Azathioprine/hydroxyurea preconditioning prior to nonmyeloablative matched sibling donor hematopoietic stem cell transplantation in adults with sickle cell disease: A prospective observational cohort study.

Nonmyeloablative, matched sibling donor hematopoietic stem cell transplantation with alemtuzumab/total body irradiation (TBI) conditioning is a curative therapy with low toxicity for adults with sickle cell disease (SCD). However, relatively low donor chimerism levels and graft rejection remain important challenges. We hypothesized that adding azathioprine/hydroxyurea preconditioning will improve donor chimerism levels and reduce graft failure rate. In this prospective cohort study, we enrolled consecutive adult patients with SCD undergoing matched sibling donor transplantation at the Amsterdam UMC. Patients received azathioprine 150 mg/day and hydroxyurea 25 mg/kg/day for 3 months prior to alemtuzumab 1 mg/kg and 300 cGy TBI conditioning. Twenty patients with SCD (median age 26 years [range 19-49], 13 females) were transplanted. Median follow-up was 46.0 months (IQR 21.8-57.9). One-year overall survival and event-free survival (graft failure or death) were both 95% (95% confidence interval 86-100). Mean donor myeloid and T-cell chimerism 1-year post-transplant were 95.2% (SD ±10.6) and 67.3% (±15.3), respectively. One patient (5%) experienced graft failure without autologous regeneration, resulting in infections and death. All other patients had a corrected SCD phenotype and were able to discontinue sirolimus. Three patients were successfully treated with alemtuzumab (1 mg/kg) after the transplant because of declining donor chimerism and cytopenias to revert impending graft rejection. Toxicity was mostly related to sirolimus and alemtuzumab. One patient developed steroid-responsive grade II intestinal acute graft-versus-host disease. Collectively, preconditioning with azathioprine/hydroxyurea prior to nonmyeloablative matched sibling donor transplantation resulted in excellent event-free survival and robust donor T-cell chimerism, enabling the successful withdrawal of sirolimus. ClinicalTrials.gov: NCT05249452.

Phase 3 randomized trial of mavorixafor, CXCR4 antagonist, in WHIM syndrome.

We investigated efficacy and safety of mavorixafor, an oral CXCR4 antagonist for participants with Warts, Hypogammaglobulinemia, Infections, and Myelokathexis (WHIM) syndrome, a rare immunodeficiency caused by CXCR4 gain-of-function variants. This randomized (1:1), double-blind, placebo-controlled, phase 3 trial enrolled participants aged ≥12 years with WHIM syndrome and absolute neutrophil count (ANC) ≤400/µL. Participants received once-daily mavorixafor or placebo for 52 weeks. Primary endpoint was time (hours) above ANC threshold ≥500/µL (TATANC; over 24 hours). Secondary endpoints included TAT absolute lymphocyte count ≥1000/µL (TATALC; defined similar to TATANC); absolute changes in white blood cell (WBC), ANC, and ALC from baseline; annualized infection rate; infection duration and total infection score (combined infection number/severity). In 31 participants (mavorixafor, n=14; placebo, n=17), mavorixafor least squares (LS) mean TATANC was 15.0 hours, placebo 2.8 hours (P<0.001). Mavorixafor LS mean TATALC was 15.8 hours, placebo 4.6 hours (P<0.001). Higher absolute WBC, ANC, and ALC levels were seen with mavorixafor than placebo at each timepoint assessed. Annualized infection rates were 60% lower with mavorixafor versus placebo (LS mean 1.7 versus 4.2; nominal P=0.007) and total infection scores were 40% lower (7.4 [95% CI, 1.6-13.2] versus 12.3 [95% CI, 7.2-17.3]). Treatment with mavorixafor reduced infection frequency, severity, duration, and antibiotic use. No discontinuations occurred due to treatment-emergent adverse events (TEAEs); no related serious TEAEs were observed. Overall, mavorixafor-treated participants showed significant increases in LS mean TATANC and TATALC, reduced infection frequency, severity/duration. Mavorixafor was well tolerated in participants with WHIM syndrome. Trial was registered at ClinicalTrials.gov NCT03995108.

Fusariosis in patients with hematological malignancies: Two case reports.

Immunosuppressed patients with hematological malignancies are at risk for invasive fungal infections (IFI), including infections with Fusarium species (spp.), which are increasingly reported. Particularly at risk are patients with acute myeloid leukemia (AML) treated with high-dose cytarabine as remission-induction therapy. Whether cytarabine increases the risk of IFI in comparison to other chemotherapy remains not fully determined. Additionally, no clear correlation between the in vitro established minimal inhibitory concentrations (MICs) of antifungal agents and clinical outcome has been established for fusariosis. To increase awareness and knowledge of invasive fusariosis, we report two cases of Fusarium spp. infections in neutropenic patients following treatment with cytarabine for AML. Despite high MICs for azoles both patients were treated with an azole in combination with liposomal amphotericin B. The combination therapy was successful in one patient, however the other patient did not survive the disseminated Fusarium infection.

ATG versus PTCy in matched unrelated donor haematopoietic stem cell transplantations with non-myeloablative conditioning.

Graft-versus-host disease (GvHD) is a serious complication of allogeneic haematopoietic stem cell transplantation (HSCT). Both anti-thymocyte globulin (ATG) and post-transplant cyclophosphamide (PTCy) are used as lymphocyte-depleting strategies, yet a systematic comparison of transplantation outcomes between these two methods in matched unrelated donors (MUD) transplantations with non-myeloablative conditioning (NMC) is lacking. Adult patients with haematological malignancies who had undergone MUD HSCT with NMC regimens between 2014 and 2021 at 2 centres in Amsterdam (ATG: n = 95, PTCy: n = 90), were included in this retrospective study. Patient characteristics were comparable between the groups. The cumulative incidence of acute GvHD grade II-IV was 48% in the ATG group compared to 21% in the PTCy group (p < 0.001). The 3-year moderate/severe chronic GvHD was similar in both groups (p = 0.69). While the relapse rate was comparable between the groups (ATG 31% vs. PTCy 34%, p = 0.94), non-relapse mortality tended to be higher in the ATG group (17% vs. 9%, p = 0.069). Overall survival was similar in both groups (p = 0.12). In conclusion, PTCy-based regimens resulted in a significantly lower rate of acute GvHD than ATG-containing regimens in MUD transplantations with NMC. Whether PTCy results in improved overall survival as compared to ATG needs to be elucidated in larger prospective studies.

Fourth mRNA COVID-19 vaccination in immunocompromised patients with haematological malignancies (COBRA KAI): a cohort study.

Background: Patients with haematological malignancies have impaired antibody responses to SARS-CoV-2 vaccination. We aimed to investigate whether a fourth mRNA COVID-19 vaccination improved antibody quantity and quality. Methods: In this cohort study, conducted at 5 sites in the Netherlands, we compared antibody concentrations 28 days after 4 mRNA vaccinations (3-dose primary series plus 1 booster vaccination) in SARS-CoV-2 naive, immunocompromised patients with haematological malignancies to those obtained by age-matched, healthy individuals who had received the standard primary 2-dose mRNA vaccination schedule followed by a first booster mRNA vaccination. Prior to and 4 weeks after each vaccination, peripheral blood samples and data on demographic parameters and medical history were collected. Concentrations of antibodies that bind spike 1 (S1) and nucleocapsid (N) protein of SARS-CoV-2 were quantified in binding antibody units (BAU) per mL according to the WHO International Standard for COVID-19 serological tests. Seroconversion was defined as an S1 IgG concentration >10 BAU/mL and a previous SARS-CoV-2 infection as N IgG >14.3 BAU/mL. Antibody neutralising activity was tested using lentiviral-based pseudoviruses expressing spike protein of SARS-CoV-2 wild-type (D614G), Omicron BA.1, and Omicron BA.4/5 variants. This study is registered with EudraCT, number 2021-001072-41. Findings: Between March 24, 2021 and May 4, 2021, 723 patients with haematological diseases were enrolled, of which 414 fulfilled the inclusion criteria for the current analysis. Although S1 IgG concentrations in patients significantly improved after the fourth dose, they remained significantly lower compared to those obtained by 58 age-matched healthy individuals after their first booster (third) vaccination. The rise in neutralising antibody concentration was most prominent in patients with a recovering B cell compartment, although potent responses were also observed in patients with persistent immunodeficiencies. 19% of patients never seroconverted, despite 4 vaccinations. Patients who received their first 2 vaccinations when they were B cell depleted and the third and fourth vaccination during B cell recovery demonstrated similar antibody induction dynamics as patients with normal B cell numbers during the first 2 vaccinations. However, the neutralising capacity of these antibodies was significantly better than that of patients with normal B cell numbers after two vaccinations. Interpretation: A fourth mRNA COVID-19 vaccination improved S1 IgG concentrations in the majority of patients with a haematological malignancy. Vaccination during B cell depletion may pave the way for better quality of antibody responses after B cell reconstitution. Funding: The Netherlands Organisation for Health Research and Development and Amsterdam UMC.

Antibody Response in Immunocompromised Patients With Hematologic Cancers Who Received a 3-Dose mRNA-1273 Vaccination Schedule for COVID-19.

Importance: It has become common practice to offer immunocompromised patients with hematologic cancers a third COVID-19 vaccination dose, but data substantiating this are scarce. Objective: To assess whether a third mRNA-1273 vaccination is associated with increased neutralizing antibody concentrations in immunocompromised patients with hematologic cancers comparable to levels obtained in healthy individuals after the standard 2-dose mRNA-1273 vaccination schedule. Design, Setting, and Participants: This prospective observational cohort study was conducted at 4 university hospitals in the Netherlands and included 584 evaluable patients spanning the spectrum of hematologic cancers and 44 randomly selected age-matched adults without malignant or immunodeficient comorbidities. Exposures: One additional mRNA-1273 vaccination 5 months after completion of the standard 2-dose mRNA-1273 vaccination schedule. Main Outcomes and Measures: Serum immunoglobulin G (IgG) antibodies to spike subunit 1 (S1) antigens prior to and 4 weeks after a third mRNA-1273 vaccination, and antibody neutralization capacity of wild-type, Delta, and Omicron variants in a subgroup of patients. Results: In this cohort of 584 immunocompromised patients with hematologic cancers (mean [SD] age, 60 [11.2] years; 216 [37.0%] women), a third mRNA-1273 vaccination was associated with median S1-IgG concentrations comparable to concentrations obtained by healthy individuals after the 2-dose mRNA-1273 schedule. The rise in S1-IgG concentration after the third vaccination was most pronounced in patients with a recovering immune system, but potent responses were also observed in patients with persistent immunodeficiencies. Specifically, patients with myeloid cancers or multiple myeloma and recipients of autologous or allogeneic hematopoietic cell transplantation (HCT) reached median S1-IgG concentrations similar to those obtained by healthy individuals after a 2-dose schedule. Patients receiving or shortly after completing anti-CD20 therapy, CD19-directed chimeric antigen receptor T-cell therapy recipients, and patients with chronic lymphocytic leukemia receiving ibrutinib were less responsive or unresponsive to the third vaccination. In the 27 patients who received cell therapy between the second and third vaccination, S1 antibodies were preserved, but a third mRNA-1273 vaccination was not associated with significantly enhanced S1-IgG concentrations except for patients with multiple myeloma receiving autologous HCT. A third vaccination was associated with significantly improved neutralization capacity per antibody. Conclusions and Relevance: Results of this cohort study support that the primary schedule for immunocompromised patients with hematologic cancers should be supplemented with a delayed third vaccination. Patients with B-cell lymphoma and allogeneic HCT recipients need to be revaccinated after treatment or transplantation. Trial Registration: EudraCT Identifier: 2021-001072-41.

Quantitative analysis of mRNA-1273 COVID-19 vaccination response in immunocompromised adult hematology patients.

Vaccination guidelines for patients treated for hematological diseases are typically conservative. Given their high risk for severe COVID-19, it is important to identify those patients that benefit from vaccination. We prospectively quantified serum immunoglobulin G (IgG) antibodies to spike subunit 1 (S1) antigens during and after 2-dose mRNA-1273 (Spikevax/Moderna) vaccination in hematology patients. Obtaining S1 IgG ≥ 300 binding antibody units (BAUs)/mL was considered adequate as it represents the lower level of S1 IgG concentration obtained in healthy individuals, and it correlates with potent virus neutralization. Selected patients (n = 723) were severely immunocompromised owing to their disease or treatment thereof. Nevertheless, >50% of patients obtained S1 IgG ≥ 300 BAUs/mL after 2-dose mRNA-1273. All patients with sickle cell disease or chronic myeloid leukemia obtained adequate antibody concentrations. Around 70% of patients with chronic graft-versus-host disease (cGVHD), multiple myeloma, or untreated chronic lymphocytic leukemia (CLL) obtained S1 IgG ≥ 300 BAUs/mL. Ruxolitinib or hypomethylating therapy but not high-dose chemotherapy blunted responses in myeloid malignancies. Responses in patients with lymphoma, patients with CLL on ibrutinib, and chimeric antigen receptor T-cell recipients were low. The minimal time interval after autologous hematopoietic cell transplantation (HCT) to reach adequate concentrations was <2 months for multiple myeloma, 8 months for lymphoma, and 4 to 6 months after allogeneic HCT. Serum IgG4, absolute B- and natural killer-cell number, and number of immunosuppressants predicted S1 IgG ≥ 300 BAUs/mL. Hematology patients on chemotherapy, shortly after HCT, or with cGVHD should not be precluded from vaccination. This trial was registered at Netherlands Trial Register as #NL9553.

Immunogenicity of a 5-dose pneumococcal vaccination schedule following allogeneic hematopoietic stem cell transplantation.

The optimal schedule of pneumococcal vaccination after allogeneic hematopoietic stem cell transplantation (allo-HSCT) remains controversial. The objective of this study was to investigate the immunogenicity of a 5-dose pneumococcal vaccination schedule in adult allo-HSCT recipients with and without immunosuppressive therapy. In this prospective cohort study, allo-HSCT recipients received four doses of the 13-valent pneumococcal conjugate vaccine (PCV13) and one dose of the 23-valent pneumococcal polysaccharide vaccine (PPSV23) starting 4-6 months after allo-HSCT. PCV13 was administered at T0, T1, T2, and T8 (T = months from enrollment) and PPSV23 at T10. Serum was collected at T0, T4, T8, T10, and T12, and IgG levels were measured for all 24 vaccine serotypes by immunoassay. The primary outcome was overall seroprotection at T12 defined as an IgG concentration ≥1.3 µg/ml for 17/24 vaccine serotypes in allo-HCST recipients with and without immunosuppressive therapy at baseline. Secondary outcomes were serotype-specific seroprotection and dynamics of IgG levels. We included 89 allo-HSCT recipients in the final analysis. Overall seroprotection was 47% (15/32) for patients without immunosuppressive therapy at baseline versus 24% (11/46) for patients with immunosuppressive therapy (p = .03). Seroprotection was higher for PCV13 serotypes (78% and 54% respectively; p = .03) and lower for PPSV23-unique serotypes (28% and 13% respectively; p = .1). IgG concentrations increased significantly over time for all 24 serotypes. Concluding, although immunogenicity of PCV13 serotypes was reasonable, the poor response to PPSV23 serotypes resulted in an insufficient overall response to pneumococcal vaccination for allo-HSCT recipients. Research into vaccination strategies with higher-valent T-cell-dependent pneumococcal vaccines is needed.

SARS-CoV-2 vaccine-induced humoral and cellular immunity in patients with hematologic malignancies.

Patients with hematologic conditions have a higher risk of severe COVID-19 and COVID-19-related death. This is related to immune deficiencies induced by hematologic conditions and/or the treatment thereof. Prospective vaccine immunogenicity studies have demonstrated that in the majority of patients, a 3-dose COVID-19 vaccination schedule leads to antibody concentrations comparable to levels obtained in healthy adults after a 2-dose schedule. In B cell depleted patients, humoral responses are poor, however vaccination did induce potent cellular immune responses. The effect of 3-dose vaccination schedules and COVID-19 booster vaccinations on the protection of patients with hematologic malignancies against severe COVID-19 and COVID-19 related death remains to be confirmed by population-based vaccine effectiveness studies.

Allogeneic hematopoietic cell transplantation in the management of GATA2 deficiency and pulmonary alveolar proteinosis.

Human hematopoiesis is critically dependent on the transcription factor GATA2. Patients with GATA2 deficiency typically present with myelodysplastic syndrome, reduced numbers of monocytes, NK cells and B cells, and/or opportunistic infections. Here, we present two families that harbor distinct GATA2 mutations with highly variable onset and course of disease. We discuss the use of allogeneic hematopoietic cell transplantation in these patients, especially as treatment for pulmonary alveolar proteinosis.

Discovery and Differential Processing of HLA Class II-Restricted Minor Histocompatibility Antigen LB-PIP4K2A-1S and Its Allelic Variant by Asparagine Endopeptidase.

Minor histocompatibility antigens are the main targets of donor-derived T-cells after allogeneic stem cell transplantation. Identification of these antigens and understanding their biology are a key requisite for more insight into how graft vs. leukemia effect and graft vs. host disease could be separated. We here identified four new HLA class II-restricted minor histocompatibility antigens using whole genome association scanning. For one of the new antigens, i.e., LB-PIP4K2A-1S, we measured strong T-cell recognition of the donor variant PIP4K2A-1N when pulsed as exogenous peptide, while the endogenously expressed variant in donor EBV-B cells was not recognized. We showed that lack of T-cell recognition was caused by intracellular cleavage by a protease named asparagine endopeptidase (AEP). Furthermore, microarray gene expression analysis showed that PIP4K2A and AEP are both ubiquitously expressed in a wide variety of healthy tissues, but that expression levels of AEP were lower in primary acute myeloid leukemia (AML). In line with that, we confirmed low activity of AEP in AML cells and demonstrated that HLA-DRB1*03:01 positive primary AML expressing LB-PIP4K2A-1S or its donor variant PIP4K2A-1N were both recognized by specific T-cells. In conclusion, LB-PIP4K2A-1S not only represents a novel minor histocompatibility antigen but also provides evidence that donor T-cells after allogeneic stem cell transplantation can target the autologous allelic variant as leukemia-associated antigen. Furthermore, it demonstrates that endopeptidases can play a role in cell type-specific intracellular processing and presentation of HLA class II-restricted antigens, which may be explored in future immunotherapy of AML.

Selective graft-versus-leukemia depends on magnitude and diversity of the alloreactive T cell response.

Patients with leukemia who receive a T cell-depleted allogeneic stem cell graft followed by postponed donor lymphocyte infusion (DLI) can experience graft-versus-leukemia (GVL) reactivity, with a lower risk of graft-versus-host disease (GVHD). Here, we have investigated the magnitude, diversity, and specificity of alloreactive CD8 T cells in patients who developed GVL reactivity after DLI in the absence or presence of GVHD. We observed a lower magnitude and diversity of CD8 T cells for minor histocompatibility antigens (MiHAs) in patients with selective GVL reactivity without GVHD. Furthermore, we demonstrated that MiHA-specific T cell clones from patients with selective GVL reactivity showed lower reactivity against nonhematopoietic cells, even when pretreated with inflammatory cytokines. Expression analysis of MiHA-encoding genes showed that similar types of antigens were recognized in both patient groups, but in patients who developed GVHD, T cell reactivity was skewed to target broadly expressed MiHAs. As an inflammatory environment can render nonhematopoietic cells susceptible to T cell recognition, prevention of such circumstances favors induction of selective GVL reactivity without development of GVHD.

Patient HLA-DP-specific CD4+ T cells from HLA-DPB1-mismatched donor lymphocyte infusion can induce graft-versus-leukemia reactivity in the presence or absence of graft-versus-host disease.

Clinical studies have demonstrated that HLA-DPB1-mismatched allogeneic stem cell transplantation (allo-SCT) is associated with a decreased risk of disease relapse and an increased risk of graft-versus-host disease (GVHD) compared with HLA-DPB1-matched SCT. In T cell-depleted allo-SCT, mismatching of HLA-DPB1 was not associated with an increased risk of severe GVHD, but a significant decreased risk of disease relapse was still observed. To investigate whether patient HLA-DP-specific CD4(+) T cell responses were frequently induced after T cell-depleted HLA-DPB1-mismatched allo-SCT and donor lymphocyte infusion (DLI), we developed a method to screen for the presence of HLA-DP-specific CD4(+) T cells using CD137 as an activation marker and analyzed 24 patient-donor combinations. The patients suffered from various B cell malignancies, multiple myeloma, and myeloid leukemias. Patient HLA-DP-specific CD4(+) T cells were detected after DLI in 13 of 18 patients who exhibited a clinical response to DLI, compared with only 1 of 6 patients without a clinical response to DLI. Eight patients developed significant GVHD. These data show that patient HLA-DP-specific CD4(+) T cells frequently occur after HLA-DPB1-mismatched T cell-depleted allo-SCT and DLI, and are associated with graft-versus-leukemia reactivity both in the presence and absence of GVHD.

Alloreactive effector T cells require the local formation of a proinflammatory environment to allow crosstalk and high avidity interaction with nonhematopoietic tissues to induce GVHD reactivity.

Based on clinical observations that donor T cells specific for minor histocompatibility antigens (MiHA) ubiquitously expressed on both hematopoietic and nonhematopoietic cells were detected in patients showing evident graft-versus-leukemia/lymphoma (GVL) reactivity with no or limited coinciding graft-versus-host disease (GVHD), we hypothesized that nonhematopoietic tissues may be relatively unsusceptible to the cytotoxic effect of MiHA-specific T cells under normal, noninflammatory conditions. To test this hypothesis, we investigated the reactivity of alloreactive T cells specific for ubiquitously expressed MiHA against skin-derived primary human fibroblasts. We demonstrated that this reactivity was not merely determined by their antigen-specificity, but was highly dependent on adhesion molecule expression. ICAM-1 expression on the fibroblasts upregulated under proinflammatory conditions and induced during cross-talk with the T cells was demonstrated to be a crucial factor facilitating formation of high avidity interactions with the T cells and subsequent efficient target cell destruction. Furthermore, we provide supporting evidence for the role of ICAM-1 in vivo by demonstrating that ICAM-1 expression on nonhematopoietic target cells was dependent on the presence of infiltrating activated T cells, as was illustrated by restricted ICAM-1 expression at the sites of T cell infiltration in skin biopsies of patients with acute GVHD (aGVHD), by the absence of ICAM-1 expression in the same biopsies in areas without T cell infiltration and by the absence of ICAM-1 expression in biopsies of patients without GVHD independent of the presence of infiltrating nonactivated T cells. In conclusion, under noninflammatory conditions, nonhematopoietic tissues are unsusceptible to the GVHD reactivity of alloreactive T cells due to their inability to establish high avidity interactions.

High-throughput characterization of 10 new minor histocompatibility antigens by whole genome association scanning.

Patients with malignant diseases can be effectively treated with allogeneic hematopoietic stem cell transplantation (allo-SCT). Polymorphic peptides presented in HLA molecules, the so-called minor histocompatibility antigens (MiHA), play a crucial role in antitumor immunity as targets for alloreactive donor T cells. Identification of multiple MiHAs is essential to understand and manipulate the development of clinical responses after allo-SCT. In this study, CD8+ T-cell clones were isolated from leukemia patients who entered complete remission after allo-SCT, and MiHA-specific T-cell clones were efficiently selected for analysis of recognition of a panel of EBV-transformed B cells positive for the HLA restriction elements of the selected T-cell clones. One million single nucleotide polymorphisms (SNP) were determined in the panel cell lines and investigated for matching with the T-cell recognition data by whole genome association scanning (WGAs). Significant association with 12 genomic regions was found, and detailed analysis of genes located within these genomic regions revealed SNP disparities encoding polymorphic peptides in 10 cases. Differential recognition of patient-type, but not donor-type, peptides validated the identification of these MiHAs. Using tetramers, distinct populations of MiHA-specific CD8+ T cells were detected, demonstrating that our WGAs strategy allows high-throughput discovery of relevant targets in antitumor immunity after allo-SCT.

HLA-DPB1 mismatching results in the generation of a full repertoire of HLA-DPB1-specific CD4+ T cell responses showing immunogenicity of all HLA-DPB1 alleles.

Clinical studies have indicated that HLA-DPB1 functions as a classical transplantation antigen in allogeneic stem cell transplantation. Mismatching for HLA-DPB1 was associated with an increased risk of graft-versus-host disease (GVHD), but also a decreased risk of disease relapse. However, specific HLA-DPB1 mismatches were associated with poor clinical outcome. It was suggested that this unfavorable effect was caused by a difference in immunogenicity between HLA-DPB1 alleles. To analyze whether immunogenicity of HLA-DPB1 mismatches could be predicted based on the presence or absence of specific amino acid sequences we developed a model to generate allo-HLA-DPB1 responses in vitro. We tested in total 48 different stimulator/responder combinations by stimulating CD4(+) T cells from 5 HLA-DPB1 homozygous individuals with the same antigen-presenting cells transduced with different allo-HLA-DPB1 molecules. HLA-DPB1 molecules used for stimulation comprised 76% to 99% of HLA-DPB1 molecules present in different ethnic populations. We show that all HLA-DPB1 mismatches as defined by allele typing resulted in high-frequency immune responses. Furthermore, we show that crossrecognition of different HLA-DPB1 molecules is a broadly observed phenomenon. We confirm previously described patterns in crossrecognition, and demonstrate that a high degree in similarity between HLA-DPB1 molecules is predictive for crossrecognition, but not for immunogenicity.