Maylene E. Wagener

Antigen-specific precursor frequency impacts T cell proliferation, differentiation, and requirement for costimulation

After a brief period of antigenic stimulation, T cells become committed to a program of autonomous expansion and differentiation. We investigated the role of antigen-specific T cell precursor frequency as a possible cell-extrinsic factor impacting T cell programming in a model of allogeneic tissue transplantation. Using an adoptive transfer system to incrementally raise the precursor frequency of antigen-specific CD8+ T cells, we found that donor-reactive T cells primed at low frequency exhibited increased cellular division, decreased development of multifunctional effector activity, and an increased requirement for CD28- and CD154-mediated costimulation relative to those primed at high frequency. The results demonstrated that recipients with low CD4+ and CD8+ donor-reactive T cell frequencies exhibited long-term skin graft survival upon CD28/CD154 blockade, whereas simultaneously raising the frequency of CD4+ T cells to ∼0.5% and CD8+ T cells to ∼5% precipitated graft rejection despite CD28/CD154 blockade. Antigenic rechallenge of equal numbers of cells stimulated at high or low frequency revealed that cells retained an imprint of the frequency at which they were primed. These results demonstrate a critical role for initial precursor frequency in determining the CD8+ T cell requirement for CD28- and CD154-mediated costimulatory signals during graft rejection.

Cutting Edge: Rapamycin Augments Pathogen-Specific but Not Graft-Reactive CD8+ T Cell Responses

Recent evidence demonstrating that exposure to rapamycin during viral infection increased the quantity and quality of Ag-specific T cells poses an intriguing paradox, because rapamycin is used in transplantation to dampen, rather than enhance, donor-reactive T cell responses. In this report, we compared the effects of rapamycin on the Ag-specific T cell response to a bacterial infection versus a transplant. Using a transgenic system in which the Ag and the responding T cell population were identical in both cases, we observed that treatment with rapamycin augmented the Ag-specific T cell response to a pathogen, whereas it failed to do so when the Ag was presented in the context of a transplant. These results suggest that the environment in which an Ag is presented alters the influence of rapamycin on Ag-specific T cell expansion and highlights a fundamental difference between Ag presented by an infectious agent as compared with an allograft.

Integrin antagonists prevent costimulatory blockade-resistant transplant rejection by CD8+ memory T cells

The success of belatacept in late‐stage clinical trials inaugurates the arrival of a new class of immunosuppressants based on costimulatory blockade, an immunosuppression strategy that disrupts essential signals required for alloreactive T‐cell activation. Despite having improved renal function, kidney transplant recipients treated with belatacept experienced increased rates of acute rejection. This finding has renewed focus on costimulatory blockade‐resistant rejection and specifically the role of alloreactive memory T cells in mediating this resistance. To study the mechanisms of costimulatory blockade‐resistant rejection and enhance the clinical efficacy of costimulatory blockade, we developed an experimental transplant system that models a donor‐specific memory CD8+ T‐cell response. After confirming that graft‐specific memory T cells mediate costimulatory blockade‐resistant rejection, we characterized the role of integrins in this rejection. The resistance of memory T cells to costimulatory blockade was abrogated when costimulatory blockade was coupled with either anti‐VLA‐4 or anti‐LFA‐1. Mechanistic studies revealed that in the presence of costimulatory blockade, anti‐VLA‐4 impaired T‐cell trafficking to the graft but not memory T‐cell recall effector function, whereas anti‐LFA‐1 attenuated both trafficking and memory recall effector function. As antagonists against these integrins are clinically approved, these findings may have significant translational potential for future clinical transplant trials.

Antigen-specific induced Foxp3+ regulatory T cells are generated following CD40/CD154 blockade

Blockade of the CD40/CD154 pathway potently attenuates T-cell responses in models of autoimmunity, inflammation, and transplantation. Indeed, CD40 pathway blockade remains one of the most powerful methods of prolonging graft survival in models of transplantation. But despite this effectiveness, the cellular and molecular mechanisms underlying the protective effects of CD40 pathway blockade are incompletely understood. Furthermore, the relative contributions of deletion, anergy, and regulation have not been measured in a model in which donor-reactive CD4+ and CD8+ T-cell responses can be assessed simultaneously. To investigate the impact of CD40/CD154 pathway blockade on graft-specific T-cell responses, a transgenic mouse model was used in which recipients containing ovalbumin-specific CD4+ and CD8+ TCR transgenic T cells were grafted with skin expressing ovalbumin in the presence or absence of anti-CD154 and donor-specific transfusion. The results indicated that CD154 blockade altered the kinetics of donor-reactive CD8+ T-cell expansion, delaying differentiation into IFN-γ+ TNF+ multifunctional cytokine producers. The eventual differentiation of cytokine-producing effectors in tolerant animals coincided with the emergence of an antigen-specific CD4+ CD25hi Foxp3+ T-cell population, which did not arise from endogenous natural Treg but rather were peripherally generated from naïve Foxp3− precursors.

Chronic Alcohol Ingestion Increases Mortality and Organ Injury in a Murine Model of Septic Peritonitis

Background Patients admitted to the intensive care unit with alcohol use disorders have increased morbidity and mortality. The purpose of this study was to determine how chronic alcohol ingestion alters the host response to sepsis in mice. Methods Mice were randomized to receive either alcohol or water for 12 weeks and then subjected to cecal ligation and puncture. Mice were sacrificed 24 hours post-operatively or followed seven days for survival. Results Septic alcohol-fed mice had a significantly higher mortality than septic water-fed mice (74% vs. 41%, p = 0.01). This was associated with worsened gut integrity in alcohol-fed mice with elevated intestinal epithelial apoptosis, decreased crypt proliferation and shortened villus length. Further, alcohol-fed mice had higher intestinal permeability with decreased ZO-1 and occludin protein expression in the intestinal tight junction. The frequency of splenic and bone marrow CD4+ T cells was similar between groups; however, splenic CD4+ T cells in septic alcohol-fed mice had a marked increase in both TNF and IFN-γ production following ex vivo stimulation. Neither the frequency nor function of CD8+ T cells differed between alcohol-fed and water-fed septic mice. NK cells were decreased in both the spleen and bone marrow of alcohol-fed septic mice. Pulmonary myeloperoxidase levels and BAL levels of G-CSF and TFG-β were higher in alcohol-fed mice. Pancreatic metabolomics demonstrated increased acetate, adenosine, xanthine, acetoacetate, 3-hydroxybutyrate and betaine in alcohol-fed mice and decreased cytidine, uracil, fumarate, creatine phosphate, creatine, and choline. Serum and peritoneal cytokines were generally similar between alcohol-fed and water-fed mice, and there were no differences in bacteremia, lung wet to dry weight, or pulmonary, liver or splenic histology. Conclusions When subjected to the same septic insult, mice with chronic alcohol ingestion have increased mortality. Alterations in intestinal integrity, the host immune response, and pancreatic metabolomics may help explain this differential response.

2B4 (CD244) induced by selective CD28 blockade functionally regulates allograft-specific CD8+ T cell responses

Blockade of CD28 signals results in the up-regulation of 2B4 on primary CD8+ effectors and plays a critical role in controlling antigen-specific CD8+ T cell responses.

LFA-1 blockade induces effector and regulatory T-cell enrichment in lymph nodes and synergizes with CTLA-4Ig to inhibit effector function

Despite encouraging results using lymphocyte function antigen-1 (LFA-1) blockade to inhibit BM and solid organ transplantation rejection in nonhuman primates and humans, the precise mechanisms underlying its therapeutic potential are still poorly understood. Using a fully allogeneic murine transplantation model, we assessed the relative distribution of total lymphocyte subsets in untreated versus anti-LFA-1-treated animals. Our results demonstrated a striking loss of naive T cells from peripheral lymph nodes, a concomitant gain in blood after LFA-1 blockade, and a shift in phenotype of the cells remaining in the node to a CD62LloCD44hi profile. We determined that this change was due to a specific enrichment of activated, graft-specific effectors in the peripheral lymph nodes of anti-LFA-1-treated mice compared with untreated controls, and not to a direct effect of anti-LFA-1 on CD62L expression. LFA-1 blockade also resulted in a dramatic increase in the frequency of CD4+ FoxP3+ regulatory T cells in graft-draining nodes. Our results suggest that the differential impact of LFA-1 blockade on the distribution of naive versus effector and regulatory T cells may underlie its ability to inhibit alloreactive T-cell responses after transplantation.

Failure to Induce Neonatal Tolerance in Mice That Lack Both IL-4 and IL-13 but Not in Those That Lack IL-4 Alone

Current evidence suggests that neonatal tolerance to a foreign Ag is the consequence of IL-4-mediated Th2 immunity rather than the thymic deletion of Ag-specific T cells. Here, we addressed the role of IL-4 in neonatal tolerance by testing whether tolerance to a minor histocompatibility Ag can be induced in newborn mice that lack IL-4 (IL-4−/−). We found that IL-4 does not play a dominant role in the induction of neonatal tolerance as newborn female IL-4−/− mice could be readily tolerized to the H-Y male Ag. In contrast, mice that lack both IL-4 and IL-13 (IL-4−/−/IL-13−/−) were resistant to the induction of neonatal tolerance, and their splenocytes produced exaggerated amounts of IFN-γ on rechallenge with the same Ag encountered during the neonatal period. These findings argue against the view that IL-4 alone is critical for the induction of neonatal tolerance and suggest that the combined actions of both IL-4 and IL-13 are essential for this process.

An anti-CD154 domain antibody prolongs graft survival and induces Foxp3(+) iTreg in the absence and presence of CTLA-4 Ig.

The use of monoclonal antibodies targeting the CD154 molecule remains one of the most effective means of promoting graft tolerance in animal models, but thromboembolic complications during early clinical trials have precluded their use in humans. Furthermore, the role of Fc‐mediated deletion of CD154‐expressing cells in the observed efficacy of these reagents remains controversial. Therefore, determining the requirements for anti‐CD154‐induced tolerance will instruct the development of safer but equally efficacious treatments. To investigate the mechanisms of action of anti‐CD154 therapy, two alternative means of targeting the CD40–CD154 pathway were used: a nonagonistic anti‐CD40 antibody and an Fc‐silent anti‐CD154 domain antibody. We compared these therapies to an Fc‐intact anti‐CD154 antibody in both a fully allogeneic model and a surrogate minor antigen model in which the fate of alloreactive cells could be tracked. Results indicated that anti‐CD40 mAbs as well as Fc‐silent anti‐CD154 domain antibodies were equivalent to Fc‐intact anti‐CD154 mAbs in their ability to inhibit alloreactive T cell expansion, attenuate cytokine production of antigen‐specific T cells and promote the conversion of Foxp3+ iTreg. Importantly, iTreg conversion observed with Fc‐silent anti‐CD154 domain antibodies was preserved in the presence of CTLA4‐Ig, suggesting that this therapy is a promising candidate for translation to clinical use.

A critical precursor frequency of donor-reactive CD4+ T cell help is required for CD8+ T cell-mediated CD28/CD154-independent rejection.

Ag-specific precursor frequency is increasingly being appreciated as an important factor in determining the kinetics, magnitude, and degree of differentiation of T cell responses, and recently was found to play a critical role in determining the relative requirement of CD8+ T cells for CD28- and CD154-mediated costimulatory signals during transplantation. We addressed the possibility that variations in CD4+ T cell precursor frequency following transplantation might affect CD4+ T cell proliferation, effector function, and provision of help for donor-reactive B cell and CD8+ T cell responses. Using a transgenic model system wherein increasing frequencies of donor-reactive CD4+ T cells were transferred into skin graft recipients, we observed that a critical CD4+ T cell threshold precursor frequency was necessary to provide help following blockade of the CD28 and CD154 costimulatory pathways, as measured by increased B cell and CD8+ T cell responses and precipitation of graft rejection. In contrast to high-frequency CD8+ T cell responses, this effect was observed even though the proliferative and cytokine responses of Ag-specific CD4+ T cells were inhibited. Thus, we conclude that an initial high frequency of donor-reactive CD4+ T cells uncouples T cell proliferative and effector cytokine production from the provision of T cell help.