Epitope Specificity of HLA Class I Alloantibodies in Indian Renal Transplant Patients: A Single-Center Study

Abstract

Background/Objectives: Epitope-based matching has emerged as a refined approach for assessing donor–recipient compatibility in renal transplantation. However, limited data are available on HLA Class I epitope distribution among Indian patients, particularly from northern India, where substantial allelic diversity is known to influence immunological risk. Methods: This retrospective analysis evaluated HLA Class I single-antigen bead (SAB) antibody data from 218 consecutive renal-transplant candidates who tested positive for anti-HLA antibodies between July 2018 and September 2024. HLA Class I epitopes were identified and analyzed using MATCH IT Antibody Software (Immucor, version 1.5.0). Demographic variables and sensitization history (previous transplant, transfusion, pregnancy) were reviewed. Results: A total of 504 distinct epitopes were identified, with 65GK and 163LG emerging as the most frequent motifs. The predominance of these epitopes mirrors the high prevalence of alleles such as HLA-A*24 and HLA-B*35 reported in North-Indian populations. The data suggest a strong influence of regional allele architecture on the immunogenic epitope landscape. Conclusions: This study provides the first baseline characterization of HLA Class I epitope distribution among northern-Indian renal-transplant candidates. The findings emphasize the need for establishing population-specific HLA epitope databases and highlight the potential of epitope-based matching to enhance donor selection and minimize immunological risk in Indian transplantation programs.

1. Introduction

Human Leukocyte Antigen (HLA) mismatching between recipient and donor can trigger the development of de novo donor-specific antibodies (DSA), ultimately resulting in poorer clinical outcomes and reduced graft survival [1]. Hence, HLA matching remains a cornerstone immunological strategy for determining organ compatibility in transplant recipients. Recent advancements in tissue typing and the development of sophisticated computer software have led to the introduction of the concept of eplets. Eplets are clusters of polymorphic amino acids located on the surface of HLA molecules. These eplets are considered functional epitopes, as they contain amino acids that can be recognized by anti-HLA antibodies, representing a specific segment of the larger amino acid structure of an HLA epitope. This approach provides a more detailed understanding of the antigen- antibody interaction [2,3,4]. HLA matching at the epitope or eplets level is now increasingly recognized as a refined method for evaluating immunologic compatibility and risk stratification in transplantation [1,3,5,6]. HLA epitopes are specialized regions of HLA molecules that interact with antibodies or the paratopes of T-cell receptors (TCRs) [2,4]. HLA epitopes exhibit two key characteristics; antigenicity, i.e., their reactivity with antibody, and immunogenicity, i.e., their ability of inducing an antibody response. In both cases, the definition of an HLA epitope relies on the context of its corresponding eplets within the structural epitope. These principles are crucial for understanding antibody-defined HLA epitopes and have significant clinical implications in HLA antibody analysis and the evaluation of mismatch acceptability and permissibility for transplant recipients [7]. In HLA antibody analysis using single-antigen bead (SAB), each detected antibody binds not to the entire HLA molecule but to a specific structural motif- known as an epitope. An epitope, typically defined by one or more amino-acid residues (eplets), may be shared across multiple alleles, leading to apparent cross-reactivity. Identification of shared epitopes helps distinguish true alloantibody targets from assay artefacts. Epitope-level interpretation refines antibody characterization, assists in immunologic risk stratification, and supports precise donor selection by focusing on the molecular determinants of antigenicity [6,8,9]. Limited information exists regarding the prevalence of epitopes or eplets across different population groups yet understanding this is crucial to provide candidates from diverse ethnic backgrounds with equitable opportunities for a well-matched organ [8]. Given the limited information available regarding common epitope occurrence in the Indian population, this study seeks to identify and analyze epitope relevant to class HLA Class I in Indian renal transplant patients, thereby contributing to the existing global epitope database and supporting improved donor selection strategies. Although the concept of HLA epitope analysis has been extensively explored in Western and East-Asian cohorts, region-specific data from India remain scarce [5]. Recent advances in molecular epitope-matching and population-based eplet analysis have underscored the clinical relevance of such approaches [4,6,10]. Building on this framework, the present study provides the first systematic evaluation of HLA Class I epitope distribution among Indian renal-transplant recipients, establishing an essential regional baseline for donor–recipient matching at the epitope level.

2. Materials and Methods

2.1. Study Design and Participants

A retrospective analysis was conducted on data from 218 consecutive renal transplant patients who tested positive for SAB class I assays between July 2018 and September 2024. Among them, 129 (59.17%) were males, and the remaining 89 (40.83%) were females. Among these, 79 (36.2%) had undergone previous transplantation, 79 (36.2%) had a transfusion history, and 29 (13.3%) had a history of pregnancy. The participants were primarily from Northern states of India. Written informed consent was obtained from all participants included in the study for sample collection and laboratory testing. They were assured that their names and photos would not be published and that all standard procedures for protecting their identities would be strictly followed.

2.2. Sample Collection and Processing

Approximately 3–4 mL of blood was collected in plain red-top vacutainer vials (without anticoagulant) for serum preparation. Samples were allowed to clot briefly at room temperature, then centrifuged at 3500× g for 5 min to separate the serum, which was subsequently processed for SAB testing.

2.3. Antibody Detection

Detection and characterization of anti-HLA Class I antibodies were processed using the LIFECODES LSA™ Class I kit (Cat. No. 265100), Immucor, Inc., GA, USA) in accordance with the manufacturer’s instructions. Processing was performed on the Luminex^® 200 platform (Luminex Corporation, Austin, TX, USA) utilizing ×MAP technology. The assay targeted class I antigens (HLA-A, HLA-B, and HLA-C), and antibody specificity, strength, and epitope types were analyzed using MATCH IT antibody software (Immucor GTI Diagnostics, Inc., Waukesha, WI, USA, version1.5.0). The cut-off value for a positive antibody was a mean fluorescence intensity (MFI) of ≥1000. All assays were performed in a designated histocompatibility and immunogenetics laboratory accredited under (ISO 15189:2012, Available at: https://www.iso.org/standard/56115.html, accessed on 1 October 2025) by the National Accreditation Board for Testing and Calibration Laboratories (NABL), located in New Delhi, North India.

2.4. Epitope Analysis

For all antibodies exceeding the threshold, epitope assignment was automatically generated by the MATCH IT antibody software based on known amino acid configurations shared among reactive HLAs. These assigned epitopes represent the predicted molecular sites of antibody binding, allowing cross-comparison of shared eplets across different Class I alleles. The occurrence of each epitope was recorded for subsequent analysis

2.5. Data Compilation and Categorization

The epitope information generated from MATCH IT antibody software was compiled for all 218 samples. Each epitope was tabulated along with its associated HLA locus (A, B, C) and its occurrence within the study cohort. Sex wise distribution was also analyzed. A total of 504 distinct epitope occurrences were identified.

Table 1. Representing the epitopes identified among the studied population (N = 218).

S. No	Epitope	Associated HLA Locus	Total Number	FEMALES (N = 89)	MALES (N = 129)
1	9D	BC	2	1	1
2	9F	AC	1	1	0
3	9T	A	1	0	1
4	9Y	ABC	1	0	1
5	11AV	BC	1	0	1
6	12M	B	3	1	2
7	14W	C	1	0	1
8	16S	C	2	1	1
9	19K	A	5	2	3
10	24T	B	2	1	1
11	30G	B	6	4	2
12	32L	B	1	1	0
13	41T	B	2	1	1
14	43R	A	6	3	3
15	44KM	A	7	2	5
16	44RM	AB	2	0	2
17	44RMA	B	2	1	1
18	44RME	A	2	1	1
19	44RT	B	6	4	2
20	45EE	B	2	0	2
21	56R	A	11	2	9
22	62EE	A	1	1	0
23	62GE	AB	9	6	3
24	62GK	A	5	2	3
25	62GRN	AB	5	3	2
26	62LQ	A	9	4	5
27	62QE	A	3	2	1
28	62RN	AB	2	1	1
29	62RR	AB	6	4	2
30	63NI	B	1	1	0
31	65GK	A	21	5	16
32	65QIA	B	9	9	0
33	65RA	AB	3	0	3
34	65RNA	AB	2	2	0
35	66I	B	3	3	0
36	66IC	B	1	1	0
37	66IF	B	11	5	6
38	66IS	B	1	1	0
39	66IY	B	1	1	0
40	66KA	A	1	1	0
41	66NM	AB	1	0	1
42	66NV	A	2	0	2
43	69AA	B	1	0	1
44	69RA	C	1	0	1
45	69TNT	B	2	0	2
46	70IAQ	B	3	2	1
47	71ATD	B	4	3	1
48	71QS	A	2	1	1
49	71SA	B	4	2	2
50	71TD	B	14	7	7
51	71TN	B	2	2	0
52	71TTS	B	1	1	0
53	73AN	C	1	0	1
54	73AS	C	1	1	0
55	73ID	A	7	3	4
56	76ANT	A	1	0	1
57	76ED	B	2	1	1
58	76ESI	A	11	7	4
59	76ESN	B	4	2	2
60	76ET	AB	1	1	0
61	76VRN	BC	1	1	0
62	77S	ABC	1	1	0
63	80I	AB	5	4	1
64	82LR	AB	8	4	4
65	90D	AC	2	2	0
66	91R	C	1	1	0
67	95V	A	1	0	1
68	97N	B	11	6	5
69	97S	B	1	1	0
70	97V	B	7	3	4
71	99Y	ABC	1	0	1
72	103M		2	1	1
73	105S	A	2	0	2
74	107W	A	2	0	2
75	109F	AB	2	0	2
76	113HD	BC	1	0	1
77	113HN	B	2	1	1
78	114H	AB	2	2	0
79	114Q	A	1	1	0
80	114R	A	1	1	0
81	116L	BC	1	1	0
82	116Y	ABC	1	0	1
83	127K	A	17	12	5
84	131S	B	3	2	1
85	138K	C	2	0	2
86	138MI	A	1	1	0
87	144K	A	2	0	2
88	144KR	A	1	1	0
89	144QL	B	12	3	9
90	144TKH	A	10	4	6
91	145HT	A	2	0	2
92	145RT	A	7	3	4
93	147L	BC	2	0	2
94	149TAH	A	1	1	0
95	150AAH	A	1	1	0
96	150AH	A	4	1	3
97	151AHA	A	10	6	4
98	151AHV	A	3	2	1
99	151ARV	AB	1	0	1
100	151H	A	1	1	0
101	152RA	C	1	0	1
102	152RR	A	4	1	3
103	152T	C	1	0	1
104	152V	AB	1	0	1
105	152W	A	1	0	1
106	156DA	BC	3	3	0
107	156QA	AC	1	0	1
108	156WA	ABC	5	0	5
109	158T	B	5	3	2
110	161D	A	4	1	3
111	162GLS	B	10	2	8
112	163EW	ABC	5	2	3
113	163L	BC	4	1	3
114	163LG	B	18	4	14
115	163LS/G	B	10	4	6
116	163LW	BC	8	6	2
117	163RG	A	4	1	3
118	163RW	A	2	0	2
119	166DG	A	6	2	4
120	170RH	AB	4	4	0
121	173K	C	5	2	3
122	177DK	B	5	3	2
123	177DT	B	2	2	0
124	177KT	C	2	0	2
125	180E	B	3	3	0
126	184A	A	1	0	1
127	184R	C	1	0	1
128	193AV	A	2	1	1
129	193LV	C	1	0	1
130	193PI	AB	2	1	1
131	193PL	C	4	2	2
132	199V	B	4	2	2
133	211T	C	2	1	1
134	245TA	B	2	1	1
135	245V	A	7	4	3
136	253Q	AC	2	2	0
137	275EL	A	1	0	1
Total			504	237	267

summarizes the complete epitope dataset.

3. Results

3.1. Overall Epitope Distribution

Analysis of SAB Class I antibody data from 218 renal-transplant recipients revealed 504 total epitope occurrences, representing 137 distinct epitopes distributed across HLA-A, -B, and -C loci as presented in Table 1. Among these, 237 epitope events were detected in females and 267 in males, indicating a comparable overall prevalence between the two groups. The most frequently identified epitopes were 65GK (n = 21), 163LG (n = 18), 127K (n = 17), 71TD (n = 14), 144QL (n = 12), 66IF (n = 11), 76ESI (n = 11), and 56R (n = 11). 11). Epitope nomenclature (e.g., 65GK, 163LG) denotes specific amino-acid positions and residues that constitute the reactive motif on the HLA molecule. The number refers to the starting amino-acid position on the HLA heavy chain, while the accompanying letters represent the residues that form the functional epitope recognized by alloantibodies. For example, 65GK corresponds to a glycine–lysine motif beginning at residue 65, and 163LG denotes a leucine–glycine motif beginning at residue 163. These high-frequency epitopes were predominantly associated with HLA-A and HLA-B loci as shown in Figure 1.

3.2. Locus-Wise Distribution

Locus-specific analysis revealed that HLA-B locus contributed the highest proportion of identified epitopes (49.6%), followed by HLA-A (35.7%) and HLA-C (14.7%) as shown in Figure 2. Numerous epitopes such as 62GE, 163EW, and 77S were detected across multiple loci (A/B/C), demonstrating the presence of shared or public epitopes responsible for cross-reactive antibody patterns.

3.3. Sex-Wise Comparison

Among the most prevalent epitopes, 127K was observed in 17 cases (12 females, 5 males), 56R in 11 cases (9 males, 2 females), and 65GK in 21 cases (16 males, 5 females). While some epitopes showed noticeable sex-related frequency differences, no epitope was exclusive to either sex, and the overall spectrum of immunogenic epitopes was broadly comparable between males and females.

4. Discussion

The primary objective of this study was to estimate the prevalence of epitopes in patients requiring renal transplantation. The analysis identified a wide spectrum of Class I HLA epitopes among Indian renal-transplant patients, dominated by recurrent motifs shared among HLA-A and -B molecules. The predominance of conserved public epitopes such as 65GK and 163LG underscores their likely contribution to cross-reactive donor-specific antibody formation in this population. This investigation is one of the first such studies conducted in India, addressing a significant gap in the existing literature from the region. Epitope analysis and matching have become critical in the field of renal transplantation. With continuous advancements in the identification and mapping of functional HLA epitopes or eplets, epitope matching is increasingly indispensable for assessing immunological risk between donors and recipients [4]. However, additional clinical data and a deeper understanding of the variations in methods for determining epitope compatibility are necessary before this approach can be widely implemented in clinical practice [3]. Recent global analyses have highlighted both conserved and population-specific trends in HLA epitope distribution. Oguz et al. examined the spectrum of Class I eplets in the Turkish population and reported several shared (public) motifs contributing to cross-reactive antibody responses [4]. Likewise, Mangiola et al. showed that the most frequent HLA Class I eplets occur at comparable rates across diverse ethnic groups, suggesting partial global conservation of immunogenic motifs [10]. In this context, our findings provide complementary data from the Indian population, positioning the observed epitope frequencies within a broader international framework and emphasizing the importance of developing population-specific epitope databases to support equitable donor-matching strategies.

Marked regional heterogeneity of HLA alleles has been well documented across northern India. Population-based analyses have shown that HLA-A*01 (25.4%), A*02 (24.8%), B*35 (20.5%), and C*07 (28.1%) are among the most prevalent alleles in this region [11]. More recent large-cohort genotyping further confirmed this diversity, reporting high frequencies of HLA-A*11 (16.4%), A*24 (14.4%), B*35 (14.2%), and C*07 (26.0%) [12,13]. These findings demonstrate significant inter-state and intra-regional variability in HLA distribution, reflecting the genetic heterogeneity of northern India. Such differences underscore the importance of establishing region-specific epitope databases to ensure accurate donor–recipient matching and equitable organ allocation across the country.

HLA-A*24:02 and HLA-A*24:03 have been reported as the most frequent anti-HLA antibodies in renal transplant patients in North India [14,15]. In this study, the 65GK epitope was found to be the most prevalent in the studied population, which is associated with HLA-A*24:02 and HLA-A*24:03. These findings align with previous publications from the region. Similarly, HLA-B*15:12 has been reported as a more prevalent anti-HLA antibody and the 163LG epitope was identified as corresponding to HLA-B*15:12 [10]. Regional allele heterogeneity likely influences the distribution of immunogenic epitopes. For example, the high frequency of HLA-A*24:02 and HLA-B*15:12 in northern India-alleles corresponding to epitopes 65GK and 163LG-supports the relationship between local allele architecture and dominant epitope patterns. These high-frequency epitopes were mainly associated with the HLA-A and HLA-B loci, suggesting that these regions play a major role in antibody responses and are important for transplant immunogenicity, potentially guiding donor–recipient matching and risk assessment. In the studied population, several other epitopes were identified, including 9Y (n = 1), 77S (n = 1), 99Y (n = 1), 116Y (n = 1), 156WA (n = 5), and 163EW (n = 5). Although these cases were not statistically significant, all these epitopes shared HLA-A, HLA-B, and HLA-C alleles and may play a critical role in donor selection on the basis of epitope matching. Additionally, gender-related differences in epitope prevalence suggest that sex may influence alloimmune responses. For example, 127K was more frequent in females, whereas 56R and 65GK were more common in males. Although no epitope was exclusive to either sex, these patterns may reflect the influence of prior sensitization events, such as pregnancies or transfusions, on antibody formation. Overall, despite these sex-specific trends, the general spectrum of immunogenic epitopes was similar between males and females, indicating that while gender can modulate specific responses, it does not drastically alter the overall immunogenic landscape in this population.

At present, HLA Matchmaker is a widely used tool for predicting epitope compatibility; however, it is not the only available method. Another promising approach is the Predicted Indirectly Recognizable HLA Epitopes (PIRCHE) score, which explains the recognition of HLA polymorphisms corresponding to epitopes by recipient CD4+ T cells [4,16,17]. Both HLA Matchmaker and PIRCHE represent advanced computational algorithms designed to predict the epitopes present in HLA molecules. However, these tools cannot definitively determine whether the predicted epitopes are antigenic (capable of binding to antibodies) or immunogenic (capable of inducing antibody production through antigen–antibody interactions) [7].

This study provides foundational data on the prevalence of immunogenic HLA Class I epitopes among Indian renal transplant recipients. The identification of common epitopes can support the development of population specific eplet-matching algorithms and improve donor–recipient compatibility assessment beyond conventional antigen-level matching.

Future research should expand this approach to include Class II epitopes and incorporate prospective correlation with clinical outcomes, such as graft survival and the occurrence of donor-specific antibodies. Additionally, integration of next generation sequencing (NGS) based HLA typing with epitope-level antibody mapping could further refine transplant immunogenicity prediction models. Establishing a national epitope database for Indian transplant candidates may ultimately enhance equitable organ allocation and personalized immunological risk assessment.

5. Conclusions

This study highlights the importance of HLA epitope analysis in renal transplantation, emphasizing its role in assessing immunological risk and optimizing donor–recipient compatibility for improved donor selection and, ultimately, better transplant outcomes.

6. Limitation

Despite its valuable insights, this study is subject to certain limitations. It is retrospective and single-center in nature and focuses exclusively on HLA Class I epitopes without incorporating Class II analysis. Clinical correlations with graft outcomes were not evaluated. Although the cohort offers important baseline information, the sample size may not fully represent the allelic diversity of the broader Indian population. Future multi-center prospective studies integrating both Class I and II epitopes and correlating them with clinical outcomes are warranted to validate and expand these findings.