Marginal Zone B Cells Are Necessary for the Formation of Anti-donor IgG After Allogeneic Sensitization.

BACKGROUND: The formation of anti-major histocompatibility complex (MHC) antibodies is a significant barrier for many patients awaiting organ transplantation. Patients with preformed anti-MHC antibodies have limited options for suitable donors, and the formation of donor-specific anti-MHC antibodies after transplantation is a harbinger of graft rejection. Despite the recognized importance of anti-MHC antibodies, the mechanisms responsible for the differentiation of B cells after exposure to allogeneic antigens are poorly understood. METHODS: To evaluate the differentiation of B cells in response to allogeneic antigen, we used a model of H-2 b C57Bl/6 sensitization with H-2 d antigen. We used a class I MHC tetramer-based approach to identify allogeneic B cells and flow cytometric crossmatch to identify allogeneic IgM and IgG. RESULTS: We found that although the formation of anti-H-2 d IgG was robust, few class-switched B cells and germinal center B cells were formed. Antigen-specific B cells did not express classical memory B-cell markers after sensitization but had an IgM + CD21 + marginal zone B-cell phenotype. The frequency of marginal zone B cells increased after sensitization. Depletion of marginal zone B cells before sensitization or skin grafting resulted in a significant diminution of anti-H-2 d IgG and fewer germinal center B cells. Adoptive transfer experiments revealed that marginal zone B cells more efficiently differentiated into germinal center B cells and anti-donor IgG-producing cells than follicular B cells. CONCLUSIONS: These results demonstrate an important role for marginal zone B cells as a reservoir of alloreactive B cells that are activated by allogeneic antigens.

Transplantation elicits a clonally diverse CD8+ T cell response that is comprised of potent CD43+ effectors.

CD8+ T cells mediate acute rejection of allografts, which threatens the long-term survival of transplanted organs. Using MHC class I tetramers, we find that allogeneic CD8+ T cells are present at an elevated naive precursor frequency relative to other epitopes, only modestly increase in number after grafting, and maintain high T cell receptor diversity throughout the immune response. While antigen-specific effector CD8+ T cells poorly express the canonical effector marker KLRG-1, expression of the activated glycoform of CD43 defines potent effectors after transplantation. Activated CD43+ effector T cells maintain high expression of the coreceptor induced T cell costimulator (ICOS) in the presence of CTLA-4 immunoglobulin (Ig), and dual CTLA-4 Ig/anti-ICOS treatment prolongs graft survival. These data demonstrate that graft-specific CD8+ T cells have a distinct response profile relative to anti-pathogen CD8+ T cells and that CD43 and ICOS are critical surface receptors that define potent effector CD8+ T cell populations that form after transplantation.

Donor specific HLA antibody in hematopoietic stem cell transplantation: Implications for donor selection.

Advances in hematopoietic stem cell transplant (HSCT) have led to changes in the approach to donor selection. Many of these new approaches result in greater HLA loci mismatching, either through the selection of haploidentical donors or permissive HLA mismatches. Although these approaches increase the potential of transplant for many patients by expanding the number of acceptable donor HLA genotypes, they add the potential barrier of donor-specific HLA antibodies (DSA). DSA presents a unique challenge in HSCT, as it can limit engraftment and lead to graft failure. However, transient reduction of HLA antibodies through desensitization treatments can limit the risk of graft failure and facilitate engraftment. Thus, the consideration of DSA in donor selection and the management of DSA prior to transplant are playing an increasingly important role in HSCT. In this review, we will discuss studies addressing the role of HLA antibodies in HSCT, the reported impact of desensitization on DSA levels, and the implications for selecting donors for patients with DSA. We found that there is a clear consensus that moderate strength DSA should be avoided, while desensitization strategies are reported to be effective in most cases at reducing DSA to amenable levels. There is limited information regarding the impact of specific characteristics of DSA, such as HLA loci or overall level of sensitization, which could further aid in donor selection for sensitized HSCT candidates.

Implementing virtual crossmatch based diagnostic management teams in human leukocyte antigen laboratories and transplant programs.

Histocompatibility testing has continuously evolved since its inception. One such advancement is the implementation of the virtual crossmatch (VXM). Recent changes to allograft allocation schemes have resulted in increased organ sharing over greater distances, resulting in expanded utilization of the VXM to assess donor: recipient compatibility. In fact, the VXM has become a major arbitrator of pre-transplant compatibility assessment prior to both deceased and living donor organ allocation. This shift in pre-transplant practice is concurrent with the US healthcare systems' move towards more inclusive and coordinated team-based management approach to disease diagnosis. Diagnostic Management Teams (DMTs) exemplify this shift in patient care. Our institution seized the opportunity to build and implement a VXM DMT to improve and streamline pre-transplant assessment. This VXM DMT is compliant with US regulatory standards and provides a consultative report containing relevant pre-transplant information, test interpretation as well as recommendations for HLA additional (if any) testing. Herein we describe the development of and experience with the VXM DMT a year after its launch.

The utility of imputation for molecular mismatch analysis in solid organ transplantation.

HLA genotyping has undergone a rapid progression in resolution since the development of DNA-based typing methods. Despite the advent of high-resolution next-generation sequencing, the bulk of solid organ genotyping is performed at intermediate resolution, which provides multiple possible two-field results for each classical HLA loci. As a result, several methodologies have been developed to impute the most likely allele-level (two-field) HLA genotype for the purposes of donor-recipient compatibility analysis. The advent of molecular mismatch analysis, however, has placed a new emphasis on the accuracy of imputation. While seminal molecular mismatch studies have relied on the imputation of intermediate resolution genotyping, several recent studies have performed analysis showing that imputation generates inaccuracies in epitope identification. While the clinical impact of these errors is not clear, it is important that these concerns do not preclude future progress in understanding the utility of molecular mismatch analysis in transplantation. In the future, advances in genotyping methods will result in routine two-field resolution that will abrogate these concerns. In the meantime, however, studies are needed in order to address the role of molecular mismatch in diverse patient populations and to carefully address the potential of molecular mismatch analysis in the context of imputation.

TIGIT regulates apoptosis of risky memory T cell subsets implicated in belatacept-resistant rejection.

Belatacept confers increased patient and graft survival in renal transplant recipients relative to calcineurin inhibitors, but is associated with an increased rate of acute rejection. Recent immunophenotypic studies comparing pretransplant T cell phenotypes of patients who reject versus those who remain stable on belatacept identified three potential "risky" memory T cell subsets that potentially underlie belatacept-resistant rejection: CD4+ CD28+ TEM , CD8+ CD28null , and CD4+ CD57+ PD1- subsets. Here, we compared key phenotypic and functional aspects of these human memory T cell subsets, with the goal of identifying additional potential targets to modulate them. Results demonstrate that TIGIT, an increasingly well-appreciated immune checkpoint receptor, was expressed on all three risky memory T cell subsets in vitro and in vivo in the presence of belatacept. Coculture of human memory CD4+ and CD8+ T cells with an agonistic anti-TIGIT mAb significantly increased apoptotic cell death of all three risky memory T cell subsets. Mechanistically, TIGIT-mediated apoptosis of risky memory T cells was dependent on FOXP3+ Treg, suggesting that agonism of the TIGIT pathway increases FOXP3+ Treg suppression of human memory T cell populations. Overall, these data suggest that TIGIT agonism could represent a new therapeutic target to inhibit belatacept-resistant rejection during transplantation.

The utility of second single antigen bead assay: Clearing the water or stirring up mud?

Though solid-phase single antigen bead (SAB) testing has provided major advances to the HLA community and organ allocation, it has not been without limitations. In particular, false-positive reactions lead to interpretative challenges and the potential to preclude a transplant if the corresponding antigens are deemed unacceptable. Two different vendor platforms are commercially available for SAB testing, one more recent than the other. The aim herein was to assess the benefit of using the newer SAB platform in situations where the primary platform yielded suspicious (specifically, false positive) reactions. Therefore, 42 serum samples with commonly encountered false-positive patterns observed in our laboratory were tested with the newer platform. Cases were classified as resolved, equivalent, or divergent based on whether the second platform produced no reactivity, the same pattern, or a distinctly different pattern compared to the primary platform, respectively. Approximately 33% of cases were resolved, 46% were equivalent, and 21% were divergent. The project revealed advantages of adding a second SAB platform to the laboratory's test menu including resolving challenging samples and including broader coverage of different alleles and unique class II alpha/beta subunit combinations. However, the challenges of validating, maintaining, and billing for another test method in the laboratory may be barriers to routine use.

CD28-Dependent CTLA-4 Expression Fine-Tunes the Activation of Human Th17 Cells.

Previous work has demonstrated that Th17 memory cells but not Th1 cells are resistant to CD28/CTLA-4 blockade with CTLA-4 Ig, leading us to investigate the individual roles of the CD28 and CTLA-4 cosignaling pathways on Th1 versus Th17 cells. We found that selective CD28 blockade with a domain antibody (dAb) inhibited Th1 cells but surprisingly augmented Th17 responses. CD28 agonism resulted in a profound increase in CTLA-4 expression in Th17 cells as compared with Th1 cells. Consistent with these findings, inhibition of the CD28 signaling protein AKT revealed that CTLA-4 expression on Th17 cells was more significantly reduced by AKT inhibition relative to CTLA-4 expression on Th17 cells. Finally, we found that FOXO1 and FOXO3 overexpression restrained high expression of CTLA-4 on Th17 cells but not Th1 cells. This study demonstrates that the heterogeneity of the CD4+ T cell compartment has implications for the immunomodulation of pathologic T cell responses.

CD45RB Status of CD8+ T Cell Memory Defines T Cell Receptor Affinity and Persistence.

The identity of CD45 isoforms on the T cell surface changes following the activation of naive T cells and impacts intracellular signaling. In this study, we find that the anti-viral memory CD8+ T pool is unexpectedly comprised of both CD45RBhi and CD45RBlo populations. Relative to CD45RBlo memory T cells, CD45RBhi memory T cells have lower affinity and display greater clonal diversity, as well as a persistent CD27hi phenotype. The CD45RBhi memory population displays a homeostatic survival advantage in vivo relative to CD45RBlo memory, and long-lived high-affinity cells that persisted long term convert from CD45RBlo to CD45RBhi. Human CD45RO+ memory is comprised of both CD45RBhi and CD45RBlo populations with distinct phenotypes, and antigen-specific memory to two viruses is predominantly CD45RBhi. These data demonstrate that CD45RB status is distinct from the conventional central/effector T cell memory classification and has potential utility for monitoring and characterizing pathogen-specific CD8+ T cell responses.

Identification of a recurrent pattern of false-positivity by Luminex HLA MHC class I single antigen bead testing.

Previously, a distinct MHC class II Luminex-single antigen bead (SAB) pattern was described and attributed to antibodies targeting denatured antigens. In this study, we describe a distinct MHC class I reactivity pattern observed in 1.8% (105/5992) of samples resulted in 2017. The pattern displays reactivity to the following Luminex-SABs: HLA-A*33:03, A*36:01, A*80:01, B*54:01, B*53:01, C*06:02, C*07:02, C*18:02, C*14:02, C*03:03, C*03:04, and C*15:02. This pattern was identified in patients with no sensitization history, negative FlowPRA results, and antibody to self-antigen(s). Epitope analysis failed to reveal a common determinant(s) to explain this pattern of reactivity. Additionally, we found this pattern to be prevalent in female patients (62%) and also those with systemic lupus erythematosus (62%). Given these findings, we speculate this pattern likely represents false-positive reactivity, possibly due to antibody targeting denatured antigens or a specific peptide, molecular mimicry, autoimmunity, or a combination thereof.

How do I implement diagnostic management teams in transfusion medicine?

Diagnostic management teams (DMTs) were conceptualized approximately twenty years ago in response to increasing subspecialization in medicine. DMTs are a collaboration between diagnostic experts and clinicians that aim to improve accurate and timely diagnosis and treatment of disease. Diagnostic experts provide their expertise in the increasingly complex realm of laboratory testing and interpretation of those test results to guide appropriate test utilization for individual patients. Not only can this approach improve patient care and safety, but DMTs also decrease healthcare costs by reducing unnecessary testing and potential diagnostic errors. Following the DMT construct and principles along with the 2015 National Academy of Medicine recommendations, our transfusion medicine (TM) service streamlined the workup and management of platelet refractory (PR) patients by developing and implementing a formal PR laboratory consult. The goals of this DMT and consult are to improve diagnostic management of PR patients and to decrease delays in providing these patients with appropriate and compatible platelet units. A comprehensive interpretation of test results is directly uploaded to the patient's electronic medical record (EMR), which is associated with a CPT code allowing for compensation for the PR evaluation. Herein we describe the development of and experience with the DMT since its implementation.

The impact of transfused blood products on deceased donor HLA typing.

Accurate deceased donor HLA typing assumes that the blood sample tested contains only DNA from the organ donor. Prior to procurement, many organ donors are transfused at least one unit of red blood cells (RBC). Non-organ donor DNA acquired from transfusions may result in incorrect and/or ambiguous HLA typing. To address this question, we investigated the impact of RBC transfusion on organ donor HLA typing by using different in vitro transfusion models: leukoreduced (LR) and non-LR RBCs. Various quantities of LR and non-LR RBCs were added to normal peripheral blood and HLA typing was performed by real time PCR. Our results show that HLA typing of deceased donors can be impacted dependent upon the type and quantity of transfused RBCs. Importantly, if LR RBCs are given, HLA typing is unlikely to be affected, precluding the need to delay typing and obtain an alternative source of donor DNA.

Out with the old, in with the new: Virtual versus physical crossmatching in the modern era.

The virtual crossmatch (VXM) is gaining acceptance as an alternative approach to assess donor:recipient compatibility prior to transplantation. In contrast to a physical crossmatch, the virtual crossmatch does not require viable donor cells but rather relies on complete HLA typing of the donor and current antibody assessment of the recipient. Thus, the VXM can be performed in minutes which allows for faster transplant decisions thereby increasing the likelihood that organs can be shipped across significant distances yet safely transplanted. Here, we present a brief review of the past 50 years of histocompatibility testing; from the original complement-dependent cytotoxicity crossmatch in 1969 to the new era of molecular HLA typing, solid-phase antibody testing and virtual crossmatching. These advancements have shaped a paradigm shift in our approach to transplantation. That is, foregoing a prospective physical crossmatch in favor of a VXM. In this review, we undertake an in-depth analysis of the pros- and cons- of physical and virtual crossmatching.Finally, we provide objective data on the selected use of the VXM which demonstrate the value of a VXM in lieu of the traditional physical crossmatch for safe and efficient organ transplantation.

Stability of anti-A blood group titers among blood group B renal transplant candidates.

BACKGROUND: 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 A2 B 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. STUDY DESIGN AND METHODS: Titers performed between January 2011 and December 2015 were assessed for 191 group B patients with two or more documented titers. RESULTS: Fifty patients (26%) were ineligible, as the first titer exceeded the cutoff of 8. Of the remaining 141 patients, 19 (13%) became ineligible as the second titer exceeded 8. Thirty-nine patients (28%) had no change in titer between samples, while 71 (50%) had a titer change that never exceeded 8. Only 12 patients (8.5% of total) experienced a titer change that affected eligibility after the second test. CONCLUSION: Although patients experience some variability in anti-A titers over time, in most cases, stability did not affect candidate eligibility. Our results indicate that regular testing beyond the second titer may be unnecessary and represent test overutilization.

(F)Utility of the physical crossmatch for living donor evaluations in the age of the virtual crossmatch.

Flow cytometric crossmatches (FCXM) are routinely performed to support living-donor renal transplantation. While long a laboratory mainstay, a physical crossmatch is costly, time consuming, and frequently poses interpretative conundrums with both false-positive and false- negative results. Given the increased utilization of the virtual crossmatch (vXM) in the deceased donor setting, our aim was to assess its utility in living donor evaluations. We reviewed 100 living donor FCXMs and retrospectively performed a vXM for each pair. Seventy-five (75) cases were concordant, (i.e., FCXM-/vXM- or FCXM+/vXM+) while 25 cases were discordant; Five were vXM+/FCXM- and 20 were FCXM+/vXM-. Since donor-specific antibodies (DSA) were not detected in the 20 FCXM+/vXM- cases, these were interpreted as false-positive, i.e., due to non-HLA antibodies. Importantly, none of these patients, when transplanted across a positive FCXM, experienced early antibody mediated rejection or subsequently developed HLA DSA. These data reveal that, for the vast majority of living donor evaluations, a vXM is an acceptable vetting procedure.

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 TNFR2(hi) 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.