Objective: The extreme polymorphism of the HLA system complicates Hardy-Weinberg Equilibrium (HWE) analyses. Low-resolution typing may create artificial homozygosity by grouping functionally distinct alleles. This study aims to determine how 2-, 4-, and 8-digit HLA typing resolutions systematically alter HWE results and to reveal mechanisms like "hidden heterozygosity". Materials and Methods: High-resolution (8-digit) genotyping for HLA-A, -B, -C, -DRB1, -DQB1, and -DPB1 was performed using Next-Generation Sequencing (NGS) in 150 healthy, unrelated donors from Central Anatolia. The 8-digit data were reduced to 2- and 4-digit levels for comparison. HWE compliance was assessed using the Markov Chain Monte Carlo (MCMC) exact test via PyPop v1.2.1 software. Results: Allele richness (k) increased systematically with resolution (e.g., HLA-B: 23 to 58 alleles), reducing observed homozygosity (e.g., HLA-C: 34 to 14 homozygotes). HWE compliance was highly resolution-sensitive. HLA-B consistently deviated from HWE at all resolutions (p<0.05). Conversely, HLA-C showed deviation only at 2-digit resolution (p<0.0001) but aligned with HWE at 4- and 8-digit levels, indicating a low-resolution artifact. Notably, HLA-DPB1 appeared compatible at lower resolutions but showed significant deviation only at the 8-digit level (p<0.0001), revealing hidden evolutionary pressure. Conclusion: Low-resolution typing causes a "dilution effect," masking allelic diversity and leading to erroneous HWE assessments. The emergence of significant deviations in loci like DPB1 only at high resolution proves that evolutionary forces are obscured by lower-resolution methods. Therefore, 8-digit typing should be the gold standard for accurate population genetics and disease association studies. Keywords: HLA antigens, gene frequency, heterozygote, genetics, population, NGS
Objective: STR-PCR is the gold standard for monitoring chimerism following haematopoietic stem cell transplantation. However, it fails to detect mixed and microchimerism due to a sensitivity limit of 1%, as well as technical issues such as stutter peaks and allelic imbalance. Despite these limitations, this study aims to compare STR-PCR with digital PCR (dPCR), which achieves 0.01% sensitivity using high-specificity markers. Materials and Methods: This study examined chimerism following an allogeneic bone marrow transplant from a TÜRKKÖK donor in a patient diagnosed with acute myeloid leukaemia (AML), using STR-PCR and digital PCR methods. The patient’s chimerism status at 1 and 2 months post-transplant was assessed using both methods with the same samples and under the same conditions. Subsequent follow-ups (on days 99 and 227) were performed using dPCR only. Results: While STR-PCR yielded a 100% donor profile in the first month, dPCR revealed the presence of microchimerism with a donor-to-recipient ratio of 99.04% to 0.96%. In the second month, STR-PCR yielded a donor to recipient ratio of 61% to 39%, while dPCR revealed a ratio of 70.73% to 29.27%. On day 99, the dPCR analysis showed a donor-to-recipient ratio of 11.24% to 88.76% and on day 227, a ratio of 12.58% to 87.42% was observed, which was consistent with clinical relapse. Discussion: This study demonstrates that STR-PCR is a reliable and informative method for monitoring HSCT chimerism. However, it cannot detect microchimerism at levels below 1%. Using the same samples, the dPCR-INDEL method detected microchimerism and, due to its quantitative accuracy, produced more reliable results than STR. Later analyses (Days 100 and 227) performed exclusively with dPCR captured the relapse process with a high recipient contribution. Keywords: Acute Myeloid Leukemia (AML), chimerism analysis, dPCR, STR-PCR
Objective: To evaluate the impact of HLA mismatch level, donor type, donor relationship, sex matching, and age group on mortality among kidney transplant recipients in a single-center cohort. Materials and Methods: A total of 365 kidney transplant cases were analyzed. Recipient and donor HLA-A, -B and -DRB1 loci were typed using sequence-specific oligonucleotide and sequence-specific primer methods. Donors were categorized as nuclear family members, other relatives, spouses, or cadavers. Total HLA mismatch (0–6) was calculated, and mortality outcomes were assessed. Statistical analyses were performed using JASP, with significance set at p<0.05. Results: HLA mismatch increased progressively with decreasing donor relatedness (ρ=0.87). Sibling donors demonstrated the lowest mean mismatch (2.29), while spouse donors had the highest (4.33). Higher mismatch levels showed a strong correlation with mortality (ρ=0.857; R²=0.97), with mortality reaching 10–25% at ≥4 mismatches. Mortality varied by donor type: 7.7% in nuclear family donors, 13.0% in other relatives or spouses, and 17.6% in cadaveric donors. Increased mortality in cadaveric transplantation appeared to reflect both immunologic and non-immunologic factors, including prolonged cold ischemia and higher rejection frequency.Sex matching influenced outcomes, with mortality rising from 9.4% in sex-matched to 13.4% in sex-mismatched pairs. Pediatric recipients had significantly lower mortality compared with adults (5.6% vs 11.6%; p=0.048). Presence of the DRB1*11 allele demonstrated a protective trend, with mortality of 7.4% in positive versus 12.1% in negative cases. Conclusion: Survival after kidney transplantation is shaped by HLA compatibility as well as donor type, sex matching, age, donor relationship, and specific allelic characteristics. Optimizing both immunologic and non-immunologic risk factors—particularly in cadaveric transplantation—remains essential for improving long-term patient outcomes. Keywords: Kidney transplantation, HLA mismatch, donor type, mortality, immunogenetics, DRB1*11
Objective: Our study was conducted to analyze the association between HLA polymorphism and end stage renal disease (ESRD) in patients and to determine the underlying mechanisms of the onset and progression of renal failure. Materials and Methods: In our study we included 120 patients on the kidney transplant waiting list and 100 unrelated donors who underwent HLA typing using the polymerase chain reaction-sequence specific primer (SSP) method at the Tissue Typing Laboratory of Demiroğlu Bilim University Gayrettepe Florence Nightingale Hospital in between 2021-2023. The frequencies of HLA-A,-B,-DRB1 alleles were compared between ESRD patients and the control group, and the relationship between ESRD risk factors, HLA allele frequency (AF), haplotype frequency (HF) was investigated. Results: Similar alleles were observed at high frequencies in both the ESRD patient and control groups, with these alleles which are HLA-A*02 (0.239/0.245), HLA-B*35 (0.225/0.183), and HLA-DRB1*11 (0.230/0.216). The most common haplotypes in patients were A*24~B*35~DR*11 (0.0495), A*02~B*51~DR*11 (0.0485), A*03~B*44~DR*04 (0.0270), A*11~B*35~DR*01 (0.0225), A*02~B*35~DR*04 (0.0225), while in controls they were A*24~B*35~DR*11 (0.0231), A*23~B*49~DR*11 (0.0192), A*02~B*51~DR*13 (0.0179), A*03~B*18~DR*11 (0.0149), A*02~B*18~DR*04 (0.0142) in the controls. For the HLA-B locus, the risk factors were B*50 (p:0.017), B*51 (p <0.0001), B*52 (p:0.023), B*55 (p:0.0017), B*57 (p:0.0017) were identified as risk factors, while B*14 (p:0.016), B*15 (p:0.002) were identified as protective factors. Furthermore, in patients with a primary diagnosis of diabetic nephropathy, A*11 (25.0%), B*51 (31.3%), DRB1*11 (25%) were found at high frequencies. A*02 (27.3%), B*35 (36.4%), DRB1*04 (22.7%) were found at high frequencies in patients with a primary diagnosis of polycystic kidney disease. Conclusion: This study has revealed the relationship between HLA polymorphism and susceptibility to ESRD in the patient population. In patients awaiting kidney transplantation, five susceptible alleles and four susceptible haplotypes were identified. These HLA alleles and haplotypes might serve as genetic markers for susceptibility to ESRD in our population. Keywords: End stage renal disease, human leukocyte antigen, haplotype, alleles frequency
Objective: The Human Leukocyte Antigen (HLA) region is characterized by strong Linkage Disequilibrium (LD). However, traditional symmetric LD metrics fail to provide information on the direction of allelic relationships. This study aims to examine the effect of HLA typing resolution on directional relationships detected by Asymmetric Linkage Disequilibrium (ALD) and to investigate whether this hierarchy is a statistical artifact or a reflection of evolutionary architecture. Materials and Methods: High-resolution (8-digit) HLA-A, -B, -C, -DRB1, -DQB1, and -DPB1 loci genotyping was performed using Next-Generation Sequencing (NGS) in 150 healthy and unrelated individuals of Central Anatolian origin. The 8-digit data were reduced to 2- and 4-digit levels to analyze three different resolution layers. Standard LD (D') and directional relationships (ALD) between locus pairs were calculated using PyPop v1.2.1 software. Results: Standard LD (D') values increased significantly with higher resolution, showing the strongest signals in C: B (D': ~0.94) and DRB1:DQB1 (D': ~0.96) pairs. ALD analysis revealed that directional dominance was consistently preserved regardless of allele number or resolution. The predictive power of HLA-C for HLA-B was systematically higher than the reverse direction (C → B dominance) at all resolutions (p<0.0001). Similarly, HLA-DQB1 predicted HLA-DRB1 significantly better than the reverse (DQB1 → DRB1 dominance; p<0.0001). These hierarchies remained stable across 2-, 4-, and 8-digit levels. Conclusion: ALD analysis is a powerful tool for revealing the hierarchical and directional structure of HLA haplotypes hidden by traditional symmetric metrics. The consistency of C → B and DQB1→DRB1 directional relationships, unaffected by typing resolution, suggests this hierarchy reflects the evolutionary history and functional integrity of the HLA region rather than statistical coincidence. These findings offer new perspectives for mapping genetic architecture and donor selection algorithms. Keywords: HLA antigens, linkage disequilibrium, haplotypes, immunogenetics, ALD
The novel allele HLA-C*12:05:03 differs from HLA-C*12:05:01 by one nucleotide change G>T in exon 5 (GTG>GTT). The findings presented in this poster were previously reported as a novel allele and published in HLA: Immune Response Genetics.
Objective: Panel-reactive antibodies (PRA) or anti-human leucocyte antigens (anti-HLAs) are produced by the patient's immune system in response to donors during transplant procedures. Antibodies to HLA are known to develop following blood transfusion, pregnancy, and a history of transplantation. Donor-specific antibodies (DSA) arising in patient serum in response to donor-specific HLA can be detected using the single antigen bead (SAB) test. The objective of this multicentre retrospective study is to analyse the distribution of anti-HLA antibodies in Turkey, mean fluorescence intensity (MFI) values, and to assess the concordance of PRA tests across centres on a national scale. Materials and Methods: The study will include adult patients and their donors who were on the living and cadaveric waiting lists and who underwent PRA screening/identification/SAB tests between 2013-2024. Data on patients' anti-HLA antibodies and demographics will be obtained from 12 tissue typing laboratories. Tests and MFI values will be recorded. The data will undergo rigorous testing for statistical significance using SPSS version 23.0, with a p-value <0.05. Results: This study will reveal the distribution of anti-HLA antibodies across Turkey for the first time, and the effects of sensitization factors will be analysed. The correlation between Class I and II antibody positivity, MFI levels, and demographic data will be evaluated. Discussion: The findings of this study will contribute to advancing laboratory consistency and establishing national test standardisation. It is hypothesized that the obtained data may also provide insights into updating the organ allocation system to include antibody testing. The comparison of PRA tests, SAB methods, and the MFI values will provide a new perspective on laboratory and clinical evaluation. This study aims to enhance the efficacy of patient-donor matching through the identification of shared criteria for pre-transplant immunological risk assessment within the national context. Keywords: MFI, PRA screening/identification, SAB, transplantation
Objective: HLA molecules and antigen presentation pathways are critical for interactions between tumor cells and T cells. Defects in these mechanisms during carcinogenesis enable immune evasion. Cancer stem cells (CSCs) contribute to recurrence and treatment resistance due to their tumorigenic capacity and resistance to conventional therapies. Targeting CSCs and restoring HLA expression may improve outcomes. This study aimed to evaluate antigen presentation components and HLA expression in pancreatic cancer and CSCs. Materials and Methods: In silico analyses assessed expression of 40 antigen presentation genes according to high and low levels of 10 CSC markers across two pancreatic cancer datasets. Survival analyses compared patients with mutated and unmutated antigen presentation genes. In vitro studies were performed using BxPC3 and CFPAC1 cell lines. CSC presence was determined using specific markers. Tumor spheres were generated, and CSC proportions, along with HLA-ABC and B2M expression, were analyzed in spheres and parental cells by flow cytometry. This study was supported by the Hacettepe University Scientific Research Projects Coordination Unit (No: 19477). Results: Differential expression of antigen presentation molecules was observed across CSC marker groups. NLRC5 expression was significantly reduced in groups with high CSC marker expression, including PROM1 (CD133), SOX2, CD24, and CD44 (p<0.05). Survival was higher in patients without mutations in antigen presentation genes, although not statistically significant (p=0.564; p=0.128). In vitro findings showed increased CSC proportions in spheroids, while B2M and HLA-ABC expression was significantly decreased (p<0.05). Conclusion: These findings indicate that impaired antigen presentation and reduced HLA expression contribute to immune evasion in pancreatic cancer and CSCs. Targeting key molecules in this pathway may improve treatment strategies and warrants further investigation. Keywords: Antigen presentation mechanism, HLA expression, cancer stem cell, pancreatic cancer
Objective: Kidney transplantation is the most effective treatment for end-stage renal disease. Soluble immune checkpoints may regulate immune responses and serve as biomarkers for monitoring and graft function prediction. Materials and Methods: In this study, serum samples collected and stored from healthy controls (n = 15) and kidney transplant recipients (n = 30) before transplantation (day 0) and after transplantation (days 3 and 7) were analyzed for levels of sCD25, s4-1BB, sCD86, active TGF-β1, sCTLA-4, sPD-L1, sPD-1, sTIM-3, sLAG-3, sGalectin-9, sCD27, and sPD-L2 using a flow cytometry–based multiplex bead assay. Kidney function parameters (creatinine and GFR calculated by CKD-EPI) were retrospectively determined at different time points (day 0, day 3, day 7, week 2, month 1, and month 3). Data were analyzed using SPSS to evaluate the post-transplant dynamics of soluble immune checkpoints and their associations with kidney function parameters. Results: In pre-transplant patients, levels of sCD25, sPD-L1, sTIM-3, sGalectin-9, sCD27, and sPD-L2 were significantly higher compared with healthy individuals. In the post-transplant period, statistically significant temporal changes were observed in the levels of sCD25, sPD-L1, sTIM-3, sGalectin-9, sCD27, sPD-L2, and sCD86. In contrast, no temporal changes were detected in the levels of s4-1BB, sLAG-3, sCTLA-4, active TGF-β1, or sPD-1, and these levels were comparable to those of healthy controls. Correlation analyses and subgroup analyses based on post-transplant GFR indicated that higher sLAG-3 and sCTLA-4 levels were associated with better kidney function, whereas higher sCD25 and sGalectin-9 levels were associated with poorer kidney function. Conclusion: Soluble immune checkpoints are linked to early kidney function and may serve as biomarkers and therapeutic targets. Larger studies are needed to confirm their clinical value in improving transplant outcomes. Keywords: Soluble immune checkpoints, kidney transplantation, transplantation immunology
Objective: One of the major challenges in achieving long-term success in heart transplantation is the presence of donor-specific antibodies (DSAs) against HLA antigens. The extent of mismatch can be associated with the likelihood of DSAs formation. These HLA-specific antibodies recognize clusters of accessible polymorphic amino acid residues, known as functional epitopes or eplets, shared by the target HLA molecules. Magnetic beads with a single HLA specificity (MagSort, One Lambda, USA) were developed to facilitate the isolation of HLA specific DSAs from complex serum samples and represent one of the most recent techniques. The aim of the study is to re-test serum samples in which the presence of DSAs was detected by LSA testing during pre- and post-transplant evaluation using MagSort, to determine the presence of the identified antibodies and to correlate these results with clinical outcomes. Materials and Methods: A total of 6 serum samples collected at different time points from 4 heart transplant patients at Baskent University Ankara Hospital. LSA Class I–II tests (before and after MagSort treatment) (One Lambda, USA) were performed on serum samples from the patients at the Baskent University Adana Dr.Turgut Noyan Medical Center. The LSA Class I–II test was performed using the Luminex method. This study was approved by Baskent University Institutional Review Board (Project no: KA25/171) and supported by Baskent University Research Fund. Results: Immunology and pathology results are summarized in the Table 1. Conclusion: The MagSort method, as a new technique, may support donor selection in the organ transplantation process by identifying antibodies defined as DSAs for recipients and/or determining changes in MFI levels. It may facilitate and enhance the accuracy of identifying shared eplets. It may enable clear identification of de novo DSAs after transplantation, thereby guiding the treatment plan. Keywords: Donor specific antibody, heart transplantation, MagSort
Objective: This study aimed to characterize the immuno-epigenetic role of the YY1–SIRT7–H3K18Ac–PD-L1 axis in renal allograft survival and to identify molecular biomarkers associated with rejection. Materials and Methods: A total of 65 participants were enrolled: 20 patients with biopsy-confirmed allograft rejection, 25 with stable graft function, and 20 healthy controls. Blood samples were collected into EDTA-containing tubes and stored at −80°C. Total RNA was isolated and complementary DNA (cDNA) synthesized for gene expression analysis by real-time polymerase chain reaction (RT-PCR), using glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and U6 small nuclear RNA (snRNA) as reference genes. Serum concentrations of SIRT7, H3K18Ac, and oxidative stress markers — superoxide dismutase (SOD) and glutathione (GSH) — were quantified by enzyme-linked immunosorbent assay (ELISA). Statistical analyses were performed using SPSS v23.0 (p < 0.05). Results: Messenger RNA levels of STAT3, CD274 (PD-L1), H3F3A, SIRT7, and YY1 were significantly elevated in the rejection group, whereas PDCD1 (PD-1) was higher in the survival group (p<0.05). Long non-coding RNAs NEAT1, MALAT1, and GAS5 were upregulated in rejection (p<0.05). Significant alterations were detected in miR-21, miR-34a, miR-125, and miR-155 (p<0.01). SOD and GSH were decreased, while malondialdehyde (MDA) and catalase were elevated (p<0.05). NEAT1 and MALAT1 were predominantly upregulated in humoral rejection, whereas GAS5 was reduced. Conclusion: The YY1–SIRT7 epigenetic axis, in conjunction with PD-L1 signaling, appears to be implicated in immune evasion and inflammatory amplification following renal transplantation, supporting the potential utility of these molecules as immuno-epigenetic biomarkers of allograft rejection. Keywords: kidney transplantation allograft rejection epigenetics PD-L1 non-coding RNA biomarkers
Objective: Single antigen bead (SAB) tests on Luminex platform are widely used for donor-specific antibody (DSA) detection. This study aimed to evaluate the frequency of anti-HLA antibody profiles based on SAB test results. Materials and Methods: A total of 317 SAB tests that were performed between January 2022 and September 2025 were included in the retrospective analysis. For patients under follow-up, only the most recent results were evaluated. Antibodies against HLA-A, HLA-B, HLA-DRB1, and HLA-DQB1 with a mean fluorescence intensity (MFI) ≥10,000 were analyzed. Results: Among 145 SAB-positive patients with MFI ≥10,000, 63 (43.4%) were positive for Class I, 42 (29%) for Class II, and 40 (27.6%) for both Class I and Class II antibodies. A total of 1,440 Class I antibodies were detected (anti-HLA-A: 541, anti-HLA-B: 899). For Class II, a total of 740 antibodies were identified (anti-DQB1: 524 [78.8%], anti-DRB1: 216 [29.1%]). The most frequent antibodies were as follows: HLA-A: A2 (12.2%), A68 (9.2%), A24 (8.9); HLA-B: B57 (6.2%), B51 (5.0%), B27 (4.8); HLA-DRB1: DR4 (27.3%), DR9 (14.3%), DR15 (7.9); HLA-DQB1: DQ6 (25.6%), DQ7 (17.2%), DQ4 (16.9). Conclusion: The distribution of detected antibodies was consistent with HLA allele frequencies reported in the Turkish population. Among Class II antibodies, the high prevalence of anti-DQB1 antibodies was particularly noteworthy. Accumulating evidence in the literature suggests that DQB1 antibodies may play a significant role in graft rejection. These findings indicate that the HLA-DQB1 locus should be included in pre-transplant compatibility assessment, especially for re-transplant candidates. Keywords: Single antigen bead assay, HLA antibody, donor specific antibody
Objective: The HLA system exhibits significant population variability due to demographic history. As a migration crossroad, Turkiye has a complex genetic structure. This study analyzes 25 years of data from a single center in West-Central Anatolia to document the evolution of regional HLA distribution between low-migration (2001-2011) and high-migration (2011-2025) periods and to evaluate its implications for transplantation immunology. Materials and Methods: In this retrospective study, HLA-A, -B, -C, -DRB1, -DQB1, and -DPB1 data from 6550 healthy donors were analyzed. The cohort was divided into two periods: 2001-2011 (n=2089) and 2011-2025 (n=4461). Genotyping was performed via PCR-SSO. Population genetics analyses, including Hardy-Weinberg Equilibrium (HWE) and Linkage Disequilibrium (LD), were conducted using PyPop software. Statistical significance of allele frequency differences was assessed using Fisher's Exact test. Results: Statistically significant frequency changes were detected in 18 alleles (p<0.05). In the post-2011 period, Middle Eastern/Asian-origin alleles (e.g., B*52, DRB1*03) increased, while European-associated alleles (e.g., B*44, DQB1*05) decreased. The high-migration cohort showed significant deviation from HWE due to excess homozygosity, indicating a "Wahlund effect" and population stratification. Furthermore, LD between HLA-C and HLA-B weakened significantly, whereas the strong LD between DRB1 and DQB1 was preserved. Specific haplotypes like A*30∼B*51∼C*16∼DRB1*13 disappeared, replaced by new ones such as A*01∼B*08∼C*07∼DRB1*03. Conclusion: Dynamic changes in the gene pool directly affect donor matching probabilities and transplantation strategies. These findings highlight the necessity of periodically updating transplantation registries and donor search algorithms to reflect the current genetic profiles of populations. Keywords: HLA Antigens, emigration and immigration, biological evolution, linkage disequilibrium, genetics population.
Objective: Hematopoietic stem cell transplantation (HSCT) is a curative treatment for hematological malignancies; however, graft-versus-host disease (GVHD) remains a major complication. While donor T cells are central to GVHD pathogenesis, natural killer (NK) cells, via activating and inhibitory receptors, may also influence disease course. This study evaluated post-transplant changes in non-KIR receptor expression on NK cells and their association with GVHD. Materials and Methods: Twenty patients (M:3, F:13) who underwent allogeneic HSCT at Hacettepe University (2021–2024) were included. The mean age was 50, with a median follow-up of 26.1 months. Eleven patients received fully matched and nine haploidentical transplants; GVHD developed in 10 patients. Peripheral blood samples were collected at day 0 and on days 30, 100, and 180. Patients were followed for at least 6 months. Mononuclear cells were isolated, and non-KIR receptor expression in NK subsets was analyzed by flow cytometry. NK cells from days 0 and 30 were co-cultured with the K562 cell line to assess CD107a, perforin, and granzyme expression. Results: Eight AML, six ALL, four MDS, and two lymphoma patients were included. NKG2A and NKG2D decreased significantly (p=0.0001; p=0.0049). NKG2C declined until day 30, then increased. NKp30, NKp44, and NKp46 decreased. ILT-2 and ILT-4 also showed significant reductions (p=0.004; p=0.0001). Cytotoxicity analysis showed increased granzyme and decreased perforin and CD107a, with only perforin reduction significant (p=0.025). Increased NKp46 and NKp30 expression was associated with reduced GVHD risk (AUC: 0.819; 95% CI: 0.576–1.000). Conclusion: Allogeneic HSCT is associated with reduced NK cell non-KIR receptor and perforin expression. Although no significant relationship was found between GVHD development and receptor profile, changes in expression levels suggest that NK cells are affected by chronic inflammation. Higher NKp46 and NKp30 expression may indicate reduced GVHD risk, supporting their potential as biomarkers and the need for larger studies. Keywords: Graft-versus-host disease (GVHD), Hematopoietic Stem Cell Transplantation (HSCT), non-KIR receptor, NK cell
Objective: Genetic variation within the HLA region plays a pivotal role in transplantation compatibility and the shaping of immune responses. Next-generation sequencing (NGS) technology enables the detection of non-coding region variants that cannot be identified by conventional methods, thereby allowing a more comprehensive assessment of HLA allelic diversity. However, the structural differences of these rare variants at the eplet level and their potential immunogenic effects on KIR-ligand epitopes, namely Bw4/Bw6 and C1/C2 groups, have not yet been fully elucidated. Materials and Methods: In this study, rare HLA alleles harboring non-coding region variants were identified among 15,350 HLA alleles characterized by NGS at the Tissue Typing Laboratory of Istanbul Faculty of Medicine. These alleles were validated and named through comparison with reference sequences in the IPD-IMGT/HLA database. The identified alleles were as follows: A02:01:01:164, A02:01:01:118, A02:06:01:15, A23:39, A24:02:01:02L, A24:314, A31:234, A02:01:01L, B44:02:01:35, B44:02:01:02S, B51:01:01:17 (in two individuals), B51:01:01:69, B51:11N, B58:01:01:10, C17:38, C02:02:02:47, C12:323, C15:171, DRB113:157, DRB114:23, DRB111:310, DRB401:03:01:02N, and DPB1*1321:01. Eplet characterization of these alleles was performed at the amino acid level by comparison with their respective reference alleles. In addition, Bw4/Bw6 epitopes of HLA-B alleles and C1/C2 epitope characteristics of HLA-C alleles were evaluated. Results: For the majority of alleles, the observed differences were confined to non-coding regions and did not involve amino acid changes within the antigen-binding domain. Consequently, their eplet profiles and Bw4/Bw6–C1/C2 epitope characteristics were found to be similar to those of the corresponding reference alleles. However, certain alleles—namely C17:38, A23:39, A24:314*, A31:234*, C12:323*, C15:171*, and DRB1*13:157*—were predicted to harbor potential amino acid substitutions within the antigen-binding groove, which could theoretically give rise to novel eplet combinations. Alleles categorized as L, N, and S were also considered to be of potential clinical relevance due to their altered expression patterns. Conclusion: Rare HLA alleles with non-coding region variants identified by NGS generally exhibit eplet and KIR-ligand (Bw4/Bw6, C1/C2) profiles similar to those of their reference alleles; however, certain variants may still possess immunogenic differences. These findings indicate that the combined evaluation of high-resolution HLA typing results at both the eplet and KIR-ligand levels may provide more precise predictive insights in transplantation immunology. Keywords: Non-coding, NGS, HLA alleles, eplet analysis,
Objective: The aim of this study was to assess serum levels of soluble immune checkpoint proteins (sICPs) in patients with systemic lupus erythematosus (SLE) and Sj & ouml;gren's syndrome (SS) and to explore their association with disease activity. Materials and Methods: This preliminary cross-sectional study included 27 patients with SLE, 23 patients with SS, and 23 healthy controls. Serum concentrations of sCD25 (soluble interleukin-2 receptor alpha [IL-2R alpha; CD25]), 4-1BB (tumor necrosis factor receptor superfamily member 9 [TNFRSF9; CD137]), B7.2 (CD86; cluster of differentiation 86), transforming growth factor beta 1 (TGF-beta 1), cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), programmed cell death protein 1 (PD-1), programmed death-ligand 1 (PD-L1), T-cell immunoglobulin and mucin-domain containing-3 (TIM-3), lymphocyte activation gene 3 (LAG-3), and Galectin-9 (Gal-9) were measured using a multiplex bead-based flow cytometric assay. Disease activity was evaluated using standard indices (Systemic Lupus Erythematosus Disease Activity Index [SLEDAI] for SLE; EULAR Sj & ouml;gren's Syndrome Disease Activity Index [ESSDAI] for SS). Group differences were analyzed, and correlations between sICP levels and clinical indices were assessed. Results: Compared with healthy controls, SLE patients had significantly elevated levels of soluble TIM-3 (sTIM-3) (p<0.0001), sLAG-3 (p<0.0001), and Gal-9 (p<0.001). A strong positive correlation was observed between sGal-9 and sTIM-3 levels (r=0.79, p<0.0001), and a moderate correlation was observed between sGal-9 and sLAG-3 levels (r=0.55, p=0.002). In SS, sICP levels did not differ significantly from controls; however, sPD-1 correlated with sPD-L1 (r=0.56, p=0.004), and sGal-9 with sTIM-3 (r=0.54, p=0.007). ESSDAI scores in SS correlated with sCD25 (r=0.65, p=0.0007), sTIM-3 (r=0.49, p=0.01), and sGal-9 (r=0.41, p=0.04). Conclusion: Our findings suggest the potential use of sICPs as biomarkers in SLE and SS. However, larger studies are needed to validate the diagnostic and prognostic roles of these soluble checkpoints.
