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The purpose of the STAR 2019 Working Group was to build on findings from the initial STAR report to further clarify the expectations, limitations, perceptions, and utility of alloimmune assays that are currently in use or in development for risk assessment in the setting of organ transplantation. The goal was to determine the precision and clinical feasibility/utility of such assays in evaluating both memory and primary alloimmune risks. The process included a critical review of biologically driven, state-of-the-art, clinical diagnostics literature by experts in the field and an open public forum in a face-to-face meeting to promote broader engagement of the American Society of Transplantation and American Society of Histocompatibility and Immunogenetics membership. This report summarizes the literature review and the workshop discussions. Specifically, it highlights (1) available assays to evaluate the attributes of HLA antibodies and their utility both as clinical diagnostics and as research tools to evaluate the effector mechanisms driving rejection; (2) potential assays to assess the presence of alloimmune T and B cell memory; and (3) progress in the development of HLA molecular mismatch computational scores as a potential prognostic biomarker for primary alloimmunity and its application in research trial design.

The regulatory circuits dictating CD8+ T cell responsiveness versus exhaustion during anti-tumor immunity are incompletely understood. Here we report that tumor-infiltrating antigen-specific PD-1+ TCF-1− CD8+ T cells express the immunosuppressive cytokine Fgl2. Conditional deletion of Fgl2 specifically in mouse antigen-specific CD8+ T cells prolongs CD8+ T cell persistence, suppresses phenotypic and transcriptomic signatures of T cell exhaustion, and improves control of the tumor. In a mouse model of chronic viral infection, PD-1+ CD8+ T cell-derived Fgl2 also negatively regulates virus-specific T cell responses. In humans, CD8+ T cell-derived Fgl2 is associated with poorer survival in patients with melanoma. Mechanistically, the dampened responsiveness of WT Fgl2-expressing CD8+ T cells, when compared to Fgl2-deficient CD8+ T cells, is underpinned by the cell-intrinsic interaction of Fgl2 with CD8+ T cell-expressed FcγRIIB and concomitant caspase 3/7-mediated apoptosis. Our results thus illuminate a cell-autonomous regulatory axis by which PD-1+ CD8+ T cells both express the receptor and secrete its ligand in order to mediate suppression of anti-tumor and anti-viral immunity. Mechanisms regulating CD8 T cell responsiveness are not fully understood. Here the authors show, using mouse genetic tools and human patient samples, that CD8 T-derived Fgl2, an immunosuppressive cytokine, binds autologous FcγRIIB receptor to induce CD8 T cell apoptosis and dampen anti-tumor or antivirus immunity. [ABSTRACT FROM AUTHOR]

Introduction: A hallmark of T cell dysregulation during sepsis is the downregulation of costimulatory molecules. CD28 is one of T cell costimulatory molecules significantly altered on memory T cells during sepsis. We recently showed that treatment with a aCD28 agonist in septic immunologically experienced mice led to improved survival. Therefore, here we aimed to identify the cell subset(s) necessary for the survival benefit observed in the context of CD28 agonism, and to further investigate the mechanism by which CD28 agonism improves sepsis survival in immunologically experienced mice. Methods: Mice received specific pathogen inoculation to generate memory T cell populations similar in frequency to that of adult humans. Once these infections were cleared and the T cell response had transitioned to the memory phase, animals were rendered septic via cecal ligation and puncture in the presence or absence of an agonistic anti-CD28 mAb. Results: Results demonstrated that CD8+ T cells, and not bulk CD4+ T cells or CD25+ regulatory T cells, were necessary for the survival benefit observed in CD28 agonist-treated septic immunologically experienced mice. Upon examination of these CD8+ T cells, we found that CD28 agonism in septic immunologically experienced mice was associated with an increase in Foxp3+ CD8+ T cells as compared to vehicle-treated controls. When CD8+ T cells were depleted in septic immunologically experienced mice in the setting of CD28 agonism, a significant increase in levels of inflammatory cytokines in the blood was observed. Discussion: Taken together, these results indicate that CD28 agonism in immunologically experienced mice effectively suppresses inflammation via a CD8+-dependent mechanism to decrease mortality during sepsis. [ABSTRACT FROM AUTHOR]

Intestinal epithelial expression of the tight junction protein claudin-2, which forms paracellular cation and water channels, is precisely regulated during development and in disease. Here, we show that small intestinal epithelial claudin-2 expression is selectively upregulated in septic patients. Similar changes occurred in septic mice, where claudin-2 upregulation coincided with increased flux across the paracellular pore pathway. In order to define the significance of these changes, sepsis was induced in claudin-2 knockout (KO) and wild-type (WT) mice. Sepsis-induced increases in pore pathway permeability were prevented by claudin-2 KO. Moreover, claudin-2 deletion reduced interleukin-17 production and T cell activation and limited intestinal damage. These effects were associated with reduced numbers of neutrophils, macrophages, dendritic cells, and bacteria within the peritoneal fluid of septic claudin-2 KO mice. Most strikingly, claudin-2 deletion dramatically enhanced survival in sepsis. Finally, the microbial changes induced by sepsis were less pathogenic in claudin-2 KO mice as survival of healthy WT mice injected with cecal slurry collected from WT mice 24 h after sepsis was far worse than that of healthy WT mice injected with cecal slurry collected from claudin-2 KO mice 24 h after sepsis. Claudin-2 upregulation and increased pore pathway permeability are, therefore, key intermediates that contribute to development of dysbiosis, intestinal damage, inflammation, ineffective pathogen control, and increased mortality in sepsis. The striking impact of claudin-2 deletion on progression of the lethal cascade activated during sepsis suggests that claudin-2 may be an attractive therapeutic target in septic patients. [ABSTRACT FROM AUTHOR]

Checkpoint inhibition using Fc-containing monoclonal antibodies has emerged as a powerful therapeutic approach to augment antitumor immunity. We recently showed that FcγRIIB, the only inhibitory IgG-Fc receptor, is expressed on a population of highly differentiated effector CD8+ T cells in the tumors of mice and humans, raising the possibility that CD8+ T cell responses may be directly modulated by checkpoint inhibitor binding to T cell–expressed FcγRIIB. Here, we show that despite exhibiting strong proliferative and cytokine responses at baseline, human FcγRIIBpos CD8+ T cells exhibited reduced responsiveness to both PD-1 and CTLA-4 checkpoint inhibition as compared with FcγRIIBneg CD8+ T cells in vitro. Moreover, frequencies of FcγRIIBpos CD8+ T cells were reduced after treatment of patients with melanoma with nivolumab in vivo. This reduced responsiveness was FcγRIIB dependent, because conditional genetic deletion of FcγRIIB on tumor-specific CD8+ T cells improved response to checkpoint blockade in B16 and LLC mouse models of cancer. The limited responsiveness of FcγRIIBpos CD8+ T cells was also dependent on an intact Fc region of the checkpoint inhibitor, in that treatment with Fc-devoid anti–PD-1 F(ab) fragments resulted in increased proliferation of FcγRIIBpos CD8+ T cells, without altering the response of FcγRIIBneg CD8+ T cells. Last, the addition of FcγRIIB blockade improved efficacy of PD-1 checkpoint inhibition in mouse models of melanoma, lung, and colon cancer. These results illuminate an FcγRIIB-mediated, cell-autonomous mechanism of CD8+ T cell suppression, which limits the efficacy of checkpoint inhibitors during antitumor immune responses in vivo. Editor's summary: Although immune checkpoint inhibitors (ICI) has been beneficial for many patients, 40% of patients still fail to respond. To investigate this, Bennion et al. evaluated Fcγ Receptor IIB (FcγRIIB), an inhibitory receptor that has recently been found on activated CD8+ T cells. The authors demonstrated that the Fc portion of the anti–programmed death 1 could bind to FcγRIIB on activated CD8+T cells in mice and reduce efficacy of immunotherapy, which was abrogated by adding an antibody against FcyRIIB to treatment. These results elucidate an unappreciated mechanism of FcγRIIB-mediated, cell-autonomous suppression, suggesting altering the Fc portion of these antibodies could better improve efficacy across patients. —Dorothy Hallberg [ABSTRACT FROM AUTHOR]

Sepsis is one of the leading causes of death worldwide. While mortality is high regardless of inciting infection or comorbidities, mortality in patients with cancer and sepsis is significantly higher than mortality in patients with sepsis without cancer. Cancer patients are also significantly more likely to develop sepsis than the general population. The mechanisms underlying increased mortality in cancer and sepsis patients are multifactorial. Cancer treatment alters the host immune response and can increase susceptibility to infection. Preclinical data also suggests that cancer, in and of itself, increases mortality from sepsis with dysregulation of the adaptive immune system playing a key role. Further, preclinical data demonstrate that sepsis can alter subsequent tumor growth while tumoral immunity impacts survival from sepsis. Checkpoint inhibition is a well-accepted treatment for many types of cancer, and there is increasing evidence suggesting this may be a useful strategy in sepsis as well. However, preclinical studies of checkpoint inhibition in cancer and sepsis demonstrate results that could not have been predicted by examining either variable in isolation. As sepsis management transitions from a 'one size fits all' model to a more individualized approach, understanding the mechanistic impact of cancer on outcomes from sepsis represents an important strategy towards delivering on the promise of precision medicine in the intensive care unit. [ABSTRACT FROM AUTHOR]

Introduction: Chronic alcohol use poses significant negative consequences to public health and, among its many biologic effects, is associated with significant T cell dysregulation within the adaptive immune system that has yet to be fully characterized. Novel, automated strategies for high dimensional flow cytometric analysis of the immune system are rapidly improving researchers' ability to detect and characterize rare cell types. Methods: Using a murine model of chronic alcohol ingestion in conjunction with viSNE and CITRUS analysis tools, we performed a machine-driven, exploratory analysis comparing rare splenic subpopulations within the conventional CD4+, regulatory CD4+ and CD8+ T cell compartments between alcohol- and waterfed animals. Results: While there were no differences in the absolute numbers of bulk CD3+ T cells, bulk CD4+ T cells, bulk CD8+ T cells, Foxp3- CD4+ conventional T cells (Tconv) or Foxp3+ CD4+ regulatory T cells (Treg), we identified populations of naïve Helios+ CD4+Tconv and naïve CD103+ CD8+ splenic T cells that were decreased in chronically alcohol exposed mice versus water-fed controls. In addition, we identified increased CD69+ Treg and decreased CD103+ effector regulatory T cell (eTreg) subsets in conjunction with increased frequency of a population that may represent a transitional phenotype between central regulatory T cell (cTreg) and eTreg. Discussion: These data provide further resolution into the character of decreased naïve T cell populations known to be present in alcohol exposed mice, as well as describe alterations in effector regulatory T cell phenotypes associated with the pathogenesis of chronic alcohol-induced immune dysfunction. [ABSTRACT FROM AUTHOR]

Introduction: Costimulation blockade targeting the CD28 pathway provides improved long-term renal allograft survival compared to calcineurin inhibitors but may be limited as CTLA-4-Ig (abatacept, belatacept) blocks both CD28 costimulation and CTLA-4 coinhibition. Directly targeting CD28 while leaving CTLA-4 intact may provide a mechanistic advantage. Fc-silent noncrosslinking CD28 antagonizing domain antibodies (dAb) are currently in clinical trials for renal transplantation. Given the current standard of care in renal transplantation at most US centers, it is likely that lymphodepletion via thymoglobulin induction therapy could be used in patients treated with CD28 antagonists. Thus, we investigated the impact of T cell depletion (TCD) on T cell phenotype following homeostatic reconstitution in a murine model of skin transplantation treated with anti-CD28dAb. Methods: Skin from BALB/cJ donors was grafted onto C56BL/6 recipients which were treated with or without 0.2mg anti-CD4 and 10mg anti-CD8 one day prior to transplant and with or without 100mg anti-CD28dAb on days 0, 2, 4, 6, and weekly thereafter. Mice were euthanized six weeks post-transplant and lymphoid cells were analyzed by flow cytometry. Results: Anti-CD28dAb reversed lymphopenia-induced differentiation of memory CD4+ T cells in the spleen and lymph node compared to TCD alone. Mice treated with TCD+anti-CD28dAb exhibited significantly improved skin graft survival compared to anti-CD28dAb alone, which was also improved compared to no treatment. In addition, the expression of CD69 was reduced on CD4+ and CD8+ T cells in the spleen and lymph node from mice that received TCD+anti-CD28dAb compared to TCD alone. While a reduced frequency of CD4+FoxP3+ T cells was observed in anti-CD28dAb treated mice relative to untreated controls, this was balanced by an increased frequency of CD8+Foxp3+ T cells that was observed in the blood and kidney of mice given TCD+anti-CD28dAb compared to TCD alone. Discussion: These data demonstrate that CD28 signaling impacts the differentiation of both CD4+ and CD8+ T cells during homeostatic reconstitution following lymphodepletion, resulting in a shift towards fewer activated memory T cells and more CD8+FoxP3+ T cells, a profile that may underpin the observed prolongation in allograft survival. [ABSTRACT FROM AUTHOR]

Background: Outcomes from Gram‐negative bacteremia (GNB) in solid organ transplant (SOT) recipients are poorly understood. Methods: This is a single center prospective cohort study comparing the clinical characteristics and outcomes of SOT recipients with GNB to immunocompetent non‐SOT patients with GNB between 1/1/2002 through 12/31/2018. Outcomes of interest included incidence of septic shock, respiratory failure, and time to death. A multivariable logistic regression model was used to determine factors associated with incidence of septic shock and respiratory failure. Time to death was evaluated using Cox proportional hazard models. Results: A total of 297 SOT and 1245 immunocompetent non‐SOT patients were included. Incidence of septic shock did not significantly differ between the groups (SOT 25.3% vs. non‐SOT 24.6%, p =.8225). Overall survival did not significantly differ by transplant status (30‐day survival: SOT 76%, 95% confidence interval [CI] 70, 92, non‐SOT 74%, 95% CI 71, 77: log rank: p =.76). SOT recipients taking three immunosuppressive medications had significantly lower odds of developing septic shock or respiratory failure requiring intubation and mechanical ventilation than those taking ≤1 agent (shock: adjusted odds ratio [aOR] 0.29, 95% CI 0.09, 0.90, p =.0316; respiratory failure: aOR 0.14, 95% CI: 0.04, 0.49, p =.0020). Conclusions: SOT recipients with GNB do not experience higher rates of septic shock, respiratory failure, or mortality than immnon‐SOT recipients with GNB. Among SOT recipients, a greater number of immunosuppressive medications may be associated with improved outcomes during GNB. Future studies are needed to understand the potential relationship between levels of immunosuppression and clinical outcome in SOT recipients with GNB. [ABSTRACT FROM AUTHOR]

Increasing evidence indicates that microbes have a large influence on immune function. Previous studies have linked pathogenic microorganisms with decreased allograft tolerance and subsequent rejection. In this issue of the JCI, Lei and colleagues demonstrate that commensal organisms also influence the host response to allograft transplantation. Using murine skin and cardiac transplant models, the authors demonstrate that allograft rejection is accelerated in mice with a normal microbiome compared with germ-free animals and antibiotic-treated mice. The increased graft rejection observed in conventional animals was due to enhanced T cell priming and was mediated through type I IFN. Together, these results suggest that altering a patient's microbial community prior to transplant could improve allograft acceptance., The microbial influence on alloimmunity The ability of microbes to modulate alloimmunity has been well appreciated for the better part of two decades. Seminal studies in the early 2000s revealed [...]