scholarly journals Accurate MHC Motif Deconvolution of Immunopeptidomics Data Reveals a Significant Contribution of DRB3, 4 and 5 to the Total DR Immunopeptidome

2021 ◽  
Author(s):  
Saghar Kaabinejadian ◽  
Carolina Barra ◽  
Bruno Alvarez ◽  
Hooman Yari ◽  
William Hildebrand ◽  
...  
Keyword(s):  
Large Scale ◽  
Vaccine Development ◽  
State Of The Art ◽  
Hla Class I ◽  
Class Ii ◽  
Ease Of Use ◽  
Hla Dr ◽  
Generation Vaccine ◽  
Major Bottleneck ◽  
Epitope Discovery

Mass spectrometry (MS) based immunopeptidomics is used in several biomedical applications including neo-epitope discovery in oncology and next-generation vaccine development. Immunopeptidome data are highly complex given the expression of multiple HLA alleles on the cell membrane and presence of co-immunoprecipitated contaminants. The absence of tools that accurately deal with these challenges is currently a major bottleneck for the large-scale application of this technique. Here, we present the MHCMotifDecon that benefits from state-of-the-art HLA class-I and class-II predictions to accurately deconvolute immunopeptidome datasets and assign individual ligands to the most likely HLA allele while discarding co-purified contaminants. We have benchmarked the tool against other state-of-the-art methods and illustrated its application on experimental datasets for HLA-DR demonstrating a previously underappreciated role for HLA-DRB3/4/5 molecules in defining HLA class II immune repertoires. With its ease of use MHCMotifDecon can efficiently guide interpretation of immunopeptidome datasets, serving the discovery of novel T cell targets.

scholarly journals The Impact of Hypomethylating Agents and BCL-2 Inhibitor on HLA Expression on THP-1 Cells

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 3990-3990
Author(s):  
Benjamin Peton ◽  
Melissa Valerio ◽  
Michiko Taniguchi ◽  
Ivan Rodriguez ◽  
Ebtsesam Nafie ◽  
...  
Keyword(s):  
Immune Escape ◽  
Hla Class I ◽  
Surface Expression ◽  
Class Ii ◽  
Hla Class Ii ◽  
Class I ◽  
Hypomethylating Agents ◽  
Hla Dr ◽  
Hla Dq ◽  
Ifn Γ

Abstract Note: BP, MV and LG, KG contributed equally Background Relapsed acute myeloid leukemia (AML) remains the most common reason for allogeneic hematopoietic cell transplant (HCT) failure. Thus, understanding AML immune escape mechanism is important for improving the odds of curing HCT patients with AML. Downregulation of HLA Class I and II expression by AML is one of the potential immune escape mechanisms. Therefore, treatment to restore HLA surface expression is crucial to prevent and treat relapse. Endogenous cytokines, such as IFN-γ, have been shown to stimulate HLA expression but are poorly tolerated by patients. However, two hypomethylating agents (HMA), decitabine (Dec) and azacitadine (Aza), that are routinely used in AML treatment are known to augment HLA expression. For AML, HMAs are often combined with venetoclax (Ven), a drug that blocks the anti-apoptotic B-cell lymphoma-2 (Bcl-2) protein. Thus, while HMAs have been reported to increase HLA expression, what is unknown is whether these agents impact individual HLA loci differently and whether Ven has any impact on HLA expression. To address these questions, we treated the THP-1 cell line with Dec, Aza or Ven and measured changes in cell-surface expression of HLA proteins by flow cytometry using locus-specific HLA mAbs. Methods THP-1 cells were incubated with IFN-γ (500 U/mL), Aza (2µM), Dec (5µM), or Ven (30nM) for 48 hours (drug concentrations were determined by earlier titration experiments). THP-1 cells are a monocytic cell line, derived from the peripheral blood of a childhood case of acute monocytic leukemia (M5 subtype), that express HLA Class I and HLA-DR but not HLA-DQ or -DP under basal conditions, although they are inducible by IFN-γ. Thus, the induction of HLA Class II expression by IFN-γ serves as a positive control. Isotype controls were included to measure background. Data is presented as the difference in MFI (delta MFI) between cells treated with a drug and those treated with diluent only. Results Treatment of THP-1 cells with either IFN-γ or Dec led to increases in Class I HLA-A, -B & -C (Figure 1) compared to untreated cells (a mean fold increase of 1.4 and 1.2, respectively). Notably, Aza did not stimulate additional HLA-C expression and induced less of an increase in HLA-A & -B expression (an increase of 1.1-fold) than IFN-γ or Dec. Treatment of THP-1 cells by Ven did not induce a change in HLA Class I expression. For Class II, IFN-γ or Dec increased HLA-DR, -DQ and -DP expression in comparison to untreated cells (Figure 1). IFN-γ induced greater HLA-DR expression compared to Dec (an increase of 2.3-fold and 1.5-fold, respectively), and both stimulated similar increases in HLA-DQ (increases of 1.5-fold and 1.4-fold, respectively) & -DP (increases of 1.9-fold and 1.5-fold, respectively). However, treatment of cells with either Aza or Ven did not lead to changes in HLA Class II expression. Discussion Previous studies have illustrated the ability of IFN-γ to induce HLA Class II expression in THP-1 cells, however, data for Dec to induce HLA Class II expression was unconfirmed. We report differences in the degree to which IFN-γ and Dec are capable of stimulating HLA-DR with IFN-γ being more potent. The inability of Aza to induce HLA Class II expression in THP-1 cells may be related to the differing drug activating pathways of the two HMAs. Indeed, there are conflicting reports as to whether Aza can stimulate HLA Class II expression. Though Ven treatment of THP-1 cells did not impact HLA expression, because it is given with HMAs, it remains to be seen what effect these drugs may have on HLA expression when administered together. Additional studies to confirm these observations in patient-derived AML blasts are ongoing. Conclusion We report that HMAs increased expression of HLA-A, -B, & -C loci and Dec but not Aza stimulated HLA-DR, -DQ, and -DP expression in THP-1 cells. Given these data, Dec may be superior in increasing HLA Class II expression post-HCT. Figure 1 Figure 1. Disclosures Marcucci: Abbvie: Other: Speaker and advisory scientific board meetings; Agios: Other: Speaker and advisory scientific board meetings; Novartis: Other: Speaker and advisory scientific board meetings. Al Malki: Neximmune: Consultancy; CareDx: Consultancy; Jazz Pharmaceuticals, Inc.: Consultancy; Rigel Pharma: Consultancy; Hansa Biopharma: Consultancy.

scholarly journals Class II HLA Epitope Level Mismatch Can Predict Chronic Graft-Versus-Host Disease and Survival Following Haploidentical Transplant Using Post-Transplant Cyclophosphamide

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 311-311
Author(s):  
Scott R Solomon ◽  
Michael T Aubrey ◽  
Cheri Anobile ◽  
Xu Zhang ◽  
Brian M Freed ◽  
...  
Keyword(s):  
Graft Versus Host Disease ◽  
Chronic Gvhd ◽  
Hla Class I ◽  
Class Ii ◽  
Class I ◽  
Transplant Outcome ◽  
Graft Versus Host ◽  
Hla Dr ◽  
Hla Dq ◽  
The Impact

Abstract Post-transplant cyclophosphamide (PTCy) has improved the outcomes and expanded the use of haploidentical hematopoietic cell transplantation (haplo-HCT). Unlike many other allogeneic HCT settings, the impact of HLA disparity on graft-versus-host disease (GVHD) and transplant outcome in this setting remains unclear. HLAMatchmaker is a computer algorithm that assesses HLA compatibility at the structural level by determining what and how many functional epitopes (eplets), defined as patches of polymorphic residues within a radius of 3.0-3.5 Ångstroms, are shared between donor and recipient. It has been useful in the identification of acceptable mismatches (mm) for alloimmunized kidney transplant candidates. In order to determine the effects of HLA class I (HLA-A, B, C) and II (HLA-DR, DQ, DP) epitope mm on transplant outcome, we retrospectively analyzed 208 consecutive donor-recipient pairs receiving haplo-HCT with PTCy for hematologic malignancy. The impact of epitope mm (GVH direction) on GVHD and survival endpoints was evaluated by Cox multivariate analysis (MVA), controlling for other significant patient, donor and transplant-related factors. Median (range) recipient and donor age was 52 (19-75) and 38 (15-73) years respectively. Patients were transplanted for AML (34%), MDS/MPS/CML (20%), ALL (17%), NHL/HD/CLL (25%). PBSC was used as the stem cell source in 66% of patients, and conditioning intensity was myeloablative in 41%. The donor was a child, sibling, or parent in 47%, 38%, and 14% respectively. Median (range) follow-up for surviving patients was 33 (7-130) months. HLA class I epitope mm had no effect on GVHD or survival. In contrast, increased HLA class II epitope mm (>16) was significantly correlated to an increased frequency of chronic GVHD (figure 1). In MVA, higher degree of class II epitope mm was associated with chronic GVHD, total (HR 1.91, p=0.012) and moderate-to-severe (HR 2.37, p=0.006). The positive effect of increased class II epitope mm on chronic GVHD was driven mostly by HLA-DQ epitope mm (HR 1.7 for >7 vs. ≤7, p=0.047) with a non-significant contribution from HLA-DP (HR 1.36 for >2 vs. ≤2, p=0.24). In contrast, increased HLA-DR epitope mm had a protective effect on chronic GVHD (HR 0.52 for >7 vs. ≤7, p=0.021). Epitope mm was not significantly associated with acute GVHD, grade 2-4 or 3-4. There was also no effect of allele-level mm at any HLA loci on acute or chronic GVHD. We next tested the impact of class I and II epitope mm on survival, including the individual impact of HLA-DR, -DQ and -DP epitope mm. Although class I epitope mm had no impact in univariate analysis, a higher number of class II epitope mm (>16) was correlated with better overall survival and the effect was primarily driven by HLA-DQ epitope mm (figure 2). To better assess the impact of class II epitope mm on survival, we analyzed this variable in the context of a previously published MVA (Solomon et al. Biol Blood Marrow Transplant. 2018;24:789-798). Controlling for other significant variables (age, race, CMV status, donor relationship, HLA-DR mm, nonpermissive HLA-DP mm, KIR receptor-ligand mm and KIR haplotype), only increased HLA-DQ epitope mm (>7) was independently associated with decreased non-relapse (HR 0.34, p=0.021) and overall mortality (HR 0.60, p=0.039). These results indicate a significant effect of class II epitope mm on chronic GVHD and survival following haplo-HCT with PTCy. Higher level of class II epitope mm and HLA-DQ epitope mm is associated with increased chronic GVHD incidence, whereas HLA-DR epitope mm is protective. Higher HLA-DQ epitope mm is independently associated with better survival, when controlling for the presence of HLA-DR allele-level mm or a nonpermissive HLA-DP mm, which have been shown previously to improve survival. Class II HLA epitope level matching provides important prognostic information in the setting of haplo-HCT and PTCy, which is not reflected by conventional allele-level matching. Disclosures Solh: Amgen: Speakers Bureau; Celgene: Speakers Bureau; ADC Therapeutics: Research Funding.

scholarly journals DEMO - A Simulation Framework for Multi-modal Commuting and Parking Optimization.

10.29007/94j5 ◽  
2019 ◽  
Author(s):  
Lara Codeca ◽  
Jérôme Härri ◽  
Jakob Erdmann
Keyword(s):  
Traffic Congestion ◽  
Large Scale ◽  
State Of The Art ◽  
Smart Cities ◽  
Ease Of Use ◽  
Sustainable Growth ◽  
Simulation Framework ◽  
Smart Mobility

In the last decade, many efforts to solve traffic congestion and sustainable growth issues are going in the direction of research and investments in smart cities and consequently smart mobility.We use the proposed simulation framework is compatible with SUMO 1.1.0. We use it to study multi-modal commuting and parking optimization issues in a state-of-the-art large-scale mobility scenario, and we intend to demonstrate the ease of use and its capabilities.

scholarly journals Immunoinformatic identification of B cell and T cell epitopes in the SARS-CoV-2 proteome

2020 ◽  
Author(s):  
Stephen N. Crooke ◽  
Inna G. Ovsyannikova ◽  
Richard B. Kennedy ◽  
Gregory A. Poland
Keyword(s):  
T Cell ◽  
B Cell ◽  
Selection Criteria ◽  
Vaccine Development ◽  
Hla Class I ◽  
Class Ii ◽  
Class I ◽  
T Cell Epitopes ◽  
B Cell Epitopes ◽  
Novel Coronavirus

AbstractA novel coronavirus (SARS-CoV-2) emerged from China in late 2019 and rapidly spread across the globe, infecting millions of people and generating societal disruption on a level not seen since the 1918 influenza pandemic. A safe and effective vaccine is desperately needed to prevent the continued spread of SARS-CoV-2; yet, rational vaccine design efforts are currently hampered by the lack of knowledge regarding viral epitopes targeted during an immune response, and the need for more in-depth knowledge on betacoronavirus immunology. To that end, we developed a computational workflow using a series of open-source algorithms and webtools to analyze the proteome of SARS-CoV-2 and identify putative T cell and B cell epitopes. Using increasingly stringent selection criteria to select peptides with significant HLA promiscuity and predicted antigenicity, we identified 41 potential T cell epitopes (5 HLA class I, 36 HLA class II) and 6 potential B cell epitopes, respectively. Docking analysis and binding predictions demonstrated enrichment for peptide binding to HLA-B (class I) and HLA-DRB1 (class II) molecules. Overlays of predicted B cell epitopes with the structure of the viral spike (S) glycoprotein revealed that 4 of 6 epitopes were located in the receptor-binding domain of the S protein. To our knowledge, this is the first study to comprehensively analyze all 10 (structural, non-structural and accessory) proteins from SARS-CoV-2 using predictive algorithms to identify potential targets for vaccine development.Significance StatementThe novel coronavirus SARS-CoV-2 recently emerged from China, rapidly spreading and ushering in a global pandemic. Despite intensive research efforts, our knowledge of SARS-CoV-2 immunology and the proteins targeted by the immune response remains relatively limited, making it difficult to rationally design candidate vaccines. We employed a suite of bioinformatic tools, computational algorithms, and structural modeling to comprehensively analyze the entire SARS-CoV-2 proteome for potential T cell and B cell epitopes. Utilizing a set of stringent selection criteria to filter peptide epitopes, we identified 41 T cell epitopes (5 HLA class I, 36 HLA class II) and 6 B cell epitopes that could serve as promising targets for peptide-based vaccine development against this emerging global pathogen.

Purified CD4 T Lymphocyte Infusion Can Result in Graft-Versus-Leukemia Reactivity without Gvhd by Recognition of Broadly Expressed Minor Histocompatibility Antigens in HLA Class-II

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 4116-4116
Author(s):  
P. van Balen ◽  
C.A.M. van Bergen ◽  
I. Jedema ◽  
S.A.P. van Luxemburg-Heijs ◽  
J.C. Harskamp ◽  
...  
Keyword(s):  
Immune Response ◽  
T Lymphocytes ◽  
T Lymphocyte ◽  
Hematopoietic Cells ◽  
Hla Class I ◽  
Class Ii ◽  
Hla Class Ii ◽  
Inflammatory Conditions ◽  
Cd8 T Lymphocytes ◽  
Hla Dr

Abstract Abstract 4116 Donor lymphocyte infusion (DLI) after allogeneic stem cell transplantation (alloSCT) can mediate curative Graft-versus-Leukemia (GVL) reactivity although frequently at the cost of Graft-versus-Host Disease (GVHD). We previously illustrated that donor CD8 T lymphocytes recognizing HLA class-I restricted minor histocompatibility antigens (MiHAs) that are broadly expressed on tissues of the recipient cause GVL associated with GVHD, whereas T lymphocytes recognizing MiHAs selectively expressed on hematopoietic cells, including the malignant cells, can selectively mediate GVL without GVHD. Since in contrast to HLA class-I, expression of HLA class-II molecules is predominantly restricted to hematopoietic cells, we hypothesized that infused purified donor CD4 T lymphocytes may selectively recognize and eliminate hematopoietic cells from the recipient resulting in GVL without GVHD. We treated a patient with CML in blastic phase in remission after intensive chemotherapy with T cell depleted alloSCT from his HLA-identical sibling donor after myelo-ablative conditioning. After donor engraftment, recipient hematopoiesis reoccurred within 3 months to 90% of CD8 T lymphocytes, 13% of CD4 T lymphocytes and 5% of myelopoiesis. As part of a clinical trial, the patient was treated with 106/kg positively selected purified donor derived CD4 T lymphocytes resulting within 19 weeks in conversion into full donor chimerism in all hematopoietic cell lineages in the total absence of GVHD. To characterize the nature of this hematopoiesis restricted immune response, in vivo activated HLA-DR positive CD4 and CD8 T lymphocytes were clonally isolated by flowcytometric cell sorting at the time of the clinical response, expanded and tested for alloreactivity on patient and donor derived hematopoietic target cells using IFNγ ELISA. From the 204 expanding CD4 T lymphocyte clones 31 clones were alloreactive, whereas none of the 66 expanding CD8 T lymphocyte clones showed alloreactivity. To further identify the fine specificity of this hematopoiesis directed HLA class-II restricted immune response, target molecules of several T lymphocyte clones were molecularly characterized using whole genome association scanning. We first performed blocking studies with HLA class-II restricted monoclonal antibodies and identified HLA-DR to be the restriction molecule. Next, a large panel of third party EBV-LCLs was retrovirally transduced with each of the possible restriction molecules being HLA-DRB1*11:01, HLA-DRB1*15:01, HLA-DRB3*02:02 and HLA-DRB5*01:01. By comparing the recognition pattern of the transduced EBV-LCLs with the 1.1 million single nucleotide polymorphisms in each EBV-LCL, we identified 3 novel MiHAs. Synthesis and analysis of the patient and donor derived allelic peptide variants further confirmed the specificity of the MiHAs as LB-KHNYN-1K in the context of HLA-DRB5*01:01, LB-CTSB-1G in HLA-DRB1*11:01 and LB-ZDHHC13-1K in HLA-DRB1*15:01. Gene expression profiles of KHNYN (located on chromosome 14), CTSB (chromosome 8) and ZDHHC13 (chromosome 11) illustrated that the genes encoding these MiHAs were not only transcribed in hematopoietic cells, but also in other tissues including GVHD target tissues. These results further illustrated that the hematopoietic specificity of the CD4 T lymphocyte response was mainly defined by the restricted expression of the HLA-DR molecules on hematopoietic cells. We conclude that purified CD4 DLI can lead to GVL without GVHD by a selective HLA class-II restricted immune response against patient hematopoiesis. By molecular characterization of 3 novel HLA-DR restricted MiHAs we illustrated that the relative specificity of HLA class-II molecules on hematopoietic cells under non inflammatory conditions was probably responsible for this effect. Since HLA class-II is predominantly expressed on hematopoietic cells only, infusion of donor CD4 T lymphocytes under non inflammatory conditions after HLA identical alloSCT can result in efficient induction of GVL without the toxicity of GVHD. Disclosures: No relevant conflicts of interest to declare.

Extensive genetic alterations of the HLA region, including homozygous deletions of HLA class II genes in B-cell lymphomas arising in immune-privileged sites

Blood ◽  
2000 ◽  
Vol 96 (10) ◽  
pp. 3569-3577 ◽  
Author(s):  
Sietske A. Riemersma ◽  
Ekaterina S. Jordanova ◽  
Roelandt F. J. Schop ◽  
Katja Philippo ◽  
Leendert H. J. Looijenga ◽  
...  
Keyword(s):  
B Cell ◽  
Loss Of Heterozygosity ◽  
B Cell Lymphoma ◽  
Hla Class I ◽  
Class Ii ◽  
Genetic Alterations ◽  
Class I ◽  
B Cell Lymphomas ◽  
Hla Dr ◽  
Homozygous Deletions

In B-cell lymphomas, loss of human leukocyte antigen (HLA) class I and II molecules might contribute to immune escape from CD8+ and CD4+ cytotoxic T cells, especially because B cells can present their own idiotype. Loss of HLA expression and the possible underlying genomic alterations were studied in 28 testicular, 11 central nervous system, and 21 nodal diffuse large B-cell lymphomas (DLCLs), the first two sites are considered as immune-privileged sites. The analysis included immunohistochemistry, loss of heterozygosity analysis, and fluorescent in situ hybridization (FISH) on interphase cells and isolated DNA fibers. Total loss of HLA-A expression was found in 60% of the extranodal cases and in 10% of the nodal cases (P < .01), whereas loss of HLA-DR expression was found in 56% and 5%, respectively (P < .01). This was accompanied by extensive loss of heterozygosity within the HLA region in the extranodal DLCLs. In 3 cases, retention of heterozygosity for D6S1666 in the class II region suggested a homozygous deletion. This finding was confirmed by interphase FISH that showed homozygous deletions in the class II genes in 11 of the 18 extranodal lymphomas but in none of the 7 nodal DLCLs (P < .001). Mapping by fiber FISH showed variable deletions that always included HLA-DQ and HLA-DR genes. Hemizygous deletions and mitotic recombinations often involving all HLA genes were found in 13 of 18 extranodal and 2 of 7 nodal lymphomas. In conclusion, a structural loss of HLA class I and II expression might help the B-cell lymphoma cells to escape from immune attack.

scholarly journals Coverage of Related Pathogenic Species by Multivalent and Cross-Protective Vaccine Design: Arenaviruses as a Model System

2010 ◽  
Vol 74 (2) ◽  
pp. 157-170 ◽  
Author(s):  
Jason Botten ◽  
John Sidney ◽  
Bianca R. Mothé ◽  
Bjoern Peters ◽  
Alessandro Sette ◽  
...  
Keyword(s):  
Large Scale ◽  
Vaccine Development ◽  
Vaccine Design ◽  
Hemorrhagic Fever ◽  
Lymphocytic Choriomeningitis ◽  
Model System ◽  
T Cell Epitopes ◽  
Fever Syndromes ◽  
Lymphocytic Choriomeningitis Virus Infection ◽  
Epitope Discovery

SUMMARY The arenaviruses are a family of negative-sense RNA viruses that cause severe human disease ranging from aseptic meningitis to hemorrhagic fever syndromes. There are currently no FDA-approved vaccines for the prevention of arenavirus disease, and therapeutic treatment is limited to the use of ribavirin and/or immune plasma for a subset of the pathogenic arenaviruses. The considerable genetic variability observed among the seven arenaviruses that are pathogenic for humans illustrates one of the major challenges for vaccine development today, namely, to overcome pathogen heterogeneity. Over the past 5 years, our group has tested several strategies to overcome pathogen heterogeneity, utilizing the pathogenic arenaviruses as a model system. Because T cells play a prominent role in protective immunity following arenavirus infection, we specifically focused on the development of human vaccines that would induce multivalent and cross-protective cell-mediated immune responses. To facilitate our vaccine development and testing, we conducted large-scale major histocompatibility complex (MHC) class I and class II epitope discovery on murine, nonhuman primate, and human backgrounds for each of the pathogenic arenaviruses, including the identification of protective HLA-restricted epitopes. Finally, using the murine model of lymphocytic choriomeningitis virus infection, we studied the phenotypic characteristics associated with immunodominant and protective T cell epitopes. This review summarizes the findings from our studies and discusses their application to future vaccine design.

GVHD in HLA-A2 Mismatched Transplantation Caused by a Combined CD8 Response Directed Against HLA-A2 and a CD4 Response Recognizing an HLA-A2 Derived Peptide in HLA-DR1.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 5164-5164
Author(s):  
Avital Amir ◽  
Menno A.W.G. van der Hoorn ◽  
Erik W.A. Marijt ◽  
Michel G.D. Kester ◽  
Roelof Willemze ◽  
...  
Keyword(s):  
Immune Response ◽  
T Cells ◽  
Variable Region ◽  
Hla Class I ◽  
Class Ii ◽  
Hla Class Ii ◽  
Mixed Chimerism ◽  
Target Cells ◽  
Activated T Cells ◽  
Hla Dr

Abstract HLA mismatched Stem Cell Transplantation (SCT) can be performed in patients with leukemia if no HLA identical donor can be found. Although HLA class I is expressed by almost all recipient cells and the frequency of allo-HLA reactive T-cells is high in all normal donors, the risk of GVHD after single locus mismatched SCT is comparable to that after HLA identical SCT. Thus, the occurrence of GVHD may not simply be explained by recognition of the mismatched HLA by allo-HLA reactive T-cells. Therefore, we characterized in detail the nature of the allo-immune response in an HLA mismatched setting. A patient with acute myeloid leukemia was treated with T-cell depleted SCT from a sibling donor who was HLA identical except for an HLA-A2 crossover. 6 Months after SCT, Donor Lymphocyte Infusion (DLI) of 2.5*10e6 CD3+ T-cells/kg was given for mixed chimerism caused by persistence of patient T-cells. No clinical response and no GVHD developed. 12 months after SCT the leukemia relapsed with 9% blasts in bone marrow, and a second DLI of 7.5*10e6 CD3+ T-cells/kg was given. The patient died of grade IV GVHD 5 weeks after DLI. durign the GVHD flow cytometry of PBMC’s showed conversion of patient to donor type T-cells. 80% Of the CD8 and 40% of the CD4 T-cells were activated, as determined by co-expression of HLA-DR. These activated T-cells were single cell sorted, non-specifically expanded, and tested for alloreactivity using cytotoxicity and cytokine production assays. 46 Out of 56 isolated CD8 clones and 7 out of 88 CD4 clones recognized patient but not donor target cells, indicating that at the time of the GVHD almost 70% of circulating T-cells were alloreactive. The response was highly polyclonal as shown by usage of at least 13 different TCR Vβs by the CD8 clones, and 6 by the CD4 clones. HLA restriction of the clones was tested with HLA blocking antibodies, a panel of HLA-typed target cells and donor EBV-LCL transduced with HLA-A2. All alloreactive CD8 clones were HLA-A2 specific. To further characterize the specificity, CD8 clones were tested against T2 cells loaded with HPLC fractions of peptides eluted from HLA-A2. Some CD8 clones recognized HLA-A2 with all different HPLC fractions, indicating peptide-independent recognition. Other clones recognized one fraction indicating peptide specificity, or several fractions indicating “promiscuous” peptide recognition. The CD4 clones were HLA-DR1 restricted and recognized donor EBV-LCL transduced with HLA-A2, indicating that the peptide recognized in HLA-DR1 was derived from the mismatched HLA-A2 molecule. Therefore, CD4 clones were tested against different peptides covering the whole HLA-A2 sequence. All clones recognized epitope 101–122 derived from a hyper variable region of HLA-A2. These results indicate that the GVHD in this HLA-A2 mismatched transplantation was caused by a combined highly polyclonal CD8 response directed against the HLA-A2 molecule and a CD4 response recognizing an HLA-A2 derived peptide presented by HLA class II. We speculate that the absence of an immune response observed after the first DLI despite high frequency of allo-HLA reactive T-cells, indicates that CD8 anti-HLA-A2 T-cells are insufficient to cause severe GVHD. We hypothesize that the rise of HLA-DR expressing leukemic blasts presenting HLA-A2 derived peptide in HLA class II triggered the CD4 response which was necessary to initiate the CD8 alloresponse resulting in this clinical outcome.

Extensive genetic alterations of the HLA region, including homozygous deletions of HLA class II genes in B-cell lymphomas arising in immune-privileged sites

Blood ◽  
2000 ◽  
Vol 96 (10) ◽  
pp. 3569-3577 ◽  
Author(s):  
Sietske A. Riemersma ◽  
Ekaterina S. Jordanova ◽  
Roelandt F. J. Schop ◽  
Katja Philippo ◽  
Leendert H. J. Looijenga ◽  
...  
Keyword(s):  
B Cell ◽  
Loss Of Heterozygosity ◽  
B Cell Lymphoma ◽  
Hla Class I ◽  
Class Ii ◽  
Genetic Alterations ◽  
Class I ◽  
B Cell Lymphomas ◽  
Hla Dr ◽  
Homozygous Deletions

Abstract In B-cell lymphomas, loss of human leukocyte antigen (HLA) class I and II molecules might contribute to immune escape from CD8+ and CD4+ cytotoxic T cells, especially because B cells can present their own idiotype. Loss of HLA expression and the possible underlying genomic alterations were studied in 28 testicular, 11 central nervous system, and 21 nodal diffuse large B-cell lymphomas (DLCLs), the first two sites are considered as immune-privileged sites. The analysis included immunohistochemistry, loss of heterozygosity analysis, and fluorescent in situ hybridization (FISH) on interphase cells and isolated DNA fibers. Total loss of HLA-A expression was found in 60% of the extranodal cases and in 10% of the nodal cases (P &lt; .01), whereas loss of HLA-DR expression was found in 56% and 5%, respectively (P &lt; .01). This was accompanied by extensive loss of heterozygosity within the HLA region in the extranodal DLCLs. In 3 cases, retention of heterozygosity for D6S1666 in the class II region suggested a homozygous deletion. This finding was confirmed by interphase FISH that showed homozygous deletions in the class II genes in 11 of the 18 extranodal lymphomas but in none of the 7 nodal DLCLs (P &lt; .001). Mapping by fiber FISH showed variable deletions that always included HLA-DQ and HLA-DR genes. Hemizygous deletions and mitotic recombinations often involving all HLA genes were found in 13 of 18 extranodal and 2 of 7 nodal lymphomas. In conclusion, a structural loss of HLA class I and II expression might help the B-cell lymphoma cells to escape from immune attack.

scholarly journals Characterization of cells emerging at the time of graft failure after bone marrow transplantation from an unrelated marrow donor

Blood ◽  
1993 ◽  
Vol 82 (3) ◽  
pp. 1023-1029 ◽  
Author(s):  
J Donohue ◽  
M Homge ◽  
NA Kernan
Keyword(s):  
T Cell ◽  
Mononuclear Cells ◽  
Hla Class I ◽  
Class Ii ◽  
Hla Class Ii ◽  
Class I ◽  
Unrelated Donor ◽  
Peripheral Blood Mononuclear ◽  
Hla Dr

Abstract To help elucidate the mechanism responsible for graft failure (GF) following a T-cell depleted bone marrow transplant (BMT) from an unrelated donor, five patients (2 chronic myelogenous leukemia, 1 acute undifferentiated leukemia, 2 myelodysplastic syndrome) who experienced this complication were studied. All patients were HLA class I identical with their donors as determined by serology and one-dimensional isoelectric focusing (IEF); two were serologically matched with their donors for HLA class II antigens, whereas three donor-recipient pairs were serologically mismatched for one HLA-DR antigen. All patients received total body irradiation (fractionated, 1,500 rads), VP-16 (750 mg/m2), and cyclophosphamide (120 mg/kg) pre-BMT and antithymocyte globulin (15 mg/kg every other day) and methylprednisolone (2 mg/kg) post-BMT. Three patients experienced primary nonengraftment and two experienced secondary GF. Peripheral blood mononuclear cells obtained from the patients at the time of GF were studied to examine their functional and phenotypic characteristics. Emerging cells were of host origin and were found to be specifically cytotoxic to donor target cells and suppressive to the in vitro growth of donor BM, especially in the cases of primary nonengraftment. Peripheral blood mononuclear cells from these patients were expanded to form T-cell lines (TcLs). The cytotoxic activities of TcLs were tested in the presence of blocking MoAbs directed against various HLA determinants in an attempt to determine if HLA antigens expressed on donor cells were the target for cytotoxicity. The observed cytotoxic activity was blocked by antibodies to HLA-B, -C (1 patient), HLA-DR (1 patient), and HLA-DQ (1 patient). In two cases, antidonor cytotoxicity could not be blocked by MoAb directed against HLA-A, -B, -C, or -DR. Phenotypic characterization of four successfully maintained TcLs showed 100% CD3+ cells with 100% CD4+ (3 patients) or 50% CD4+/50% CD8+ (1 patient). In two of the three patients with 100% CD4+ cells, antidonor cytotoxicity was blocked by an anti-HLA class II MoAb. In contrast to our previous findings in cases of GF following T-cell-depleted HLA nonidentical family member BMT in which host T cells were CD8+ and cytotoxicity was directed against HLA class I antigens, our present study indicates host T cells emerging at the time of GF following BMT from an HLA class I IEF-identical unrelated donor can be of the CD4+ subset and seem to be capable of recognizing antigenic disparities in the HLA class II region.

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