Expanding the repertoire reveals recurrent, cryptic and hematopoietic HLA class I minor histocompatibility antigens.

Allogeneic stem cell transplantation (alloSCT) is a curative treatment for hematological malignancies. After HLA-matched alloSCT, anti-tumor immunity is caused by donor T cells recognizing polymorphic peptides, designated minor histocompatibility antigens (MiHAs), that are presented by HLA on malignant patient cells. However, T cells often target MiHAs on healthy non-hematopoietic tissues of patients, thereby inducing side effects known as Graft-versus-Host Disease. Here, we aimed to identify the dominant repertoire of HLA-I-restricted MiHAs to enable strategies to predict, monitor or modulate immune responses after alloSCT. To systematically identify novel MiHAs by genome-wide association screening, T-cell clones were isolated from 39 transplanted patients and tested for reactivity against 191 EBV-B cell lines of the 1000 Genomes Project. By discovering 81 new MiHAs, we more than doubled the antigen repertoire to 159 MiHAs and demonstrated that, despite many genetic differences between patients and donors, often the same MiHAs are targeted in multiple patients. Furthermore, we showed that one quarter of the antigens are cryptic, i.e. translated from unconventional open reading frames, for example long non-coding RNAs, showing that these antigen types are relevant targets in natural immune responses. Finally, using single cell RNA-seq data, we analyzed tissue expression of MiHA-encoding genes to explore their potential role in clinical outcome, and characterized 11 new hematopoietic-restricted MiHAs as potential targets for immunotherapy. In conclusion, we expanded the repertoire of HLA-I-restricted MiHAs and identified recurrent, cryptic and hematopoietic-restricted antigens, which are fundamental to predict, follow or manipulate immune responses to improve clinical outcome after alloSCT.

Risk factors for graft-versus-host-disease after donor lymphocyte infusion following T-cell depleted allogeneic stem cell transplantation.

Introduction: Unmodified donor lymphocyte infusions (DLI) after allogeneic stem cell transplantation (alloSCT) can boost the beneficial Graft-versus-Leukemia (GvL) effect but may also induce severe Graft-versus-Host-Disease (GvHD). To improve the balance between GvL and GvHD, it is crucial to identify factors that influence the alloreactivity of DLI. Methods: We investigated the effects of the presence of patient-derived antigen-presenting cells at time of DLI as estimated by the bone marrow (BM) chimerism status, lymphopenia as measured by the absolute lymphocyte count (ALC) at time of DLI, and the presence of a viral infection (de novo or reactivation) close to DLI on the risk of GvHD after DLI. The cohort consisted of patients with acute leukemia or myelodysplastic syndrome who prophylactically or pre-emptively received DLI as standard care after alemtuzumab-based alloSCT. In patients at high risk for relapse, DLI was administered at 3 months after alloSCT (n=88) with a dose of 0.3x106 or 0.15x106 T cells/kg in case of a related or unrelated donor, respectively. All other patients (n=76) received 3x106 or 1.5x106 T cells/kg, respectively, at 6 months after alloSCT. Results: For both DLIs, patients with reduced-intensity conditioning and an unrelated donor had the highest risk of GvHD. For DLI given at three months, viral infection within 1 week before and 2 weeks after DLI was an additional significant risk factor (hazard ratio (HR) 3.66 compared to no viral infection) for GvHD. At six months after alloSCT, viral infections were rare and not associated with GvHD. In contrast, mixed BM chimerism (HR 3.63 for ≥5% mixed chimerism compared to full donor) was an important risk factor for GvHD after DLI given at six months after alloSCT. ALC of <1000x106/l showed a trend for association with GvHD after this DLI (HR 2.05 compared to ≥1000x106/l, 95% confidence interval 0.94-4.45). Furthermore, the data suggested that the presence of a viral infection close to the DLI at three months or ≥5% mixed chimerism at time of the DLI at six months correlated with the severity of GvHD, thereby increasing their negative impact on the current GvHD-relapse-free survival. Conclusion: These data demonstrate that the risk factors for GvHD after DLI depend on the setting of the DLI.

Mapping AML heterogeneity - multi-cohort transcriptomic analysis identifies novel clusters and divergent ex-vivo drug responses.

Subtyping of acute myeloid leukaemia (AML) is predominantly based on recurrent genetic abnormalities, but recent literature indicates that transcriptomic phenotyping holds immense potential to further refine AML classification. Here we integrated five AML transcriptomic datasets with corresponding genetic information to provide an overview (n = 1224) of the transcriptomic AML landscape. Consensus clustering identified 17 robust patient clusters which improved identification of CEBPA-mutated patients with favourable outcomes, and uncovered transcriptomic subtypes for KMT2A rearrangements (2), NPM1 mutations (5), and AML with myelodysplasia-related changes (AML-MRC) (5). Transcriptomic subtypes of KMT2A, NPM1 and AML-MRC showed distinct mutational profiles, cell type differentiation arrests and immune properties, suggesting differences in underlying disease biology. Moreover, our transcriptomic clusters show differences in ex-vivo drug responses, even when corrected for differentiation arrest and superiorly capture differences in drug response compared to genetic classification. In conclusion, our findings underscore the importance of transcriptomics in AML subtyping and offer a basis for future research and personalised treatment strategies. Our transcriptomic compendium is publicly available and we supply an R package to project clusters to new transcriptomic studies.

Accurate prediction of HLA class II antigen presentation across all loci using tailored data acquisition and refined machine learning.

Accurate prediction of antigen presentation by human leukocyte antigen (HLA) class II molecules is crucial for rational development of immunotherapies and vaccines targeting CD4+ T cell activation. So far, most prediction methods for HLA class II antigen presentation have focused on HLA-DR because of limited availability of immunopeptidomics data for HLA-DQ and HLA-DP while not taking into account alternative peptide binding modes. We present an update to the NetMHCIIpan prediction method, which closes the performance gap between all three HLA class II loci. We accomplish this by first integrating large immunopeptidomics datasets describing the HLA class II specificity space across all loci using a refined machine learning framework that accommodates inverted peptide binders. Next, we apply targeted immunopeptidomics assays to generate data that covers additional HLA-DP specificities. The final method, NetMHCIIpan-4.3, achieves high accuracy and molecular coverage across all HLA class II allotypes.

Joint models quantify associations between immune cell kinetics and allo-immunological events after allogeneic stem cell transplantation and subsequent donor lymphocyte infusion.

Alloreactive donor-derived T-cells play a pivotal role in alloimmune responses after allogeneic hematopoietic stem cell transplantation (alloSCT); both in the relapse-preventing Graft-versus-Leukemia (GvL) effect and the potentially lethal complication Graft-versus-Host-Disease (GvHD). The balance between GvL and GvHD can be shifted by removing T-cells via T-cell depletion (TCD) to reduce the risk of GvHD, and by introducing additional donor T-cells (donor lymphocyte infusions [DLI]) to boost the GvL effect. However, the association between T-cell kinetics and the occurrence of allo-immunological events has not been clearly demonstrated yet. Therefore, we investigated the complex associations between the T-cell kinetics and alloimmune responses in a cohort of 166 acute leukemia patients receiving alemtuzumab-based TCD alloSCT. Of these patients, 62 with an anticipated high risk of relapse were scheduled to receive a prophylactic DLI at 3 months after transplant. In this setting, we applied joint modelling which allowed us to better capture the complex interplay between DLI, T-cell kinetics, GvHD and relapse than traditional statistical methods. We demonstrate that DLI can induce detectable T-cell expansion, leading to an increase in total, CD4+ and CD8+ T-cell counts starting at 3 months after alloSCT. CD4+ T-cells showed the strongest association with the development of alloimmune responses: higher CD4 counts increased the risk of GvHD (hazard ratio 2.44, 95% confidence interval 1.45-4.12) and decreased the risk of relapse (hazard ratio 0.65, 95% confidence interval 0.45-0.92). Similar models showed that natural killer cells recovered rapidly after alloSCT and were associated with a lower risk of relapse (HR 0.62, 95%-CI 0.41-0.93). The results of this study advocate the use of joint models to further study immune cell kinetics in different settings.

Competitive Repopulation and Allo-Immunologic Pressure Determine Chimerism Kinetics after T Cell-Depleted Allogeneic Stem Cell Transplantation and Donor Lymphocyte Infusion.

After allogeneic stem cell transplantation (alloSCT), patient-derived stem cells that survived the pretransplantation conditioning compete with engrafting donor stem cells for bone marrow (BM) repopulation. In addition, donor-derived alloreactive T cells present in the stem cell product may favor establishment of complete donor-derived hematopoiesis by eliminating patient-derived lymphohematopoietic cells. T cell-depleted alloSCT with sequential transfer of potentially alloreactive T cells by donor lymphocyte infusion (DLI) provides a unique opportunity to selectively study how competitive repopulation and allo-immunologic pressure influence lymphohematopoietic recovery. This study aimed to determine the relative contribution of competitive repopulation and donor-derived anti-recipient alloimmunologic pressure on the establishment of lymphohematopoietic chimerism after alloSCT. In this retrospective cohort study of 281 acute leukemia patients treated according to a protocol combining alemtuzumab-based T cell-depleted alloSCT with prophylactic DLI, we investigated engraftment and quantitative donor chimerism in the BM and immune cell subsets. DLI-induced increase of chimerism and development of graft-versus-host disease (GVHD) were analyzed as complementary indicators for donor-derived anti-recipient alloimmunologic pressure. Profound suppression of patient immune cells by conditioning sufficed for sustained engraftment without necessity for myeloablative conditioning or development of clinically significant GVHD. Although 61% of the patients without any DLI or GVHD showed full donor chimerism (FDC) in the BM at 6 months after alloSCT, only 24% showed FDC in the CD4+ T cell compartment. In contrast, 75% of the patients who had received DLI and 83% of the patients with clinically significant GVHD had FDC in this compartment. In addition, 72% of the patients with mixed hematopoiesis receiving DLI converted to complete donor-derived hematopoiesis, of whom only 34% developed clinically significant GVHD. Our data show that competitive repopulation can be sufficient to reach complete donor-derived hematopoiesis, but that some alloimmunologic pressure is needed for the establishment of a completely donor-derived T cell compartment, either by the development of GVHD or by administration of DLI. We illustrate that it is possible to separate the graft-versus-leukemia effect from GVHD, as conversion to durable complete donor-derived hematopoiesis following DLI did not require induction of clinically significant GVHD.

Human T cells recognize HLA-DP-bound peptides in two orientations.

Human leukocyte antigen (HLA) molecules present small peptide antigens to T cells, thereby allowing them to recognize pathogen-infected and cancer cells. A central dogma over the last 50+ y is that peptide binding to HLA molecules is mediated by the docking of side chains of particular amino acids in the peptide into pockets in the HLA molecules in a conserved N- to C-terminal orientation. Whether peptides can be presented in a reversed C- to N-terminal orientation remains unclear. Here, we performed large-scale identification of peptides bound to HLA-DP molecules and observed that in addition to peptide binding in an N- to C-terminal orientation, in 9 out of 14 HLA-DP allotypes, reverse motifs are found, compatible with C- to N-terminal peptide binding. Moreover, we isolated high-avidity human cytomegalovirus (CMV)-specific HLA-DP-restricted CD4+ T cells from the memory repertoire of healthy donors and demonstrate that such T cells recognized CMV-derived peptides bound to HLA-DPB1*01:01 or *05:01 in a reverse C- to N-terminal manner. Finally, we obtained a high-resolution HLA-DPB1*01:01-CMVpp65(142-158) peptide crystal structure, which is the molecular basis for C- to N-terminal peptide binding to HLA-DP. Our results point to unique features of HLA-DP molecules that substantially broaden the HLA class II bound peptide repertoire to combat pathogens and eliminate cancer cells.

[Work-related asthma in clinical practice]. / Werkgerelateerd astma in de klinische praktijk.

Work-related asthma is an underestimated problem. More awareness is needed for early identification of work-related lung diseases to prevent permanent damage in patients with lung diseases. Work-related asthma can lead to an increase in the burden of disease, the number of exacerbations and hospital admissions. Therefore, exposure to substances with irritating and/or sensitizing properties should be regarded as an additional treatable trait in asthma treatment. The number of occupations where such exposures plays a role is large and diverse. Inquiring about the profession, activities or future career choice of the (asthma) patient during the first consultation is of great importance for the correct diagnosis and treatment. Cooperation between pulmonologist, lung nurse, ENT specialist, dermatologist, company doctor and occupational hygienist can contribute to this. A regional organization with a 'multidisciplinary consultation' for occupational lung diseases is important to guarantee optimal care and advice. The clinical case series demonstrate work-related asthma in clinical practice.

Promiscuity of Peptides Presented in HLA-DP Molecules from Different Immunogenicity Groups Is Associated With T-Cell Cross-Reactivity.

In the context of HLA-DP-mismatched allogeneic stem cell transplantation, mismatched HLA-DP alleles can provoke profound allo-HLA-DP-specific immune responses from the donor T-cell repertoire leading to graft-versus-leukemia effect and/or graft-versus-host disease in the patient. The magnitude of allo-HLA-DP-specific immune responses has been shown to depend on the specific HLA-DP disparity between donor and patient and the immunogenicity of the mismatched HLA-DP allele(s). HLA-DP peptidome clustering (DPC) was developed to classify the HLA-DP molecules based on similarities and differences in their peptide-binding motifs. To investigate a possible categorization of HLA-DP molecules based on overlap of presented peptides, we identified and compared the peptidomes of the thirteen most frequently expressed HLA-DP molecules. Our categorization based on shared peptides was in line with the DPC classification. We found that the HLA-DP molecules within the previously defined groups DPC-1 or DPC-3 shared the largest numbers of presented peptides. However, the HLA-DP molecules in DPC-2 segregated into two subgroups based on the overlap in presented peptides. Besides overlap in presented peptides within the DPC groups, a substantial number of peptides was also found to be shared between HLA-DP molecules from different DPC groups, especially for groups DPC-1 and -2. The functional relevance of these findings was illustrated by demonstration of cross-reactivity of allo-HLA-DP-reactive T-cell clones not only against HLA-DP molecules within one DPC group, but also across different DPC groups. The promiscuity of peptides presented in various HLA-DP molecules and the cross-reactivity against different HLA-DP molecules demonstrate that these molecules cannot be strictly categorized in immunogenicity groups.

Permissive HLA-DPB1 mismatches in HCT depend on immunopeptidome divergence and editing by HLA-DM.

In hematopoietic cell transplantation (HCT), permissive HLA-DPB1 mismatches between patients and their unrelated donors are associated with improved outcomes compared with nonpermissive mismatches, but the underlying mechanism is incompletely understood. Here, we used mass spectrometry, T-cell receptor-ß (TCRß) deep sequencing, and cellular in vitro models of alloreactivity to interrogate the HLA-DP immunopeptidome and its role in alloreactive T-cell responses. We find that permissive HLA-DPB1 mismatches display significantly higher peptide repertoire overlaps compared with their nonpermissive counterparts, resulting in lower frequency and diversity of alloreactive TCRß clonotypes in healthy individuals and transplanted patients. Permissiveness can be reversed by the absence of the peptide editor HLA-DM or the presence of its antagonist, HLA-DO, through significant broadening of the peptide repertoire. Our data establish the degree of immunopeptidome divergence between donor and recipient as the mechanistic basis for the clinically relevant permissive HLA-DPB1 mismatches in HCT and show that permissiveness is dependent on HLA-DM-mediated peptide editing. Its key role for harnessing T-cell alloreactivity to HLA-DP highlights HLA-DM as a potential novel target for cellular and immunotherapy of leukemia.

HA-1H T-Cell Receptor Gene Transfer to Redirect Virus-Specific T Cells for Treatment of Hematological Malignancies After Allogeneic Stem Cell Transplantation: A Phase 1 Clinical Study.

Graft-vs.-leukemia (GVL) reactivity after HLA-matched allogeneic stem cell transplantation (alloSCT) is mainly mediated by donor T cells recognizing minor histocompatibility antigens (MiHA). If MiHA are targeted that are exclusively expressed on hematopoietic cells of recipient origin, selective GVL reactivity without severe graft-vs.-host-disease (GVHD) may occur. In this phase I study we explored HA-1H TCR gene transfer into T cells harvested from the HA-1H negative stem-cell donor to treat HA-1H positive HLA-A*02:01 positive patients with high-risk leukemia after alloSCT. HA-1H is a hematopoiesis-restricted MiHA presented in HLA-A*02:01. Since we previously demonstrated that donor-derived virus-specific T-cell infusions did not result in GVHD, we used donor-derived EBV and/or CMV-specific T-cells to be redirected by HA-1H TCR. EBV and/or CMV-specific T-cells were purified, retrovirally transduced with HA-1H TCR, and expanded. Validation experiments illustrated dual recognition of viral antigens and HA-1H by HA-1H TCR-engineered virus-specific T-cells. Release criteria included products containing more than 60% antigen-specific T-cells. Patients with high risk leukemia following T-cell depleted alloSCT in complete or partial remission were eligible. HA-1H TCR T-cells were infused 8 and 14 weeks after alloSCT without additional pre-conditioning chemotherapy. For 4/9 included patients no appropriate products could be made. Their donors were all CMV-negative, thereby restricting the production process to EBV-specific T-cells. For 5 patients a total of 10 products could be made meeting the release criteria containing 3-280 × 106 virus and/or HA-1H TCR T-cells. No infusion-related toxicity, delayed toxicity or GVHD occurred. One patient with relapsed AML at time of infusions died due to rapidly progressing disease. Four patients were in remission at time of infusion. Two patients died of infections during follow-up, not likely related to the infusion. Two patients are alive and well without GVHD. In 2 patients persistence of HA-1H TCR T-cells could be illustrated correlating with viral reactivation, but no overt in-vivo expansion of infused T-cells was observed. In conclusion, HA-1H TCR-redirected virus-specific T-cells could be made and safely infused in 5 patients with high-risk AML, but overall feasibility and efficacy was too low to warrant further clinical development using this strategy. New strategies will be explored using patient-derived donor T-cells isolated after transplantation transduced with HA-1H-specific TCR to be infused following immune conditioning.

Immunopeptidome Analysis of HLA-DPB1 Allelic Variants Reveals New Functional Hierarchies.

HLA-DP alleles can be classified into functional T cell epitope (TCE) groups. TCE-1 and TCE-2 are clearly defined, but TCE-3 still represents an heterogeneous group. Because polymorphisms in HLA-DP influence the presented peptidome, we investigated whether the composition of peptides binding in HLA-DP may be used to refine the HLA-DP group classification. Peptidomes of human HLA-DP-typed B cell lines were analyzed with mass spectrometry after immunoaffinity chromatography and peptide elution. Gibbs clustering was performed to identify motifs of binding peptides. HLA-DP peptide-binding motifs showed a clear association with the HLA-DP allele-specific sequences of the binding groove. Hierarchical clustering of HLA-DP immunopeptidomes was performed to investigate the similarities and differences in peptidomes of different HLA-DP molecules, and this clustering resulted in the categorization of HLA-DP alleles into 3-DP peptidome clusters (DPC). The peptidomes of HLA-DPB1*09:01, -10:01, and -17:01 (TCE-1 alleles) and HLA-DPB1*04:01, -04:02, and -02:01 (TCE-3 alleles) were separated in two maximal distinct clusters, DPC-1 and DPC-3, respectively, reflecting their previous TCE classification. HLA-DP alleles categorized in DPC-2 shared certain similar peptide-binding motifs with DPC-1 or DPC-3 alleles, but significant differences were observed for other positions. Within DPC-2, divergence between the alleles was observed based on the preference for different peptide residues at position 9. In summary, immunopeptidome analysis was used to unravel functional hierarchies among HLA-DP alleles, providing new molecular insights into HLA-DP classification.

Generation and infusion of multi-antigen-specific T cells to prevent complications early after T-cell depleted allogeneic stem cell transplantation-a phase I/II study.

Prophylactic infusion of selected donor T cells can be an effective method to restore specific immunity after T-cell-depleted allogeneic stem cell transplantation (TCD-alloSCT). In this phase I/II study, we aimed to reduce the risk of viral complications and disease relapses by administrating donor-derived CD8pos T cells directed against cytomegalovirus (CMV), Epstein-Barr virus (EBV) and adenovirus antigens, tumor-associated antigens (TAA) and minor histocompatibility antigens (MiHA). Twenty-seven of thirty-six screened HLA-A*02:01pos patients and their CMVpos and/or EBVpos donors were included. Using MHC-I-Streptamers, 27 T-cell products were generated containing a median of 5.2 × 106 cells. Twenty-four products were administered without infusion-related complications at a median of 58 days post alloSCT. No patients developed graft-versus-host disease during follow-up. Five patients showed disease progression without coinciding expansion of TAA/MiHA-specific T cells. Eight patients experienced CMV- and/or EBV-reactivations. Four of these reactivations were clinically relevant requiring antiviral treatment, of which two progressed to viral disease. All resolved ultimately. In 2/4 patients with EBV-reactivations and 6/8 patients with CMV-reactivations, viral loads were followed by the expansion of donor-derived virus target-antigen-specific T cells. In conclusion, generation of multi-antigen-specific T-cell products was feasible, infusions were well tolerated and expansion of target-antigen-specific T cells coinciding viral reactivations was illustrated in the majority of patients.

The allogeneic HLA-DP-restricted T-cell repertoire provoked by allogeneic dendritic cells contains T cells that show restricted recognition of hematopoietic cells including primary malignant cells.

Stem cell grafts from 10/10 HLA-matched unrelated donors are often mismatched for HLA-DP. In some patients, donor T-cell responses targeting the mismatched HLA-DP allele(s) have been found to induce a specific graft-versus-leukemia effect without coinciding graft-versus-host disease, whereas in other cases significant graft-versus-host disease occurred. Cell-lineage-specific recognition patterns within the allogeneic HLA-DP-specific donor T-cell repertoire could explain the differential clinical effects mediated by donor T cells after HLA-DP-mismatched allogeneic stem cell transplantation. To unravel the composition of the HLA-DP T-cell repertoire, donor T-cell responses were provoked by in vitro stimulation with allogeneic HLA-DP-mismatched monocyte-derived dendritic cells. A strategy including depletion of reactivity against autologous dendritic cells allowed efficient identification and enrichment of allo-reactive T cells upon stimulation with HLA-DP-mismatched dendritic cells. In this study we elucidated that the allogeneic HLA-DP-restricted T-cell repertoire contained T cells with differential cell-lineage-specific recognition profiles. As expected, some of the allogeneic HLA-DP-restricted T cells showed broad recognition of a variety of hematopoietic and non-hematopoietic cell types expressing the targeted mismatched HLA-DP allele. However, a significant proportion of the allogeneic HLA-DP-restricted T cells showed restricted recognition of hematopoietic cells, including primary malignant cells, or even restricted recognition of only myeloid cells, including dendritic cells and primary acute myeloid leukemia samples, but not of other hematopoietic and non-hematopoietic cell types. These data demonstrate that the allogeneic HLA-DP-specific T-cell repertoire contains T cells that show restricted recognition of hematopoietic cells, which may contribute to the specific graft-versus-leukemia effect without coinciding graft-versus-host disease.

Dissecting Genetic Control of HLA-DPB1 Expression and Its Relation to Structural Mismatch Models in Hematopoietic Stem Cell Transplantation.

HLA expression levels have been suggested to be genetically controlled by single nucleotide polymorphisms (SNP) in the untranslated regions (UTR), and expression variants have been associated with the outcome of chronic viral infection and hematopoietic stem cell transplantation (HSCT). In particular, the 3'UTR rs9277534-G/A SNP in HLA-DPB1 has been associated with graft-versus-host-disease after HSCT (Expression model); however its relevance in different immune cells and its mode of action have not been systematically addressed. In addition, there is a strong though not complete overlap between the rs9277534-G/A SNP and structural HLA-DPB1 T cell epitope (TCE) groups which have also been associated with HSCT outcome (TCE Structural model). Here we confirm and extend previous findings of significantly higher HLA-DPB1 expression in B cell lines, unstimulated primary B cells, and monocytes homozygous for rs9277534-G compared to those homozygous for rs9277534-A. However, these differences were abrogated by interferon-γ stimulation or differentiation into dendritic cells. We identify at least seven 3'UTR rs9277534-G/A haplotypes differing by a total of 37 SNP, also characterized by linkage to length variants of a short tandem repeat (STR) in intron 2 and TCE group assignment. 3'UTR mapping did not show any significant differences in post-transcriptional regulation assessed by luciferase assays between two representative rs9277534-G/A haplotypes for any of eight overlapping fragments. Moreover, no evidence for alternative splicing associated with the intron 2 STR was obtained by RT-PCR. In an exemplary cohort of 379 HLA-DPB1 mismatched donor-recipient pairs, risk prediction by the Expression model and the Structural TCE model was 36.7% concordant, with the majority of discordances due to non-applicability of the Expression model. HLA-DPB1 from different TCE groups expressed in the absence of the 3'UTR at similar levels by transfected HeLa cells elicited significantly different mean alloreactive CD4+ T-cell responses, as assessed by CD137 upregulation assays in 178 independent cultures. Taken together, our data provide new insights into the cell type-specific and mechanistic basis of the association between the rs9277534-G/A SNP and HLA-DPB1 expression, and show that, despite partial overlap between both models in HSCT risk-prediction, differential alloreactivity determined by the TCE structural model occurs independently from HLA-DPB1 differential expression.

Alloreactive T Cell Receptor Diversity against Structurally Similar or Dissimilar HLA-DP Antigens Assessed by Deep Sequencing.

T cell alloreactivity is mediated by a self-human leukocyte antigen (HLA)-restricted T cell receptor (TCR) repertoire able to recognize both structurally similar and dissimilar allogeneic HLA molecules (i.e., differing by a single or several amino acids in their peptide-binding groove). We hypothesized that thymic selection on self-HLA molecules could have an indirect impact on the size and diversity of the alloreactive response. To test this possibility, we used TCR Vß immunophenotyping and immunosequencing technology in a model of alloreactivity between self-HLA selected T cells and allogeneic HLA-DPB1 (DPB1) differing from self-DPB1*04:02 by a single (DPB1*02:01) or several (DPB1*09:01) amino acids in the peptide-binding groove. CD4+ T cells from three different self-DPB1*04:01,*04:02 individuals were stimulated with HeLa cells stably transduced with the relevant peptide processing machinery, co-stimulatory molecules, and HLA-DP. Flow cytometric quantification of the DPB1-specific T cell response measured as upregulation of the activation marker CD137 revealed significantly lower levels of alloreactivity against DPB1*02:01 compared with DPB1*09:01 (mean CD4+CD137+ frequency 35.2 ± 9.9 vs. 61.5 ± 7.7%, respectively, p < 0.0001). These quantitative differences were, however, not reflected by differences in the breadth of the alloreactive response at the Vß level, with both alloantigens eliciting specific responses from all TCR-Vß specificities tested by flow cytometry, albeit with higher levels of reactivity from most Vß specificities against DPB1*09:01. In line with these observations, TCRB-CDR3 immunosequencing showed no significant differences in mean clonality of sorted CD137+CD4+ cells alloreactive against DPB1*02:01 or DPB1*09:01 [0.39 (0.36-0.45) and 0.39 (0.30-0.46), respectively], or in the cumulative frequencies of the 10 most frequent responding clones (55-67 and 58-62%, respectively). Most of the clones alloreactive against DPB1*02:01 (68.3%) or DPB1*09:01 (75.3%) were characterized by low-abundance (i.e., they were not appreciable among the pre-culture T cells). Interestingly, however, their cumulative frequency was lower against DPB1*02:01 compared with DPB1*09:01 (mean cumulative frequency 35.3 vs. 50.6%, respectively). Our data show that, despite lower levels of alloreactivity, a similar clonal diversity can be elicited by structurally similar compared with structurally dissimilar HLA-DPB1 alloantigens and demonstrate the power of TCRB immunosequencing in unraveling subtle qualitative changes not appreciable by conventional methods.

Tissue Damage Caused by Myeloablative, but Not Non-Myeloablative, Conditioning before Allogeneic Stem Cell Transplantation Results in Dermal Macrophage Recruitment without Active T-Cell Interaction.

Introduction: Conditioning regimens preceding allogeneic stem cell transplantation (alloSCT) can cause tissue damage and acceleration of the development of graft-versus-host disease (GVHD). T-cell-depleted alloSCT with postponed donor lymphocyte infusion (DLI) may reduce GVHD, because tissue injury can be restored at the time of DLI. In this study, we investigated the presence of tissue injury and inflammation in skin during the period of hematologic recovery and immune reconstitution after alloSCT. Methods: Skin biopsies were immunohistochemically stained for HLA class II, CD1a, CD11c, CD40, CD54, CD68, CD86, CD206, CD3, and CD8. HLA class II-expressing cells were characterized as activated T-cells, antigen-presenting cells (APCs), or tissue repairing macrophages. In sex-mismatched patient and donor couples, origin of cells was determined by multiplex analysis combining XY-FISH and fluorescent immunohistochemistry. Results: No inflammatory environment due to pretransplant conditioning was detected at the time of alloSCT, irrespective of the conditioning regimen. An increase in HLA class II-positive macrophages and CD3 T-cells was observed 12-24 weeks after myeloablative alloSCT, but these macrophages did not show signs of interaction with the co-localized T-cells. In contrast, during GVHD, an increase in HLA class II-expressing cells coinciding with T-cell interaction was observed, resulting in an overt inflammatory reaction with the presence of activated APC, activated donor T-cells, and localized upregulation of HLA class II expression on epidermal cells. In the absence of GVHD, patient derived macrophages were gradually replaced by donor-derived macrophages although patient-derived macrophages were detectable even 24 weeks after alloSCT. Conclusion: Conditioning regimens cause tissue damage in the skin, but this does not result in a local increase of activated APC. In contrast to the inflamed situation in GVHD, when interaction takes place between activated APC and donor T-cells, the tissue damage caused by myeloablative alloSCT results in dermal recruitment of HLA class II-positive tissue repairing macrophages co-existing with increased numbers of patient- and donor-derived T-cells, but without signs of specific interaction and initiation of an immune response. Thus, the local skin damage caused by the conditioning regimen appears to be insufficient as single factor to provoke GVHD induction.

CD4 Donor Lymphocyte Infusion Can Cause Conversion of Chimerism Without GVHD by Inducing Immune Responses Targeting Minor Histocompatibility Antigens in HLA Class II.

Under non-inflammatory conditions HLA class II is predominantly expressed on hematopoietic cells. Therefore, donor CD4 T-cells after allogeneic stem cell transplantation (alloSCT) may mediate graft-vs.-leukemia reactivity without graft-vs.-host disease (GVHD). We analyzed immune responses in four patients converting from mixed to full donor chimerism without developing GVHD upon purified CD4 donor lymphocyte infusion (DLI) from their HLA-identical sibling donor after T-cell depleted alloSCT. In vivo activated T-cells were clonally isolated after CD4 DLI. Of the alloreactive T-cell clones, 96% were CD4 positive, illustrating the dominant role of CD4 T-cells in the immune responses. We identified 9 minor histocompatibility antigens (MiHA) as targets for alloreactivity, of which 8 were novel HLA class II restricted MiHA. In all patients, MiHA specific CD4 T-cells were found that were capable to lyse hematopoietic cells and to recognize normal and malignant cells. No GVHD was induced in these patients. Skin fibroblasts forced to express HLA class II, were recognized by only two MiHA specific CD4 T-cell clones. Of the 7 clones that failed to recognize fibroblasts, two targeted MiHA were encoded by genes not expressed in fibroblasts, presentation of one MiHA was dependent on HLA-DO, which is absent in fibroblasts, and T-cells recognizing the remaining 4 MiHA had an avidity that was apparently too low to recognize fibroblasts, despite clear recognition of hematopoietic cells. In conclusion, purified CD4 DLI from HLA-identical sibling donors can induce conversion from mixed to full donor chimerism with graft-vs.-malignancy reactivity, but without GVHD, by targeting HLA class II restricted MiHA.