P012 An HLA Typing for every JIA?

Abstract Background Among the genetic susceptibility factors of Juvenile Idiopathic Arthritis (JIA), the HLA class I and II genes are the most frequently involved. These HLA genes are extensively investigated, highlighting the role of self/non-self-balance in auto-immune and auto-inflammatory diseases. Methods We carried out a retrospective study of HLA class I and/or class II genes (HLA B and HLA DR genes) of 12 children. Genotyping was performed through microlymphocytotoxicity for class I (HLA B5 and B27) and through indirect immunofluorescence and PCR for class II (HLA DR4) Results The summarized results depict: 4 patients have the HLA B27 + 3 patients have the HLA B5+ 1 patient had the HLA DR JIA comprises a broad spectrum influenced by both genetic and environmental factors. The International League of Rheumatology Associations (ILAR) has defined seven categories of JIA consisting of a myriad of pediatric auto-immune and auto-inflammatory diseases. The HLA genomic region is also associated with a wide range of auto-immune diseases, encoding the HLA-DR, HLA-DQ, and HLA-DP proteins involved in the peptide’s presentations to HLA -class II-restricted CD4 + helper T cells. These mechanisms are involved in the pathogenesis of various diseases such as rheumatoid arthritis. Juvenile arthritis has also been associated with a single nucleotide polymorphism in the regulatory region of the interleukin-6 gene, close to the HLA coding region. Conclusion Genetic exploration of the HLA system in JIA, more accessible and less expensive than other targeted genetic typing, can be a helpful tool. Its usage ought to be encouraged and expanded in clinical practice.

Download Full-text

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

Blood ◽

10.1182/blood-2021-145801 ◽

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.

Download Full-text

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

Blood ◽

10.1182/blood-2018-99-114122 ◽

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.

Download Full-text

Evidence for HLA-related susceptibility for stroke in children with sickle cell disease

Blood ◽

10.1182/blood.v95.11.3562 ◽

2000 ◽

Vol 95 (11) ◽

pp. 3562-3567 ◽

Cited By ~ 47

Author(s):

Lori A. Styles ◽

Carolyn Hoppe ◽

William Klitz ◽

Elliott Vichinsky ◽

Bertram Lubin ◽

...

Keyword(s):

Linkage Disequilibrium ◽

Cerebral Infarction ◽

Sickle Cell ◽

Hla Class I ◽

Class Ii ◽

Hla Typing ◽

Class I ◽

Increased Risk ◽

Mri Scans ◽

Comparison Of The Results

Abstract Cerebral infarction occurs in one quarter of all children with sickle cell anemia (SCA). There is an increased risk of stroke in siblings with SCA, suggesting genetic factors may influence risk of stroke. The authors investigated whether HLA type was associated with risk of stroke in children with SCA. Fifty-three patients with SCA underwent complete HLA typing at both HLA class I (HLA-A, B) and HLA class II (HLA-DR, DQ, DP) loci. Of the 53 patients, 22 had magnetic resonance imagining (MRI)–documented evidence of cerebral infarction, and the remaining 31 patients had negative MRI scans. Comparison of the results of HLA typing between the SCA patients with a positive and those with a negative MRI documented that the 2 groups differed with respect to the class I HLA-B (P = .012), and the class II HLA-DRB1 (P = .0008) and DQB1 (P = .029). Susceptibility associations at the HLA-DRB1 locus included both DR3 alleles, where DRB1*0301 and *0302 were both associated with an increased risk of stroke. Protective associations were found in the DR2 group, where DRB1*1501 was protective for stroke. DQB1*0201, which is in linkage disequilibrium with DRB1*0301, was also associated with stroke. Similarly, DQB1*0602, in linkage disequilibrium with DRB1*1501, was protective. Specific HLA alleles may influence the risk of stroke in children with SCA. HLA typing may prove useful in identifying SCA patients at higher risk for stroke.

Download Full-text

Product Attributes and Donor Factors in Reported Cases of Transfusion-Associated Graft Versus Host Disease: A Systematic Review

Blood ◽

10.1182/blood.v124.21.2883.2883 ◽

2014 ◽

Vol 124 (21) ◽

pp. 2883-2883

Author(s):

Ilana Kopolovic ◽

Jackie Ostro ◽

Christine Cserti ◽

Walter Sunny Dzik ◽

Hidacki Tsubota ◽

...

Keyword(s):

Storage Time ◽

Hla Antigens ◽

Hla Class I ◽

Class Ii ◽

Hla Typing ◽

Class I ◽

Blood Component ◽

Blood Components ◽

Donor Factors ◽

Related Donor

Abstract INTRODUCTION: Transfusion-associated graft-vs-host disease (TA-GVHD) is a rare and often fatal complication of transfusion of cellular blood products. The relative contributions of product and donor factors to the risk of TA-GVHD remain uncertain. METHODS: Systematic review of all reported cases of TA-GVHD in the published literature prior to Oct 2013, without language restrictions. Cases attributed to granulocyte transfusions, passenger lymphocyte syndrome, or GVHD following stem-cell transplant (unless traced to blood components rather than the graft) were excluded. Data collected included patient demographics and health information, details of transfusion event(s) and blood component(s), clinicolaboratory features of the TA-GVHD presentation and outcome, and results of human leukocyte antigen (HLA) and chimerism studies. HLA typing was evaluated where reported for both donor (product) and recipient at either class I or class II loci. Donor/recipient pairs were categorized as D=0 when there were no identified donor HLA antigens foreign to the recipient, or D>0 when donor cells contained one or more HLA antigens not found in the recipient. This classification applied separately to HLA class I and class II loci for each case. RESULTS: After removing duplicates, 2130 citations discovered by the search were examined by two independent reviewers, with 394 identified as publications of interest for complete review. An additional 21 publications were found from the initial review, for a total of 415 publications. Of these, 195 publications described 348 unique cases for inclusion. Component: The component implicated in TA-GVHD was identified in 248 (71%) cases: Red cells (RBC) in 132 (38%); whole blood (WB) in 92 (26%); platelets in 20 (6%); buffy- coat product in 2 (0.6%); and plasma and plasma-reduced blood in one case each. In 100 (29%) cases, the blood component was either not specified or not identified among several potentially responsible components. Storage: Component storage time was reported in 158 (45%) cases. Of these, the implicated product was either described as “fresh” or </=10 days old in 148 (94%). 10 (6%) cases reported a storage time >10 days (maximum 14 days). Related donor: In 63 cases, the donor was either related (n=61) or deliberately HLA-matched (n=2) to the recipient, while in 113 cases the donor was unrelated. The remaining cases either reported a “possible” related donor or did not report the donor-recipient relationship. Leukoreduction/Irradiation: Leukoreduction status was reported in 135 (39%) cases. Of these, the implicated product was leukoreduced in 23 (17%) (10 bedside, 2 pre-storage, 11 not specified). The product was irradiated in 9 cases. HLA: HLA typing of recipient and donor, by serological or molecular techniques, was available for 84 cases (74 cases Class I, 62 Class II). Among patients with HLA data available, 20 (24%) had an underlying diagnosis warranting irradiation by current standards, while 64 (76%) did not. The category of D=0 was found in 47 (64%) of cases with reported class I typing; 44 (71%) of cases with reported class II typing; and 60 (71%) overall (Figure 1). There were 9 cases in which the category of D=0 could be ruled out for both HLA I and II. In the remaining 15 cases, the category of D=0 at either HLA I or II could not be definitively ruled in or out based on reported data. When considering those in whom the presence or absence of D=0 could be definitely determined, while D=0 at either HLA class I or class II was present in 55 of 57 (96%) of recipients without an indication for blood component irradiation, D=0 was present in only 5 of 12 (42%) of recipients with an indication for irradiation, p< 0.0001 (Table 1). CONCLUSIONS: The most common components implicated in TA-GVHD were WB and RBC. Most units were non-leukoreduced and stored for <10 days. Most cases of TA-GVHD occurred in recipients without a standard indication for irradiation. The absence of a foreign donor antigen at either HLA class I or class II occurred in a large majority of cases and was significantly more common in TA-GVHD among recipients without an indication for irradiation compared with those in whom irradiation would be indicated, suggesting that this donor-recipient relationship is the predominant risk factor in the development of TA-GVHD. Policies for irradiating cellular blood components based solely on the diagnosis of the recipient may fail to address all relevant risk factors for TA-GVHD. Figure 1 Figure 1. Table 1. Table 1. Disclosures No relevant conflicts of interest to declare.

Download Full-text

Evidence for HLA-related susceptibility for stroke in children with sickle cell disease

Blood ◽

10.1182/blood.v95.11.3562.011k49_3562_3567 ◽

2000 ◽

Vol 95 (11) ◽

pp. 3562-3567

Author(s):

Lori A. Styles ◽

Carolyn Hoppe ◽

William Klitz ◽

Elliott Vichinsky ◽

Bertram Lubin ◽

...

Keyword(s):

Linkage Disequilibrium ◽

Cerebral Infarction ◽

Sickle Cell ◽

Hla Class I ◽

Class Ii ◽

Hla Typing ◽

Class I ◽

Increased Risk ◽

Mri Scans ◽

Comparison Of The Results

Cerebral infarction occurs in one quarter of all children with sickle cell anemia (SCA). There is an increased risk of stroke in siblings with SCA, suggesting genetic factors may influence risk of stroke. The authors investigated whether HLA type was associated with risk of stroke in children with SCA. Fifty-three patients with SCA underwent complete HLA typing at both HLA class I (HLA-A, B) and HLA class II (HLA-DR, DQ, DP) loci. Of the 53 patients, 22 had magnetic resonance imagining (MRI)–documented evidence of cerebral infarction, and the remaining 31 patients had negative MRI scans. Comparison of the results of HLA typing between the SCA patients with a positive and those with a negative MRI documented that the 2 groups differed with respect to the class I HLA-B (P = .012), and the class II HLA-DRB1 (P = .0008) and DQB1 (P = .029). Susceptibility associations at the HLA-DRB1 locus included both DR3 alleles, where DRB1*0301 and *0302 were both associated with an increased risk of stroke. Protective associations were found in the DR2 group, where DRB1*1501 was protective for stroke. DQB1*0201, which is in linkage disequilibrium with DRB1*0301, was also associated with stroke. Similarly, DQB1*0602, in linkage disequilibrium with DRB1*1501, was protective. Specific HLA alleles may influence the risk of stroke in children with SCA. HLA typing may prove useful in identifying SCA patients at higher risk for stroke.

Download Full-text

A simplified HLA typing procedure by a panel of HLA class I and class II monoclonal antibodies

Human Immunology ◽

10.1016/0198-8859(93)90056-7 ◽

1993 ◽

Vol 36 (1) ◽

pp. 55

Author(s):

M. Lias ◽

C.-T. Deng ◽

S. Etessami ◽

J. Loon ◽

M. Chen ◽

...

Keyword(s):

Monoclonal Antibodies ◽

Hla Class I ◽

Class Ii ◽

Hla Typing ◽

Class I ◽

Typing Procedure

Download Full-text

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 ◽

10.1182/blood.v96.10.3569.h8003569_3569_3577 ◽

2000 ◽

Vol 96 (10) ◽

pp. 3569-3577 ◽

Cited By ~ 6

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.

Download Full-text

Quantification of Allele-Specific HLA Expression with Nanopore Long-Read Sequencing

Blood ◽

10.1182/blood-2020-140902 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 42-43

Author(s):

Andrew E. O. Hughes ◽

Maureen C. Montgomery ◽

Chang Liu ◽

Eric T. Weimer

Keyword(s):

Significant Variation ◽

Hla Class I ◽

Class Ii ◽

Hla Typing ◽

Class I ◽

Specific Expression ◽

Allele Specific Expression ◽

Hla Genes ◽

Long Read ◽

Allele Specific

Human leukocyte antigen (HLA) typing plays a critical role in evaluating donor-recipient compatibility prior to hematopoietic cell transplantation (HCT) to minimize the risk of rejection and graft versus host disease (GVHD). Compared to traditional sequence-based methods for HLA typing, next-generation sequencing offers significant advantages in terms of accuracy, turnaround time, and cost (Weimer et al., JMD, 2016). Nevertheless, an intrinsic limitation of DNA-based typing is that it does not quantify HLA gene expression, which has been implicated in clinical outcomes (Petersdorf et al., Blood, 2014; Petersdorf et al., NEJM, 2015). Previously, we demonstrated simultaneous HLA class I genotyping and gene-level expression analysis by RNA-seq using nanopore long-read sequencing (Montgomery et al., JMD, 2020). Given that mismatches in both class I and class II HLA genes-as well as the relative expression of individual alleles-impact donor-recipient compatibility, we sought to build on our previous work by quantifying allele-specific expression of both class I and class II HLA loci in donor lymphocytes. For this study, mRNA was isolated from peripheral blood lymphocytes from 12 donors. Barcoded cDNA libraries were prepared and sequenced on MinION flow cells (R9.4.1) using MinKNOW (v3.1.13) to a median depth of 1.6x106reads. Basecalling and demultiplexing were performed with Albacore (v2.3.4) or Guppy (v2.3.1), and adapter trimming was performed with Porechop (v0.2.3). Processed reads were aligned to the international ImMunoGeneTics project (IMGT) HLA database (v3.41.0) using minimap2 (v2.17). Reads mapping to individual HLA loci were realigned to allele-specific references using subject HLA types determined by Athlon (v1.0) or Illumina sequencing. In parallel, library size factors were estimated by aligning reads to GRCh38, counting reads in genes with HTseq (v0.12.4), and using trimmed mean of M-values normalization. As shown in Fig. 1, we observed higher expression of HLA class I genes compared to class II (median 593 vs. 150, p < 0.001, Mann-Whitney U test), a pattern consistent with a mixture of primarily T cells, which express class I genes, as well as B cells, which express both class I and II. Within class I genes, we observed the highest expression of HLA-B, followed by HLA-A, and HLA-C (median 663, 578, and 459, respectively). Within class II, we observed the highest expression of HLA-DPB1, followed by HLA-DRB1, and HLA-DQB1 (median 281, 266, and 104, respectively). Importantly, we observed significant variation in expression both between and within alleles of individual HLA genes, suggesting that HLA type alone does not accurately predict HLA expression. We next analyzed HLA-DPB1 specifically, given reports that the risk of GVHD in HCT recipients with HLA-DPB1mismatched donors is modulated by HLA-DPB1 expression (Petersdorf et al., NEJM, 2015). Of note, HLA-DPB1 expression is linked to a single nucleotide polymorphism, rs9277534, which can be imputed from HLA-DPB1 type (Meurer et al., Front Immunol, 2018). Accordingly, we analyzed HLA-DPB1 expression conditioned on rs9277534 genotype. Although we observed lower HLA-DPB1 expression for the 'A' allele compared to 'G' (median 220 vs. 265), consistent with the reported association, this difference was not statistically significant (p = 0.22, Mann-Whitney U test). Furthermore, we observed significant variation in expression among 'A' alleles, with normalized counts ranging from 57 to 408 (vs. 191 to 367 for 'G' alleles). In this study, we demonstrate the feasibility of quantifying allele-specific expression of both class I and class II HLA genes with nanopore long-read sequencing. Taken together, our results reveal extensive variation in the expression of class I and class II HLA loci, even after accounting for individual allele types and known markers of expression. These results emphasize the potential value of methods, such as nanopore sequencing, for directly quantifying allele-specific HLA expression to develop improved risk prediction models that can inform the evaluation of donor-recipient immunocompatibility. Disclosures No relevant conflicts of interest to declare.

Download Full-text

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 ◽

10.1182/blood.v96.10.3569 ◽

2000 ◽

Vol 96 (10) ◽

pp. 3569-3577 ◽

Cited By ~ 129

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 < .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.

Download Full-text

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

Blood ◽

10.1182/blood.v82.3.1023.1023 ◽

1993 ◽

Vol 82 (3) ◽

pp. 1023-1029 ◽

Cited By ~ 33

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.

Download Full-text