Scott M. Krummey

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.

High CTLA-4 expression on Th17 cells results in increased sensitivity to CTLA-4 coinhibition and resistance to belatacept.

The CD28/cytotoxic T‐lymphocyte antigen 4 (CTLA‐4) blocker belatacept selectively inhibits alloreactive T cell responses but is associated with a high incidence of acute rejection following renal transplantation, which led us to investigate the etiology of belatacept–resistant graft rejection. T cells can differentiate into functionally distinct subsets of memory T cells that collectively enable protection against diverse classes of pathogens and can cross‐react with allogeneic antigen and mediate graft rejection. T helper 17 (Th17) cells are a pro‐inflammatory CD4+ lineage that provides immunity to pathogens and are pathogenic in autoimmune disease. We found that T helper 1 (Th1) and Th17 memory compartments contained a similar frequency of divided cells following allogeneic stimulation. Compared to Th1 cells, Th17 memory cells expressed significantly higher levels of the coinhibitory molecule CTLA‐4. Stimulation in the presence of belatacept inhibited Th1 responses but augmented Th17 cells due to greater sensitivity to coinhibition by CTLA‐4. Th17 cells from renal transplant recipients were resistant to ex vivo CD28/CTLA‐4 blockade with belatacept, and an elevated frequency of Th17 memory cells was associated with acute rejection during belatacept therapy. These data highlight important differences in costimulatory and coinhibitory requirements of CD4+ memory subsets, and demonstrate that the heterogeneity of pathogen‐derived memory has implications for immunomodulation strategies.

Heterogeneity within T Cell Memory: Implications for Transplant Tolerance

Adaptive immunity in both mouse and man results in the generation of immunological memory. Memory T cells are both friend and foe to transplant recipients, as they are intimately involved and in many cases absolutely required for the maintenance of protective immunity in the face immunosuppression, yet from the evidence presented herein they clearly constitute a formidable barrier for the successful implementation of tolerance induction strategies in transplantation. This review describes the experimental evidence demonstrating the increased resistance of memory T cells to many distinct tolerance induction strategies, and outlines recent advances in our knowledge of the ways in which alloreactive memory T cells arise in previously untransplanted individuals. Understanding the impact of alloreactive memory T cell specificity, frequency, and quality might allow for better donor selection in order to minimize the donor-reactive memory T cell barrier in an individual transplant recipient, thus allowing stratification of relative risk of alloreactive memory T cell mediated rejection, and conversely increase the likelihood of successful establishment of tolerance. However, further research into the molecular and cellular pathways involved in alloreactive memory T cell-mediated rejection is required in order to design new strategies to overcome the memory T cell barrier, without critically impairing protective immunity.

Braking Bad: Novel Mechanisms of CTLA-4 Inhibition of T Cell Responses

The coinhibitory receptor cytotoxic T‐lymphocyte antigen 4 (CTLA‐4) is a master regulator of T cell responses and its function is critical in models of transplant tolerance. The CD28/CTLA‐4 pathway is also an important therapeutic target, as the costimulation blocker belatacept was recently approved for use following renal transplantation. While the traditional model of CTLA‐4 coinhibition focuses on its ability to directly counteract CD28 costimulation, recently this paradigm has significantly broadened. Recent work has uncovered the ability of CTLA‐4 to act as a cell‐extrinsic coinhibitory molecule on CD4+ T cell effectors. While it has been appreciated that CTLA‐4 is required for FoxP3+ regulatory T cell (Treg) suppression, current studies have elucidated important differences in the function of CTLA‐4 on Tregs compared to effectors. CTLA‐4 expression patterns also differ by T cell subset, with Th17 cells expressing significantly higher levels of CTLA‐4. Thus, in contrast to the traditional model of CTLA‐4 as a negative receptor to counter CD28 costimulation, recent work has begun to define CTLA‐4 as a global regulator of T cell responses with subset‐specific functions. Future studies must continue to uncover the molecular mechanisms that govern CTLA‐4 function. These novel findings have implications for novel strategies to maximize the regulatory potential of CTLA‐4 during allogeneic T cell responses.

Candida-Elicited Murine Th17 Cells Express High CTLA-4 Compared with Th1 Cells and Are Resistant to Costimulation Blockade

Effector and memory T cells may cross-react with allogeneic Ags to mediate graft rejection. Whereas the costimulation properties of Th1 cells are well studied, relatively little is known about the costimulation requirements of microbe-elicited Th17 cells. The costimulation blocker CTLA-4 Ig has been ineffective in the treatment of several Th17-driven autoimmune diseases and is associated with severe acute rejection following renal transplantation, leading us to investigate whether Th17 cells play a role in CD28/CTLA-4 blockade-resistant alloreactivity. We established an Ag-specific model in which Th1 and Th17 cells were elicited via Mycobacterium tuberculosis and Candida albicans immunization, respectively. C. albicans immunization elicited a higher frequency of Th17 cells and conferred resistance to costimulation blockade following transplantation. Compared with the M. tuberculosis group, C. albicans–elicited Th17 cells contained a higher frequency of IL-17+IFN-γ+ producers and a lower frequency of IL-10+ and IL-10+IL-17+ cells. Importantly, Th17 cells differentially regulated the CD28/CTLA-4 pathway, expressing similarly high CD28 but significantly greater amounts of CTLA-4 compared with Th1 cells. Ex vivo blockade experiments demonstrated that Th17 cells are more sensitive to CTLA-4 coinhibition and therefore less susceptible to CTLA-4 Ig. These novel insights into the differential regulation of CTLA-4 coinhibition on CD4+ T cells have implications for the immunomodulation of pathologic T cell responses during transplantation and autoimmunity.

SAP Facilitates Recruitment and Activation of LCK at NTB-A Receptors during Restimulation-Induced Cell Death

Upon TCR restimulation, activated, cycling T cells can undergo a self-regulatory form of apoptosis known as restimulation-induced cell death (RICD). We previously demonstrated that RICD is impaired in T cells from patients with X-linked lymphoproliferative disease, which lack SLAM-associated protein (SAP) expression. Both SAP and the specific SLAM receptor NK, T, and B cell Ag (NTB-A) are required for RICD, but the mechanism by which these molecules promote a strong, proapoptotic signal through the TCR remains unclear. In this article, we show that the Src-family kinase LCK, but not FYN, associates with NTB-A in activated human T cells. This association increased after TCR restimulation in a SAP-dependent manner, requiring both immunoreceptor tyrosine-based switch motifs in the NTB-A cytoplasmic tail. Both NTB-A–associated LCK phosphorylation and kinase activity were enhanced in restimulated T cells, amplifying proximal TCR signaling. In contrast, TCR-induced LCK association with NTB-A, as well as phosphorylation and kinase activity, was reduced in T cells from patients with X-linked lymphoproliferative disease or normal T cells transfected with SAP-specific small interfering RNA, consistent with RICD resistance. Collectively, our data reveal how SAP nucleates a previously unknown signaling complex involving NTB-A and LCK to potentiate RICD of activated human T cells.

Low-Affinity Memory CD8+ T Cells Mediate Robust Heterologous Immunity

Heterologous immunity is recognized as a significant barrier to transplant tolerance. Whereas it has been established that pathogen-elicited memory T cells can have high or low affinity for cross-reactive allogeneic peptide–MHC, the role of TCR affinity during heterologous immunity has not been explored. We established a model with which to investigate the impact of TCR-priming affinity on memory T cell populations following a graft rechallenge. In contrast to high-affinity priming, low-affinity priming elicited fully differentiated memory T cells with a CD45RBhi status. High CD45RB status enabled robust secondary responses in vivo, as demonstrated by faster graft rejection kinetics and greater proliferative responses. CD45RB blockade prolonged graft survival in low affinity–primed mice, but not in high affinity–primed mice. Mechanistically, low affinity–primed memory CD8+ T cells produced more IL-2 and significantly upregulated IL-2Rα expression during rechallenge. We found that CD45RBhi status was also a stable marker of priming affinity within polyclonal CD8+ T cell populations. Following high-affinity rechallenge, low affinity–primed CD45RBhi cells became CD45RBlo, demonstrating that CD45RB status acts as an affinity-based differentiation switch on CD8+ T cells. Thus, these data establish a novel mechanism by which CD45 isoforms tune low affinity–primed memory CD8+ T cells to become potent secondary effectors following heterologous rechallenge. These findings have direct implications for allogeneic heterologous immunity by demonstrating that despite a lower precursor frequency, low-affinity priming is sufficient to generate memory cells that mediate potent secondary responses against a cross-reactive graft challenge.

Enhanced Requirement for TNFR2 in Graft Rejection Mediated by Low-Affinity Memory CD8 + T Cells during Heterologous Immunity

The affinity of a TCR binding to peptide:MHC profoundly impacts the phenotype and function of effector and memory cell differentiation. Little is known about the effect of low-affinity priming on memory cell generation and function, which is particularly important in heterologous immunity, when microbe-specific T cells cross-react with allogeneic Ag and mediate graft rejection. We found that low-affinity–primed memory CD8+ T cells produced high levels of TNF ex vivo in response to heterologous rechallenge compared with high-affinity–primed memory T cells. Low-affinity secondary effectors significantly upregulated TNFR2 on the cell surface and contained a higher frequency of TNFR2hi proliferating cells. Low-affinity–primed secondary effectors concurrently downregulated TNF production. Importantly, blockade of TNFR2 attenuated graft rejection in low- but not high-affinity–primed animals. These data establish a functional connection between TNF signaling and TCR-priming affinity and have implications for the immunomodulation of pathogenic T cell responses during transplantation.

New insights into T-cell cosignaling in allograft rejection and survival.

Purpose of reviewStimulatory and inhibitory receptor signaling (cosignaling) on T cells is a critical component of T-cell responses that mediate graft rejection. The blockade of cosignaling pathways is an attractive strategy for preventing allogeneic T-cell responses. Here, we review the new studies that provide critical insight into the well studied CD28–CTLA-4 and CD40–CD40L cosignaling pathways, as well as the identification of novel cosignaling receptors that play a role in allogeneic T-cell responses. Recent findingsRecently, it has been appreciated that the CD28–CTLA-4 pathway has unique roles on specific T-cell subsets, particularly on forkhead box P3 (FoxP3)+ regulatory T cell (Treg) and T helper 17 (Th17) cells. New insight has been provided into the mechanism by which CD40–CD154 blockade elicits FoxP3+ Treg conversion and memory T cells elicit CD40-independent alloantibody responses. Finally, several novel cosignaling pathways have been demonstrated to be important to graft-specific T cells, including CD160, signaling lymphocytic activation molecule family member 2B4, T-cell Ig mucin 4, and the Notch receptor. SummaryRecent work has provided more granular understanding of the CD28–CTLA-4 and CD40–CD154 pathways on T-cell subsets, and provided important insight into the generation and maintenance of FoxP3+ Treg. This information, as well as the characterization of novel transplantation-relevant cosignaling pathways, has implications for the modulation of alloreactive T-cell responses.

Stability of anti-A blood group titers among blood group B renal transplant candidates: STABILITY OF ANTI-A BLOOD GROUP TITERS

As deceased donor kidney allocation is based in part on blood type compatibility, group B candidates are disadvantaged due to their disproportionate representation on the wait list compared to the group B donor pool. To mitigate this discrepancy, group B candidates can receive group A2 or A2B donor kidneys if their anti‐A titers are below a predetermined cutoff. Currently, eligibility is reverified quarterly to UNet based on individual center protocols, which can vary due to a lack of set guidelines for monitoring ABO titers in these patients. Our goal was to assess the stability of anti‐A titers in blood group B renal transplant candidates over time to provide data that could aid in the development of standardized ABO titer protocols.