Michelle L. Miller

High TCR Stimuli Prevent Induced Regulatory T Cell Differentiation in a NF-κB–Dependent Manner

The concentration of Ag or mitogenic stimuli is known to play an important role in controlling the differentiation of naive CD4+ T cells into different effector phenotypes. In particular, whereas TCR engagement at low Ag doses in the presence of TGF-β and IL-2 can promote differentiation of Foxp3-expressing induced regulatory T cells (iTregs), high levels of Ag have been shown in vitro and in vivo to prevent Foxp3 upregulation. This tight control of iTreg differentiation dictated by Ag dose most likely determines the quality and duration of an immune response. However, the molecular mechanism by which this high-dose inhibition of Foxp3 induction occurs is not well understood. In this study, we demonstrate that when cells are in the presence of CD28 costimulation, TCR-dependent NF-κB signaling is essential for Foxp3 inhibition at high doses of TCR engagement in mouse T cells. Prevention of Foxp3 induction depends on the production of NF-κB–dependent cytokines by the T cells themselves. Moreover, T cells that fail to upregulate Foxp3 under iTreg-differentiating conditions and high TCR stimulation acquire the capacity to make TNF and IFN-γ, as well as IL-17 and IL-9. Thus, NF-κB helps T cells control their differentiation fate in a cell-intrinsic manner and prevents peripheral iTreg development under conditions of high Ag load that may require more vigorous effector T cell responses.

Lessons and Limits of Mouse Models

Seminal studies in rabbits and rodent transplantation models by Peter Medawar revealed that cellular processes, rather than humoral antibodies, are central to the acute rejection of transplanted organs, and much of basic transplantation research continues to be focused on the biology and control of these cells, which were subsequently shown to be T cells. However, the success of current immunosuppression at controlling T-cell-mediated rejection has resulted in an increasing awareness of antibody-mediated rejection in the clinic. This, in turn, has fueled an emerging interest in the biology of allospecific antibodies, the B cells that produce these antibodies, and the development of mouse models that allow their investigation. Here we summarize some of the more widely used mouse models that have been developed to study the immunobiology of alloreactivity, transplantation rejection and tolerance, and used to identify therapeutic strategies that modulate these events.

Spontaneous restoration of transplantation tolerance after acute rejection

Transplantation is a cure for end-stage organ failure but, in the absence of pharmacological immunosuppression, allogeneic organs are acutely rejected. Such rejection invariably results in allosensitization and accelerated rejection of secondary donor-matched grafts. Transplantation tolerance can be induced in animals and a subset of humans, and enables long-term acceptance of allografts without maintenance immunosuppression. However, graft rejection can occur long after a state of transplantation tolerance has been acquired. When such an allograft is rejected, it has been assumed that the same rules of allosensitization apply as to non-tolerant hosts and that immunological tolerance is permanently lost. Using a mouse model of cardiac transplantation, we show that when Listeria monocytogenes infection precipitates acute rejection, thus abrogating transplantation tolerance, the donor-specific tolerant state re-emerges, allowing spontaneous acceptance of a donor-matched second transplant. These data demonstrate a setting in which the memory of allograft tolerance dominates over the memory of transplant rejection.

Delayed Cytotoxic T Lymphocyte–Associated Protein 4–Immunoglobulin Treatment Reverses Ongoing Alloantibody Responses and Rescues Allografts From Acute Rejection

Antibody‐mediated rejection has emerged as the leading cause of late graft loss in kidney transplant recipients, and inhibition of donor‐specific antibody production should lead to improved transplant outcomes. The fusion protein cytotoxic T lymphocyte–associated protein 4–immunoglobulin (CTLA4‐Ig) blocks T cell activation and consequently inhibits T‐dependent B cell antibody production, and the current paradigm is that CTLA4‐Ig is effective with naïve T cells and less so with activated or memory T cells. In this study, we used a mouse model of allosensitization to investigate the efficacy of continuous CTLA4‐Ig treatment, initiated 7 or 14 days after sensitization, for inhibiting ongoing allospecific B cell responses. Delayed treatment with CTLA4‐Ig collapsed the allospecific germinal center B cell response and inhibited alloantibody production. Using adoptively transferred T cell receptor transgenic T cells and a novel approach to track endogenous graft‐specific T cells, we demonstrate that delayed CTLA4‐Ig minimally inhibited graft‐specific CD4+ and T follicular helper responses. Remarkably, delaying CTLA4‐Ig until day 6 after transplantation in a fully mismatched heart transplant model inhibited alloantibody production and prevented acute rejection, whereas transferred hyperimmune sera reversed the effects of delayed CTLA4‐Ig. Collectively, our studies revealed the unexpected efficacy of CTLA4‐Ig for inhibiting ongoing B cell responses even when the graft‐specific T cell response was robustly established.

Basal NF-κB controls IL-7 responsiveness of quiescent naïve T cells.

Significance T lymphocytes are white blood cells that recognize and fight pathogens. Maintenance of sufficient numbers of T cells is essential to prevent susceptibility to infections. Survival of quiescent T cells is maintained, in part, by the interaction between the soluble factor (IL-7 produced by various stromal cells) and the IL-7 receptor (IL-7R) expressed on the surface of T cells. Here, we show that naïve T cells have basal nuclear levels of the transcription factor NF-κB and that is key to maintain IL-7R expression in T cells and for their survival. Our results imply that antiinflammatory therapies targeting NF-κB may affect the pool of naïve T cells required to control infections. T cells are essential for immune defenses against pathogens, such that viability of naïve T cells before antigen encounter is critical to preserve a polyclonal repertoire and prevent immunodeficiencies. The viability of naïve T cells before antigen recognition is ensured by IL-7, which drives expression of the prosurvival factor Bcl-2. Quiescent naïve T cells have low basal activity of the transcription factor NF-κB, which was assumed to have no functional consequences. In contrast to this postulate, our data show that basal nuclear NF-κB activity plays an important role in the transcription of IL-7 receptor α-subunit (CD127), enabling responsiveness of naïve T cells to the prosurvival effects of IL-7 and allowing T-cell persistence in vivo. Moreover, we show that this property of basal NF-κB activity is shared by mouse and human naïve T cells. Thus, NF-κB drives a distinct transcriptional program in T cells before antigen encounter by controlling susceptibility to IL-7. Our results reveal an evolutionarily conserved role of NF-κB in T cells before antigenic stimulation and identify a novel molecular pathway that controls T-cell homeostasis.

Erosion of Transplantation Tolerance After Infection.

Recent clinical studies suggest that operational allograft tolerance can be persistent, but long‐term surviving allografts can be rejected in a subset of patients, sometimes after episodes of infection. In this study, we examined the impact of Listeria monocytogenes (Lm) infection on the quality of tolerance in a mouse model of heart allograft transplantation. Lm infection induced full rejection in 40% of tolerant recipients, with the remaining experiencing a rejection crisis or no palpable change in their allografts. In the surviving allografts on day 8 postinfection, graft‐infiltrating cell numbers increased and exhibited a loss in the tolerance gene signature. By day 30 postinfection, the tolerance signature was broadly restored, but with a discernible reduction in the expression of a subset of 234 genes that marked tolerance and was down‐regulated at day 8 post‐Lm infection. We further demonstrated that the tolerant state after Lm infection was functionally eroded, as rejection of the long‐term surviving graft was induced with anti‐PD‐L1 whereas the same treatment had no effect in noninfected tolerant mice. Collectively, these observations demonstrate that tolerance, even if initially robust, exists as a continuum that can be eroded following bystander immune responses that accompany certain infections.

Tracking of TCR-Transgenic T Cells Reveals That Multiple Mechanisms Maintain Cardiac Transplant Tolerance in Mice

Solid organ transplantation tolerance can be achieved following select transient immunosuppressive regimens that result in long‐lasting restraint of alloimmunity without affecting responses to other antigens. Transplantation tolerance has been observed in animal models following costimulation or coreceptor blockade therapies, and in a subset of patients through induction protocols that include donor bone marrow transplantation, or following withdrawal of immunosuppression. Previous data from our lab and others have shown that proinflammatory interventions that successfully prevent the induction of transplantation tolerance in mice often fail to break tolerance once it has been stably established. This suggests that established tolerance acquires resilience to proinflammatory insults, and prompted us to investigate the mechanisms that maintain a stable state of robust tolerance. Our results demonstrate that only a triple intervention of depleting CD25+ regulatory T cells (Tregs), blocking programmed death ligand‐1 (PD‐L1) signals, and transferring low numbers of alloreactive T cells was sufficient to break established tolerance. We infer from these observations that Tregs and PD‐1/PD‐L1 signals cooperate to preserve a low alloreactive T cell frequency to maintain tolerance. Thus, therapeutic protocols designed to induce multiple parallel mechanisms of peripheral tolerance may be necessary to achieve robust transplantation tolerance capable of maintaining one allograft for life in the clinic.

Adoptive Transfer of Tracer‐Alloreactive CD4+ T Cell Receptor Transgenic T Cells Alters the Endogenous Immune Response to an Allograft

T cell receptor transgenic (TCR‐Tg) T cells are often used as tracer populations of antigen‐specific responses to extrapolate findings to endogenous T cells. The extent to which TCR‐Tg T cells behave purely as tracer cells or modify the endogenous immune response is not clear. To test the impact of TCR‐Tg T cell transfer on endogenous alloimmunity, recipient mice were seeded with CD4+ or CD8+ TCR‐Tg or polyclonal T cells at the time of cardiac allograft transplantation. Only CD4+ TCR‐Tg T cells accelerated rejection and, unexpectedly, led to a dose‐dependent decrease in both transferred and endogenous T cells infiltrating the graft. In contrast, recipients of CD4+ TCR‐Tg T cells exhibited enhanced endogenous donor‐specific CD8+ T cell activation in the spleen and accelerated alloantibody production. Introduction of CD4+ TCR‐Tg T cells also perturbed the intragraft accumulation of innate cell populations. Transferred CD4+ TCR‐Tg T cells alter many aspects of endogenous alloimmunity, suggesting that caution should be used when interpreting experiments using these adoptively transferred cells because the overall nature of allograft rejection may be altered. These results also may have implications for adoptive CD4+ T cell immunotherapy in tumor and infectious clinical settings because cell infusion may have additional effects on natural immune responses.

Active immunotherapy for TNF-mediated inflammation using self-assembled peptide nanofibers

Active immunotherapies raising antibody responses against autologous targets are receiving increasing interest as alternatives to the administration of manufactured antibodies. The challenge in such an approach is generating protective and adjustable levels of therapeutic antibodies while at the same time avoiding strong T cell responses that could lead to autoimmune reactions. Here we demonstrate the design of an active immunotherapy against TNF-mediated inflammation using short synthetic peptides that assemble into supramolecular peptide nanofibers. Immunization with these materials, without additional adjuvants, was able to break B cell tolerance and raise protective antibody responses against autologous TNF in mice. The strength of the anti-TNF antibody response could be tuned by adjusting the epitope content in the nanofibers, and the T-cell response was focused on exogenous and non-autoreactive T-cell epitopes. Immunization with unadjuvanted peptide nanofibers was therapeutic in a lethal model of acute inflammation induced by intraperitoneally delivered lipopolysaccharide, whereas formulations adjuvanted with CpG showed comparatively poorer protection that correlated with a more Th1-polarized response. Additionally, immunization with peptide nanofibers did not diminish the ability of mice to clear infections of Listeria monocytogenes. Collectively this work suggests that synthetic self-assembled peptides can be attractive platforms for active immunotherapies against autologous targets.

Cutting Edge: Engineering Active IKKβ in T Cells Drives Tumor Rejection

Acquired dysfunction of tumor-reactive T cells is one mechanism by which tumors can evade the immune system. Identifying and correcting pathways that contribute to such dysfunction should enable novel anticancer therapy design. During cancer growth, T cells show reduced NF-κB activity, which is required for tumor rejection. Impaired T cell–intrinsic NF-κB may create a vicious cycle conducive to tumor progression and further T cell dysfunction. We hypothesized that forcing T cell–intrinsic NF-κB activation might break this cycle and induce tumor elimination. NF-κB was activated in T cells by inducing the expression of a constitutively active form of the upstream activator IκB kinase β (IKKβ). T cell–restricted constitutively active IKKβ augmented the frequency of functional tumor-specific CD8+ T cells and improved tumor control. Transfer of constitutively active IKKβ–transduced T cells also boosted endogenous T cell responses that controlled pre-established tumors. Our results demonstrate that driving T cell–intrinsic NF-κB can result in tumor control, thus identifying a pathway with potential clinical applicability.