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Novel allele, DPB1*1049:01, is a one nucleotide variant of DPB1*03:01:01:01. [ABSTRACT FROM AUTHOR]

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:γ2aFc 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:γ2aFc 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 naı¨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:γ2aFc multimers are robust and reliable reagents for the analysis of MHC class II-restricted T cells. [Copyright &y& Elsevier]

Aim: Many leukemia patients referred to St. Jude for HSCT have been previously transplanted. Buccal samples may be a mixture of donor and recipient-derived cells and this may complicate HLA typing. In addition, due to HLA disparity between various types of donors, there may be competition for engraftment of subsequent transplants. Methods: HLA Typing SSP. HLA Typing SSO. HLA Typing SBT. HLA Antibody ID. Results: The patient is a female with infant ALL, who received a 6/6 matched unrelated cord blood transplant. Following relapse, she was referred to St. Jude for haploidentical HSCT and NK therapy. HLA typing of the patient’s buccal sample was in agreement with family studies and with referred typing at all loci, with the exception of DPB1. DPB1 typing by SSO, SBT and SSP were inconclusive. Manual analysis of SSP suggested that multiple alleles were present in this sample, two of which were not present in either parent. Indeed, FISH analysis using XY probes showed that 96.5% of blood cells were of donor origin. The patient received a haploidentical transplant and NK infusion from her mother, and exhibited early engraftment. However, the patient’s chimerism began to show a decrease in donor 2 (mother) and a reappearance of donor 1 (cord), beginning at day 41 post-transplant. She was given 3 donor lymphocyte infusions (DLI) from her mother but continued to lose the graft. Tests of the patient’s serum for anti-class I and class II HLA antibodies were negative. The patient was given a stem cell boost from her mother, but continues to exhibit mixed donor chimerism. Conclusions: The DPB1 alleles complicating HLA typing of the buccal sample likely represent those derived from the previous cord donor. Although the cord unit was well-matched with the patient, it is unlikely that donor and recipient also shared both DPB1 alleles. Finally, although the patient did not have detectable antibodies to HLA, the competition between the cord donor and haploidentical donor may reflect a T cell-mediated response. [Copyright &y& Elsevier]

Aim: A hematopoietic stem cell transplantation program in Santiago, Chile affiliated with St. Jude Children’s Research Hospital referred 190 patients and their family members for HLA typing between 2000 and 2011. Methods: These individuals were studied for HLA specificities by the use of sequence-specific oligonucleotide primers (SSP), sequence-specific oligonucleotide probes (SSO) and DNA sequencing. Haplotype assignment could be made unambiguously in all cases from family studies. Results: The expectation of the referring center was that this group consisted largely of those of western European origin. HLA typing of the large proportion of this referred group indicated that although 70 - 80 % was similar to populations found in western Europe, a substantial number demonstrated alleles not found in western Europeans and which were initially described in native South American populations. For example, of 35 patient samples positive for HLA-A∗68, 34 (97%) were found to be HLA-A∗68:01:02 by DNA sequencing, a variation described in native South American populations. HLA-A∗68:23 was identified in 8 unrelated individuals in this group of 719 patients and family members. This would indicate that this unusual allele would meet the criteria for a common and well-documented allele in this population. Alleles of HLA-A∗02, A∗24, as well as other A∗68 alleles not commonly seen in those of western European heritage were also found. Eleven HLA-B∗35, six HLA-B∗39 and five HLA-B∗51 alleles first described in native South American populations were also found in HLA typing of this group from Chile. HLA-DRB1∗04:07 was shown in 87 and HLA-DRB1∗08:02 in 55 of these 719 individuals. Conclusions: Unrelated donor registries in Argentina and Brazil have recently formed cooperative agreements with the National Marrow Donor Program and can be expected to be beneficial for those patients from South America requiring unrelated hematopoietic stem cell donors. [Copyright &y& Elsevier]

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.