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A pediatric patient with acute myeloid leukemia was referred to our institution for investigational therapy after disease relapse following a mismatched unrelated donor hematopoietic cell transplant (HCT). Prior to second HCT, the patient's serum was negative for antibodies to class I and class II HLA. Eight days after receiving a maternal donor haploidentical transplant, the patient became platelet refractory and highly sensitized to multiple class I HLA. Serum from the patient's mother was positive for the strongest antibodies present in the patient, suggesting the antibodies were donor-derived. Patient sera showed magnified and expanded sensitization over time in the context of 100% donor chimerism and despite undetectable circulating B cells. Escalating sensitization suggests active transfer of rituximab-resistant antibody-producing passenger lymphocytes from a haploidentical donor to a transplant recipient at the time of progenitor cell infusion. Evaluation of donor sensitization status may be a consideration prior to HLA mismatched HCT., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Akshay Sharma has received consultant fees from Spotlight Therapeutics, Medexus Inc., Vertex Pharmaceuticals, Sangamo Therapeutics, and Editas Medicine. He is a medical monitor for the RCI BMT CSIDE clinical trial, for which receives financial compensation. He has also received research funding from CRISPR Therapeutics and honoraria from Vindico Medical Education. Dr. Sharma is the St. Jude Children’s Research Hospital site principal investigator for clinical trials of genome editing of sickle cell disease sponsored by Vertex Pharmaceuticals/CRISPR Therapeutics (NCT03745287), Novartis Pharmaceuticals (NCT04443907), and Beam Therapeutics (NCT05456880). The industry sponsors provide funding for the clinical trials, which includes salary support paid to Dr Sharma’s institution. Dr Sharma has no direct financial interest in these therapies. Shane Cross has received consultant fees for Lexicomp (Wolters Kluwer). The other authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved.)

Background: Relapse remains a challenge after transplantation in pediatric patients with hematological malignancies. Myeloablative regimens used for disease control are associated with acute and long-term adverse effects. We used a CD45RA-depleted haploidentical graft for adoptive transfer of memory T cells combined with NK-cell addback and hypothesized that maximizing the graft-versus-leukemia (GVL) effect might allow for reduction in intensity of conditioning regimen., Methods: In this phase II clinical trial (NCT01807611), 72 patients with hematological malignancies (complete remission (CR)1: 25, ≥ CR2: 28, refractory disease: 19) received haploidentical CD34 + enriched and CD45RA-depleted hematopoietic progenitor cell grafts followed by NK-cell infusion. Conditioning included fludarabine, thiotepa, melphalan, cyclophosphamide, total lymphoid irradiation, and graft-versus-host disease (GVHD) prophylaxis consisted of a short-course sirolimus or mycophenolate mofetil without serotherapy., Results: The 3-year overall survival (OS) and event-free-survival (EFS) for patients in CR1 were 92% (95% CI:72-98) and 88% (95% CI: 67-96); ≥ CR2 were 81% (95% CI: 61-92) and 68% (95% CI: 47-82) and refractory disease were 32% (95% CI: 11-54) and 20% (95% CI: 6-40). The 3-year EFS for all patients in morphological CR was 77% (95% CI: 64-87) with no difference amongst recipients with or without minimal residual disease (P = 0.2992). Immune reconstitution was rapid, with mean CD3 and CD4 T-cell counts of 410/μL and 140/μL at day + 30. Cumulative incidence of acute GVHD and chronic GVHD was 36% and 26% but most patients with acute GVHD recovered rapidly with therapy. Lower rates of grade III-IV acute GVHD were observed with NK-cell alloreactive donors (P = 0.004), and higher rates of moderate/severe chronic GVHD occurred with maternal donors (P = 0.035)., Conclusion: The combination of a CD45RA-depleted graft and NK-cell addback led to robust immune reconstitution maximizing the GVL effect and allowed for use of a submyeloablative, TBI-free conditioning regimen that was associated with excellent EFS resulting in promising long-term outcomes in this high-risk population. The trial is registered at ClinicalTrials.gov (NCT01807611)., (© 2024. The Author(s).)

HLA loss in hematologic malignancies is rare at diagnosis but may occur in leukemic cells at disease relapse. Although HLA mismatched hematopoietic cell transplant (HCT) is useful in exploiting graft vs. leukemia (GvL) effects, alloreactive T cells may exert immune pressure on leukemic cells, leading to immune escape. Roughly 10-30% of relapses after HLA mismatched hematopoietic cell transplant (HCT) involve loss of recipient-specific HLA genes, thereby rendering leukemic cells resistant to donor GvL effects. The use of alloreactive T cells, i.e., donor lymphocyte infusions (DLI) to control relapse following HCT will not be effective in the context of recipient-specific HLA loss and will instead carry all the risk of graft versus host disease (GvHD). Additionally, the source of the lost HLA, maternal or paternal, is important in informing the best donor choice for rescue HCT. The growing body of evidence, together with developments in laboratory testing for genomic loss of HLA, has highlighted the importance of routine testing for HLA loss in patients who relapse post HLA-mismatched HCT. Assessment of HLA loss allows transplant centers to make quick decisions regarding the most appropriate therapies and/or alternative donor selection for rescue HCT., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved.)

Novel allele, DPB1*1049:01, is a one nucleotide variant of DPB1*03:01:01:01., (© 2020 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Type 1 diabetes is a T cell-mediated autoimmune disease, characterized by lymphocytic infiltration of the pancreatic islets. It is currently thought that islet antigen specificity is not a requirement for islet entry and that diabetogenic T cells can recruit a heterogeneous bystander T cell population. We tested this assumption directly by generating T cell receptor (TCR) retrogenic mice expressing two different T cell populations. By combining diabetogenic and nondiabetogenic or nonautoantigen-specific T cells, we demonstrate that bystander T cells cannot accumulate in the pancreatic islets. Autoantigen-specific T cells that accumulate in islets, but do not cause diabetes, were also unaffected by the presence of diabetogenic T cells. Additionally, 67% of TCRs cloned from nonobese diabetic (NOD) islet-infiltrating CD4(+) T cells were able to mediate cell-autonomous islet infiltration and/or diabetes when expressed in retrogenic mice. Therefore, islet entry and accumulation appears to be a cell-autonomous and tightly regulated event and is governed by islet antigen specificity.

Objective: Type 1 diabetes is mediated by T-cell entry into pancreatic islets and destruction of insulin-producing beta-cells. The relative contribution of T-cells specific for different autoantigens is largely unknown because relatively few have been assessed in vivo., Research Design and Methods: We generated mice possessing a monoclonal population of T-cells expressing 1 of 17 T-cell receptors (TCR) specific for either known autoantigens (GAD65, insulinoma-associated protein 2 (IA2), IA2beta/phogrin, and insulin), unknown islet antigens, or control antigens on a NOD.scid background using retroviral-mediated stem cell gene transfer and 2A-linked multicistronic retroviral vectors (referred to herein as retrogenic [Rg] mice). The TCR Rg approach provides a mechanism by which T-cells with broad phenotypic differences can be directly compared., Results: Neither GAD- nor IA2-specific TCRs mediated T-cell islet infiltration or diabetes even though T-cells developed in these Rg mice and responded to their cognate epitope. IA2beta/phogrin and insulin-specific Rg T-cells produced variable levels of insulitis, with one TCR producing delayed diabetes. Three TCRs specific for unknown islet antigens produced a hierarchy of insulitogenic and diabetogenic potential (BDC-2.5 > NY4.1 > BDC-6.9), while a fourth (BDC-10.1) mediated dramatically accelerated disease, with all mice diabetic by day 33, well before full T-cell reconstitution (days 42-56). Remarkably, as few as 1,000 BDC-10.1 Rg T-cells caused rapid diabetes following adoptive transfer into NOD.scid mice. CONCLUSIONS; Our data show that relatively few autoantigen-specific TCRs can mediate islet infiltration and beta-cell destruction on their own and that autoreactivity does not necessarily imply pathogenicity.

TCR transgenic mice are valuable tools for dissecting the role of autoantigen-specific T cells in the pathogenesis of type 1 diabetes but are time-consuming to generate and backcross onto congenic strains. To circumvent these limitations, we developed a new approach to rapidly generate mice expressing TCR using retroviral-mediated stem cell gene transfer and a novel picornavirus-like 2A peptide to link the TCR alpha- and beta-chains in a single retroviral vector. We refer to these as retrogenic (Rg) mice to avoid confusion with conventional transgenic mice. Our approach was validated by demonstrating that Rg nonobese diabetic (NOD)-scid mice expressing the diabetogenic TCRs, BDC2.5 and 4.1, generate clonotype-positive T cells and develop diabetes. We then expressed three TCR specific for either glutamate decarboxylase (GAD) 206-220 or GAD 524-538 or for hen egg lysozyme 11-25 as a control in NOD, NOD-scid, and B6.H2(g7) mice. Although T cells from these TCR Rg mice responded to their respective Ag in vitro, the GAD-specific T cells exhibited a naive, resting phenotype in vivo. However, T cells from Rg mice challenged with Ag in vivo became activated and developed into memory cells. Neither of the GAD-reactive TCR accelerated or protected mice from diabetes, nor did activated T cells transfer or protect against diabetes in NOD-scid recipients, suggesting that GAD may not be a primary target for diabetogenic T cells. Generation of autoantigen-specific TCR Rg mice represents a powerful approach for the analysis of a wide variety of autoantigens.

MHC tetramers have proven to be powerful reagents for the analysis of MHC class I-restricted T cells. However, generating similarly reliable reagents for MHC class II-restricted T cells has been elusive. Here we evaluated the utility of MHC class II:gamma2aFc multimers, which contain the MHC class II extracellular domains, with or without recombinantly attached peptides, dimerized via a fos-jun leucine zipper and attached to the hinge of murine IgG2a. We have successfully generated 24 multimers in either myeloma or Drosophila melanogaster S2 cells, with an average yield of 7 mg/L. 'Empty' MHC class II:gamma2aFc multimers were effectively used in peptide binding assays. Similar versions that contained recombinantly attached peptides stimulated T cells in an antigen-specific, MHC-restricted manner, and identified antigen-specific nai;ve and effector T cells by flow cytometry. Furthermore, we have successfully used these reagents to stain T cells generated following viral infection. Thus, MHC class II:gamma2aFc multimers are robust and reliable reagents for the analysis of MHC class II-restricted T cells.

Peptides bind to MHC class II molecules with a defined periodicity such that the peptide-flanking residues (PFRs) P-1 and P11, which lie outside the core binding sequence (P1-P9), are solvent exposed and accessible to the TCR. Using a novel MHC class II:peptide binding assay, we defined the binding register for nine immunogenic epitopes to formally identify the flanking residues. Seven of the nine epitopes, restricted by H-2A(k), H-2A(g7), or H-2E(k), were found to generate T cells that were completely dependent on either P-1 or P11, with dependency on P-1 favored over P11. Such PFR dependency appears to be influenced by the type of amino acid exposed, in that residues that can form salt bridges or hydrogen bonds are favored over small or hydrophobic residues. Peptides containing alanine substitutions at P-1 or P11 in place of PFRs that mediate dependency were considerably less immunogenic and mediated a substantially reduced in vitro recall response to the native protein, inferring that PFR recognition increases immunogenicity. Our data suggest that PFR recognition is a common event characteristic of all MHC class II-restricted T cell responses. This key feature, which is not shared by MHC class I-restricted responses, may underlie the broad functional diversity displayed by MHC class II-restricted T cells.