Lymphopenic Recipients Research Articles

Natural killer cells and cytotoxic T lymphocytes are required to clear solid tumor in a patient-derived xenograft.

Existing patient-derived xenograft (PDX) mouse models of solid tumors lack a fully tumor donor–matched, syngeneic, and functional immune system. We developed a model that overcomes these limitations by engrafting lymphopenic recipient mice with a fresh, undisrupted piece of solid tumor, whereby tumor-infiltrating lymphocytes (TILs) persisted in the recipient mice for several weeks. Successful tumor engraftment was achieved in 83% to 89% of TIL-PDX mice, and these were seen to harbor exhausted immuno-effector as well as functional immunoregulatory cells persisting for at least 6 months postengraftment. Combined treatment with interleukin-15 stimulation and immune checkpoint inhibition resulted in complete or partial tumor response in this model. Further, depletion of cytotoxic T lymphocytes and/or natural killer cells before combined immunotherapy revealed that both cell types were required for maximal tumor regression. Our TIL-PDX model provides a valuable resource for powerful mechanistic and therapeutic studies in solid tumors.

Recipient expression of C-type lectin receptor Mincle promotes rapid T cell homeostatic recovery following antibody mediated lymphoablation

Abstract Antibody-mediated lymphocyte depletion is widely used in transplantation. We recently showed that T cell recovery in mouse heart allograft recipients treated with anti-thymocyte globulin (mATG) depends on B cells activated via CD40 and is mediated by B cell-derived IL-1β and IL-6. Mincle (Clec-4e) is a FcRγ-coupled C-type lectin receptor that binds ligands released by pathogens or damaged cells and signals through the Card-9 pathway. While Mincle functions are well characterized in myeloid cells, its significance in B cells remains to be determined. Our goal was to investigate the role of Mincle in initiating B cell cytokine production in lymphopenic heart allograft recipients. The NanoString and RT-PCR analyses of B cells showed that mATG depletion resulted in B cell up-regulation of Mincle and Card-9 gene expression. Flow cytometry confirmed cell surface Mincle expression in B cells from mATG-treated recipients but not from naïve mice. Treatment with anti-CD154 mAb in addition to mATG decreased the B cell expression of Mincle, IL-1β, IL-6 and TNF-α. Moreover, B cell IL-1β and TNF-α expression were reduced in mATG treated Mincle KO allograft recipients compared to WT controls. Most importantly, Mincle KO recipients treated with mATG had severely impaired T cell recovery and significantly prolonged allograft survival compared to WT recipients. We propose that B cells activation via CD40 results in up-regulation of cell surface Mincle. Endogenous ligands released from necrotic graft cells bind to Mincle thus inducing B cells to secrete proinflammatory cytokines and facilitate CD8+ T cell proliferation. These data identify Mincle as a novel therapeutic target to improve the efficacy of lymphoablation in allograft recipients.

Marginal zone B cells induce T cell homeostatic proliferation following antibody-mediated lymphoablation

Abstract Antibody-mediated lymphoablation is commonly used in clinical transplantation. Using mouse model of heart transplantation and murine anti-thymocyte globulin (mATG) lymphoablation, we previously showed that B cell-derived IL-1β and IL-6 are critical mediators of homeostatic T cell proliferation. While marginal zone (MZ) B cells are best known as important sensors and amplifiers of inflammation, their role in depletion-induced lymphopenia has not been previously addressed. The goal of this study was to test the contribution of distinct B cell subsets to proinflammatory cytokine production and T cell homeostatic reconstitution. B6 (H-2b) recipients of BALB/c (H-2d) heart allografts were treated with mATG (d. 0, 4). At d. 8 posttransplant, the proportion of IL-1β, IL-6 and TNF-α secreting cells was significantly higher within MZ vs follicular (FO) B cell subset. Depletion of both FO and MZ B cells with anti-CD20 mAb in addition to mATG (d. -3, 11) significantly diminished T cell reconstitution and prolonged heart allograft survival compared to mATG treatment alone. Notably, specific MZ B cell depletion with anti-VLA4 and anti-LFA1α mAbs (d. -4, -2) resulted in similarly impaired T cells recovery along with improved heart allograft survival. Consistent with these findings, mATG treated Notch2flox/flox CD19Cre/+ mice, which lack MZ B cells but contain normal numbers of FO B cells, had delayed T cell recovery and prolonged heart allograft survival compared to WT recipients. These results show the critical role of MZ B cells in stimulating homeostatic T cell proliferation in lymphopenic heart allograft recipients and suggest that transient removal of MZ B cells may improve the efficacy of lymphoablative induction therapies.

Persistent Dengue Infection in an Immunosuppressed Patient Reveals the Roles of Humoral and Cellular Immune Responses in Virus Clearance

Detailed understanding of the roles of humoral and cellular immune responses in sterilizing dengue virus (DENV) infection in humans is required to inform effective vaccine development. We report an unusual case of persistent DENV infection in a lymphopenic renal transplant recipient who was therapeutically immunosuppressed to prevent organ rejection. Following resolution of symptomatic dengue, this patient remained positive for DENV3 RNA in the blood for 4months and viruric up to 9months post-infection despite demonstrable levels of serum neutralizing antibodies throughout this period. Full resolution of DENV infection instead coincided with recovery of CD8+ Tcell counts during reversal from lymphopenia. Taken collectively, our observations suggest a role for cellular immunity in sterilizing DENV infection in humans. Any dengue vaccine should thus be able to induce both humoral and cellular immunity that respectively prevent symptomatic infection and enable effective viral clearance.

Induction of acute graft vs. host disease in lymphopenic mice

Allogeneic hematopoietic stem cell transplantation (HSCT) is a potentially life-saving treatment for refractory/relapsing hematological malignancies, blood disorders or autoimmune diseases. However, approximately 40–50% of patients undergoing allogeneic HSCT will develop a multi-organ, inflammatory disorder called acute graft vs. host disease (aGVHD). Experimental and clinical studies suggest that intestinal injury due to toxic, pre-transplant conditioning protocols (e.g. lethal irradiation and/or chemotherapy) may play a major role in the development of aGVHD. However, recent studies from our laboratory suggest that this may not be the case. The objective of this study was to quantify and compare the onset and severity of aGVHD induced by the adoptive transfer of allogeneic T cells into untreated lymphopenic mice. Four million allogeneic or syngeneic CD4+CD62L+CD25− T cells were transferred (i.p.) into NK cell-depleted RAG1-/- mice or RAG2-/-IL2rγ-/-double knock-out (DKO) mice and assessed daily for signs of aGVHD. We found that adoptive transfer of allogeneic but not syngeneic T cells into NK cell-depleted RAG1-/- or DKO mice induced many of the clinical and histological features of aGVHD including weight loss, inflammatory cytokine production and tissue inflammation. In addition, adoptive transfer of allogeneic T cells into each recipient induced severe anemia as well as dramatic reductions in bone marrow and spleen cellularity. Taken together, we conclude that allogeneic CD4+ T cells are both necessary and sufficient to induce aGVHD in lymphopenic recipients in the absence of toxic, pre-transplant conditioning.

CD4+ T Cells are both necessary and sufficient to induce acute graft vs. host disease in lymphopenic recipients

Abstract Allogeneic hematopoietic stem cell transplantation (HSCT) is a potentially life-saving treatment for refractory/relapsing hematological malignancies, blood disorders or autoimmune diseases. However, approximately 40–50% of patients undergoing allogeneic HSCT will develop a multi-organ, inflammatory disorder called acute graft vs. host disease (aGVHD). Experimental and clinical studies suggest that intestinal injury due to toxic, pre-transplant conditioning protocols (e.g. lethal irradiation and/or chemotherapy) may play a major role in the development of aGVHD. However, recent studies from our laboratory as well as others, suggest that this may not be the case. Objective We wished to quantify and compare the onset and severity of aGVHD induced by the adoptive transfer of allogeneic T cells into untreated lymphopenic mice. Methods Four million allogeneic or syngeneic CD4+CD62L+CD25− T cells were transferred (i.p.) into NK cell-depleted RAG1−/− mice or RAG2−/−IL2rγ−/− double knock-out (DKO) mice and assessed daily for signs of aGVHD. Results We found that adoptive transfer of allogeneic but not syngeneic T cells into NK cell-depleted RAG1−/− or DKO mice induced many of the clinical and histological features of aGVHD including weight loss, inflammatory cytokine production and tissue inflammation. In addition, adoptive transfer of allogeneic T cells into each recipient induced severe anemia as well as dramatic reductions in bone marrow and spleen cellularity. Conclusions We conclude that naïve allogeneic CD4+T cells are both necessary and sufficient to induce aGVHD in lymphopenic recipients in the absence of toxic, pre-transfer conditioning.

Nod2 Deficiency Augments Th17 Responses and Exacerbates Autoimmune Arthritis.

Arthritis in a genetically susceptible SKG strain of mice models a theoretical paradigm wherein autoimmune arthritis arises because of interplay between preexisting autoreactive T cells and environmental stimuli. SKG mice have a point mutation in ZAP-70 that results in attenuated TCR signaling, altered thymic selection, and spontaneous production of autoreactive T cells that cause arthritis following exposure to microbial β-glucans. In this study, we identify Nod2, an innate immune receptor, as a critical suppressor of arthritis in SKG mice. SKG mice deficient in Nod2 (Nod2-/-SKG) developed a dramatically exacerbated form of arthritis, which was independent of sex and microbiota, but required the skg mutation in T cells. Worsened arthritis in Nod2-/-SKG mice was accompanied by expansion of Th17 cells, which to some measure coproduced TNF, GM-CSF, and IL-22, along with elevated IL-17A levels within joint synovial fluid. Importantly, neutralization of IL-17A mitigated arthritis in Nod2-/-SKG mice, indicating that Nod2-mediated protection occurs through suppression of the Th17 response. Nod2 deficiency did not alter regulatory T cell development or function. Instead, Nod2 deficiency resulted in an enhanced fundamental ability of SKG CD4+ T cells (from naive mice) to produce increased levels of IL-17 and to passively transfer arthritis to lymphopenic recipients on a single-cell level. These data reveal a previously unconsidered role for T cell-intrinsic Nod2 as an endogenous negative regulator of Th17 responses and arthritogenic T cells. Based on our findings, future studies aimed at understanding a negative regulatory function of Nod2 within autoreactive T cells could provide novel therapeutic strategies for treatment of patients with arthritis.

NK Cells Regulate CD8 T Cell-Mediated Allogeneic Rejection in Immunocompetent Recipients across an MHC Class I Mismatched Barrier

Non-T natural killer (NK) cells are a population of granular lymphoid cells TcR- CD3− CD122+ (NK1.1+ in C57BL/6 mice and DX5+ in Balb/c mice). These innate lymphoid cells mature in the bone marrow and are responsible for killing viral-infected and transformed cells in a non-MHC restricted manner, without the need of prior sensitization due to a recognition mechanism based on the array of germline-encoded inhibitory and activating receptors expressed on the cell surface of NK cells [1]. NK cells modulate CD8 T cell expansion during homeostatic proliferation in response to lymphopenic conditions by killing activated T cells and competing for IL-15 with the proliferating CD8 effector T cells [2]. This NK cell behavior may respond to an adaptive evolutionary mechanism to prevent aggressive anti-viral immune responses during the acute phase of infection and thus attenuate immunopathology [3]. We set up an experimental mouse model of alloreactivity across an MHC class I barrier to evaluate the role of NK cells in CD8 T cell-mediated rejection of bm1 skin allografts in naïve B6 recipients. In this transplant setting, host CD8 T cells are the main effectors T cells involved in the rejection of class I MHC mismatched skin allografts preferentially through the direct pathway of antigen presentation with limited help from CD4 T cells primed by the indirect pathway of antigen presentation. As opposed to previous studies done in lymphopenic immunodeficient recipients [2], depletion of NK cells was performed in immunocompetent C57BL/6 recipients with or without CD8 T depletion, the former displaying homeostatic proliferation in response to lymphopenia due to antibody-mediated elimination of CD8 T cells. We observed that depletion of NK cells in both CD8-repleted and CD8-depleted naïve C57BL/6 immunocompetent mice accelerated significantly bm1 graft rejection compared to non-NK-depleted controls (p<0.0005) (Figure 1). Our data claims for precaution in solid organ transplantation under inducing protocols of extensive depletion of leukocytes, such as ATG or alentuzumab. The immunotherapeutic interventions that include the removal of NK cells may accelerate graft rejection and promote aggressive CD8 T cell cytotoxic responses, which could be refractory to current immunosuppression. It also suggests that enhancing NK-cell functional activity combined with CD8 T cell depletion under tolerogenic regimen that tolerizes CD4 T cells at the time of transplantation may contribute to allogeneic donor-specific tolerance induction by elimination of the direct pathway of antigen presentation and by regulating the recovery of CD8 T cells.Bibliographic

Nod2 is a negative regulator of arthritogenic T cell responses in SKG mice

Abstract Nod2 is a pattern recognition receptor known for its role in antimicrobial immunity. Intriguingly, NOD2 appears to have a role outside of infection, as a single point mutation in this molecule leads to 100% incidence of an inflammatory arthritis called Blau Syndrome. Here we investigate the role of Nod2 in regulation of arthritogenic CD4+ T cells in SKG mice. SKG mice have a mutation in ZAP-70 that results in altered thymic selection and spontaneous production of arthritogenic T cells. Nod2−/− SKG mice have exacerbated arthritis and increased numbers of CD4+IL-17+ (Th17) cells that co-produce TNF and GM-CSF compared to SKG mice. Additionally, Nod2−/− SKG mice have augmented levels of IL-17A in their joint synovial fluid. Exacerbated disease in Nod2−/− SKG mice is independent of sex or SKG-mediated dysbiosis as shown by co-housing studies, but dependent on the skg-mutation. Naïve (non-arthritic) Nod2−/− SKG mice have similar numbers of conventional CD4+ T cells with unaltered Treg frequency or function compared to SKG mice. However, CD4+ T cells isolated from naïve Nod2−/− SKG mice produce increased levels of pro-inflammatory cytokines (IL-17, IFNγ) in response to TCR-dependent or -independent stimulus. Furthermore, transfer of CD4+ T cells isolated from naïve Nod2−/−SKG mice into lymphopenic (Nude) recipients results in worsened arthritis compared to an analogous transfer of SKG-CD4+ T cells. These data suggest a previously unappreciated function of Nod2 as a negative regulator of the proinflammatory capacity and pathogenesis of autoreactive CD4+ T cells in SKG mice. Understanding how Nod2 participates in immune tolerance mechanisms will contribute to our knowledge of the pathophysiology of autoimmunity and arthritis.

CD4+CD25+ T Regulatory Cells in Transplantation Tolerance: 25 Years On.

In the 1970s, the capacity of T cells to inhibit immunity and those from transplant tolerant hosts to transfer alloantigen-specific suppression to lymphopenic recipients was described. CD4 T suppressor cells that ex vivo reverted to effector cells were described in the 1980s. Their antigen-specific suppressor function could be preserved by stimulation by specific donor alloantigen and cytokines from activated lymphocytes. This led to the finding that alloantigen-specific T suppressor cells express IL-2 receptor (CD25) and that IL-2 in part promotes their survival.Whether these alloantigen-specific CD4CD25FOXP3 regulatory T (Treg) cells are progeny of thymic-derived CD4CD25FOXP3 Treg (tTreg) cells or are induced from peripheral effector CD4CD25FOXP3T cells (iTreg) cells is still debated.In vitro studies of antigen specific Treg has been difficult as they die in the absence of cytokines produced by immune activated cells. The antigen-specific CD4CD25 T cells that control rejection in tolerant hosts differ from the naive tTreg. Thymic-derived Treg cells are not antigen-specific, and very high ratios are required to suppress rejection. Thymic-derived Treg cells can be expanded ex vivo and are currently being tested in trials to control allograft rejection and graft versus host disease.Thymic-derived Treg cells with specific receptors for alloantigen are activated by either IL-2 or IL-4 but rapidly become dependent on other cytokines, respectively, IFN-γ or IL-12 if activated by IL-2, or IL-5 if activated by IL-4. The Th1 and Th2 pathways for activation of tTreg produce more potent antigen-specific Treg that only suppress specific donor rejection. After 25 years, much remains unknown about antigen-specific CD4CD25FOXP3 Treg-mediating transplant tolerance.

Dopamine Receptor D3 Signaling on CD4+ T Cells Favors Th1- and Th17-Mediated Immunity.

Dopamine receptor D3 (DRD3) expressed on CD4(+) T cells is required to promote neuroinflammation in a murine model of Parkinson's disease. However, how DRD3 signaling affects T cell-mediated immunity remains unknown. In this study, we report that TCR stimulation on mouse CD4(+) T cells induces DRD3 expression, regardless of the lineage specification. Importantly, functional analyses performed in vivo using adoptive transfer of OVA-specific OT-II cells into wild-type recipients show that DRD3 deficiency in CD4(+) T cells results in attenuated differentiation of naive CD4(+) T cells toward the Th1 phenotype, exacerbated generation of Th2 cells, and unaltered Th17 differentiation. The reciprocal regulatory effect of DRD3 signaling in CD4(+) T cells favoring Th1 generation and impairing the acquisition of Th2 phenotype was also reproduced using in vitro approaches. Mechanistic analysis indicates that DRD3 signaling evokes suppressor of cytokine signaling 5 expression, a negative regulator of Th2 development, which indirectly favors acquisition of Th1 phenotype. Accordingly, DRD3 deficiency results in exacerbated eosinophil infiltration into the airways of mice undergoing house dust mite-induced allergic response. Interestingly, our results show that, upon chronic inflammatory colitis induced by transfer of naive CD4(+) T cells into lymphopenic recipients, DRD3 deficiency not only affects Th1 response, but also the frequency of Th17 cells, suggesting that DRD3 signaling also contributes to Th17 expansion under chronic inflammatory conditions. In conclusion, our findings indicate that DRD3-mediated signaling in CD4(+) T cells plays a crucial role in the balance of effector lineages, favoring the inflammatory potential of CD4(+) T cells.

Long-term CD4 lymphopenia is associated with accelerated decline of kidney allograft function

Persistent CD4 T-cell lymphopenia after kidney transplantation has been associated with an increased occurrence of opportunistic infections, malignancies and even mortality, but studies have focussed only on the first few years after kidney transplantation. In this study, we investigated the risk factors and clinical significance of long-term profound CD4 lymphopenia detected ≥10 years after renal transplantation. Between 2007 and 2010, 6206 CD4 T-cell counts, including 1507 counts <300/mm(3), were identified in an active cohort of 1876 kidney transplant patients. We identified 27 HIV-negative lymphopenic kidney transplant recipients out of 513 patients with graft survival over 10 years. We compared this cohort to 54 non-lymphopenic controls matched for the date of kidney transplantation. The prevalence of CD4 lymphopenia 10 years after transplantation was 5.3%. CD4 T-cell lymphopenia was associated with significantly lower thymic output and with B-cell lymphopenia (P < 0.05). The duration of pre-transplant dialysis, but not the use of lymphopenic induction or recipient age, was significantly associated with a persistent CD4 lymphopenia (6.1 versus 3.0 years, P = 0.008). CD4 lymphopenia was associated with a higher frequency of cancer (50 versus 29.6%, P = 0.047). Most strikingly, long-term lymphopenia was significantly and independently associated with an accelerated decline in renal allograft function (P = 0.005), despite a similar rate of biopsy-proven acute rejection and comparable immunosuppression. Our study shows an association between long-term CD4 T-cell lymphopenia in kidney recipients and malignancy and an accelerated decline of kidney allograft function.

Abstract 3153: Critical roles of chemo-resistant effector and regulatory T cells in cancer immunotherapy during hemostatic proliferation

Abstract Antitumor immunity has been well established to be augmented by cytotoxic lymphodepletion therapies. Adoptively transferred naïve and effector T cells proliferate extensively and show enhanced antitumor effects during homeostatic proliferation when they were adoptively transferred into tumor-bearing hosts that were lymphodepleted with cytotoxic agents or by whole body irradiation. Although the impact of lymphodepletion on transferred donor T cells has been well evaluated, its influence on recipient T cells is largely unknown. The current study demonstrates that both regulatory T cells (Tregs) and effector CD8+ T cells from lymphopenic recipients play critical roles in the development of antitumor immunity after lymphodepletion. Cyclophosphamide (CPA) treatment depleted lymphocytes more efficiently than other cytotoxic agents, such as fludarabine, cisplatin, etoposide, paclitaxel or gemcitabine; however, the percentage of CD4+CD25+Foxp3+ Tregs was significantly increased in CPA-treated lymphopenic mice. Depletion of these chemo-resistant Tregs following CPA treatment and transfer of naïve CD4+ T cells augmented the antitumor immunity and significantly suppressed tumor progression. Further analyses revealed that recipient CD8+ T cells were responsible for this augmentation. Using Rag2−/− mice or depletion of recipient CD8+ T cells after CPA treatment abrogated the augmentation of antitumor effects in CPA-treated reconstituted mice. The transfer of donor CD4+ T cells enhanced the proliferation of CD8+ T cells and the priming of tumor-specific CD8+ T cells originating from the lymphopenic recipients. These results highlight the importance of the recipient cells surviving cytotoxic regimens in cancer immunotherapies. Citation Format: Ko Sato, Satoshi Watanabe, Yu Saida, Tomohiro Tanaka, Junko Baba, Aya Ohtsubo, Satoshi Shoji, Daisuke Ishikawa, Rie Kondo, Masaaki Okajima, Satoru Miura, Junta Tanaka, Hiroshi Kagamu, Hirohisa Yoshizawa, Ichiei Narita. Critical roles of chemo-resistant effector and regulatory T cells in cancer immunotherapy during hemostatic proliferation. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3153. doi:10.1158/1538-7445.AM2015-3153

Critical Roles of Chemoresistant Effector and Regulatory T Cells in Antitumor Immunity after Lymphodepleting Chemotherapy.

Antitumor immunity is augmented by cytotoxic lymphodepletion therapies. Adoptively transferred naive and effector T cells proliferate extensively and show enhanced antitumor effects in lymphopenic recipients. Although the impact of lymphodepletion on transferred donor T cells has been well evaluated, its influence on recipient T cells is largely unknown. The current study demonstrates that both regulatory T cells (Tregs) and effector CD8(+) T cells from lymphopenic recipients play critical roles in the development of antitumor immunity after lymphodepletion. Cyclophosphamide (CPA) treatment depleted lymphocytes more efficiently than other cytotoxic agents; however, the percentage of CD4(+)CD25(+) Foxp3(+) Tregs was significantly increased in CPA-treated lymphopenic mice. Depletion of these chemoresistant Tregs following CPA treatment and transfer of naive CD4(+) T cells augmented the antitumor immunity and significantly suppressed tumor progression. Further analyses revealed that recipient CD8(+) T cells were responsible for this augmentation. Using Rag2(-/-) mice or depletion of recipient CD8(+) T cells after CPA treatment abrogated the augmentation of antitumor effects in CPA-treated reconstituted mice. The transfer of donor CD4(+) T cells enhanced the proliferation of CD8(+) T cells and the priming of tumor-specific CD8(+) T cells originating from the lymphopenic recipients. These results highlight the importance of the recipient cells surviving cytotoxic regimens in cancer immunotherapies.

Lymphopenia-induced proliferation in the absence of functional Autoimmune regulator (Aire) induces colitis in mice

Autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED) is caused by mutations in Autoimmune regulator (Aire), a transcriptional regulator of negative selection in thymus. However, Aire is also expressed in periphery, but the full range of Aire's peripheral function is unknown. Here, we transferred lymphocytes from wildtype donors into lymphopenic recipients with or without functional Aire. Following cell proliferation thus took place in Aire-sufficient or deficient environment. The wildtype lymphocytes hyperproliferated and induced disease in lymphopenic Aire−/− but not in Aire+/+ recipients. The disease was characterized by diarrhea, inflammation, and colitis, and in some recipients pancreatitis, gastritis, and hepatitis was also found. Our results identify Aire as an important regulator of peripheral T cell homeostasis in gastrointestinal tissues. Given a suitable trigger the absence of peripheral Aire leads to dysregulated T cell proliferation and disease.

T cells generated in the absence of a thoracic thymus fail to establish homeostasis.

Cervical thymus mimics the thoracic thymus in supporting T-cell development and exists in a subset of mice and humans. Importantly, it remains unknown whether the cervical thymus can generate T cells that are self-tolerant in the complete absence of signals from the thoracic thymus. Using a fetal liver reconstitution model in thoracic thymectomized RAG(-/-) mice, we found that T cells could be generated without contribution from the thoracic thymus. However, these mice had decreased T cells, increased proportions of effector memory T cells and Treg phenotype cells, increased serum IgG1/2b, and increased frequency of T cells expressing IFN-γ, IL-17 or IL-10. Half of the mice that received a thoracic thymectomy and fetal liver cells, unlike sham surgery controls, developed substantial morbidity with age. Disease was associated with lymphopenia-driven activation rather than inherent defects in the cervical thymus, as both thoracic and cervical thymocytes could generate disease in lymphopenic recipients. Administration of the homeostatic cytokine IL-7 caused a rapid, transient increase in T-cell numbers and reduced the time to disease onset. Together the data suggests that the cervical thymus can function in the complete absence of the thoracic thymus; however, the T cells generated do not establish homeostasis.

T-bet controls intestinal chronic inflammation via regulation of IL-10 production by CD4 T cells (MUC8P.806)

Abstract The dynamics of pro- and anti-inflammatory cytokine secretion by CD4 T cells is critical for the maintenance of intestinal homeostasis. Disruption of this equilibrium can result in chronic inflammation in the intestine, causing inflammatory bowel disease (IBD). Among the various subsets of CD4 T cells, the Th1 and Th17 subsets are increased in IBD patients and are proposed to be pathogenic. Therefore, understanding the mechanisms by which these cells are regulated is important to expand current treatments of IBD. Using the CD4 T cell dependent adoptive transfer model of colitis, we have investigated the Th1-associated transcription factor, T-bet, in the context of intestinal inflammation. Loss of T-bet results in Th17 skewed immune responses; however, despite wild-type Th17 CD4 T cells being colitogenic, transfer of T-bet deficient naïve CD4 T cells fails to induce colitis in a lymphopenic recipient. Surprisingly, we show that IL-10 production is significantly increased in CD4 T cells in the absence of T-bet. Furthermore, deficiency of both T-bet and IL-10 negates the protective effects of T-bet deficiency alone, as recipients of T-bet-/-IL-10-/- CD4 T cells develop colitis with similar weight loss and histological damage to recipients of wild-type CD4 T cells. Therefore, our data suggests that T-bet plays crucial role in the maintenance of intestinal homeostasis by regulating the intricate balance of effector and regulatory cytokines, in particular IL-10 levels.

PD-1 deletion restores susceptibility to experimental autoimmune encephalomyelitis in miR-155-deficient mice

MiR-155 (-/-) mice are highly resistant to experimental autoimmune encephalomyelitis (EAE), while Pdcd1 (-/-) mice develop a more severe form of the disease. To determine the conflicting roles of these two molecules in the disease, we generated miR-155 (-/-) Pdcd1 (-/-) double knockout (DKO) mice. We found that ablation of programmed cell death protein 1 (PD-1) expression in miR-155-deficient mice restored the susceptibility to EAE. The increased severity of the disease in DKO mice was accompanied by an enhanced T-cell infiltration into the brain as well as an increased production of pro-inflammatory cytokines IFN-γ and IL-17. Furthermore, the major contribution of the DKO to EAE was T-cell intrinsic since adoptive transfer of CD4(+) T cells from DKO donors promoted the disease in lymphopenic recipients. These results define PD-1 deficiency in miR-155 (-/-) mice as a promoting factor of autoimmune inflammation by increasing antigen-driven T-cell expansion and infiltration.

TGF-β Signalling Is Required for CD4+ T Cell Homeostasis But Dispensable for Regulatory T Cell Function

Author SummaryTGF-β is a cytokine thought to be critical for the maintenance and function of tolerance in the immune system. In many studies the disruption of TGF-β signalling in CD4+ T cells (a type of white blood cell that coordinates immune responses) has resulted in autoimmune syndromes. We show here that the induced removal of this cytokine's receptor from these specialised blood cells results in an astonishingly mild outcome. Contrary to expectations, the number of regulatory T cells is actually increased, and we find that these cells are not dependent on TGF-β signalling. We also show that removal of the receptor from mature CD4+ T cells does not lead to lethal autoinflammation; only when we removed the receptor during development of the cells did we see the characteristic lethal multi-organ inflammation reported previously in constitutive models of TGF-β receptor ablation. In summary, our findings indicate that although TGF-β regulates maintenance of mature CD4+ T cells, its signals are dispensable for immune tolerance within this cell population.

Vascular Endothelial Growth Factor-D–Mediated Blockade of Regulatory T Cells within Tumors Is Induced by Hematopoietic Stem Cell Transplantation

Lymphopenia-induced homeostatic proliferation of T cells after autologous hematopoietic stem cell transplantation (HSCT) skews the T cell repertoire by engaging tumor-associated Ags, leading to an induction of antitumor immunity. However, how HSCT alters the immunosuppressive microenvironment in the tumors is unknown. In this study, we first analyzed the kinetics of regulatory T cells (Tregs) in the tumors after syngeneic HSCT. Unexpectedly, the frequency of CD4⁺ cells expressing Foxp3 was increased in the spleens, whereas the frequency was clearly decreased in the tumors after HSCT. The origin of reconstituted CD4⁺ and Foxp3⁺ cells in the tumors was mainly from the expansion of transferred splenic T cells. Then, to examine the mechanism of Treg suppression after HSCT, we isolated CD11c⁺ cells from tumors. A large amount of Treg-inhibitory cytokine IL-6 was secreted from the CD11c⁺ cells in the tumors, but not in the spleens in the recipient mice. Furthermore, to understand what factor affects the activity of CD11c⁺ cells in the tumors after HSCT, we analyzed the expression of various cytokines/chemokines with mouse cytokine Ab arrays, and noticed that VEGF-D concentration was increased in the tumors in the early period after HSCT. The CD11c⁺ cells produced IL-6 in response to VEGF-D stimulation, and an administration of VEGF receptor-3 neutralizing Ab significantly suppressed the production of IL-6 from CD11c⁺ cells accompanied with the increase of Tregs in the tumors of HSCT recipients. Autologous HSCT creates an environment that strongly supports the enhancement of antitumor immunity in reconstituted lymphopenic recipients through the suppression of Tregs.