Specificity of HLA monoclonal antibodies and their use to determine HLA expression on lymphocytes and peripheral blood stem cells.

HLA Class I and II expression are known to differ locus-to-locus, however, HLA expression on the cell-surface is frequently reported as the total amount of HLA Class I or II antigens. This is despite evidence that indicates the differential expression of HLA can influence patient outcomes post-transplantation. Although numerous commercially available HLA monoclonal antibodies (mAbs) exist to characterize HLA expression, there is currently a lack of detailed information regarding their reactivities to HLA specificities. The specificities of locus-specific HLA mAbs (nine Class I and four Class II mAbs) were evaluated by two solid-phase Luminex single antigen bead assays. The reactivity patterns of these mAbs were then confirmed by flow cytometry using lymphocytes and PBSCs (peripheral blood stem cells). Out of the 13 HLA mAbs tested, only four (one Class I and three Class II mAbs) displayed intra-locus reactivity without also reacting to inter-locus specificities. Epitope analysis revealed the presence of shared epitopes across numerous HLA loci, explaining much of the observed inter-locus reactivity. The specificity of the HLA mAbs seen in solid-phase assays was confirmed against PBSCs and lymphocytes by flow cytometry. Using this method, we observed differences in the cell surface expression of HLA-C, HLA-DR, HLA-DQ, and HLA-DP between PBSCs and lymphocytes. Our results emphasize the need to characterize the reactivity patterns of HLA mAbs using solid-phase assays before their use on cells. Through understanding the reactivity of these HLA mAbs, the cellular expression of HLA can be more accurately assessed in downstream assays.

Non-Expressed Donor KIR3DL1 Alleles May Represent a Risk Factor for Relapse after T-Replete Haploidentical Hematopoietic Stem Cell Transplantation.

KIR3DL1 alleles are expressed at different levels on the natural killer (NK) cell surface. In particular, the non-expressed KIR3DL1*004 allele appears to be common in Caucasian populations. However, the overall distribution of non-expressed KIR3DL1 alleles and their clinical relevance after T-replete haploidentical hematopoietic stem cell transplantation (hHSCT) with post-transplant cyclophosphamide remain poorly documented in European populations. In a cohort of French blood donors (N = 278), we compared the distribution of expressed and non-expressed KIR3DL1 alleles using next-generation sequencing (NGS) technology combined with multi-color flow cytometry. We confirmed the predominance of the non-expressed KIR3DL1*004 allele. Using allele-specific constructs, the phenotype and function of the uncommon KIR3DL1*019 allotype were characterized using the Jurkat T cell line and NKL transfectants. Although poorly expressed on the NK cell surface, KIR3DL1*019 is retained within NK cells, where it induces missing self-recognition of the Bw4 epitope. Transposing our in vitro observations to a cohort of hHSCT patients (N = 186) led us to observe that non-expressed KIR3DL1 HSC grafts increased the incidence of relapse in patients with myeloid diseases. Non-expressed KIR3DL1 alleles could, therefore, influence the outcome of hHSCT.

Functional SARS-CoV-2-specific T cells of donor origin in allogeneic stem cell transplant recipients of a T-cell-replete infusion: A prospective observational study.

In the current post-pandemic era, recipients of an allogeneic hematopoietic stem cell transplant (HCT) deserve special attention. In these vulnerable patients, vaccine effectiveness is reduced by post-transplant immune-suppressive therapy; consequently, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) disease (COVID-19) is often associated with elevated morbidity and mortality. Characterizing SARS-CoV-2 adaptive immunity transfer from immune donors to HCT recipients in the context of immunosuppression will help identify optimal timing and vaccination strategies that can provide adequate protection to HCT recipients against infection with evolving SARS-CoV-2 variants. We performed a prospective observational study (NCT04666025 at ClinicalTrials.gov) to longitudinally monitor the transfer of SARS-CoV-2-specific antiviral immunity from HCT donors, who were either vaccinated or had a history of COVID-19, to their recipients via T-cell replete graft. Levels, function, and quality of SARS-CoV-2-specific immune responses were longitudinally analyzed up to 6 months post-HCT in 14 matched unrelated donor/recipients and four haploidentical donor/recipient pairs. A markedly skewed donor-derived SARS-CoV-2 CD4 T-cell response was measurable in 15 (83%) recipients. It showed a polarized Th1 functional profile, with the prevalence of central memory phenotype subsets. SARS-CoV-2-specific IFN-γ was detectable throughout the observation period, including early post-transplant (day +30). Functionally experienced SARS-CoV-2 Th1-type T cells promptly expanded in two recipients at the time of post-HCT vaccination and in two others who were infected and survived post-transplant COVID-19 infection. Our data suggest that donor-derived SARS-CoV-2 T-cell responses are functional in immunosuppressed recipients and may play a critical role in post-HCT vaccine response and protection from the fatal disease. Clinical trial registration: clinicaltrials.gov, identifier NCT04666025.

Hematopoietic stem cell donor vaccination with cytomegalovirus triplex augments frequencies of functional and durable cytomegalovirus-specific T cells in the recipient: A novel strategy to limit antiviral prophylaxis.

To enhance protective cytomegalovirus (CMV)-specific T cells in immunosuppressed recipients of an allogeneic hematopoietic cell transplant (HCT), we evaluated post-HCT impact of vaccinating healthy HCT donors with Triplex. Triplex is a viral vectored recombinant vaccine expressing three immunodominant CMV antigens. The vector is modified vaccinia Ankara (MVA), an attenuated, non-replicating poxvirus derived from the vaccinia virus strain Ankara. It demonstrated tolerability and immunogenicity in healthy adults and HCT recipients, in whom it also reduced CMV reactivation. Here, we report feasibility, safety, and immunological outcomes of a pilot phase 1 trial (NCT03560752 at ClinicalTrials.gov) including 17 CMV-seropositive recipients who received an HCT from a matched related donor (MRD) vaccinated with 5.1 × 108 pfu/ml of Triplex before cell harvest (median 15, range 11-28 days). Donor and recipient pairs who committed to participation in the trial resulted in exceptional adherence to the protocol. Triplex was well-tolerated with limited adverse events in donors and recipients, who all engrafted with full donor chimerism. On day 28 post-HCT, levels of functional vaccinia- and CMV-specific CD137+ CD8+ T cells were significantly higher (p < .0001 and p = .0174, respectively) in recipients of Triplex vaccinated MRD than unvaccinated MRD (control cohort). Predominantly, central and effector memory CMV-specific T-cell responses continued to steadily expand through 1-year follow-up. CMV viremia requiring antivirals developed in three recipients (18%). In summary, this novel approach represents a promising strategy applicable to different HCT settings for limiting the use of antiviral prophylaxis, which can impair and delay CMV-specific immunity, leading to CMV reactivation requiring treatment.

Seventy-five years of service: an overview of the College of American Pathologists' proficiency testing program in histocompatibility and identity testing.

The Histocompatibility and Identity Testing Committee offers an overview of the College of American Pathologists' (CAP) Proficiency Testing (PT) program, commemorating its significant 75th anniversary in 2024. The CAP PT program has undergone significant growth and evolution over the years, ultimately achieving Centers for Medicare and Medicaid Services approval. In 1979, CAP's partnership with the American Association for Clinical Histocompatibility Testing marked a pivotal moment, leading to the creation of the first proficiency testing survey in 1980. This laid the foundation for various PT programs managed by the CAP Histocompatibility and Identity Testing Committee, including HLA antibody testing, HLA molecular typing, engraftment monitoring, parentage/relationship testing, HLA disease associations and drug risk, and HLA-B27 typing. Each program's distinctive considerations, grading methodologies, and future prospects are detailed here, highlighting the continual evolution of histocompatibility and identity testing PT to support emerging technologies and evolving laboratory practices in the field.

Treatment of allosensitized patients receiving allogeneic transplantation.

Donor-specific anti-HLA antibodies (DSAs) are a major cause of engraftment failure in patients receiving haploidentical stem cell transplantation (HaploSCT). Effective treatments are needed for these patients, who often have no other donor options and/or are in need to proceed urgently to transplantation. We studied a multimodality treatment with alternate-day plasma exchange (PE), rituximab, intravenous γ globulin (IVIg) and an irradiated donor buffy coat for patients with DSAs at 2 institutions. Thirty-seven patients with a median age of 51 years were treated with this desensitization protocol. Treatment outcomes were compared with a control group of HaploSCT patients without DSAs (n = 345). The majority of patients in the DSA group were female (83.8% vs 37.1% in controls, P < .001) and received stem cells from a child as the donor (67.6% vs 44.1%, P = .002). Mean DSA level before and after desensitization was 10 198 and 5937 mean fluorescence intensity (MFI), respectively, with mean differences of 4030 MFI. Fourteen of 30 tested patients (46.7%) had C1q positivity, while 8 of 29 tested patients (27.6%) remained positive after desensitization. In multivariable analysis, patients with initial DSA > 20 000 MFI and persistent positive C1q after desensitization had a significantly lower engraftment rate, which resulted in significantly higher non-relapse mortality and worse overall survival (OS) than controls, whereas graft outcome and survival of patients with initial DSA < 20 000 MFI and those with negative C1q after treatment were comparable with controls. In conclusion, treatment with PE, rituximab, IVIg, and donor buffy coat is effective in promoting engraftment in patients with DSAs ≤20 000 MFI.

Cytokine gene polymorphisms are associated with response to blinatumomab in B-cell acute lymphoblastic leukemia.

Blinatumomab is a bispecific T cell-engaging antibody approved for treatment of relapsed/refractory (r/r) ALL, with 40%-50% complete response (CR)/CR with incomplete count recovery (CRi). Cytokine release syndrome (CRS) as a major adverse effect after blinatumomab therapy. Here, we evaluated the possible association between single-nucleotide polymorphisms (SNPs) in cytokine genes, disease response, and CRS in r/r ALL patients who received blinatumomab between 2012 and 2017 at our center (n = 66), using patients' archived DNA samples. With a median duration of 9.5 months (range: 1-37), 37 patients (56.1%) achieved CR/CRi, 54 (81.8%) experienced CRS (G1: n = 35, G2: n = 14, G3: n = 5), and 9 (13.6%) developed neurotoxicity. By multivariable analysis, after adjusting for high disease burden, one SNP on IL2 (rs2069762), odds ratio (OR) = 0.074 (95% CI: NE-0.43, P = .01) and one SNP on IL17A (rs4711998), OR = 0.28 (95% CI: 0.078-0.92, P = .034) were independently associated with CR/CRi. None of the analyzed SNPs were associated with CRS. To our knowledge, this is the first study demonstrating a possible association between treatment response to blinatumomab and SNPs. Our hypothesis-generated data suggest a potential role for IL-17 and IL-2 in blinatumomab response and justify a larger confirmatory study, which may lead to personalized blinatumomab immunotherapy for B-ALL.

Centromeric KIR AA Individuals Harbor Particular KIR Alleles Conferring Beneficial NK Cell Features with Implications in Haplo-Identical Hematopoietic Stem Cell Transplantation.

We have recently shown a broad disparity of Natural Killer (NK) cell responses against leukemia highlighting good and bad responders resting on the Killer cell Immunoglobulin-like Receptors (KIR) and HLA genetics. In this study, we deeply studied KIR2D allele expression, HLA-C recognition and functional effect on NK cells in 108 blood donors in combining high-resolution KIR allele typing and multicolor flow cytometry. The KIR2DL1*003 allotype is associated with centromeric (cen) AA motif and confers the highest NK cell frequency, expression level and strength of KIR/HLA-C interactions compared to the KIR2DL1*002 and KIR2DL1*004 allotypes respectively associated with cenAB and BB motifs. KIR2DL2*001 and *003 allotypes negatively affect the frequency of KIR2DL1+ and KIR2DL3+ NK cells. Altogether, our data suggest that cenAA individuals display more efficient KIR2DL alleles (L1*003 and L3*001) to mount a consistent frequency of KIR2DL+ NK cells and to confer an effective NK cell responsiveness. The transposition of our in vitro observations in the T-replete haplo-identical HSCT context led us to observe that cenAA HSC grafts limit significantly the incidence of relapse in patients with myeloid diseases after T-replete haplo-identical HSCT. As NK cells are crucial in HSCT reconstitution, one could expect that the consideration of KIR2DL1/2/3 allelic polymorphism could help to refine scores used for HSC donor selection.

Posttransplant Cyclophosphamide and Antithymocyte Globulin versus Posttransplant Cyclophosphamide as Graft-versus-Host Disease Prophylaxis for Peripheral Blood Stem Cell Haploidentical Transplants: Comparison of T Cell and NK Effector Reconstitution.

A higher incidence of graft-versus-host disease (GVHD) has been observed after haploidentical hematopoietic stem cell transplantation (h-HSCT) with posttransplant cyclophosphamide (PTCY) using peripheral blood stem cells (PBSC) as a source of graft. Moreover, combining PTCY with antithymocyte globulin (ATG) may help to reduce GVHD incidence. In this study, early immune reconstitution, especially of T and NK cell compartments, was compared after both types of transplant (PTCY versus PTCY + ATG) investigate their influence on patient outcomes. This retrospective study included 58 adults who received a reduced intensity conditioning to PBSC h-HSCT with cyclosporine and mycophenolate mofetyl + PTCY (n = 32) or PTCY + ATG (n = 26) as GVHD prophylaxis. Both groups shared similar characteristics except for the median number of CD3+ T cells infused, significantly higher for PTCY + ATG patients. Blood samples from all patients were collected three times a week from day 0 until day 30 then at day 60 and day 90/100 to evaluate T and NK cells reconstitution by flow cytometry. The results show that PTCY + ATG versus PTCY alone significantly limits the occurrence of acute grade 2-4 GVHD after reduced intensity conditioning PBSC h-HSCT, perhaps because of the combined effect of T and NK cell reconstitution. Indeed, although a slower T cell reconstitution with PTCY + ATG may limit GVHD occurrence, the quicker reconstitution of some NK cell subtypes may help with avoiding relapse. Larger prospective studies are needed to better determine which NK cell subsets may influence the incidence of relapse after h-HSCT and optimize donor selection.

Genetic and Molecular Basis of Heterogeneous NK Cell Responses against Acute Leukemia.

Natural killer (NK) cells are key cytotoxic effectors against malignant cells. Polygenic and polymorphic Killer cell Immunoglobulin-like Receptor (KIR) and HLA genes participate in the structural and functional formation of the NK cell repertoire. In this study, we extensively investigated the anti-leukemic potential of NK cell subsets, taking into account these genetic parameters and cytomegalovirus (CMV) status. Hierarchical clustering analysis of NK cell subsets based on NKG2A, KIR, CD57 and NKG2C markers from 68 blood donors identified donor clusters characterized by a specific phenotypic NK cell repertoire linked to a particular immunogenetic KIR and HLA profile and CMV status. On the functional side, acute lymphoblastic leukemia (ALL) was better recognized by NK cells than acute myeloid leukemia (AML). However, a broad inter-individual disparity of NK cell responses exists against the same leukemic target, highlighting bad and good NK responders. The most effective NK cell subsets against different ALLs expressed NKG2A and represented the most frequent subset in the NK cell repertoire. In contrast, minority CD57+ or/and KIR+ NK cell subsets were more efficient against AML. Overall, our data may help to optimize the selection of hematopoietic stem cell donors on the basis of immunogenetic KIR/HLA for ALL patients and identify the best NK cell candidates in immunotherapy for AML.

Protective effect of HLA-DPB1 mismatch remains valid in reduced-intensity conditioning unrelated donor hematopoietic cell transplantation.

A mismatch at HLA-DPB1 locus is associated with higher acute GVHD and lower relapse rate after myeloablative (MAC) allogeneic hematopoietic cell transplantation (alloHCT). Also, in MAC setting, mismatch permissiveness and expression level impact alloHCT outcomes. However, in reduced intensity conditioning (RIC), DP mismatch effect on transplant outcomes is unknown. We retrospectively evaluated DP mismatch influence (number, permissiveness, and expression) on HCT outcomes in 310 patients with high-resolution typing (HLA-A, -B, -C, -DRB1, -DQB1, and -DPB1), who underwent RIC HCT. By multivariable analysis, 11/12 had better overall survival (OS) and relapse vs. 12/12 (HR = 1.61 and 2.02; p = 0.04 and 0.01, respectively) and better OS vs. 10/12 (HR = 1.68; p = 0.02). Within the 11/12, nonpermissive (NoPR) mismatch was associated with higher risk of grade II-IV acute GVHD (HR = 1.97; p = 0.005) and nonrelapse mortality (HR = 2.13; p = 0.02) vs. permissive (PR). Grouping 11/12 based on the DP expression conferred higher mortality (HR = 3.78; p = 0.003) when low expressers received a graft from high expressers (AG) vs. low expressers (AA). Better OS was achieved in PR 11/12, when expression was low in patient and donor (AA) vs. all other combinations. Therefore, in RIC HCT, a single-DP mismatch has a protective role, especially in permissive setting, when donor and recipient are low expressers.

Long-term Outcome of Allogeneic Hematopoietic Stem Cell Transplantation From Unrelated Donor Using Tacrolimus/Sirolimus-based GvHD Prophylaxis: Impact of HLA Mismatch.

BACKGROUND: While tacrolimus and sirolimus (T/S)-based graft-versus-host disease (GvHD) prophylaxis has been effective in preventing acute GvHD post hematopoietic cell transplantation (HCT), its efficacy and long-term outcome in matched (MUD) and mismatched unrelated donor (mMUD) setting is not well defined. METHODS: Herein, we evaluated a consecutive case-series of 482 patients who underwent unrelated donor HCT (2005-2013) with T/S-based GvHD prophylaxis. RESULTS: With a median follow-up of 6.2 years (range = 2.4-11.3), the 5-year overall survival (OS) and relapse/progression-free survival were 47.5% (95% confidence interval [CI]: 43.0-52.0) and 43.6% (95% CI: 39.1-48.1), respectively; and the 5-year cumulative incidence of nonrelapse mortality (NRM) and relapse were 24.9%, and 31.5%, respectively. In this cohort, mMUD was associated with worse OS (39.0% versus 50.7% at 5 y; P = 0.034), primarily due to greater risk of NRM (33.5% versus 21.7%; P = 0.038). While rates of relapse, acute (II-IV or III-IV) or chronic GvHD (limited or extensive) were not different, death caused by chronic GvHD (20.8% versus 12.8%; P = 0.022) and infection (33.0% versus 18.1%; P < 0.01) were significantly greater in mMUD. In multivariable analysis, high-risk disease (hazard ratio [HR] = 2.21, 95% CI: 1.16-4.23; P < 0.01) and mMUD (HR = 1.55, 95% CI: 1.15-2.08; P = 0.004) were independent predictive factors for OS. CONCLUSIONS: T/S-based GvHD prophylaxis is an effective and acceptable GvHD prophylactic regimen. However, survival after mMUD remained poor, possibly related to the severity of chronic GvHD.

Influence of donor KIR genotypes on reduced relapse risk in acute myelogenous leukemia after hematopoietic stem cell transplantation in patients with CMV reactivation.

Multiple retrospective studies have demonstrated an association between cytomegalovirus (CMV) reactivation and reduced risk of AML relapse. However, the potential mechanism explaining this association remains elusive. We investigated a homogeneous cohort of 288 adult patients with AML in remission who received allogeneic hematopoietic stem cell transplantation (HCT) from matched sibling/unrelated donors between 1995 and 2011. The 5-year cumulative incidence of relapse was greater in patients without CMV reactivation compared with those with reactivation (30.2% vs. 12.1%, p = 0.001) in a landmark analysis. In multivariate analyses CMV reactivation was independently associated with reduced relapse risk (HR: 0.49 [0.25-0.95], p = 0.036) and increased non-relapse mortality (26.5% vs. 13.1%, p = 0.002) resulting in similar 5-year overall survival (64.5% vs. 59.1%, p = 0.8). In further subgroup analyses the protective effect of CMV reactivation was significant in patients who received HCT from donors with KIR Bx compared to KIR AA (11.7% vs. 29.5%, p = 0.01). Likewise, the protective effect of CMV reactivation was more significant when the donors had 2DS1 activating KIR (11.5% vs. 30.7%, p = 0.05) compared with those without 2DS1 (14.3% vs. 27.5%, p = 0.12). Our data independently confirmed the association between CMV reactivation and AML relapse, suggesting the involvement of donor KIR genotypes and NK cell-mediated graft-versus-leukemia effect.

HLA alleles and haplotypes observed in 263 US families.

The 17th International HLA and Immunogenetics Workshop (IHIW) conducted a project entitled "The Study of Haplotypes in Families by NGS HLA". We investigated the HLA haplotypes of 1017 subjects in 263 nuclear families sourced from five US clinical immunogenetics laboratories, primarily as part of the evaluation of related donor candidates for hematopoietic stem cell and solid organ transplantation. The parents in these families belonged to five broad groups - African (72 parents), Asian (115), European (210), Hispanic (118) and "Other" (11). High-resolution HLA genotypes were generated for each subject using next-generation sequencing (NGS) HLA typing systems. We identified the HLA haplotypes in each family using HaplObserve, software that builds haplotypes in families by reviewing HLA allele segregation from parents to children. We calculated haplotype frequencies within each broad group, by treating the parents in each family as unrelated individuals. We also calculated standard measures of global linkage disequilibrium (LD) and conditional asymmetric LD for each ethnic group, and used untruncated and two-field allele names to investigate LD patterns. Finally we demonstrated the utility of consensus DNA sequences in identifying novel variants, confirming them using HLA allele segregation at the DNA sequence level.

The association between HLA and non-Hodgkin lymphoma subtypes, among a transplant-indicated population.

Several studies have implicated HLA in non-Hodgkin lymphoma (NHL) subtype etiology. However, NHL patients indicated for stem cell transplants are underrepresented in these reports. We therefore evaluated the association between HLA and NHL subtypes among a transplant-indicated population. One thousand three hundred and sixty-six NHL patients HLA-typed and indicated for transplant at the City of Hope National Medical Center (Duarte, CA) were compared to 10,271 prospective donors. Odds ratios and 95% confidence intervals were calculated for HLA haplotype and alleles, adjusted for sex and age. The HLA-A*0201∼C*0602∼B*1302∼DRB1*0701∼DQB1*0201 haplotype was significantly associated with follicular lymphoma (FL) risk among Caucasians. Several haplotypes were associated with diffuse large B-cell lymphoma (DLBCL) risk among Caucasians, including the previously implicated DLBCL risk loci, HLA-B*0801. The HLA-A*0101 allele was also observed to be associated with mantle cell lymphoma (MCL) risk. Our results support the association between previously reported susceptibility loci and FL and suggest potentially new DLBCL and MCL risk loci.