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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. [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]

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. [ABSTRACT FROM AUTHOR]