041	2014
Disease exacerbation of multiple sclerosis is characterized by loss of terminally differentiated autoregulatory CD8+ T cells. Clinical immunology (Orlando, Fla.)\| PubMed ID: 24657764	2014
Neuroantigen-specific autoregulatory CD8+ T cells inhibit autoimmune demyelination through modulation of dendritic cell function. PloS one\| PubMed ID: 25144738	2014

Sterling B. Ortega

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