retroviral-transduction-bone-marrow-progenitor-cells-to-generate-t

Retroviral Transduction of Bone Marrow Progenitor Cells to Generate T-cell Receptor Retrogenic Mice

T cell receptor (TCR) signaling is essential in the development and differentiation of T cells in the thymus and periphery, respectively.  The vast array ...

streamlined-single-cell-tcr-isolation-generation-retroviral-vectors

Streamlined Single Cell TCR Isolation and Generation of Retroviral Vectors for <em>In Vitro </em>and <em>In Vivo</em> Expression of Human TCRs

Although, several methods for sequencing of paired T cell receptor (TCR) alpha and beta chains from single T cells have been developed, none so far have ...

Baylor College of Medicine

Baylor College of Medicine, Texas Children's Hospital

Disruption of intestinal barrier function, followed by increased antigen load, may possibly trigger joint inflammation. In seronegative spondyloarthritis (SpA) both gut inflammation and altered intestinal permeability have been reported. We evaluated the influence of Helicobacter pylori and nonsteroidal antiinflammatory drugs (NSAID) on gastrointestinal (GI) permeability in SpA. Twenty SpA patients (7 women, mean age 47 +/- 13 SD yrs), 30 patients with endoscopic gastritis (EndG; 17 women, mean age 48 +/- 14 yrs), and 35 healthy controls (16 women, mean age 40 +/- 15 yrs) were studied. No patient was undergoing antisecretory therapy. In the SpA group, 8 patients were chronically taking NSAID and 12 took NSAID occasionally, none during the month before the study. All subjects were assessed for gastroduodenal (sucrose) and intestinal (lactulose/mannitol) permeability test and H. pylori status (urea breath test).

Effect of Helicobacter pylori and eradication therapy on gastrointestinal permeability. Implications for patients with seronegative spondyloarthritis.

XIncreasing evidence supports a role for CD8+ T cells in multiple sclerosis. In an attempt to isolate the contribution of CD8+ T cells in a murine model of MS, we immunized mice with a dominant CD8 epitope MOG37-46, a truncated version of MOG35-55. The data presented here show mild disease induced with MOG37-46, characterized by lower clinical scores, a decrease in CNS infiltration and a decrease in microglial activation. CD8+ T cells reactive to MOG37-46 are pro-inflammatory and traffic to the CNS; however, the presence of CD4+ T cells elicits more severe disease and sustained inflammation of the CNS.

Pathogenic MOG-reactive CD8+ T cells require MOG-reactive CD4+ T cells for sustained CNS inflammation during chronic EAE.

Type 1 diabetes is a T cell-mediated autoimmune disease, characterized by lymphocytic infiltration of the pancreatic islets. It is currently thought that islet antigen specificity is not a requirement for islet entry and that diabetogenic T cells can recruit a heterogeneous bystander T cell population. We tested this assumption directly by generating T cell receptor (TCR) retrogenic mice expressing two different T cell populations. By combining diabetogenic and nondiabetogenic or nonautoantigen-specific T cells, we demonstrate that bystander T cells cannot accumulate in the pancreatic islets. Autoantigen-specific T cells that accumulate in islets, but do not cause diabetes, were also unaffected by the presence of diabetogenic T cells. Additionally, 67% of TCRs cloned from nonobese diabetic (NOD) islet-infiltrating CD4(+) T cells were able to mediate cell-autonomous islet infiltration and/or diabetes when expressed in retrogenic mice. Therefore, islet entry and accumulation appears to be a cell-autonomous and tightly regulated event and is governed by islet antigen specificity.

T cell islet accumulation in type 1 diabetes is a tightly regulated, cell-autonomous event.

Foxp3(+) regulatory T cells (T(regs)) contribute significantly to the maintenance of peripheral tolerance, but they ultimately fail in autoimmune diseases. The events that lead to T(reg) failure in controlling autoreactive effector T cells (T(effs)) during autoimmunity are not completely understood. In this review, we discuss possible mechanisms for this subversion as they relate to type 1 diabetes (T1D) and multiple sclerosis (MS). Recent studies emphasize firstly, the role of inflammatory cytokines, such as IL-6, in inhibiting or subverting T(reg) function; secondly, the issue of T(reg) plasticity; thirdly, the possible resistance of autoimmune T cells to T(reg)-mediated control; and fourthly, T(reg)-associated inhibitory cytokines TGFbeta, IL-10 and IL-35 in facilitating T(reg) suppressive activity and promoting T(reg) generation. These recent advances place a large emphasis on the local tissue specific inflammatory environment as it relates to T(reg) function and disease development.

Regulatory T cells and inhibitory cytokines in autoimmunity.

To fully examine the functionality of a regulatory T cell (T(reg)) population, one needs to assess their ability to suppress in a variety of in vivo models. We describe five in vivo models that examine the suppressive capacity of T(regs) upon different target cell types. The advantages and disadvantages of each model including resources, time, and technical expertise required to execute each model are also described.

In vivo Treg suppression assays.

Lymphocyte activation gene-3 (LAG-3; CD223) is a CD4 homolog that is required for maximal regulatory T cell function and for the control of CD4(+) and CD8(+) T cell homeostasis. Lag3(-)(/)(-) NOD mice developed substantially accelerated diabetes with 100% incidence. Adoptive transfer experiments revealed that LAG-3 was primarily responsible for limiting the pathogenic potential of CD4(+) T cells and, to a lesser extent, CD8(+) T cells. Lag3(-)(/)(-) mice exhibited accelerated, invasive insulitis, corresponding to increased CD4(+) and CD8(+) T cell islet infiltration and intraislet proliferation. The frequencies of islet Ag-reactive chromogranin A-specific CD4(+) T cells and islet specific glucose-6-phosphatase-specific CD8(+) T cells were significantly increased in the islets of Lag3(-)(/)(-) mice, suggesting an early expansion of pathogenic clones that is normally restrained by LAG-3. We conclude that LAG-3 is necessary for regulating CD4(+) and CD8(+) T cell function during autoimmune diabetes, and thus may contribute to limiting autoimmunity in disease-prone environments.

Cutting edge: accelerated autoimmune diabetes in the absence of LAG-3.

The destruction of beta cells in type 1 diabetes in humans and in autoimmune diabetes in the NOD mouse model is a consequence of chronic islet inflammation in the pancreas. The T cell-driven autoimmune response is initiated by environmental triggers which are influenced by the state of intestinal homeostasis and the microbiota. The disease process can be separated into two phases: firstly, initiation of mild, controlled, long-term infiltration and secondly, propagation of invasive inflammation which quickly progresses to beta cell deletion and autoimmune diabetes. In this review, we will discuss the cellular and molecular triggers that might be required for these two phases in the context of other issues including the unique anatomical location of pancreas, the location of T cell priming, the requirements for islet entry, and the events that ultimately drive beta cell destruction and the onset of diabetes.

T cell-driven initiation and propagation of autoimmune diabetes.

The T-cell receptor (TCR) is unique in its complexity. It determines not only positive (life) and negative (death) selection in the thymus, but also mediates proliferation, anergy, differentiation, cytotoxicity and cytokine production in the periphery. Through its association with six CD3 signalling chains (εγ, δε and ζζ), the TCR is capable of recognizing an extensive variety of antigenic peptides, from both pathogens and self-antigens, and translating these interactions into multiple signalling pathways that mediate diverse T-cell developmental and functional responses. The analysis of TCR biology has been revolutionized by the development of TCR transgenic mice, which express a single clonotypic T-cell population, with diverse specificities and genetic backgrounds. However, they are time consuming to generate and characterize, limiting the analysis of large numbers of TCR over a short period of time in multiple genetic backgrounds. The recent development of TCR retrogenic technology resolves these limitations and could in time have a similarly important impact on our understanding of T-cell development and function. In this review, we will discuss the advantages and limitations of retrogenic technology compared with the generation and use of TCR transgenic mice for studying all aspects of T-cell biology.

T-cell receptor retrogenic mice: a rapid, flexible alternative to T-cell receptor transgenic mice.

Interleukin (IL)-35 is a newly identified inhibitory cytokine used by T regulatory cells to control T cell-driven immune responses. However, the therapeutic potential of native, biologically active IL-35 has not been fully examined. Expression of the heterodimeric IL-35 cytokine was targeted to β-cells via the rat insulin promoter (RIP) II. Autoimmune diabetes, insulitis, and the infiltrating cellular populations were analyzed. Ectopic expression of IL-35 by pancreatic β-cells led to substantial, long-term protection against autoimmune diabetes, despite limited intraislet IL-35 secretion. Nonobese diabetic RIP-IL35 transgenic mice exhibited decreased islet infiltration with substantial reductions in the number of CD4(+) and CD8(+) T cells, and frequency of glucose-6-phosphatase catalytic subunit-related protein-specific CD8(+) T cells. Although there were limited alterations in cytokine expression, the reduced T-cell numbers observed coincided with diminished T-cell proliferation and G1 arrest, hallmarks of IL-35 biological activity. These data present a proof of principle that IL-35 could be used as a potent inhibitor of autoimmune diabetes and implicate its potential therapeutic utility in the treatment of type 1 diabetes.

Prevention of autoimmune diabetes by ectopic pancreatic β-cell expression of interleukin-35.

Regulatory T (Treg) cells, driven by the Foxp3 transcription factor, are responsible for limiting autoimmunity and chronic inflammation. We showed that a well-characterized Foxp3(gfp) reporter mouse, which expresses an N-terminal GFP-Foxp3 fusion protein, is a hypomorph that causes profoundly accelerated autoimmune diabetes on a NOD background. Although natural Treg cell development and in vitro function are not markedly altered in Foxp3(gfp) NOD and C57BL/6 mice, Treg cell function in inflammatory environments was perturbed and TGF-β-induced Treg cell development was reduced. Foxp3(gfp) was unable to interact with the histone acetyltransferase Tip60, the histone deacetylase HDAC7, and the Ikaros family zinc finger 4, Eos, which led to reduced Foxp3 acetylation and enhanced K48-linked polyubiquitylation. Collectively this results in an altered transcriptional landscape and reduced Foxp3-mediated gene repression, notably at the hallmark IL-2 promoter. Loss of controlled Foxp3-driven epigenetic modification leads to Treg cell insufficiency that enables autoimmunity in susceptible environments.

Loss of epigenetic modification driven by the Foxp3 transcription factor leads to regulatory T cell insufficiency.

The use of retrogenic mice offers a rapid and flexible approach to T cell receptor (TCR)-transgenic mice. By transducing bone marrow progenitor cells with a retrovirus that encodes a given TCR-α/β subunit, TCR-retrogenic mice can be generated in as few as 4-6 weeks, whereas conventional TCR transgenics can take 6 months or longer. In this updated protocol, we have increased the efficiency of the bone marrow transduction and bone marrow reconstitution compared with our previously published protocol. The main departure from the previous protocol is the implementation of spin transduction with the viral supernatant instead of coculture with the viral producer cell line. The changes in this protocol improve bone marrow viability, increase consistency of the bone marrow transduction and bone marrow engraftment, and they reduce the ratio of bone marrow donor mice to bone marrow recipients.

Generation of T cell receptor-retrogenic mice: improved retroviral-mediated stem cell gene transfer.

Autoreactive T cells infiltrating the target organ can possess a broad TCR affinity range. However, the extent to which such biophysical parameters contribute to T cell pathogenic potential remains unclear. In this study, we selected eight InsB9-23-specific TCRs cloned from CD4(+) islet-infiltrating T cells that possessed a relatively broad range of TCR affinity to generate NOD TCR retrogenic mice. These TCRs exhibited a range of two-dimensional affinities (∼10(-4)-10(-3) μm(4)) that correlated with functional readouts and responsiveness to activation in vivo. Surprisingly, both higher and lower affinity TCRs could mediate potent insulitis and autoimmune diabetes, suggesting that TCR affinity does not exclusively dictate or correlate with diabetogenic potential. Both central and peripheral tolerance mechanisms selectively impinge on the diabetogenic potential of high-affinity TCRs, mitigating their pathogenicity. Thus, TCR affinity and multiple tolerance mechanisms converge to shape and broaden the diabetogenic T cell repertoire, potentially complicating efforts to induce broad, long-term tolerance.

TCR Affinity and Tolerance Mechanisms Converge To Shape T Cell Diabetogenic Potential.

Single-cell paired TCR identification is a powerful tool, but has been limited in its previous incompatibility with further functional analysis. The current protocol describes a method to clone and functionally evaluate in vivo TCRs derived from single antigen-responsive human T cells and monoclonal T cell lines. We have improved upon current PCR-based TCR sequencing protocols by developing primers that allow amplification of human TCRα and TCRβ variable regions, while incorporating specific restriction cut sites for direct subcloning into the template retroviral vector. This streamlined approach for generating human:mouse chimeric TCR vectors allows for rapid TCR expression in humanized-retrogenic (hu-Rg) mice through retroviral mediated stem cell gene transfer. Using widely available techniques and equipment, this method is easily adaptable by most laboratories. This is the first TCR identification protocol that is efficiently combined with subsequent in vivo TCR expression.

Rapid identification and expression of human TCRs in retrogenic mice.

Inhibitory receptors (IRs) are pivotal in controlling T cell homeostasis because of their intrinsic regulation of conventional effector T (Tconv) cell proliferation, viability, and function. However, the role of IRs on regulatory T cells (Tregs) remains obscure because they could be required for suppressive activity and/or limit Treg function. We evaluated the role of lymphocyte activation gene 3 (LAG3; CD223) on Tregs by generating mice in which LAG3 is absent on the cell surface of Tregs in a murine model of type 1 diabetes. Unexpectedly, mice that lacked LAG3 expression on Tregs exhibited reduced autoimmune diabetes, consistent with enhanced Treg proliferation and function. Whereas the transcriptional landscape of peripheral wild-type (WT) and Lag3-deficient Tregs was largely comparable, substantial differences between intra-islet Tregs were evident and involved a subset of genes and pathways that promote Treg maintenance and function. Consistent with these observations, Lag3-deficient Tregs outcompeted WT Tregs in the islets but not in the periphery in cotransfer experiments because of enhanced interleukin-2-signal transducer and activator of transcription 5 signaling and increased Eos expression. Our study suggests that LAG3 intrinsically limits Treg proliferation and function at inflammatory sites, promotes autoimmunity in a chronic autoimmune-prone environment, and may contribute to Treg insufficiency in autoimmune disease.

LAG3 limits regulatory T cell proliferation and function in autoimmune diabetes.

Type 1 diabetes is a T cell-mediated autoimmune disease that is characterized by Ag-specific targeting and destruction of insulin-producing β cells. Although multiple studies have characterized the pathogenic potential of β cell-specific T cells, we have limited mechanistic insight into self-reactive autoimmune T cell development and their escape from negative selection in the thymus. In this study, we demonstrate that ectopic expression of insulin epitope B:9-23 (InsB9-23) by thymic APCs is insufficient to induce deletion of high- or low-affinity InsB9-23-reactive CD4(+) T cells; however, we observe an increase in the proportion and number of thymic and peripheral Foxp3(+) regulatory T cells. In contrast, the MHC stable insulin mimetope (InsB9-23 R22E) efficiently deletes insulin-specific T cells and prevents escape of high-affinity thymocytes. Collectively, these results suggest that Ag dose and peptide-MHC complex stability can lead to multiple fates of insulin-reactive CD4(+) T cell development and autoimmune disease outcome.

ArticleTotal : 2	Year
Publication title Cited by 11	2016
Publication title Cited by 3	2017

Article	Year
Effect of Helicobacter pylori and eradication therapy on gastrointestinal permeability. Implications for patients with seronegative spondyloarthritis. The Journal of rheumatology\| PubMed ID: 15693091	2005
Pathogenic MOG-reactive CD8+ T cells require MOG-reactive CD4+ T cells for sustained CNS inflammation during chronic EAE. Journal of neuroimmunology\| PubMed ID: 19540601	2009
T cell islet accumulation in type 1 diabetes is a tightly regulated, cell-autonomous event. Immunity\| PubMed ID: 19818656	2009
Regulatory T cells and inhibitory cytokines in autoimmunity. Current opinion in immunology\| PubMed ID: 19854631	2009
In vivo Treg suppression assays. Methods in molecular biology (Clifton, N.J.)\| PubMed ID: 21287333	2011
Cutting edge: accelerated autoimmune diabetes in the absence of LAG-3. Journal of immunology (Baltimore, Md. : 1950)\| PubMed ID: 21873518	2011
T cell-driven initiation and propagation of autoimmune diabetes. Current opinion in immunology\| PubMed ID: 22056379	2011
T-cell receptor retrogenic mice: a rapid, flexible alternative to T-cell receptor transgenic mice. Immunology\| PubMed ID: 22348644	2012
Prevention of autoimmune diabetes by ectopic pancreatic β-cell expression of interleukin-35. Diabetes\| PubMed ID: 22427377	2012
Loss of epigenetic modification driven by the Foxp3 transcription factor leads to regulatory T cell insufficiency. Immunity\| PubMed ID: 22579476	2012
Generation of T cell receptor-retrogenic mice: improved retroviral-mediated stem cell gene transfer. Nature protocols\| PubMed ID: 24008379	2013
TCR Affinity and Tolerance Mechanisms Converge To Shape T Cell Diabetogenic Potential. Journal of immunology (Baltimore, Md. : 1950)\| PubMed ID: 24943217	2014
Rapid identification and expression of human TCRs in retrogenic mice. Journal of immunological methods\| PubMed ID: 27589924	2016
LAG3 limits regulatory T cell proliferation and function in autoimmune diabetes. Science immunology\| PubMed ID: 28783703	2017
Ectopic Expression of Self-Antigen Drives Regulatory T Cell Development and Not Deletion of Autoimmune T Cells. Journal of immunology (Baltimore, Md. : 1950)\| PubMed ID: 28835461	2017

Maria Bettini

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