The unnecessary use of short tandem repeat testing on bone marrow samples in patients after 1 year following allogeneic hematopoietic stem cell transplant.

OBJECTIVES: To determine whether the information provided by short tandem repeat (STR) testing and bone marrow (BM) biopsy specimens following hematopoietic stem cell transplant (HSCT) provides redundant information, leading to test overutilization, without additional clinical benefit. METHODS: Cases with synchronous STR and flow cytometric immunophenotyping (FCI) testing, as part of the BM evaluation, were assessed for STR/FCI concordance. RESULTS: Of 1199 cases (410 patients), we found the overall concordance between STR and FCI was 93%, with most cases (1063) classified as STR-/FCI-. Of all discordant cases, 75 (6%) were STR+/FCI-, with only 5 (6.7%) cases best explained as identification of disease relapse. Eight cases were STR-/FCI+, representing relapsed/residual disease. Analysis of cases 1 year or more from transplant (54% of all cases) indicated only 9 (1.5%) were STR+/FCI-, and none uniquely identified relapse. CONCLUSIONS: These data suggest that STR analysis performed 1 year or more post-HSCT does not identify unknown cases of relapse. Furthermore, while STR testing is critical for identifying graft failure/rejection within the first year posttransplant, FCI appears superior to STR at detecting late relapses with low-level disease. Therefore, STR testing from patients 1 year or more post-HSCT may be unnecessary, as BM biopsy evaluation is sufficient to identify disease relapse.

Clinical recommendations for posttransplant assessment of anti-HLA (Human Leukocyte Antigen) donor-specific antibodies: A Sensitization in Transplantation: Assessment of Risk consensus document.

Although anti-HLA (Human Leukocyte Antigen) donor-specific antibodies (DSAs) are commonly measured in clinical practice and their relationship with transplant outcome is well established, clinical recommendations for anti-HLA antibody assessment are sparse. Supported by a careful and critical review of the current literature performed by the Sensitization in Transplantation: Assessment of Risk 2022 working group, this consensus report provides clinical practice recommendations in kidney, heart, lung, and liver transplantation based on expert assessment of quality and strength of evidence. The recommendations address 3 major clinical problems in transplantation and include guidance regarding posttransplant DSA assessment and application to diagnostics, prognostics, and therapeutics: (1) the clinical implications of positive posttransplant DSA detection according to DSA status (ie, preformed or de novo), (2) the relevance of posttransplant DSA assessment for precision diagnosis of antibody-mediated rejection and for treatment management, and (3) the relevance of posttransplant DSA for allograft prognosis and risk stratification. This consensus report also highlights gaps in current knowledge and provides directions for clinical investigations and trials in the future that will further refine the clinical utility of posttransplant DSA assessment, leading to improved transplant management and patient care.

A Primer on Chimerism Analysis: A Straightforward, Thorough Review.

Short tandem repeat (STR) analysis to assess chimerism is a critical aspect of routine care particularly in patients facing stem cell transplants but is also relevant in other clinical scenarios. STR analysis provides a means to assess donor and recipient cellular origins in a patient, and, as such, can inform engraftment, rejection, and relapse status in stem cell transplant recipients. In this review of STR testing, the most commonly used method to assess chimerism, its background, procedural details, and clinical utility are discussed.

CMV Status Drives Distinct Trajectories of CD4+ T Cell Differentiation.

Cytomegalovirus (CMV) is one of the most commonly recognized opportunistic pathogens and remains the most influential known parameter in shaping an individual's immune system. As such, T cells induced by CMV infection could have a long-term impact on subsequent immune responses. Accumulating evidence indicates that memory T cells developed during past bacterial and viral infection can cross-react with unrelated pathogens, including transplant antigens, and can alter responses to de novo infections, vaccines, cancers, or rejection. Therefore, careful examination of T cell responses elicited by CMV is warranted to understand their potentially beneficial or harmful roles in future major immune events. Our detailed exploration of the distribution, phenotype, TCR repertoire and transcriptome of CD4+ T cells within CMV seropositive healthy individuals using high-dimensional flow cytometry and single cell multi-omics sequencing reveals that CMV seropositivity has highly significant age-independent effects, leading to a reduction in CD4+ naïve T cells and an expansion of CD4+ effector memory T cells and CD45RA+ effector memory T cells. These induced CD4+ effector memory T cells undergo a specific differentiation trajectory resulting in a subpopulation of CD57+CD27-CD28-CD244+ CD4+ T cells with cytotoxic function and TCR oligoclonality for optimal controlled coexistence with cytomegalovirus. Through gene set enrichment analysis, we found that this subpopulation is similar to virus-specific CD8+ T cells and T cells that mediate acute rejection in patients using tacrolimus and belatacept, a selective costimulation blocker. Together, these data suggest that memory CD4+ T cells induced by cytomegalovirus are formed via a distinct differentiation program to acquire cytotoxic function and can be potentially detrimental to transplant patients adopting costimulation blockade immunosuppressive regimen.

FcγRIIB is a T cell checkpoint in antitumor immunity.

In the setting of cancer, T cells upregulate coinhibitory molecules that attenuate TCR signaling and lead to the loss of proliferative capacity and effector function. Checkpoint inhibitors currently in clinical use have dramatically improved mortality from melanoma yet are not effective in all patients, suggesting that additional pathways may contribute to suppression of tumor-specific CD8+ T cell responses in melanoma. Here, we show that FcγRIIB, an inhibitory Fc receptor previously thought to be exclusively expressed on B cells and innate immune cells, is upregulated on tumor-infiltrating effector CD8+ T cells in an experimental melanoma model and expressed on CD8+ T cells in patients with melanoma. Genetic deficiency of Fcgr2b resulted in enhanced tumor-infiltrating CD8+ T cell responses and significantly reduced tumor burden. Adoptive transfer experiments of Fcgr2b-/- tumor antigen-specific T cells into FcγRIIB-sufficient hosts resulted in an increased frequency of tumor-infiltrating CD8+ T cells with greater effector function. Finally, FcγRIIB was expressed on CD8+ memory T cells isolated from patients with melanoma. These data illuminate a cell-intrinsic role for the FcγRIIB checkpoint in suppressing tumor-infiltrating CD8+ T cells.

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.

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.

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.

When rubber meets the road: how innate features of adaptive immune cells play critical roles in transplant alloimmunity.

PURPOSE OF REVIEW: Studies on adaptive cells have largely focused on features that are specific to adaptive immunity. However, adaptive cells utilize innate cell features to modulate their responses, and this area of T and B-cell biology is understudied. This review will highlight recent work done to understand how innate features of adaptive immune cells modulate alloimmunity. RECENT FINDINGS: Over the past year, research has shown that T-cell-expressed danger-associated molecular patterns, Toll-like receptors, complement receptors, and Fc receptors regulate T-cell alloimmunity in a cell-intrinsic manner. Further, IL-17 and p40 of IL-12 have been implicated in the migration of T cells into allografts. Lastly, innate B cells, specifically B1 cells, have been shown to produce clinically relevant autoantibody associated with poor graft outcome. SUMMARY: These data provide evidence that innate features are utilized by adaptive immune cells to control adaptive alloimmunity.

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.

Influence of T Cell Coinhibitory Molecules on CD8+ Recall Responses.

T cell co-signaling molecules play an important role in fine-tuning the strength of T cell activation during many types of immune responses, including infection, cancer, transplant rejection, and autoimmunity. Over the last few decades, intense research into these cosignaling molecules has provided rich evidence to suggest that cosignaling molecules may be harnessed for the treatment of immune-related diseases. In particular, coinhibitory molecules such as programmed-death 1, 2B4, BTLA, TIGIT, LAG-3, TIM-3, and CTLA-4 inhibit T cell responses by counteracting TCR and costimulatory signals, leading to the inhibition of proliferation and effector function and the downregulation of activation and adhesion molecules at the cell surface. While many reviews have focused on the role of coinhibitory molecules in modifying primary CD8+ T cell responses, in this review, we will consider the complex role of coinhibitory molecules in altering CD8+ T cell recall potential. As memory CD8+ T cell responses are critical for protective memory responses in infection and cancer and contribute to potentially pathogenic memory responses in transplant rejection and autoimmunity, understanding the role of coinhibitory receptor control of memory T cells may illuminate important aspects of therapeutically targeting these pathways.

Improving T-cell expansion and function for adoptive T-cell therapy using ex vivo treatment with PI3Kδ inhibitors and VIP antagonists.

Adoptive therapy with ex vivo-expanded genetically modified antigen-specific T cells can induce remissions in patients with relapsed/refractory cancer. The clinical success of this therapy depends upon efficient transduction and expansion of T cells ex vivo and their homing, persistence and cytotoxicity following reinfusion. Lower rates of ex vivo expansion and clinical response using anti-CD19 chimeric antigen receptor (CAR) T cells have been seen in heavily pretreated lymphoma patients compared with B-cell acute lymphoblastic leukemia patients and motivate the development of novel strategies to enhance ex vivo T cell expansion and their persistence in vivo. We demonstrate that inhibition of phosphatidylinositol 3-kinase δ (PI3Kδ) and antagonism of vasoactive intestinal peptide (VIP) signaling partially inhibits the terminal differentiation of T cells during anti-CD3/CD28 bead-mediated expansion (mean, 54.4% CD27+CD28+ T cells vs 27.4% in control cultures; P < .05). This strategy results in a mean of 83.7% more T cells cultured from lymphoma patients in the presence of PI3Kδ and VIP antagonists, increased survival of human T cells from a lymphoma patient in a murine xenograft model, enhanced cytotoxic activity of antigen-specific human CAR T cells and murine T cells against lymphoma, and increased transduction and expansion of anti-CD5 human CAR T cells. PI3Kδ and VIP antagonist-expanded T cells from lymphoma patients show reduced terminal differentiation, enhanced polyfunctional cytokine expression, and preservation of costimulatory molecule expression. Taken together, synergistic blockade of these pathways is an attractive strategy to enhance the expansion and functional capacity of ex vivo-expanded cancer-specific T cells.

Targeted loss of SHP1 in murine thymocytes dampens TCR signaling late in selection.

SHP1 is a tyrosine phosphatase critical to proximal regulation of TCR signaling. Here, analysis of CD4-Cre SHP1(fl/fl) conditional knockout thymocytes using CD53, TCRß, CD69, CD4, and CD8α expression demonstrates the importance of SHP1 in the survival of post selection (CD53(+) ), single-positive thymocytes. Using Ca(2+) flux to assess the intensity of TCR signaling demonstrated that SHP1 dampens the signal strength of these same mature, postselection thymocytes. Consistent with its dampening effect, TCR signal strength was also probed functionally using peptides that can mediate selection of the OT-I TCR, to reveal increased negative selection mediated by lower-affinity ligand in the absence of SHP1. Our data show that SHP1 is required for the survival of mature thymocytes and the generation of the functional T-cell repertoire, as its absence leads to a reduction in the numbers of CD4(+) and CD8(+) naïve T cells in the peripheral lymphoid compartments.