Disability related to chronic graft -versus-host disease after alternative donor hematopoietic cell transplantation.

We determined the incidence of disability related to chronic graft-versus-host disease (bronchiolitis obliterans, grade ≥2 keratoconjunctivitis sicca, sclerotic features or esophageal stricture) for three categories of alternative donor: cord blood, haplorelated marrow or peripheral blood with post-transplant cyclophosphamide, and unrelated single HLA-allele mismatched peripheral blood. Among 396 consecutive hematopoietic cell transplant recipients, 129 developed chronic graft-versus-host disease with 3-year cumulative incidences of 8% for cord blood, 24% for haplorelated grafts, and 55% for unrelated single HLA-allele mismatched peripheral blood. Disability rates were significantly lower for cord blood [hazard ratio (HR) 0.13; 95% confidence interval (CI): 0.1-0.4] and for the haplorelated group (HR 0.31; 95% CI: 0.1-0.7) compared to the rate in the group transplanted with unrelated single HLA-allele mismatched peripheral blood. Cord blood recipients were also >2-fold more likely to return to work/school within 3 years from the onset of chronic graft-versus-host disease (HR 2.54; 95% CI: 1.1-5.7, P=0.02), and the haplorelated group trended similarly (HR 2.38; 95% CI: 1.0-5.9, P=0.06). Cord blood recipients were more likely to discontinue immunosuppression than were recipients of unrelated single HLA-allele mismatched peripheral blood (HR 3.96; 95% CI: 1.9-8.4, P=0.0003), similarly to the haplorelated group (HR 4.93; 95% CI: 2.2-11.1, P=0.0001). Progression-free survival and non-relapse mortality did not differ between groups grafted from different types of donors. Our observations that, compared to recipients of unrelated single HLA-allele mismatched peripheral blood, recipients of cord blood and haplorelated grafts less often developed disability related to chronic graft-versus-host disease, and were more likely to resume work/school, should help better counseling of pre-hematopoietic cell transplant candidates.

HLA-B leader genotypes in a clinical population.

The -21 dimorphism in the leader sequences of HLA-B exon 1 is associated with risk of graft-versus-host disease (GVHD), relapse and overall survival after unrelated donor hematopoietic cell transplantation (HCT), haploidentical HCT and cord blood transplantation. Consideration of the leader dimorphism in the prospective selection of allogeneic donors for HCT may help to lower risks for patients, but requires understanding of the frequencies of the leader in patients and candidate transplant donors. We defined the frequencies of the HLA-B leader, and its association to HLA-B Bw4/Bw6 and C1/C2 KIR epitopes. Sequence variants of rs1050458 of exon 1 position -21 for 11,126 haplotypes were analyzed from high resolution HLA typing of over 5500 study subjects. HLA typing was performed by TruSight/AlloSeq NGS and analyzed using TruSight/AlloSeq Assign software. HLA-B Bw4/Bw6 and C1/C2 KIR epitopes were defined based on established sequence alignments and nomenclature. Alleles at rs1050458 of HLA-B exon 1 were validated as dimorphic: rs1050458-C or -T variants encoding threonine (T) or methionine (M) at anchor position 2 (P2) of nonameric HLA-B leader peptides, respectfully. No additional variants were observed. Among study subjects, 70% of HLA-B haplotypes encoded T-leader and 30% encoded M-leader sequences. The genotype frequencies of TT, MT, and MM were consistent among patient, related, and unrelated donor groups. The associations of M/T leader, Bw4/Bw6, and C1/C2 enhanced understanding of the Class I features involved in the innate immune response. A population of patients and transplant donors confirms the rs1050458 leader dimorphism and its association with HLA-B Bw4/Bw6 and C1/C2 KIR features.

Identification of the rs9277534 HLA-DP expression marker by next generation sequencing for the selection of unrelated donors for hematopoietic cell transplantation.

Mismatching of an unrelated donor against a high-expression HLA-DPB1 recipient allele is associated with a high risk of graft-versus-host disease and mortality. The Seattle Cancer Care Alliance (SCCA) and Fred Hutchinson Cancer Research Center transplant program employs an algorithm to match for HLA-A, B, C, DRB1, DQB1 and DPB1 alleles (12/12) and to avoid, whenever possible, donor mismatching against a recipient high-expression HLA-DPB1 allele. HLA-DPB1 expression is associated with the rs9277534 A/G polymorphism located in the 3'UTR of the HLA-DPB1 gene. Next generation sequencing of HLA-DPB1 using the Illumina TruSight HLA V2 Sequencing Panel and Conexio Assign software analyses provides information on rs9277534 variants without the need for any additional SNP testing. Here we present the molecular location of rs9277534 in NGS data and discuss the challenges to resolve HLA-DPB1 ambiguities.

Elevated T regulatory cells in long-term stable transplant tolerance in rhesus macaques induced by anti-CD3 immunotoxin and deoxyspergualin.

Regulatory T cells (Tregs) are implicated in immune tolerance and are variably dependent on IL-10 for in vivo function. Brief peritransplant treatment of multiple nonhuman primates (NHP) with anti-CD3 immunotoxin and deoxyspergualin has induced stable (5-10 years) rejection-free tolerance to MHC-mismatched allografts, which associated with sustained elevations in serum IL-10. In this study, we demonstrate that resting and activated PBMC from long-term tolerant NHP recipients are biased to secrete high levels of IL-10, compared with normal NHP PBMC. Although IL-10-producing CD4+ Tregs (type 1 regulatory cells (TR1)/IL-10 Tregs) were undetectable (<0.5%) in normal rhesus monkeys, 7.5 +/- 1.7% of circulating CD4+ T cells of tolerant rhesus recipients expressed IL-10. In addition to this >15-fold increase in Tr1/IL-10 Tregs, the tolerant monkeys exhibited a nearly 3-fold increase in CD4+CD25+ Tregs, 8.1 +/- 3.0% of CD4 T cells vs 2.8 +/- 1.4% in normal cohorts (p < 0.02). The frequency of CD4+CD25+IL-10+ cells was elevated 5-fold in tolerant vs normal NHP (1.8 +/- 0.9% vs 0.4 +/- 0.2%). Rhesus CD4+CD25+ Tregs exhibited a memory phenotype, and expressed high levels of Foxp3 and CTLA-4 compared with CD4+CD25- T cells. Also, NHP CD4+CD25+ Tregs proliferated poorly after activation and suppressed proliferation of CD4+CD25- effector T cells, exhibiting regulatory properties similar to rodent and human CD4+CD25+ Tregs. Of note, depletion of CD4+CD25+ Tregs restored indirect pathway antidonor responses in tolerant NHP. Our study demonstrates an expanded presence of Treg populations in tolerant NHP recipients, suggesting that these adaptations may be involved in maintenance of stable tolerance.