Signaling through the Inhibitory Fc Receptor FcγRIIB Induces CD8+ T Cell Apoptosis to Limit T Cell Immunity.

Effector CD8+ T cells are important mediators of adaptive immunity, and receptor-ligand interactions that regulate their survival may have therapeutic potential. Here, we identified a subset of effector CD8+ T cells that expressed the inhibitory fragment crystallizable (Fc) receptor FcγRIIB following activation and multiple rounds of division. CD8+ T cell-intrinsic genetic deletion of Fcgr2b increased CD8+ effector T cell accumulation, resulting in accelerated graft rejection and decreased tumor volume in mouse models. Immunoglobulin G (IgG) antibody was not required for FcγRIIB-mediated control of CD8+ T cell immunity, and instead, the immunosuppressive cytokine fibrinogen-like 2 (Fgl2) was a functional ligand for FcγRIIB on CD8+ T cells. Fgl2 induced caspase-3/7-mediated apoptosis in Fcgr2b+, but not Fcgr2b-/-, CD8+ T cells. Increased expression of FcγRIIB correlated with freedom from rejection following withdrawal from immunosuppression in a clinical trial of kidney transplant recipients. Together, these findings demonstrate a cell-intrinsic coinhibitory function of FcγRIIB in regulating CD8+ T cell immunity.

Characterization of the Antibody Response to SARS-CoV-2 Infection in COVID-19 Transplant versus Nontransplant Recipients by Ig-MS.

Solid organ transplant recipients with immunosuppressant regimens to prevent rejection are less able to mount effective immune responses to pathogenic infection. Here, we apply a recently reported mass spectrometry-based serological approach known as Ig-MS to characterize immune responses against infection with SARS-CoV-2 in cohorts of transplant recipients and immunocompetent controls, both at a single early time point following COVID-19 diagnosis as well as over the course of one-month postdiagnosis. We found that the antibody repertoires generated by transplant recipients against SARS-CoV-2 do not differ significantly compared to immunocompetent individuals with regard to repertoire titer, clonality, or glycan composition. Importantly, our study is the first to characterize the evolution of antibody glycan profiles in transplant recipients with COVID-19 disease, presenting evidence that the evolution of glycan composition in these immunocompromised individuals is similar to that in immunocompetent people.

Sensitization in transplantation: Assessment of Risk 2022 Working Group Meeting Report.

The Sensitization in Transplantation: Assessment of Risk workgroup is a collaborative effort of the American Society of Transplantation and the American Society of Histocompatibility and Immunogenetics that aims at providing recommendations for clinical testing, highlights gaps in current knowledge, and proposes areas for further research to enhance histocompatibility testing in support of solid organ transplantation. This report provides updates on topics discussed by the previous Sensitization in Transplantation: Assessment of Risk working groups and introduces 2 areas of exploration: non-human leukocyte antigen antibodies and utilization of human leukocyte antigen antibody testing measurement to evaluate the efficacy of antibody-removal therapies.

Donor-specific antibodies after heart transplantation for Fontan-associated protein-losing enteropathy.

BACKGROUND: Despite ubiquitous exposure to sensitizing events, most Fontan PLE patients have low panel reactive antibodies (PRA). To assess whether they are at risk for donor-specific antibody (DSA) memory response following heart transplantation (HT) when their PLE resolves, DSA profiles, incidence of rejection, and graft outcomes in Fontan recipients with and without PLE were compared. METHODS: Patient characteristics, appearance of newly detected DSA (nDSA), and graft outcomes were compared between patients with and without PLE using Wilcoxon rank-sum and Chi-squared tests. DSA burden was quantified using titers and time to nDSA, incidence of rejection, and graft outcomes were compared using Kaplan-Meier curves and the log-rank test. RESULTS: Characteristics of patients with and without PLE were similar. Lymphocyte and albumin levels were lower in the PLE group, and flow PRA were comparable. Graft failure, CAV, and ACR were similar between the two groups, but AMR occurred more frequently in the PLE group (p = .03). Nearly 50% of PLE patients experienced class II nDSA by 1-year post-HT, compared to 30% of non-PLE patients, but this difference was statistically not significant. Antibody burden did not differ between groups. CONCLUSIONS: In this cohort, PLE was associated with AMR within the first-year post-HT, despite no significant difference in nDSA. Small patient numbers limited statistical comparison of nDSA in this cohort. PLE may be a risk factor for AMR post-HT, and the possibility of a clinically important DSA memory response remains. Larger studies are necessary to better understand these preliminary findings.

Impact of SIRPα polymorphism on transplant outcomes in HLA-identical living donor kidney transplantation.

Signal-regulatory protein α (SIRPα), a polymorphic inhibitory membrane-bound receptor, and its ligand CD47 have recently been implicated in the modulation of innate immune allorecognition in murine models. Here, we investigate the potential impact of SIRPα donor-recipient mismatches on graft outcomes in human kidney transplantation. To eliminate the specific role of HLA-matching in alloresponse, we genotyped the two most common variants of SIRPα in a cohort of 55 HLA-identical, biologically-related, donor-recipient pairs. 69% of pairs were SIRPα identical. No significant differences were found between donor-recipient SIRPα-mismatch status and T cell-mediated rejection/borderline changes (25.8% vs. 25%) or slow graft function (15.8% vs. 17.6%). A trend towards more graft failure (GF) (23.5% vs. 5.3%, P = .06), interstitial inflammation (50% vs. 23%, P = .06) and significant changes in peritubular capillaritis (ptc) (25% vs. 0%, P = .02) were observed in the SIRPα-mismatched group. Unexpectedly, graft-versus-host (GVH) SIRPα-mismatched pairs exhibited higher rates of GF and tubulitis (38% vs. 5%, P = .031 and .61 ± .88 vs. 0, P = .019; respectively). Whether the higher prevalence of ptc in SIRPα-mismatched recipients and the higher rates of GF in GVH SIRPα-mismatched pairs represent a potential role for SIRPα in linking innate immunity and alloimmune rejection requires further investigation in larger cohorts.

Memory T cell-mediated rejection is mitigated by FcγRIIB expression on CD8+ T cells.

Donor-reactive memory T cells generated via heterologous immunity represent a potent barrier to long-term graft survival following transplantation because of their increased precursor frequency, rapid effector function, altered trafficking patterns, and reduced reliance on costimulation signals for activation. Thus, the identification of pathways that control memory T cell survival and secondary recall potential may provide new opportunities for therapeutic intervention. Here, we discovered that donor-specific effector/memory CD8+ T cell populations generated via exposure to acute vs latent vs chronic infections contain differential frequencies of CD8+ T cells expressing the inhibitory Fc receptor FcγRIIB. Results indicated that frequencies of FcγRIIB-expressing CD8+ donor-reactive memory T cells inversely correlated with allograft rejection. Furthermore, adoptive T cell transfer of Fcgr2b-/- CD8+ T cells resulted in an accumulation of donor-specific CD8+ memory T cells and enhanced recall responses, indicating that FcγRIIB functions intrinsically to limit T cell CD8+ survival in vivo. Lastly, we show that deletion of FcγRIIB on donor-specific CD8+ memory T cells precipitated costimulation blockade-resistant rejection. These data therefore identify a novel cell-intrinsic inhibitory pathway that functions to limit the risk of memory T cell-mediated rejection following transplantation and suggest that therapeutic manipulation of this pathway could improve outcomes in sensitized patients.

The quest to decipher HLA immunogenicity: Telling friend from foe.

Molecular mismatch load analysis was recently introduced as a means for performing risk stratification following organ transplantation. However, although good correlation was demonstrated between molecular mismatch load and generation of de novo donor-specific HLA antibody (DSA), quite a few exceptions exist, and the underlying factors that define HLA immunogenicity remain unclear. Herein, we present a new paradigm to interrogate differences between molecular mismatches that lead to the generation of de novo DSA and those that do not (the 2MM1DSA cohort). Specifically, patients transplanted across 2 HLA-DQ mismatches, who formed de novo DSA only to one mismatch (foe) but not the other (friend), provide a unique environment in which patient-specific factors that affect the immune response other than immunogenicity, such as infection and immunosuppression, can be controlled for. It further permits focusing on mismatches uniquely exhibited by the de novo DSA allele, rather than mismatches shared by both DSA and non-DSA alleles. This concept paper illustrates several examples, highlights the need for center-specific or population-specific cutoff values for posttransplant risk stratification, and mostly argues that if there is no direct correlation between molecular mismatch load and immunogenicity, then molecular mismatch load must not be adopted as an approach for equitable organ allocation.

Prognostic tools to assess candidacy for and efficacy of antibody-removal therapy.

Currently, the ability to predict or monitor the efficacy of HLA antibody-removal therapies is deficient. We previously reported that titration studies are a consistent and accurate means of assessing antibody strength. To test whether titration studies can also predict which patients are better candidates for desensitization, we studied 38 patients from 3 centers (29 receiving plasmapheresis/low-dose intravenous immunoglobulin [IVIg]; 9 patients receiving high-dose IVIg). For patients undergoing plasmapheresis/low-dose IVIg, antibody titer reduction correlated with number of treatment cycles for both class I and II antibodies but only up to approximately 4 cycles. Reduction in titer slowed with additional cycles, suggesting a limit to the efficacy of this approach. Furthermore, initial titer (predesensitization) can guide the selection of candidates for successful antibody-removal treatment. In our experience, patients with antibodies at an initial titer >1:512 could not be reduced to the goal of a negative lymphocyte crossmatch, corresponding to a 1:16 titer, despite a significant increase in the number of treatment cycles. Change in mean fluorescence intensity (MFI) value did not correlate with success of treatment if initial MFI values were >10 000, likely due to single antigen bead saturation. Overall, we present a potential prognostic tool to predict candidacy and a monitoring tool to assess efficacy of desensitization treatment.

CD40/CD154 blockade inhibits dendritic cell expression of inflammatory cytokines but not costimulatory molecules.

Blockade of the CD40/CD154 pathway remains one of the most effective means of promoting graft survival following transplantation. However, the effects of CD40/CD154 antagonism on dendritic cell (DC) phenotype and functionality following transplantation remain incompletely understood. To dissect the effects of CD154/CD40 blockade on DC activation in vivo, we generated hematopoietic chimeras in mice that expressed a surrogate minor Ag (OVA). Adoptive transfer of OVA-specific CD4(+) and CD8(+) T cells led to chimerism rejection, which was inhibited by treatment with CD154 blockade. Surprisingly, CD154 antagonism did not alter the expression of MHC and costimulatory molecules on CD11c(+) DCs compared with untreated controls. However, DCs isolated from anti-CD154-treated animals exhibited a significant reduction in inflammatory cytokine secretion. Combined blockade of inflammatory cytokines IL-6 and IL-12p40 attenuated the expansion of Ag-specific CD4(+) and CD8(+) T cells and transiently inhibited the rejection of OVA-expressing cells. These results suggest that a major effect of CD154 antagonism in vivo is an impairment in the provision of signal three during donor-reactive T cell programming, as opposed to an impact on the provision of signal two. We conclude that therapies designed to target inflammatory cytokines during donor-reactive T cell activation may be beneficial in attenuating these responses and prolonging graft survival.

A blueprint for electronic utilization of ambiguous molecular HLA typing data in organ allocation systems and virtual crossmatch.

Virtual crossmatch (VXM) compares a transplant candidate's unacceptable antigens to the HLA typing of the donor before an organ offer is accepted and, in selected cases, supplant a prospective physical crossmatch. However, deceased donor typing can be ambiguous, leading to uncertainty in compatibility prediction. We have developed a prototype web application that utilizes ambiguous HLA molecular typing data to predict which unacceptable antigens are present in the donor HLA genotype as donor-specific antibodies (DSA). The application compares a candidate's listed unacceptable antigens to computed probabilities of all possible two-field donor HLA alleles and UNOS antigens. The VIrtual CrossmaTch for mOleculaR HLA typing (VICTOR) tool can be accessed at http://www.transplanttoolbox.org/victor. We reanalyzed historical VXM cases where a transplant center's manual interpretation of molecular typing results influenced offer evaluation. We found that interpretation of ambiguous donor molecular typing data using imputation could one day influence VXM decisions if the DSA predictions were rigorously validated. Standardized interpretation of molecular typing data, if applied to the match run, could also change which offers are made. HLA typing ambiguity has been an underappreciated source of immunological risk in organ transplantation. The VICTOR tool can serve as a testbed for development of allocation policies with the aim of decreasing offers refused due to HLA incompatibility.

Minimal data reporting standards for serological testing for histocompatibility.

Histocompatibility testing, and HLA antibody screening in particular, varies in practice among laboratories. Currently, standards are lacking regarding the reporting of testing methods in publications. It is essential that scientific methods are rigorously and transparently described upon publication, so that results can be accurately interpreted and independently corroborated. Additionally, this would allow work groups to compile diverse data to achieve clinically significant conclusions from meta-analyses. These efforts are hindered when there is a paucity of method descriptions and where variability in serum treatment, protocol modifications, and assay thresholds affecting assay interpretation are known to exist. Thus, the ASHI Science and Technology Initiatives Committee (ASHI STIC) undertook the task of formulating recommendations for reporting HLA antibody testing by solid phase assays, and the associated HLA typing required for interpretation, in scientific publications. Herein we put forth standards for minimum information about HLA antibody testing methods reported in histocompatibility publications.

Assessing the potential of angiotensin II type 1 receptor and donor specific anti-endothelial cell antibodies to predict long-term kidney graft outcome.

Endothelial cell antigens have been reported as potential targets for antibodies in the context of organ transplantation, leading to increased risk for graft failure. Serum samples from 142 consecutive living donor kidney recipients were tested for the presence of antibodies to angiotensin II - type 1 receptor (AT1R), donor endothelial cells, and donor HLA. Graft survival was monitored for five years post-transplant, and secondary outcomes, including biopsy-proven rejection, proteinuria, biopsy-proven vasculopathy, and renal function based on serum creatinine were also assessed for the first two to three years. AT1R antibody levels were positive (>17U/mL) in 11.3%, 18.8% and 8.1% of patients pre-transplant, post-transplant and at time of indication biopsy, respectively. XM-ONE assay was positive in 17.6% of patients pre-transplant (7 IgG+; 15 IgM+; 3 IgG+/IgM+). Overall, 4 patients experienced antibody-mediated rejection (AMR), 31 borderline cellular rejection (BCR), 19 cellular rejection (CR) and 3 mixed AMR and CR within the first 24months. While pre-existing and de novo donor-specific HLA antibodies were associated with graft failure and many secondary outcomes, no statistical association was found for either anti-endothelial or anti-AT1R antibodies, indicating that these tests may not be the best predictors of graft outcome in living donor renal transplantation.

HLA Epitopes - Are We Ready for Clinical Prime Time? Historic Perspective and Future Needs.

A significant barrier to long-term transplant success is the development of de novo donor-specific human leukocyte antigen (HLA) antibodies. The best approach to minimize the risk of developing such antibodies is an HLA identical transplant, but the likelihood of finding such an organ is very low. The alternative is to identify "permissible mismatches" - HLA antigen mismatches that are less likely to induce an immune response. In the past few years, it has become clear that matching at the "epitope level" is the likely solution; however, we are still struggling with how to define HLA epitopes. Herein, we provide a short review of the development of the epitope concept within the HLA field, with the hope that it sheds light on present knowledge. We follow with our personal opinion on where the future is leading us.

Viral Escape Mutant Epitope Maintains TCR Affinity for Antigen yet Curtails CD8 T Cell Responses.

T cells have the remarkable ability to recognize antigen with great specificity and in turn mount an appropriate and robust immune response. Critical to this process is the initial T cell antigen recognition and subsequent signal transduction events. This antigen recognition can be modulated at the site of TCR interaction with peptide:major histocompatibility (pMHC) or peptide interaction with the MHC molecule. Both events could have a range of effects on T cell fate. Though responses to antigens that bind sub-optimally to TCR, known as altered peptide ligands (APL), have been studied extensively, the impact of disrupting antigen binding to MHC has been highlighted to a lesser extent and is usually considered to result in complete loss of epitope recognition. Here we present a model of viral evasion from CD8 T cell immuno-surveillance by a lymphocytic choriomeningitis virus (LCMV) escape mutant with an epitope for which TCR affinity for pMHC remains high but where the antigenic peptide binds sub optimally to MHC. Despite high TCR affinity for variant epitope, levels of interferon regulatory factor-4 (IRF4) are not sustained in response to the variant indicating differences in perceived TCR signal strength. The CD8+ T cell response to the variant epitope is characterized by early proliferation and up-regulation of activation markers. Interestingly, this response is not maintained and is characterized by a lack in IL-2 and IFNγ production, increased apoptosis and an abrogated glycolytic response. We show that disrupting the stability of peptide in MHC can effectively disrupt TCR signal strength despite unchanged affinity for TCR and can significantly impact the CD8+ T cell response to a viral escape mutant.

Novel insights into anti-CD40/CD154 immunotherapy in transplant tolerance.

Since the discovery of the CD40-CD154 costimulatory pathway and its critical role in the adaptive immune response, there has been considerable interest in therapeutically targeting this interaction with monoclonal antibodies in transplantation. Unfortunately, initial promise in animal models gave way to disappointment in clinical trials following a number of thromboembolic complications. However, recent mechanistic studies have identified the mechanism of these adverse events, as well as detailed a myriad of interactions between CD40 and CD154 on a wide variety of immune cell types and the critical role of this pathway in generating both humoral and cell-mediated alloreactive responses. This has led to resurgence in interest and the potential resurrection of anti-CD154 and anti-CD40 antibodies as clinically viable therapeutic options.