T-cell tolerance induced by repeated antigen stimulation: Selective loss of Foxp3−conventional CD4 T cells and induction of CD4 T-cell anergy

Abstract Abstract 693 Introduction: Breakdown of humoral tolerance to red blood cell (RBC) antigens can result in autoimmune hemolytic anemia (AIHA), a severe and potentially fatal disease. The pathogenesis of AIHA is poorly understood. To investigate the baseline biology of tolerance to self-antigens expressed on RBCs, we utilized a murine transgenic mouse with RBC-specific expression of a model antigen consisting of a triple fusion protein of hen egg lysozyme (HEL), ovalbumin (Ova), and human blood group molecule Duffy; HEL-OVA-Duffy (HOD mouse). Methods: Wild-type C57BL/6 (B6) mice or HOD mice (on a B6 background) were immunized with HEL/CFA or OVA/CFA to test immune responses to antigens contained within HOD. Some animals were immunized with peptides as opposed to whole protein. Anti-HOD antibodies were quantified by indirect immunofluorescence using HOD RBCs as targets. Anti-HEL IgG was quantified by ELISA and anti-HEL secreting B cells were enumerated by ELISPOT. CD4+ T cell responses were assessed by tetramer staining and tetramer pull-down assays using I-Ab-OVA-329-337/326-334. T cell tolerance was specifically broken by adoptive transfer of OT-II CD4+ T cells into HOD mice (OT-II T cells recognize OVA323-339 presented by I-Ab). Effects of HOD antigen expression on B cell development were evaluated by crossing the HOD mouse with an anti-HEL BCR knockin mouse (SwHEL mouse) that is capable of normal class switching. Results: Immunization of B6 mice with OVA/CFA induced high titer antibodies reactive with HOD RBCs; in contrast, no anti-HOD was detected in HOD mice immunized with OVA/CFA. Similarly, no anti-HEL was detected in HOD mice immunized with HEL/CFA, whereas wild-type B6 mice had high anti-HEL titers (p<0.05). These data demonstrate overall humoral tolerance to the HOD antigen. Using pull-down assays, OVA-tetramer reactive T cells were detected in both B6 and HOD mice, with similar endogenous frequencies (mean numbers are 40 and 53 T cells, respectively; at least 6 mice analyzed), suggesting that central tolerance did not eliminate HOD reactive T cells. However, upon immunization with OVA peptide, B6 but not HOD mice had a detectable expansion of OVA-tetramer reactive CD4+ T cells, indicating that peripheral tolerance was preventing HOD autoreactive CD4+ T cells from participating in an immune response. To assess B cell tolerance to the HOD antigen, T cell tolerance was circumvented through adoptive transfer or OTII splenocytes (specific for the OVA323-339 peptide) into HOD mice. Anti-HEL autoantibodies were detected in HOD mice but not control B6 mice (p<0.001). Antibody production correlated with a 10–20 fold increase of anti-HEL antibody secreting cells, as determined by ELISPOT. Autoantibody production in HOD mice was not due to passenger B cells from the OTII donor, an artifact of excess CD4+ T cell number, or bystander activation as no autoantibodies were observed upon adoptive transfer with OTIIs on a Rag knockout background, irrelevant CD4+ T cells from SMARTA mice, or activated CD4+ T cells from TCR75 mice. To test the effects of HOD antigen expression on development of autoreactive B cells, HOD mice were crossed with SwHEL BCR transgenic mice (that express anti-HEL) and the F1 mice were analyzed. HEL-reactive B cells were visualized using multimeric HEL conjugated to allophycocyanin. In HOD-SwHEL+ mice, approximately 46±14% of immature bone marrow B cells were reactive with HEL, compared to 15±12% in HOD+SwHEL+ mice (p=0.043, 3 independent experiments, 5 mice total). Conclusions: These data demonstrate that tolerance to an RBC specific antigen is complete in the CD4+ T cell, but not the B cell compartment. CD4+ T cell tolerance appears to be more an effect of peripheral tolerance than central deletion, as OVA-tetramer reactive CD4+ T cells were visible in HOD mice but did not activate upon immunization with their cognate antigen. In contrast, while the HODxSwHEL F1 mice demonstrate that some B cell tolerance to HOD occurs, the induction of autoantibodies by introducing CD4+ autoreactive T cells (OT-II) demonstrates that B cell tolerance to the HOD antigen is incomplete in HOD mice. Together, these data suggest that a breakdown in T cell tolerance is all that is required for the pathogenesis of AIHA. As the T cell tolerance appears not to be deletional, it is predicted that environmental factors leading to a breakdown in peripheral tolerance of CD4+ T cells would be sufficient to induce AIHA. Disclosures: Zimring: Immucor Inc,: Research Funding.

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Mechanisms of early peripheral CD4 T-cell tolerance induction by anti-CD154 monoclonal antibody and allogeneic bone marrow transplantation: evidence for anergy and deletion but not regulatory cells

Blood ◽

10.1182/blood-2003-08-2642 ◽

2004 ◽

Vol 103 (11) ◽

pp. 4336-4343 ◽

Cited By ~ 87

Author(s):

Josef Kurtz ◽

Juanita Shaffer ◽

Ariadne Lie ◽

Natalie Anosova ◽

Gilles Benichou ◽

...

Keyword(s):

Monoclonal Antibody ◽

Bone Marrow ◽

T Cells ◽

Bone Marrow Transplantation ◽

T Cell ◽

Cd4 T Cells ◽

Marrow Transplantation ◽

Cd4 T Cell ◽

T Cell Tolerance ◽

Cell Tolerance

Abstract Anti-CD154 (CD40L) monoclonal antibody (mAb) plus bone marrow transplantation (BMT) in mice receiving CD8 cell-depleting mAb leads to long-term mixed hematopoietic chimerism and systemic donor-specific tolerance through peripheral and central deletional mechanisms. However, CD4+ T-cell tolerance is demonstrable in vitro and in vivo rapidly following BMT, before deletion of donor-reactive CD4 cells is complete, suggesting the involvement of other mechanisms. We examined these mechanisms in more detail. Spot enzyme-linked immunosorbent (ELISPOT) analysis revealed specific tolerization (within 4 to 15 days) of both T helper 1 (Th1) and Th2 cytokine responses to the donor, with no evidence for cytokine deviation. Tolerant lymphocytes did not significantly down-regulate rejection by naive donor-reactive T cells in adoptive transfer experiments. No evidence for linked suppression was obtained when skin expressing donor alloantigens in association with third-party alloantigens was grafted. T-cell receptor (TCR) transgenic mixing studies revealed that specific peripheral deletion of alloreactive CD4 T cells occurs over the first 4 weeks following BMT with anti-CD154. In contrast to models involving anti-CD154 without BMT, BMT with anti-CD154 leads to the rapid induction of anergy, followed by deletion of pre-existing donor-reactive peripheral CD4+ T cells; the rapid deletion of these cells obviates the need for a regulatory cell population to suppress CD4 cell-mediated alloreactivity. (Blood. 2004;103:4336-4343)

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T–cell anergy and peripheral T–cell tolerance

Philosophical Transactions of the Royal Society B Biological Sciences ◽

10.1098/rstb.2001.0844 ◽

2001 ◽

Vol 356 (1409) ◽

pp. 625-637 ◽

Cited By ~ 23

Author(s):

Robert Lechler ◽

Jian-Guo Chai ◽

Federica Marelli-Berg ◽

Giovanna Lombardi

Keyword(s):

T Cells ◽

T Cell ◽

T Cell Activation ◽

Cell Contact ◽

T Cell Tolerance ◽

Cell Tolerance ◽

T Cell Anergy ◽

Cell Anergy ◽

Anergic T Cells

The discovery that T–cell recognition of antigen can have distinct outcomes has advanced understanding of peripheral T–cell tolerance, and opened up new possibilities in immunotherapy. Anergy is one such outcome, and results from partial T–cell activation. This can arise either due to subtle alteration of the antigen, leading to a lower–affinity cognate interaction, or due to a lack of adequate co–stimulation. The signalling defects in anergic T cells are partially defined, and suggest that T–cell receptor (TCR) proximal, as well as downstream defects negatively regulate the anergic T cell's ability to be activated. Most importantly, the use of TCR–transgenic mice has provided compelling evidence that anergy is an in vivo phenomenon, and not merely an in vitro artefact. These findings raise the question as to whether anergic T cells have any biological function. Studies in rodents and in man suggest that anergic T cells acquire regulatory properties; the regulatory effects of anergic T cells require cell to cell contact, and appear to be mediated by inhibition of antigen–presenting cell immunogenicity. Close similarities exist between anergic T cells, and the recently defined CD4 + CD25 + population of spontaneously arising regulatory cells that serve to inhibit autoimmunity in mice. Taken together, these findings suggest that a spectrum of regulatory T cells exists. At one end of the spectrum are cells, such as anergic and CD4 + CD25 + T cells, which regulate via cell–to–cell contact. At the other end of the spectrum are cells which secrete antiinflammatory cytokines such as interleukin 10 and transforming growth factor–β. The challenge is to devise strategies that reliably induce T–cell anergy in vivo , as a means of inhibiting immunity to allo– and autoantigens.

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Disruption of STAT3 Signaling in Antigen Presenting Cells Represents a Novel Strategy To Overcome Tumor-Induced Antigen-Specific CD4+ T-Cell Tolerance.

Blood ◽

10.1182/blood.v104.11.109.109 ◽

2004 ◽

Vol 104 (11) ◽

pp. 109-109

Author(s):

Fengdong Cheng ◽

Hongwei Wang ◽

Alex G. Cuenca ◽

Pedro Horna ◽

Lldefonso Suarez ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Signaling Pathway ◽

Tumor Antigen ◽

Cd4 T Cells ◽

Cd4 T Cell ◽

T Cell Tolerance ◽

Cell Tolerance ◽

Tumor Bearing ◽

Stat3 Signaling

Abstract Tumor antigen-specific T-cell tolerance imposes a significant barrier to the development of effective therapeutic cancer vaccines. Bone marrow-derived antigen presenting cells (APCs) are critical in the induction of this unresponsive state. Recently, we have identified STAT3 signaling in APCs as an important regulatory pathway that determines the functional outcome of antigen-specific CD4+ T-cells in response to cognate antigen. Indeed, while disruption of this signaling pathway in APCs led to effective T cell priming, enhanced STAT3 activity resulted in the induction of T cell unresponsiveness1. Given the above results, we explored in this study whether disruption of STAT3 signaling in APCs may preserve the responsiveness of antigen-specific CD4+ T-cells during the growth of a tumor that induces antigen specific T-cell tolerance. First, mice with a genetic disruption of Stat3 in macrophages, neutrophils and a sub-population of myeloid DCs (LysMcre/Stat3flox/− mice) or control C57BL/6 mice were given subcutaneously 1x106 B16 melanoma tumor cells engineered to express Ovalbumin as a model tumor antigen (B16-OVA). Four days later, naive CD4+ T-cells (1x106) specific for a MHC class II-restricted epitope of Ovalbumin (OT-II cells) were adoptively transferred intravenously into tumor bearing mice as well as into tumor-free controls. Two weeks later animals were sacrificed and antigen-specific CD4+ T-cell responses to in vitro re-stimulation with OVA-peptide were evaluated. As expected, antigen-specific T cells re-isolated from tumor-bearing C57BL/6 mice were fully tolerant (lack of HA-specific proliferation and cytokine production). In sharp contrast, anti-OVA CD4+ T-cells isolated from tumor bearing LysMcre/Stat3flox/− mice remained fully functional as determined by their capacity to proliferate and produce IL-2 and IFN-gamma in response to cognate OVA-peptide. The demonstration that tumor-induced antigen-specific CD4+ T-cell tolerance occurs in mice with an intact STAT3 signaling in APCs, but not in mice with genetic disruption of this signaling pathway, led us next to evaluate the efficacy of pharmacologic inhibitors of STAT3 in preventing and or overcoming tumor-induced T-cell tolerance. In vivo treatment of tumor bearing mice with Tyrphostin AG490 (0.5 mg/ i.p. /twice a day x 5 days), a well-known inhibitor of STAT3 signaling, also resulted in preservation of the responsiveness of tumor-antigen specific CD4+ T-cells. Furthermore, in vitro treatment of APCs with this compound led to effective priming of naive antigen-specific T cells and breaking of antigen-specific T-cell anergy. More recently, we have evaluated the efficacy of a novel STAT3 inhibitor, compound 295558, which efficiently inhibits the DNA-binding activity of STAT3. Treatment of DCs or macrophages with this specific inhibitor led to the generation of inflammatory APCs capable of restoring the responsiveness of tolerized CD4+ T-cells isolated from tumor bearing mice. Taken together, our findings establish a critical role for STAT3 signaling in the induction of tolerance to tumor antigens in vivo. Inhibition of this signaling pathway in APCs provides a novel molecular target to overcome the remarkable barrier that tolerance to tumor antigens imposes to cancer vaccination strategies.

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Correction: T‐cell tolerance induced by repeated antigen stimulation: Selective loss of Foxp3−conventional CD4 T cells and induction of CD4 T‐cell anergy

European Journal of Immunology ◽

10.1002/eji.200990034 ◽

2009 ◽

Vol 39 (6) ◽

pp. 1682-1682

Author(s):

Lena Eroukhmanoff ◽

Cecilia Oderup ◽

Fredrik Ivars

Keyword(s):

T Cells ◽

Cd4 T Cells ◽

Cell Tolerance ◽

Antigen Stimulation ◽

Selective Loss ◽

Cell Anergy

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Interleukin-10 induces a long-term antigen-specific anergic state in human CD4+ T cells.

Journal of Experimental Medicine ◽

10.1084/jem.184.1.19 ◽

1996 ◽

Vol 184 (1) ◽

pp. 19-29 ◽

Cited By ~ 549

Author(s):

H Groux ◽

M Bigler ◽

J E de Vries ◽

M G Roncarolo

Keyword(s):

T Cells ◽

T Cell ◽

Cd4 T Cells ◽

Interleukin 10 ◽

Necrosis Factor Alpha ◽

T Cell Anergy ◽

Factor Alpha ◽

Cell Anergy ◽

Anergic T Cells ◽

Macrophage Colony

Human CD4+ T cells, activated by allogeneic monocytes in a primary mixed lymphocyte reaction in the presence of exogenous interleukin (IL) 10, specifically failed to proliferate after restimulation with the same alloantigens. A comparable state of T cell unresponsiveness could be induced by activation of CD4+ T cells by cross-linked anti-CD3 monoclonal antibodies (mAbs) in the presence of exogenous IL-10. The anergic T cells failed to produce IL-2, IL-5, IL-10, interferon gamma, tumor necrosis factor alpha, and granulocyte/macrophage colony-stimulating factor. The IL-10-induced anergic state was long-lasting. T cell anergy could not be reversed after restimulation of the cells with anti-CD3 and anti-CD28 mAbs, although CD3 and CD28 expression was normal. In addition, restimulation of anergized T cells with anti-CD3 mAbs induced normal Ca2+ fluxes and resulted in increased CD3, CD28, and class II major histocompatibility complex expression, indicating that calcineurin-mediated signaling occurs in these anergic cells. However, the expression of the IL-2 receptor alpha chain was not upregulated, which may account for the failure of exogenous IL-2 to reverse the anergic state. Interestingly, anergic T cells and their nonanergic counterparts showed comparable levels of proliferation and cytokine production after activation with phorbol myristate acetate and Ca2+ ionophore, indicating that a direct activation of a protein kinase C-dependent pathway can overcome the tolerizing effect of IL-10. Taken together, these data demonstrate that IL-10 induces T cell anergy and therefore may play an important role in the induction and maintenance of antigen-specific T cell tolerance.

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T1212 Upregulation of E3 Ubiquitin Ligases Related to T Cell Anergy in CD4+ T Cells Isolated from Patients with Ulcerative Colitis in Remission

Gastroenterology ◽

10.1016/s0016-5085(08)62369-0 ◽

2008 ◽

Vol 134 (4) ◽

pp. A-508

Author(s):

Satoshi Egawa ◽

Hideki Iijima ◽

Shinichiro Shinzaki ◽

Sachiko Nakajima ◽

Jumpei Kondo ◽

...

Keyword(s):

Ulcerative Colitis ◽

T Cells ◽

T Cell ◽

Cd4 T Cells ◽

Ubiquitin Ligases ◽

E3 Ubiquitin Ligases ◽

T Cell Anergy ◽

Cell Anergy

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T cell anergy in perinatal mice is promoted by T reg cells and prevented by IL-33

Journal of Experimental Medicine ◽

10.1084/jem.20182002 ◽

2019 ◽

Vol 216 (6) ◽

pp. 1328-1344 ◽

Cited By ~ 8

Author(s):

Jonatan Tuncel ◽

Christophe Benoist ◽

Diane Mathis

Keyword(s):

T Cells ◽

T Cell ◽

Cd4 T Cells ◽

High Expression ◽

T Cell Anergy ◽

Tissue Antigens ◽

Cell Anergy ◽

Deficient Mice

Perinatal T cells broadly access nonlymphoid tissues, where they are exposed to sessile tissue antigens. To probe the outcome of such encounters, we examined the defective elimination of self-reactive clones in Aire-deficient mice. Nonlymphoid tissues were sequentially seeded by distinct waves of CD4+ T cells. Early arrivers were mostly Foxp3+ regulatory T (T reg) cells and metabolically active, highly proliferative conventional T cells (T conv cells). T conv cells had unusually high expression of PD-1 and the IL-33 receptor ST2. As T conv cells accumulated in the tissue, they gradually lost expression of ST2, ceased to proliferate, and acquired an anergic phenotype. The transition from effector to anergic state was substantially faster in ST2-deficient perinates, whereas it was abrogated in IL-33–treated mice. A similar dampening of anergy occurred after depletion of perinatal T reg cells. Attenuation of anergy through PD-1 blockade or IL-33 administration promoted the immediate breakdown of tolerance and onset of multiorgan autoimmunity. Hence, regulating IL-33 availability may be critical in maintaining T cell anergy.

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Regulatory T Cells Are Not Required for Prevention of RBC-Specific Autoantibody Generation

Blood ◽

10.1182/blood.v124.21.568.568 ◽

2014 ◽

Vol 124 (21) ◽

pp. 568-568

Author(s):

Krystalyn E. Hudson ◽

James C. Zimring

Keyword(s):

T Cells ◽

T Cell ◽

Regulatory T Cells ◽

Cd4 T Cells ◽

T Cell Tolerance ◽

Cell Tolerance ◽

Tolerance Mechanisms ◽

Antibody Treatment ◽

Isotype Control

Introduction: Loss of humoral tolerance to red blood cell (RBC) antigens may lead to the generation of pathogenic autoantibodies and result in autoimmune hemolytic anemia (AIHA), a severe and potentially fatal disease. Failure of tolerance to RBC antigens occurs with considerable frequency (1-3 cases/1,000 adults) and prevalence of AIHA is as high as 30% in persons with compromised B and/or T cell tolerance mechanisms. However, RBC-specific tolerance mechanisms are poorly understood. To elucidate the immune tolerances to RBC autoantigens, we utilized HOD mice. The HOD mouse expresses an RBC-specific transgene consisting of hen egg lysozyme (HEL), ovalbumin (OVA), and Duffy. Using the HOD model, we previously demonstrated B cell tolerance to RBC-specific HOD antigen is incomplete; however, T cell tolerance is stringent. HOD mice have similar detectable frequencies of HOD-specific CD4+ T cells compared to B6 mice. Although present, autoreactive HOD-specific CD4+ T cells are non-functional. Circumventing T cell tolerance by adoptive transfer, HOD mice make high titer anti-HOD autoantibodies in vivo. Thus, despite the presence of autoreactive B cells, no HOD-reactive antibodies are detectable unless CD4+ T cells are given, indicating T cell tolerance is a stopgap to autoimmunity. Methods: Leukocytes from C57BL/6 (B6) and HOD mice were harvested and OVA-specific CD4+ T cell responses were assessed by tetramer-pulldown assays with pooled tetramers I-Ab-OVA 329-337/326-334. Isolated cells were stained for surface and intracellular markers and analyzed via flow cytometry. For in vivo analysis, mice were treated with 300ug anti-CD25 (clone PC-61) depleting antibody or isotype control; a subset of antibody-treated mice was immunized with OVA/CFA. Antibodies bound to HOD RBCs were determined by direct antibody test. Anti-HOD antibodies were quantified by indirect immunofluorescence using HOD RBCs as targets. Results: Tetramer pull-down assays revealed similar numbers of OVA-reactive CD4+ T cells from HOD and B6 mice (mean 56 and 40, respectively, p = 0.3). However, cell surface and intracellular marker staining demonstrated that HOD mice had higher numbers of OVA-tetramer reactive CD4+ T cells that express regulatory markers CD25 and FoxP3, and exhaustion marker PD1 as compared to control B6 mice. Inhibitory CTLA4 expression was not detectable on OVA-reactive CD4+ T cells from HOD or B6 mice. To test whether regulatory T cells were required for RBC-specific immune tolerance, HOD and B6 mice were treated with CD25 depleting antibody or isotype control antibody. Anti-CD25 antibody treated mice had a significant reduction of CD25+ cells 4 days post treatment (p < 0.001, 2 independent experiments). Similarly, there was a significant reduction in FoxP3+CD25+CD4+ T cells (Tregs) in anti-CD25 treated mice (p < 0.001), compared to isotype. Mice received weekly injections of anti-CD25 or isotype antibody to maintain depletion for one month. A subset of mice received an OVA/CFA immunization. Sustained CD25+ depletion did not result in anti-HOD autoantibody generation. Further, there was no change in the endogenous frequency of OVA-reactive CD4+ T cells between HOD and B6 mice, regardless of antibody treatment. Similarly, HOD mice treated with depletion (or isotype) antibody and immunized with OVA/CFA did not make detectable anti-HOD autoantibodies. Consistent with lack of detectable autoantibodies, no expansion of OVA-tetramer reactive CD4+ T cells was observed in HOD mice. In contrast, B6 mice (treated with anti-CD25 or isotype antibody) had a detectable expansion of OVA-specific CD4+ T cells as a result of immunization. Conclusions: The data demonstrate a phenotypic difference between the OVA-reactive CD4+ T cells from HOD and B6 mice, with an increase in number of Tregs detectable in HOD mice. Administration of anti-CD25 antibody significantly reduced the number of overall CD25+ cells and Tregs. Prolonged depletion of these cellular subsets did not elicit autoantibodies in HOD mice. Further, immunization of CD25 depleted mice with a strong immune stimulus (OVA/CFA, known to expand OVA-reactive T cells in B6 mice), did not induce anti-HOD autoantibodies nor did it expand OVA-specific autoreactive CD4+ T cells in HOD mice. Together, these data demonstrate that CD25+ cells are not required for the maintenance of RBC-specific T cell tolerance and suggest a role for other regulatory mechanisms. Disclosures No relevant conflicts of interest to declare.

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