High-throughput profiling of the T cell receptor delta CDR3 repertoire reveals species-specific patterns in cattle (Bos taurus) and water buffalo(Bubalus bubalis)

High-throughput profiling of the T cell receptor delta CDR3 repertoire reveals species-specific patterns in cattle (Bos taurus) and water buffalo(Bubalus bubalis) | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article High-throughput profiling of the T cell receptor delta CDR3 repertoire reveals species-specific patterns in cattle (Bos taurus) and water buffalo(Bubalus bubalis) Yueheng Zhang, Fengli Wu, Long Ma, Xinsheng Yao, Jun Li This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8686230/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background γδ T cells constitute a substantial proportion of lymphocytes in ruminants, and the diversity of their immune receptors is critical for understanding species-specific immune functions. The complementarity-determining region 3 (CDR3) of the T cell receptor delta chain (TRD) is a key structural determinant of γδ T cell antigen recognition; however, systematic comparative analyses of TRD immune repertoire characteristics across different bovine species remain limited. In this study, high-throughput sequencing and comprehensive analysis of the TRD CDR3 immune repertoire were performed in 7 cattle (Bos taurus) and 5 water buffalo (Bubalus bubalis). Results The results demonstrated good consistency in sequencing depth and data quality between the two groups. Diversity analysis revealed that the cattle TCRδ CDR3 repertoire exhibited higher clonal evenness and overall diversity than that of buffalo. Clonotype composition analysis showed that both species were dominated by medium- to high-frequency clonotypes, whereas buffalo relied more heavily on a limited number of highly expanded clonotypes. V(D)J gene usage analysis identified pronounced species-specific preferences in TRDV gene usage, with high intra-group consistency but low inter-species correlation; in contrast, TRDJ gene usage was highly conserved between the two species. The CDR3 length distributions in both groups displayed similar bell-shaped patterns, suggesting structural constraints during evolution, while K-mer and motif analyses revealed differences in CDR3 microstructural features between species. Furthermore, shared clonotype analysis indicated a limited number of public CDR3 amino acid sequences between cattle and buffalo, with highly abundant shared clonotypes enriched only in a small subset of individuals. Conclusions Collectively, this study provides a systematic characterization of the similarities and differences in the TRD CDR3 immune repertoires of cattle and water buffalo, offering fundamental data and a comparative perspective for understanding the mechanisms shaping γδ TCR diversity and their potential immunological functions in high γδ T cell species. γδ T cells TCR CDR3 repertoire V(D)J recombination cattle water buffalo Figures Figure 1 Figure 2 Figure 3 Figure 4 Background T cells are broadly classified into two major subsets, γδ T cells and αβ T cells, based on the composition of their antigen receptors. αβ T cells represent the central effector population of the adaptive immune system, whereas γδ T cells possess characteristics of both innate and adaptive immunity and are therefore regarded as “bridging cells” between the two immune arms[ 1 ]. The γδ T cell receptor (TCR) enables γδ T cells to recognize non-peptidic and stress-induced antigens in a major histocompatibility complex (MHC)-independent manner, allowing them to function as rapid immune sentinels at the interface of innate and adaptive immunity[ 2 – 4 ]. Although γδ T cells constitute a minor fraction of peripheral lymphocytes in most species, they represent a dominant lymphocyte subset in cattle, accounting for approximately 60% of circulating T cells in neonatal calves[ 2 ]. This striking species-specific difference in γδ T cell abundance suggests that bovine γδ T cells may fulfill immune surveillance and defense functions distinct from those observed in other mammals. The functional competence of γδ T cells is largely dependent on their TCR repertoire, particularly the highly diverse complementarity-determining region 3 (CDR3) of the δ chain (TRD), which plays a critical role in antigen recognition and the sensing of tissue stress signals [ 5 , 6 ]. With the advancement of high-throughput sequencing (HTS) technologies, research focusing on the γδ TCR repertoire, especially the CDR3 repertoire of the TRD, has garnered increasing attention[ 7 , 8 ]. At the functional level, the length, sequence composition, and junctional diversity of the TRD CDR3 region are recognized as critical determinants governing the antigen recognition specificity of γδ T cells. The TRD CDR3 regions of ruminants typically exhibit greater length and structural diversity. Moreover, their V–D–J recombination patterns and nucleotide insertion/deletion profiles exert a profound impact on the spatial conformation of CDR3, thereby shaping a unique antigen-binding interface[ 7 ]. In humans, the diversity of the TRD CDR3 repertoire is markedly higher than that of the TCR γ chain (TRG) repertoire, with a low degree of sharing among individuals, which reflects the central role of the δ chain in the functional differentiation of γδ T cells[ 5 ]. Furthermore, the V, D, J gene usage and CDR3 characteristics of the TRD repertoire undergo dynamic alterations across distinct developmental stages and immune statuses, indicating that the TRD repertoire is synergistically regulated by both developmental programs and antigen selection pressure[ 9 , 10 ]. Recent studies have further demonstrated that the characteristics of the TRD repertoire hold substantial biological significance in tumor-associated immunity. In solid tumors such as colorectal cancer, the number of functional TRD-rearranged reads decreases markedly during the transition from normal tissues to tumor tissues, suggesting that γδ T cells may be involved in the immune surveillance process during the early stages of tumorigenesis[ 11 ]. Meanwhile, the physicochemical properties of TRD CDR3 amino acids are closely correlated with tumor progression stages, implying that the structural attributes of TRD CDR3 may modulate the anti-tumor functions of γδ T cells[ 11 ]. In other digestive system tumors, both the TRD repertoire and the global TCR repertoire exhibit distinct tissue specificity and disease-associated recombination biases, reflecting the functional heterogeneity of γδ T cells in different tumor immune microenvironments[ 12 ]. These findings further highlight the TRD repertoire as a critical entry point for elucidating the functional status and immune regulatory mechanisms of γδ T cells. Although preliminary investigations into the TRD repertoire have been conducted in humans, mice, sheep, camels, and other species[ 13 , 14 ], research focusing on the TRD CDR3 diversity, V(D)J gene usage characteristics, and repertoire structural divergence remains remarkably limited in bovids-particularly in cattle and buffalo, two species that exhibit high similarities in γδ T cell proportions, immune niches, and evolutionary backgrounds yet possess distinct phylogenetic differences. Therefore, systematic characterization of the TRD repertoire features in cattle and buffalo will not only deepen the understanding of γδ T cell immune diversity and evolutionary rules in ruminants but also provide critical fundamental data to elucidate the specific immune functions of γδ T cells in species with high γδ T cell abundances. Methods Sample collection and DNA extraction We collected a total of 12 samples in this study, including spleen tissues from 5 healthy adult buffalo and 7 healthy adult cattle. All samples were obtained from a commercial abattoir located in Nanning, Guangxi, China. Immediately after collection, the spleen tissues were snap-frozen in liquid nitrogen to prevent degradation. Genomic DNA was extracted using the DNeasy Blood & Tissue Kit (QIAGEN) following the instructions of the manufacturer. We evaluated the integrity, concentration, and quality of the extracted DNA via 1% agarose gel electrophoresis and an Agilent 2100 Bioanalyzer. Then, Qualified DNA samples were aliquoted and stored at -20°C until subsequent experiments. Primer design and synthesis Based on the annotation results of the TRD locus,we designed amplification primers targeting the TCR δ CDR3 repertoire for cattle and buffalo, respectively. All forward primers were anchored in the conserved terminal regions of TRDV genes; a total of 5 gene-specific forward primers were designed, corresponding to the TRDV1-TRDV5 subgroups. Reverse primers were targeted to the conserved terminal regions of TRDJ genes, with 4 gene-specific reverse primers constructed to match the TRDJ1-TRDJ4 segments. All primers were synthesized by Sangon Biotech (Shanghai) Co., Ltd. Detailed sequences of the amplification primers are provided in Additional file 1. TCR δ CDR3 repertoire construction and sequencing Multiplex Polymerase Chain Reaction (PCR) was performed to amplify the target TCR δ CDR3 fragments. We prepared the PCR reaction system with a total volume of 50 µL, containing 25µL of PCR Master Mix, 2.8 µL each of the forward and reverse primer mixtures, 1µg of genomic DNA template, and the remaining volume was supplemented with ddH 2 O. The PCR protocol was set as follows, with an initial denaturation step at 94°C for 3 min, followed by 35 cycles of denaturation at 94°C for 1 min, annealing at 56°C for 1 min and extension at 72°C for 1 min, then a final extension at 72°C for 10 min and subsequent incubation at 4°C. The PCR products were analyzed by 2% agarose gel electrophoresis to assess the brightness and specificity of the target bands. The purified and recovered target fragments were sent to BGI Genomics Co., Ltd (BGI Genomics) for quality inspection. Qualified samples were subjected to HTS on the DNBseq platform. HTS data analysis of the TCR δ CDR3 repertoire After sequencing by BGI Genomics, raw sequence data of the TRD CDR3 repertoire were generated for all 12 samples. We uploaded the data in FASTQ format to MiXCR and aligned them against the V, D, J, and C genes in the background reference library, then outputted the CDR3 clonotype sequences of each sample. We calculated the total number of reads, the number of successfully aligned reads, and the number of reads used for clonotype construction for each sample. Non-functional sequences and sequences lacking canonical CDR3 start/stop residues were filtered out to determine the final number of TRD chains. The total number of unique CDR3 sequences obtained per sample after HTS met the required sequencing depth and were suitable for subsequent characteristic analysis of the CDR3 region. Clonotype proportion distribution and diversity analysis All statistical analyses were performed in the R 4.5.2 software environment, using major packages including immunarch, ggpubr, and ggplot2. Firstly, we calculated the number of unique clonotypes for both the cattle group and buffalo group, followed by the calculation of the proportions of top clonotypes and rare clonotypes. To comprehensively evaluate the diversity of the TRD repertoires between the two groups, multiple diversity metrics were used for further analysis, including D50 index, Gini-Simpson index and Hill index, so as to fully assess the richness and evenness of clonotypes in the two groups. V/J gene usage and correlation analysis Based on the data generated by MiXCR, the usage frequencies of V/J genes in each sample were calculated and expressed as percentages. For genes with significant differences, pairwise comparisons were performed using the Kruskal-Wallis H test, with P < 0.05 considered statistically significant. Subsequently, we analyzed the correlation of V/J gene usage within and between groups, applying the Spearman correlation coefficient to measure these associations and plotting heatmaps for visualization. The heatmaps were displayed according to the gradient of correlation coefficient values, which intuitively reflected the similarities in gene usage patterns between the two species and among samples. CDR3 amino acid length distribution Leveraging the data generated by MiXCR, we extracted all valid TCR δ CDR3 amino acid sequences and determined the amino acid length of each individual sequence.To minimize the impact of extreme values, only CDR3 sequences with lengths spanning 10 to 40 amino acids were retained, a filtering step that ensured the reliability of subsequent analyses.We quantified the distribution frequencies of CDR3 amino acid lengths across the two groups and then constructed length distribution histograms to visualize the variation trends directly. By comparing the peak values and distribution ranges of CDR3 lengths between the two groups, the fundamental structural characteristics that distinguish the CDR3 sequences of the two species were clearly revealed. K-mers and motif Analysis To further explore the characteristic differences in CDR3 amino acid sequences, we selected 5-amino-acid short peptide fragments for analysis, counted the top 10 high-frequency fragments in each group, calculated their respective frequencies, and finally plotting stacked bar charts to compare inter-species disparities. Subsequently, motif analysis was performed based on amino acid composition, with the maximum number of motifs set to 5. By integrating the physicochemical properties of amino acids, such as hydrophobicity and polarity, we analyzed the composition characteristics, occurrence frequencies, and arrangement patterns of amino acids in different motifs, aiming to explore the potential association between species-specific motifs and immune functions. TCR δ CDR3 repertoire overlap analysis To evaluate the overlap of TCR δ CDR3 repertoires among the 12 samples, we quantified the number of shared clonotypes, and further calculated the proportion and quantity of shared clonotypes between each pair of samples. Thereafter, we plotted a heatmap of inter-sample overlap to visually display the shared characteristics of clonotypes within and between groups. With UpSet plots generated via the UpSetR package, we presented the number of specific clonotypes unique to the cattle group, the buffalo group, and those common to both in the form of waterfall charts. In this way, the uniqueness and commonality of TRD repertoires between the two species were clearly identified, thereby providing evidence for revealing species-specific immune characteristics. Clonotype tracking Three categories of amino acid sequences were selected for tracking analysis, including the 5 CDR3 amino acid sequences shared most frequently across all samples, the top 5 sequences with the highest shared abundance within the buffalo group, and the 5 most prevalent shared sequences in the cattle group. We quantified the clonal frequency and proportion of each target sequence in the corresponding sample set, and plotted bar charts of clonotype frequency distribution to compare expression discrepancies of these sequences across different samples. By analyzing the conserved characteristics and variation patterns of CDR3 sequences between the two species, this study provides a molecular basis for clarifying the mechanisms underlying species-specific immune responses. Results Data processing and quality control of TRD CDR3 repertoires Raw sequence data of TRD CDR3 repertoires from 12 samples (7 cattle samples and 5 buffalo samples) were processed, with sequence alignment and quality control carried out using MiXCR. After a series of processes including low-quality sequence filtering and sequence trimming, high-quality CDR3 clonotype data were generated and used for subsequent analyses (Table 1 ). Table 1 Summary statistics of TRD CDR3 repertoire for individual Buffalo and Cattle samples. Species Sample Total reads Successfully aligned reads Reads used in clonotypes Final clonotype count TRD chains Cattle C1 8154462 7423646 (91.04%) 5654847 (69.35%) 369414 368360 (99.71%) C2 8107152 6099183 (75.23%) 4119621 (50.81%) 190206 189961 (99.87%) C3 8137606 6608920 (81.21%) 4924724 (60.52%) 406037 405571 (99.89%) C4 8119303 7440230 (91.64%) 6297407 (77.56%) 374205 373569 (99.83%) C5 8063013 7277426 (90.26%) 6247908 (77.49%) 446402 445883 (99.88%) C6 8031451 7447628 (92.73%) 5755381 (71.66%) 161129 160483 (99.6%) C7 8116415 6568898 (80.93%) 4803053 (59.18%) 402179 400587 (99.6%) Buffalo B1 8115425 5401709 (66.56%) 3100477 (38.2%) 113358 113358 (100%) B2 8075301 6940735 (85.95%) 5726841(70.92%) 335655 335655 (100%) B3 8057834 4125104 (51.19%) 1901781 (23.6%) 165452 165452 (100%) B4 8003251 7016187 (87.67%) 5758459 (71.95%) 296555 296555 (100%) B5 8075327 6669232 (82.59%) 5784335 (71.63%) 442366 442366 (100%) TRD represents the T-cell receptor delta chain; C denotes cattle samples (n = 7); B denotes buffalo samples (n = 5). Raw sequencing reads of all samples exceeded 8,000,000, with the cattle group ranging from 8,031,451 to 8,154,462 and the buffalo group from 8,003,251 to 8,115,425, demonstrating comparable initial sequencing depth between the two groups. The final clonotype counts of the cattle group spanned from 161,129 to 446,402, while those of the buffalo group ranged from 113,358 to 442,366. A relatively large intra-group variation was observed, and the clonotype counts of the cattle group were slightly higher than those of the buffalo group. Notably, the proportion of TRD chains reached 100% in all buffalo samples, whereas the corresponding proportion in the cattle group ranged from 99.6% to 99.89%. This discrepancy was primarily due to the difference in background reference libraries applied for the two groups: the reference library for buffalo was constructed in-house based on the germline genes annotated by our research team, while the library for cattle was directly adopted from the one provided by IMGT. Therefore, a small number of TRA chains were detected in the cattle group, which is consistent with the characteristic nested structure of the mammalian TRA/D gene locus where TRA and TRD chains are located on the same chromosome. Clonotype and diversity analysis of TCR δ CDR3 repertoires Statistical analysis of the unique clonotype counts in the buffalo and cattle groups showed that the cattle group had 71,990 − 170,0067 unique clonotypes while the buffalo group had 51,202 − 150,363. The number of unique clonotypes in the cattle group was slightly higher than that in the buffalo group, with significant intra-group variation observed. However, no statistically significant difference was detected between the two groups (Fig. 1 A).The diversity of TCR δ CDR3 repertoires in the two groups was evaluated using three metrics, namely the D50 index(Fig. 1 B), Hill index(Fig. 1 C), and Gini-Simpson index(Fig. 1 D). The results consistently demonstrated that the TCR δ CDR3 repertoire diversity of the cattle group was higher than that of the buffalo group. Specifically, the higher D50 index of the cattle group indicated a more uniform clonotype distribution, while the Hill index and Gini-Simpson index further verified that the cattle group had a more even clonotype distribution and richer diversity. Subsequent analyses were performed to characterize the proportion of rare clonotypes and top clonotypes in the two groups. For rare clonotypes, both the cattle and buffalo groups exhibited a low proportion of low-frequency clonotypes, with clonotypes present in fewer than 30 copies accounting for less than 10% of the total in each group(Fig. 1 E). Moreover, the two groups shared a highly similar distribution pattern of clonotypes across different abundance levels (Fig. 1 F). Regarding top clonotypes, the top 10 most abundant clonotypes accounted for a higher proportion in the buffalo group than in the cattle group(Fig. 1 G), and the cumulative proportion of the top 100 clonotypes exceeded 50% in both groups (Fig. 1 H). These findings indicated that both buffalo and cattle repertoires were dominated by medium-to-high frequency clonotypes, with the buffalo group relying more heavily on a small number of highly abundant clonotypes. The result was consistent with the aforementioned conclusion, further confirming that the cattle group had a more uniform clonotype distribution and higher repertoire diversity. V and J gene usage and correlation analysis of TCR δ CDR3 repertoires IMGT has documented 55 TRDV genes in cattle, among which 52 were detected in this experiment. Meanwhile, 57 V genes were identified in this study out of the 65 annotated V genes in buffalo. Analysis of V gene usage frequencies showed that most V genes were utilized at low frequencies. In the buffalo group, TRDV1-13 exhibited the highest usage frequency (nearing 10%), which was significantly higher than that in the cattle group. In contrast, the cattle group preferentially used TRDV1-1, TRDV1-15 and TRDV1-33 at high frequencies, with TRDV1-33 having the highest frequency (approximately 9%); the usage frequencies of these three genes were all significantly higher than those in the buffalo group. Several V genes, such as TRDV1-10, TRDV1-12, TRDV1-18 and TRDV1-2, were expressed at low frequencies in both groups, yet significant inter-group differences were still observed (Fig. 2 A). Collectively, V gene usage displayed distinct species-specific preferences between the two groups. Correlation analysis of V gene usage indicated a strong positive correlation among samples within each group, whereas the inter-group correlation was weak. These results demonstrated that V gene usage differed between groups and possessed species-specific characteristics (Fig. 2 B). Four TRDJ genes of cattle have been recorded in IMGT, and our research team annotated four TRDJ genes in buffalo. The same four TRDJ genes were detected in both cattle and buffalo, and all of them were expressed across all samples. Analysis of J gene usage frequencies revealed that both groups predominantly utilized TRDJ1 and TRDJ4 at high frequencies. Among these, TRDJ1 was the most frequently used (close to 60%), and no significant difference was found between the two groups. TRDJ2 and TRDJ3 were expressed at low frequencies in both groups but still showed significant inter-group differences (Fig. 2 C). Correlation analysis of J gene usage showed a strong positive correlation both within and between the two groups, suggesting that J gene usage was not species-specific and was highly conserved across species (Fig. 2 D). Length distribution, K-mers and motif sequence analysis of TCR δ CDR3 repertoires Analysis revealed that the CDR3 length distribution of both groups exhibited a highly consistent bell-shaped pattern, with lengths mainly concentrated between 15 and 30 amino acids and a peak at 23 amino acids for each group. No significant difference was observed in clonotype abundance across different lengths between the two groups (Fig. 3 A). These results indicated that the CDR3 length distribution was conserved between the two species. To further analyze the K-mers of the TCR δ CDR3 repertoires, the top 10 high-frequency 5-mer sequences were selected for frequency analysis. The results showed that the buffalo and cattle groups shared some K-mers while also displaying species-specific preferences. Among all 12 samples, DKLIF and TDKLI were the most frequently utilized K-mers. In the analysis of the top 10 high-frequency K-mers, QYPLI and YPLIF were predominantly present in the buffalo group, with both sequences accounting for over 30% of the total in buffalo5; by contrast, NPLIF and QNPLI were mainly detected in the cattle group, with each sequence representing more than 30% of the total in cattle4. The remaining K-mers showed minimal differences between the two groups (Fig. 3 B). Subsequently, motif sequence analysis was performed based on amino acid composition. It was found that polar amino acids and hydrophobic amino acids exhibited relatively high frequencies in the core regions, which constituted the basic amino acid composition characteristics of CDR3 sequences. Amino acids including glycine (G), arginine (R), leucine (L) and valine (V)-which belong to polar, basic and hydrophobic categories-were detected in all samples, with G showing the highest frequency. In addition, the proportion of V at position 2 was higher in the buffalo group than in the cattle group, and individual differences were observed in the proportion of L at position 3 within the buffalo group (Fig. 3 C). These findings suggested that the order and arrangement of amino acid sequences differed significantly between species and among individuals. Consensus amino acid sequences and Overlap analysis of TCR δ CDR3 repertoire Analysis of conserved amino acid sequences in the TCR δ CDR3 repertoires of the two groups showed that 1,168 unique sequences were identified in the cattle group and 615 in the buffalo group, with 1,194 sequences shared between the two groups (Fig. 4 A). Analysis of conserved clonotype overlap revealed that the extent of clonotype sharing among individuals within the cattle group was higher than that within the buffalo group. For instance, the overlap values between cattle4 and cattle6, as well as between cattle5 and cattle6, exceeded 2,000. By contrast, only a few individual pairs, such as buffalo4 and buffalo1, exhibited relatively high overlap in the buffalo group. When comparing between the two groups, the overall overlap values were low, but several cross-group pairs, including buffalo1 and cattle6, buffalo4 and cattle6, and buffalo4 and cattle4, showed higher overlap values than those observed within either group (Fig. 4 B). Analysis of shared amino acid sequences across samples demonstrated that 18,259 sequences were shared by 2 out of the 12 samples, whereas only 4 sequences were conserved across 11 samples (Fig. 4 C). Within the buffalo group, 2,894 sequences were shared by 2 samples, while merely 13 sequences were present in all 5 samples (Fig. 4 D). In the cattle group, 8,779 sequences were shared by 2 samples, and only 24 sequences were detected across all 7 samples (Fig. 4 E). Tracking of the top five most frequently shared amino acid sequences across all samples indicated that buffalo1, buffalo4, cattle4 and cattle6 had relatively high numbers of shared sequences, with CATHLHGEQSGKLIF being the most prevalent shared sequence (Fig. 4 F). When focusing on the top five most frequently shared sequences within the buffalo group, buffalo1, buffalo4, cattle4 and cattle6 were again found to harbor abundant shared sequences, and CATHLHGEQSGKLIF remained the most dominant shared sequence (Fig. 4 G). For the cattle group-specific top five most frequently shared sequences, cattle1, cattle2 and buffalo4 showed high levels of sequence sharing, with CATMDHSRARGTQTDKVIF identified as the most commonly shared sequence (Fig. 4 H). These results suggested that highly abundant shared clonotypes were enriched only in a small subset of individuals. Discussion γδ T cells account for an extremely high proportion in ruminants, a feature that is significantly different from that observed in other γδ low species such as humans and mice. Early studies have indicated that γδ T cells in ruminants including sheep and cattle represent the dominant subsets in both peripheral blood and tissues, a characteristic that may reflect the unique surveillance requirements of the ruminant immune system in response to external stimuli[ 3 , 4 , 15 ]. In addition, γδ TCRs are capable of recognizing non-classical antigens and acting as a bridge between innate and adaptive immunity, endowing these cells with a critical role in the rapid response to pathogenic challenges[ 3 ]. This study systematically compared the characteristics of TCRδ CDR3 immune repertoires between two bovine species, cattle and buffalo. Multiple dimensions including clonal diversity, V(D)J gene usage, CDR3 structural features and shared clonotypes were investigated, which revealed remarkable differences between the two species against the backdrop of their highly similar γδ T cell populations. Overall, although both species exhibited a TCR δ CDR3 repertoire architecture dominated by medium-to-high frequency clonotypes, the clonotype distribution in cattle was more homogeneous, with an overall diversity level higher than that in buffalo. These observations suggest that γδ T cells of different bovine species may adopt distinct immune strategies to combat external antigen stimulation, even when they occupy similar immune niches. The basic structure and composition of the TRD locus are the root causes of the complexity of the TRD repertoire. In bovids, the TRD gene pool is extremely large, containing a large number of TRDV1 subgroup genes and multiple D, J genes, a phenomenon of amplification that is prevalent in γδ high species[ 7 , 16 ]. Specifically, the TRD locus exhibits abundant TRAV/TRDV members, including different subgroups such as TRDV1, TRDV2, TRDV3 and TRDV4, in the annotation of multiple genome versions, along with multiple TRDD and TRDJ segments. These provide a rich genetic basis for high CDR3 diversity[ 16 , 17 ]. Such gene amplification is not only prominent in cattle but also shows a similar pattern in other ruminants like sheep, indicating an evolutionary commonality in the topological expansion of TRDV1 among ruminant species[ 7 ]. In this study, we observed that the cattle group used a total of 52 V genes, among which 47 belonged to the V1 family, accounting for 90.4%. In contrast, 57 V genes were detected in the buffalo group, with 53 from the V1 family, representing a proportion of 93.0%. This result further confirms the amplification characteristic of the TRDV1 subgroup in bovids. Notably, both the total number of TRDV genes used and the proportion of the V1 family in buffalo were slightly higher than those in cattle, suggesting that the TRD repertoire in buffalo may have richer diversity. Meanwhile, the V1 family genes dominated in both species, highlighting their important role in γδ T cell receptor assembly and conforming to the evolutionary common feature of TRDV1 topological expansion in ruminants. Additionally, studies have found that the expansion of TRDV1 + γδ T cells in ruminants may be attributed to the unique surveillance requirements of their immune systems [ 17 ]. In terms of V gene usage patterns, cattle and buffalo exhibited distinct species-specific preferences. Correlation analysis of TRDV usage between cattle and buffalo showed a high degree of consistency within each group but low correlation between the two groups, suggesting that TRDV gene usage is driven by species-specific regulatory mechanisms. Although both buffalo and cattle retain a large TRDV1 gene pool[ 17 ], the frequency and usage preference of their specific gene members are not completely consistent between the two species. This may reflect the different selective pressures experienced by the two species during long-term independent evolution and ecological adaptation. In addition, different V gene usage patterns may also be associated with their immune responses to the same pathogens and the outcomes of infection. In a Schistosoma japonicum infection model, after infection, cattle (Bos taurus) are more prone to developing hepatic white granulomas and significant inflammatory cell infiltration, whereas buffalo (Bubalus bubalis) exhibit relatively milder inflammatory responses[ 18 ]. Similarly, during Mycobacterium bovis infection, although both species can produce antigen-specific IFN‑γ and TNF‑α cytokine responses, differences in cytokine expression patterns have been observed between buffalo and cattle, indicating interspecific variations in immune responses[ 19 ]. In contrast to the species-specific differences observed in V gene usage, TRDJ genes exhibited higher conservation between cattle and buffalo. Both groups predominantly used TRDJ1 and TRDJ4, with an extremely high correlation across the two species. Such conservation in J gene usage also shows similar cross-species stability in the β chain locus of αβ TCR. Previous immune repertoire and comparative genomics analyses have demonstrated that TRBJ segments in TCR β repertoires of multiple mammalian species, including bank voles, mice and humans, display significant sequence conservation and play a critical role in shaping the terminal structure of CDR3[ 20 – 22 ]. This conservation may be attributed to the functional constraints of J genes in forming the structural framework of the CDR3 terminal region after V(D)J recombination. Specifically, J segments provide the structural backbone of the CDR3 region, enabling the variable region of the entire receptor to stably bind to D/V segments and maintain the overall three-dimensional conformation. This functional characteristic is supported by comparative analyses of gene loci across various species[ 22 ]. The TCR δ CDR3 length distribution in both cattle and buffalo showed a typical bell-shaped pattern, with lengths concentrated in the range of 15–30 amino acids. This range is wider than that of the TCR β chain CDR3 in αβ high species such as humans and mice, which usually falls between 12 and 15 amino acids[ 23 ]. This phenomenon may be attributed to the fact that the TRD gene pool of ruminants including cattle and sheep contains a large number of TRDV1 subgroups and multiple D/J genes, which provides a genetic basis for generating CDR3 regions with diverse and variable lengths[ 7 , 16 , 17 ]. For example, in sheep, the usage of different numbers of TRDD genes directly leads to a linear increase in CDR3 length, with the longest CDR3 even exceeding approximately 26 amino acids[ 7 ]. This mechanism may also apply to cattle and buffalo, enabling them to generate longer and more diverse CDR3 repertoires that can better adapt to the immune task of broad-spectrum antigen recognition. In this study, the number of shared amino acid sequences between the TRD CDR3 repertoires of cattle and buffalo was relatively small, and highly abundant shared clonotypes were only enriched among a small number of individuals. This phenomenon not only reflects the immunological essence of the γδ TCR repertoire but may also be associated with the differences accumulated between the two species during long-term evolution and genomic divergence. Previous studies have demonstrated that the human γδ T cell receptor repertoire exhibits a prominent private clonotype dominance among different individuals. In contrast, public clonotypes shared across individuals are mostly associated with the relatively conserved rearrangement structures in Vδ2 + γδ T cells, and their occurrence tends to be related to generation probability, V(D)J recombination preferences and expansion status rather than driven by a single antigen. For example, deep sequencing data have indicated that multiple public clonotypes exist in adult Vγ9Vδ2 + repertoires, whereas other γδ subsets such as Vδ1 + are dominated by private clonotypes[ 9 , 24 , 25 ]. In addition, cattle (Bos taurus) and buffalo (Bubalus bubalis) both belong to the tribe Bovini, yet their genomes exhibit significant differences in phylogenetic history and chromosomal structure. Comparative genomics studies have shown that the two species have undergone multiple structural chromosomal rearrangement events since diverging from their common ancestor. These genomic changes help to explain the potential differences in rearrangement preferences near specific gene loci[ 26 , 27 ]. Finally, the divergence time between cattle and buffalo can be traced back to millions of years ago. The two species have experienced distinct natural and artificial selection pressures under their respective ecological and domestication backgrounds, which may fundamentally affect the rearrangement tendency and generation probability distribution of the TRD locus[ 28 ]. Conclusions In summary, the present study comprehensively characterized the species-specific differences in the TRD immune repertoires of cattle and buffalo from multiple perspectives, including repertoire architecture, clonotype distribution, diversity indices, V/J gene usage and sequence features. Moreover, an interpretive framework was provided by integrating existing studies on the TRD genome and its functions in ruminants. These findings not only enrich the understanding of the mechanisms underlying immune diversity in γδ high species but also lay a foundation for further investigations into the roles of γδ T cells in pathogen recognition, vaccine responses and species-specific immune adaptability. Abbreviations CDR3 Complementarity-determining region 3 TRD T cell receptor delta chain TCR T cell receptor MHC Major histocompatibility complex HTS High-throughput sequencing TRG TCR γ chain PCR Polymerase Chain Reaction Declarations Ethics approval and consent to participate This study was approved by the Ethics Committee of Zunyi Medical University (Approval No. ZMU21-2203-111). All procedures involving animals were performed in accordance with institutional and national guidelines. Consent for publication Not applicable Competing Interests Authors declare that they have no competing interests. Funding The Science and Technology Program of Guizhou Province (ZK[2023]) and Zunyi Medical University Doctoral Initiation Fund ([F950]) funded this study. Author Contribution Y.Z. and F.W. contributed equally to this work and share first authorship. J.L. and X.Y. designed the study, Y.Z., F.W.,performed the experiments, and analyzed the data. L.M. assisted with data analysis and figure preparation. J.L. supervised the study, provided resources, and wrote the manuscript. All authors read and approved the final manuscript. Acknowledgement We thank to IMGT for sharing the full VDJC annotation for cattle. Data Availability The data supporting the findings of this study have been deposited in Zenodo and are openly available at https://zenodo.org/records/18346729. The raw sequencing data have been uploaded to the NCBI with BioProject accession number: PRJNA1417106. References Yirsaw A, Baldwin CL. Goat gammadelta T cells. Dev Comp Immunol. 2021;114:103809. Guzman E, Price S, Poulsom H, Hope J. Bovine gammadelta T cells: cells with multiple functions and important roles in immunity. Vet Immunol Immunopathol. 2012;148(1–2):161–7. Guerra-Maupome M, Slate JR, McGill JL. Gamma Delta T Cell Function in Ruminants. Vet Clin North Am Food Anim Pract. 2019;35(3):453–69. Baldwin CL, Damani-Yokota P, Yirsaw A, Loonie K, Teixeira AF, Gillespie A. Special features of gammadelta T cells in ruminants. Mol Immunol. 2021;134:161–9. Chen H, Zou M, Teng D, Zhang J, He W. Characterization of the diversity of T cell receptor gammadelta complementary determinant region 3 in human peripheral blood by Immune Repertoire Sequencing. J Immunol Methods. 2017;443:9–17. Suen TK, Al B, Scarpa A, Dorhoi A, Netea MG, Placek K. A dual nature of gammadelta T cell immune memory responses. Elife 2025, 14. Piccinni B, Massari S, Caputi Jambrenghi A, Giannico F, Lefranc MP, Ciccarese S, Antonacci R. Sheep (Ovis aries) T cell receptor alpha (TRA) and delta (TRD) genes and genomic organization of the TRA/TRD locus. BMC Genomics. 2015;16:709. Herzig CT, Blumerman SL, Baldwin CL. Identification of three new bovine T-cell receptor delta variable gene subgroups expressed by peripheral blood T cells. Immunogenetics. 2006;58(9):746–57. Deng L, Harms A, Ravens S, Prinz I, Tan L. Systematic pattern analyses of Vdelta2(+) TCRs reveal that shared public Vdelta2(+) gammadelta T cell clones are a consequence of rearrangement bias and a higher expansion status. Front Immunol. 2022;13:960920. Kallemeijn MJ, Kavelaars FG, van der Klift MY, Wolvers-Tettero ILM, Valk PJM, van Dongen JJM, Langerak AW. Next-Generation Sequencing Analysis of the Human TCRgammadelta + T-Cell Repertoire Reveals Shifts in Vgamma- and Vdelta-Usage in Memory Populations upon Aging. Front Immunol. 2018;9:448. Huda TI, Nguyen D, Sahoo A, Song JJ, Gutierrez AF, Chobrutskiy BI, Blanck G. Adaptive Immune Receptor Distinctions Along the Colorectal Polyp-Tumor Timelapse. Clin Colorectal Cancer. 2024;23(4):402–11. Yuan C, Wang B, Wang H, Wang F, Li X, Zhen Y. T-cell receptor dynamics in digestive system cancers: a multi-layer machine learning approach for tumor diagnosis and staging. Front Immunol. 2025;16:1556165. Antonacci R, Mineccia M, Lefranc MP, Ashmaoui HM, Lanave C, Piccinni B, Pesole G, Hassanane MS, Massari S, Ciccarese S. Expression and genomic analyses of Camelus dromedarius T cell receptor delta (TRD) genes reveal a variable domain repertoire enlargement due to CDR3 diversification and somatic mutation. Mol Immunol. 2011;48(12–13):1384–96. Ciccarese S, Lefranc MP, Perrone GCM, D'Addabbo P, Pierri CL. Three-Dimensional Modeling of Camelus dromedarius T Cell Receptor Gamma (TRG)_Delta (TRD)/CD1D Complex Reveals Different Binding Interactions Depending on the TRD CDR3 Length. Antibodies (Basel) 2025, 14(2). Hein WR, Mackay CR. Prominence of gamma delta T cells in the ruminant immune system. Immunol Today. 1991;12(1):30–4. Herzig CT, Lefranc MP, Baldwin CL. Annotation and classification of the bovine T cell receptor delta genes. BMC Genomics. 2010;11:100. Connelley TK, Degnan K, Longhi CW, Morrison WI. Genomic analysis offers insights into the evolution of the bovine TRA/TRD locus. BMC Genomics. 2014;15(1):994. Yang J, Fu Z, Hong Y, Wu H, Jin Y, Zhu C, Li H, Lu K, Shi Y, Yuan C, et al. The Differential Expression of Immune Genes between Water Buffalo and Yellow Cattle Determines Species-Specific Susceptibility to Schistosoma japonicum Infection. PLoS ONE. 2015;10(6):e0130344. Flores-Villalva S, De Matteis G, Grandoni F, Scata MC, Donniacuo A, Schiavo L, Franzoni G, Mazzone P, Elnaggar M, De Carlo E, et al. Polyfunctionality of CD4(+) T lymphocytes in buffaloes and cattle: comparative antigen-specific cytokine responses in bovine tuberculosis infection. Front Immunol. 2025;16:1608065. Migalska M, Sebastian A, Radwan J. Profiling of the TCRbeta repertoire in non-model species using high-throughput sequencing. Sci Rep. 2018;8(1):11613. Glusman G, Rowen L, Lee I, Boysen C, Roach JC, Smit AF, Wang K, Koop BF, Hood L. Comparative genomics of the human and mouse T cell receptor loci. Immunity. 2001;15(3):337–49. Freeman JD, Warren RL, Webb JR, Nelson BH, Holt RA. Profiling the T-cell receptor beta-chain repertoire by massively parallel sequencing. Genome Res. 2009;19(10):1817–24. Hui L, Wu F, Xu Y, Yang G, Luo Q, Li Y, Ma L, Yao X, Li J. The T-cell receptor beta chain CDR3 insights of bovine liver immune repertoire under heat stress. Anim Biosci. 2024;37(12):2178–88. Fichtner AS, Ravens S, Prinz I. Human gammadelta TCR Repertoires in Health and Disease. Cells 2020, 9(4). Okamoto M, Motomura H. Navigobius asayake, a new species of ptereleotrine goby (Gobioidei: Microdesmidae) from Kagoshima, southern Japan. Zootaxa. 2018;4526(3):373–80. Pistucci R, Cascone I, Iannuzzi A, Albarella S, Kowal-Mierzwa W, Zannotti M, Iannuzzi L, Parma P. Comparative analysis of cattle (Bos taurus, 2n = 60) and river buffalo (Bubalus bubalis, 2n = 50) genome assemblies reveals two evolutionary conserved inversions and invalid centromere-telomere orientation of some autosomes. Anim Genet. 2025;56(4):e70031. Li W, Bickhart DM, Ramunno L, Iamartino D, Williams JL, Liu GE. Genomic structural differences between cattle and River Buffalo identified through comparative genomic and transcriptomic analysis. Data Brief. 2018;19:236–9. Santhosh A, Vohra V, Gandham RK, Alex R, Gowane G. De-novo assembled mitochondrial genome of Bhadawari buffalo (Bubalus bubalis) reveals close divergence to Egyptian buffalo. BMC Vet Res. 2025;21(1):202. Additional Declarations No competing interests reported. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8686230","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":617605580,"identity":"16b38755-f6ae-4664-ba70-dbe2bae8b2eb","order_by":0,"name":"Yueheng Zhang","email":"","orcid":"","institution":"Zunyi Medical University","correspondingAuthor":false,"prefix":"","firstName":"Yueheng","middleName":"","lastName":"Zhang","suffix":""},{"id":617605581,"identity":"2da22b7c-8932-46f4-b6f0-868718dd37fa","order_by":1,"name":"Fengli Wu","email":"","orcid":"","institution":"Zunyi Medical University","correspondingAuthor":false,"prefix":"","firstName":"Fengli","middleName":"","lastName":"Wu","suffix":""},{"id":617605582,"identity":"c32de0ef-7e00-4227-abc5-b37a3efc3921","order_by":2,"name":"Long Ma","email":"","orcid":"","institution":"Zunyi Medical University","correspondingAuthor":false,"prefix":"","firstName":"Long","middleName":"","lastName":"Ma","suffix":""},{"id":617605583,"identity":"4fe0d6e9-a6d6-4478-b40e-bf70b04458d3","order_by":3,"name":"Xinsheng Yao","email":"","orcid":"","institution":"Zunyi Medical University","correspondingAuthor":false,"prefix":"","firstName":"Xinsheng","middleName":"","lastName":"Yao","suffix":""},{"id":617605584,"identity":"b69c5365-f206-46dd-9c83-524bb349e8ac","order_by":4,"name":"Jun Li","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAx0lEQVRIiWNgGAWjYBACAzBZIcHD2N7Y+PAD8VrO2Mgw9xxuNpYgWgtjW5oN+4z0NgEeYrSYSyQf/szDdpiHd+bDNgYJBjs53QYCWixnpKVJ8/Ac5pGcndj2oIAh2djsACGH3cgxY+aROMxjODux3UCC4UDiNiK0GH/mMTjMY3/zYJsED5FaDKR5EtJ4GGcwEqvlzLM0yTkHbHgYexKBgWxAjF+OJx/+8PafhD1j+/GHDz9U2MkR1IJuAmnKR8EoGAWjYBTgAAChMUFbolDLwwAAAABJRU5ErkJggg==","orcid":"","institution":"Zunyi Medical University","correspondingAuthor":true,"prefix":"","firstName":"Jun","middleName":"","lastName":"Li","suffix":""}],"badges":[],"createdAt":"2026-01-24 11:23:38","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8686230/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8686230/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":106355351,"identity":"c97e16c6-da0c-47bf-ad11-235558ccb404","added_by":"auto","created_at":"2026-04-07 18:29:42","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":281255,"visible":true,"origin":"","legend":"\u003cp\u003eClonotype and diversity of TRD chains between the cattle and buffalo groups.\u003c/p\u003e\n\u003cp\u003e(A) Differential analysis of unique clonotypes between cattle and buffalo; (B) the estimation of repertoire diversity utilized in D50 index; (C) the estimation of repertoire diversity utilized in Gini-Simpson index; (D) the estimation of repertoire diversity utilized in Hill index; (E) the rare clonal proportion; (F) the difference of rare clonal proportion with clonotype counts between the cattle and buffalo groups; (G) the top clonal proportion; (H) the difference of top clonal proportion with clonotype indices between the cattle and buffalo groups.\u003c/p\u003e","description":"","filename":"OnlineFigure1.png","url":"https://assets-eu.researchsquare.com/files/rs-8686230/v1/89054707a68488a4a7de2d9a.png"},{"id":106404542,"identity":"17ec26ff-f0f0-445e-8fb6-8a1af12b9c2c","added_by":"auto","created_at":"2026-04-08 09:16:12","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":279623,"visible":true,"origin":"","legend":"\u003cp\u003eThe usage and correlation of V and J genes of the TCR δ CDR3 repertoires.\u003c/p\u003e\n\u003cp\u003e(A) statistical analysis of the V gene usage between the cattle and buffalo groups; (B) the correlation of V gene usage among all samples; (C) statistical analysis of the J gene usage between the cattle and buffalo groups; (D) the correlation of J gene usage among all samples.(Kruskal-wallis H Test, * represents P\u0026lt;0.05, ** represents P\u0026lt;0.01, *** represents P\u0026lt;0.001)\u003c/p\u003e","description":"","filename":"OnlineFigure2.png","url":"https://assets-eu.researchsquare.com/files/rs-8686230/v1/f1361dbee0aa7ddcd0c16984.png"},{"id":106404498,"identity":"b9525fa8-4ac7-410d-bf59-d997e5c21882","added_by":"auto","created_at":"2026-04-08 09:16:07","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":362884,"visible":true,"origin":"","legend":"\u003cp\u003eThe length distribution, Kmers and motif sequences of the TCR δ CDR3 repertoires.\u003c/p\u003e\n\u003cp\u003e(A) the length distribution of CDR3; (B) the distribution of top 10 5-mers in sequence length; (C) position self-information matrix from sequence motif analysis.\u003c/p\u003e","description":"","filename":"OnlineFigure3.png","url":"https://assets-eu.researchsquare.com/files/rs-8686230/v1/e269caf1de7050512c3907a3.png"},{"id":106355355,"identity":"37260ec6-1ebb-408a-bf5a-ad5d4c8f3547","added_by":"auto","created_at":"2026-04-07 18:29:42","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":302171,"visible":true,"origin":"","legend":"\u003cp\u003eThe shared amino acid sequence and repertoire overlap of the TCR δ CDR3 repertoires.\u003c/p\u003e\n\u003cp\u003e(A) number of shared and amino acid aequences between cuffalo and cattle; (B) heatmap of TRD repertoire overlap; (C) number of shared amino acid aequences among all samples across different numbers of shared groups; (D) number of shared amino acid aequences in the buffalo groups across different numbers of shared groups; (E) number of shared amino acid aequences in the cattle groups across different numbers of shared groups; (F) tracking analysis of shared clones among all samples; (G) tracking analysis of shared clones for the top 5 amino acid sequences in the buffalo groups; (H) tracking analysis of shared clones for the top 5 amino acid sequences in the cattle groups.\u003c/p\u003e","description":"","filename":"OnlineFigure4.png","url":"https://assets-eu.researchsquare.com/files/rs-8686230/v1/93c7767702a5cf1766bc57d5.png"},{"id":106974501,"identity":"da349f32-6e94-4ea5-b261-d6c047d8f672","added_by":"auto","created_at":"2026-04-15 10:32:45","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2566627,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8686230/v1/dd158b2c-b53b-4d80-9fc1-96a4cb600ec1.pdf"},{"id":106404399,"identity":"0e05f357-d37f-4639-9960-ecb34c20a917","added_by":"auto","created_at":"2026-04-08 09:15:56","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":16329,"visible":true,"origin":"","legend":"","description":"","filename":"Additionalfile1.docx","url":"https://assets-eu.researchsquare.com/files/rs-8686230/v1/2e4ce052cb41b0c452308cb0.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"High-throughput profiling of the T cell receptor delta CDR3 repertoire reveals species-specific patterns in cattle (Bos taurus) and water buffalo(Bubalus bubalis)","fulltext":[{"header":"Background","content":"\u003cp\u003eT cells are broadly classified into two major subsets, γδ T cells and αβ T cells, based on the composition of their antigen receptors. αβ T cells represent the central effector population of the adaptive immune system, whereas γδ T cells possess characteristics of both innate and adaptive immunity and are therefore regarded as \u0026ldquo;bridging cells\u0026rdquo; between the two immune arms[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. The γδ T cell receptor (TCR) enables γδ T cells to recognize non-peptidic and stress-induced antigens in a major histocompatibility complex (MHC)-independent manner, allowing them to function as rapid immune sentinels at the interface of innate and adaptive immunity[\u003cspan additionalcitationids=\"CR3\" citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Although γδ T cells constitute a minor fraction of peripheral lymphocytes in most species, they represent a dominant lymphocyte subset in cattle, accounting for approximately 60% of circulating T cells in neonatal calves[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. This striking species-specific difference in γδ T cell abundance suggests that bovine γδ T cells may fulfill immune surveillance and defense functions distinct from those observed in other mammals. The functional competence of γδ T cells is largely dependent on their TCR repertoire, particularly the highly diverse complementarity-determining region 3 (CDR3) of the δ chain (TRD), which plays a critical role in antigen recognition and the sensing of tissue stress signals [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eWith the advancement of high-throughput sequencing (HTS) technologies, research focusing on the γδ TCR repertoire, especially the CDR3 repertoire of the TRD, has garnered increasing attention[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. At the functional level, the length, sequence composition, and junctional diversity of the TRD CDR3 region are recognized as critical determinants governing the antigen recognition specificity of γδ T cells. The TRD CDR3 regions of ruminants typically exhibit greater length and structural diversity. Moreover, their V\u0026ndash;D\u0026ndash;J recombination patterns and nucleotide insertion/deletion profiles exert a profound impact on the spatial conformation of CDR3, thereby shaping a unique antigen-binding interface[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. In humans, the diversity of the TRD CDR3 repertoire is markedly higher than that of the TCR γ chain (TRG) repertoire, with a low degree of sharing among individuals, which reflects the central role of the δ chain in the functional differentiation of γδ T cells[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Furthermore, the V, D, J gene usage and CDR3 characteristics of the TRD repertoire undergo dynamic alterations across distinct developmental stages and immune statuses, indicating that the TRD repertoire is synergistically regulated by both developmental programs and antigen selection pressure[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eRecent studies have further demonstrated that the characteristics of the TRD repertoire hold substantial biological significance in tumor-associated immunity. In solid tumors such as colorectal cancer, the number of functional TRD-rearranged reads decreases markedly during the transition from normal tissues to tumor tissues, suggesting that γδ T cells may be involved in the immune surveillance process during the early stages of tumorigenesis[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Meanwhile, the physicochemical properties of TRD CDR3 amino acids are closely correlated with tumor progression stages, implying that the structural attributes of TRD CDR3 may modulate the anti-tumor functions of γδ T cells[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. In other digestive system tumors, both the TRD repertoire and the global TCR repertoire exhibit distinct tissue specificity and disease-associated recombination biases, reflecting the functional heterogeneity of γδ T cells in different tumor immune microenvironments[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. These findings further highlight the TRD repertoire as a critical entry point for elucidating the functional status and immune regulatory mechanisms of γδ T cells.\u003c/p\u003e \u003cp\u003eAlthough preliminary investigations into the TRD repertoire have been conducted in humans, mice, sheep, camels, and other species[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e], research focusing on the TRD CDR3 diversity, V(D)J gene usage characteristics, and repertoire structural divergence remains remarkably limited in bovids-particularly in cattle and buffalo, two species that exhibit high similarities in γδ T cell proportions, immune niches, and evolutionary backgrounds yet possess distinct phylogenetic differences. Therefore, systematic characterization of the TRD repertoire features in cattle and buffalo will not only deepen the understanding of γδ T cell immune diversity and evolutionary rules in ruminants but also provide critical fundamental data to elucidate the specific immune functions of γδ T cells in species with high γδ T cell abundances.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eSample collection and DNA extraction\u003c/h2\u003e \u003cp\u003eWe collected a total of 12 samples in this study, including spleen tissues from 5 healthy adult buffalo and 7 healthy adult cattle. All samples were obtained from a commercial abattoir located in Nanning, Guangxi, China. Immediately after collection, the spleen tissues were snap-frozen in liquid nitrogen to prevent degradation. Genomic DNA was extracted using the DNeasy Blood \u0026amp; Tissue Kit (QIAGEN) following the instructions of the manufacturer. We evaluated the integrity, concentration, and quality of the extracted DNA via 1% agarose gel electrophoresis and an Agilent 2100 Bioanalyzer. Then, Qualified DNA samples were aliquoted and stored at -20\u0026deg;C until subsequent experiments.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003ePrimer design and synthesis\u003c/h3\u003e\n\u003cp\u003eBased on the annotation results of the TRD locus,we designed amplification primers targeting the TCR δ CDR3 repertoire for cattle and buffalo, respectively. All forward primers were anchored in the conserved terminal regions of TRDV genes; a total of 5 gene-specific forward primers were designed, corresponding to the TRDV1-TRDV5 subgroups. Reverse primers were targeted to the conserved terminal regions of TRDJ genes, with 4 gene-specific reverse primers constructed to match the TRDJ1-TRDJ4 segments. All primers were synthesized by Sangon Biotech (Shanghai) Co., Ltd. Detailed sequences of the amplification primers are provided in Additional file 1.\u003c/p\u003e\n\u003ch3\u003eTCR δ CDR3 repertoire construction and sequencing\u003c/h3\u003e\n\u003cp\u003eMultiplex Polymerase Chain Reaction (PCR) was performed to amplify the target TCR δ CDR3 fragments. We prepared the PCR reaction system with a total volume of 50 \u0026micro;L, containing 25\u0026micro;L of PCR Master Mix, 2.8 \u0026micro;L each of the forward and reverse primer mixtures, 1\u0026micro;g of genomic DNA template, and the remaining volume was supplemented with ddH\u003csub\u003e2\u003c/sub\u003eO. The PCR protocol was set as follows, with an initial denaturation step at 94\u0026deg;C for 3 min, followed by 35 cycles of denaturation at 94\u0026deg;C for 1 min, annealing at 56\u0026deg;C for 1 min and extension at 72\u0026deg;C for 1 min, then a final extension at 72\u0026deg;C for 10 min and subsequent incubation at 4\u0026deg;C. The PCR products were analyzed by 2% agarose gel electrophoresis to assess the brightness and specificity of the target bands. The purified and recovered target fragments were sent to BGI Genomics Co., Ltd (BGI Genomics) for quality inspection. Qualified samples were subjected to HTS on the DNBseq platform.\u003c/p\u003e\n\u003ch3\u003eHTS data analysis of the TCR δ CDR3 repertoire\u003c/h3\u003e\n\u003cp\u003eAfter sequencing by BGI Genomics, raw sequence data of the TRD CDR3 repertoire were generated for all 12 samples. We uploaded the data in FASTQ format to MiXCR and aligned them against the V, D, J, and C genes in the background reference library, then outputted the CDR3 clonotype sequences of each sample. We calculated the total number of reads, the number of successfully aligned reads, and the number of reads used for clonotype construction for each sample. Non-functional sequences and sequences lacking canonical CDR3 start/stop residues were filtered out to determine the final number of TRD chains. The total number of unique CDR3 sequences obtained per sample after HTS met the required sequencing depth and were suitable for subsequent characteristic analysis of the CDR3 region.\u003c/p\u003e\n\u003ch3\u003eClonotype proportion distribution and diversity analysis\u003c/h3\u003e\n\u003cp\u003eAll statistical analyses were performed in the R 4.5.2 software environment, using major packages including immunarch, ggpubr, and ggplot2. Firstly, we calculated the number of unique clonotypes for both the cattle group and buffalo group, followed by the calculation of the proportions of top clonotypes and rare clonotypes. To comprehensively evaluate the diversity of the TRD repertoires between the two groups, multiple diversity metrics were used for further analysis, including D50 index, Gini-Simpson index and Hill index, so as to fully assess the richness and evenness of clonotypes in the two groups.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eV/J gene usage and correlation analysis\u003c/h2\u003e \u003cp\u003eBased on the data generated by MiXCR, the usage frequencies of V/J genes in each sample were calculated and expressed as percentages. For genes with significant differences, pairwise comparisons were performed using the Kruskal-Wallis H test, with P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 considered statistically significant. Subsequently, we analyzed the correlation of V/J gene usage within and between groups, applying the Spearman correlation coefficient to measure these associations and plotting heatmaps for visualization. The heatmaps were displayed according to the gradient of correlation coefficient values, which intuitively reflected the similarities in gene usage patterns between the two species and among samples.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eCDR3 amino acid length distribution\u003c/h3\u003e\n\u003cp\u003eLeveraging the data generated by MiXCR, we extracted all valid TCR δ CDR3 amino acid sequences and determined the amino acid length of each individual sequence.To minimize the impact of extreme values, only CDR3 sequences with lengths spanning 10 to 40 amino acids were retained, a filtering step that ensured the reliability of subsequent analyses.We quantified the distribution frequencies of CDR3 amino acid lengths across the two groups and then constructed length distribution histograms to visualize the variation trends directly. By comparing the peak values and distribution ranges of CDR3 lengths between the two groups, the fundamental structural characteristics that distinguish the CDR3 sequences of the two species were clearly revealed.\u003c/p\u003e\n\u003ch3\u003eK-mers and motif Analysis\u003c/h3\u003e\n\u003cp\u003eTo further explore the characteristic differences in CDR3 amino acid sequences, we selected 5-amino-acid short peptide fragments for analysis, counted the top 10 high-frequency fragments in each group, calculated their respective frequencies, and finally plotting stacked bar charts to compare inter-species disparities. Subsequently, motif analysis was performed based on amino acid composition, with the maximum number of motifs set to 5. By integrating the physicochemical properties of amino acids, such as hydrophobicity and polarity, we analyzed the composition characteristics, occurrence frequencies, and arrangement patterns of amino acids in different motifs, aiming to explore the potential association between species-specific motifs and immune functions.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eTCR δ CDR3 repertoire overlap analysis\u003c/h2\u003e \u003cp\u003eTo evaluate the overlap of TCR δ CDR3 repertoires among the 12 samples, we quantified the number of shared clonotypes, and further calculated the proportion and quantity of shared clonotypes between each pair of samples. Thereafter, we plotted a heatmap of inter-sample overlap to visually display the shared characteristics of clonotypes within and between groups. With UpSet plots generated via the UpSetR package, we presented the number of specific clonotypes unique to the cattle group, the buffalo group, and those common to both in the form of waterfall charts. In this way, the uniqueness and commonality of TRD repertoires between the two species were clearly identified, thereby providing evidence for revealing species-specific immune characteristics.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eClonotype tracking\u003c/h2\u003e \u003cp\u003eThree categories of amino acid sequences were selected for tracking analysis, including the 5 CDR3 amino acid sequences shared most frequently across all samples, the top 5 sequences with the highest shared abundance within the buffalo group, and the 5 most prevalent shared sequences in the cattle group. We quantified the clonal frequency and proportion of each target sequence in the corresponding sample set, and plotted bar charts of clonotype frequency distribution to compare expression discrepancies of these sequences across different samples. By analyzing the conserved characteristics and variation patterns of CDR3 sequences between the two species, this study provides a molecular basis for clarifying the mechanisms underlying species-specific immune responses.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\n \u003ch2\u003eData processing and quality control of TRD CDR3 repertoires\u003c/h2\u003e\n \u003cp\u003eRaw sequence data of TRD CDR3 repertoires from 12 samples (7 cattle samples and 5 buffalo samples) were processed, with sequence alignment and quality control carried out using MiXCR. After a series of processes including low-quality sequence filtering and sequence trimming, high-quality CDR3 clonotype data were generated and used for subsequent analyses (Table \u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u0026nbsp;\u003c/p\u003e\u0026nbsp;\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eSummary statistics of TRD CDR3 repertoire for individual Buffalo and Cattle samples.\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\" colname=\"c1\"\u003e\n \u003cp\u003eSpecies\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colname=\"c2\"\u003e\n \u003cp\u003eSample\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colname=\"c3\"\u003e\n \u003cp\u003eTotal reads\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colname=\"c4\"\u003e\n \u003cp\u003eSuccessfully aligned reads\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colname=\"c5\"\u003e\n \u003cp\u003eReads used in clonotypes\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colname=\"c6\"\u003e\n \u003cp\u003eFinal clonotype count\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colname=\"c7\"\u003e\n \u003cp\u003eTRD chains\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colname=\"c1\"\u003e\n \u003cp\u003eCattle\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c2\"\u003e\n \u003cp\u003eC1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\n \u003cp\u003e8154462\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\n \u003cp\u003e7423646 (91.04%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\n \u003cp\u003e5654847 (69.35%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\n \u003cp\u003e369414\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c7\"\u003e\n \u003cp\u003e368360 (99.71%)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c2\"\u003e\n \u003cp\u003eC2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\n \u003cp\u003e8107152\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\n \u003cp\u003e6099183 (75.23%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\n \u003cp\u003e4119621 (50.81%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\n \u003cp\u003e190206\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c7\"\u003e\n \u003cp\u003e189961 (99.87%)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c2\"\u003e\n \u003cp\u003eC3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\n \u003cp\u003e8137606\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\n \u003cp\u003e6608920 (81.21%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\n \u003cp\u003e4924724 (60.52%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\n \u003cp\u003e406037\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c7\"\u003e\n \u003cp\u003e405571 (99.89%)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c2\"\u003e\n \u003cp\u003eC4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\n \u003cp\u003e8119303\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\n \u003cp\u003e7440230 (91.64%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\n \u003cp\u003e6297407 (77.56%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\n \u003cp\u003e374205\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c7\"\u003e\n \u003cp\u003e373569 (99.83%)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c2\"\u003e\n \u003cp\u003eC5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\n \u003cp\u003e8063013\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\n \u003cp\u003e7277426 (90.26%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\n \u003cp\u003e6247908 (77.49%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\n \u003cp\u003e446402\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c7\"\u003e\n \u003cp\u003e445883 (99.88%)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c2\"\u003e\n \u003cp\u003eC6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\n \u003cp\u003e8031451\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\n \u003cp\u003e7447628 (92.73%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\n \u003cp\u003e5755381 (71.66%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\n \u003cp\u003e161129\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c7\"\u003e\n \u003cp\u003e160483 (99.6%)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c2\"\u003e\n \u003cp\u003eC7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\n \u003cp\u003e8116415\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\n \u003cp\u003e6568898 (80.93%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\n \u003cp\u003e4803053 (59.18%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\n \u003cp\u003e402179\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c7\"\u003e\n \u003cp\u003e400587 (99.6%)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colname=\"c1\"\u003e\n \u003cp\u003eBuffalo\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c2\"\u003e\n \u003cp\u003eB1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\n \u003cp\u003e8115425\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\n \u003cp\u003e5401709 (66.56%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\n \u003cp\u003e3100477 (38.2%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\n \u003cp\u003e113358\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c7\"\u003e\n \u003cp\u003e113358 (100%)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c2\"\u003e\n \u003cp\u003eB2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\n \u003cp\u003e8075301\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\n \u003cp\u003e6940735 (85.95%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\n \u003cp\u003e5726841(70.92%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\n \u003cp\u003e335655\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c7\"\u003e\n \u003cp\u003e335655 (100%)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c2\"\u003e\n \u003cp\u003eB3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\n \u003cp\u003e8057834\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\n \u003cp\u003e4125104 (51.19%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\n \u003cp\u003e1901781 (23.6%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\n \u003cp\u003e165452\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c7\"\u003e\n \u003cp\u003e165452 (100%)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c2\"\u003e\n \u003cp\u003eB4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\n \u003cp\u003e8003251\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\n \u003cp\u003e7016187 (87.67%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\n \u003cp\u003e5758459 (71.95%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\n \u003cp\u003e296555\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c7\"\u003e\n \u003cp\u003e296555 (100%)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c2\"\u003e\n \u003cp\u003eB5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\n \u003cp\u003e8075327\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\n \u003cp\u003e6669232 (82.59%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\n \u003cp\u003e5784335 (71.63%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\n \u003cp\u003e442366\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c7\"\u003e\n \u003cp\u003e442366 (100%)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003cp\u003e\u003c/p\u003e\n \u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n \u003cp\u003eTRD represents the T-cell receptor delta chain; C denotes cattle samples (n\u0026thinsp;=\u0026thinsp;7); B denotes buffalo samples (n\u0026thinsp;=\u0026thinsp;5).\u003c/p\u003e\n \u003cp\u003eRaw sequencing reads of all samples exceeded 8,000,000, with the cattle group ranging from 8,031,451 to 8,154,462 and the buffalo group from 8,003,251 to 8,115,425, demonstrating comparable initial sequencing depth between the two groups. The final clonotype counts of the cattle group spanned from 161,129 to 446,402, while those of the buffalo group ranged from 113,358 to 442,366. A relatively large intra-group variation was observed, and the clonotype counts of the cattle group were slightly higher than those of the buffalo group.\u003c/p\u003e\n \u003cp\u003eNotably, the proportion of TRD chains reached 100% in all buffalo samples, whereas the corresponding proportion in the cattle group ranged from 99.6% to 99.89%. This discrepancy was primarily due to the difference in background reference libraries applied for the two groups: the reference library for buffalo was constructed in-house based on the germline genes annotated by our research team, while the library for cattle was directly adopted from the one provided by IMGT. Therefore, a small number of TRA chains were detected in the cattle group, which is consistent with the characteristic nested structure of the mammalian TRA/D gene locus where TRA and TRD chains are located on the same chromosome.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\n \u003ch2\u003eClonotype and diversity analysis of TCR \u0026delta; CDR3 repertoires\u003c/h2\u003e\n \u003cp\u003eStatistical analysis of the unique clonotype counts in the buffalo and cattle groups showed that the cattle group had 71,990\u0026thinsp;\u0026minus;\u0026thinsp;170,0067 unique clonotypes while the buffalo group had 51,202\u0026thinsp;\u0026minus;\u0026thinsp;150,363. The number of unique clonotypes in the cattle group was slightly higher than that in the buffalo group, with significant intra-group variation observed. However, no statistically significant difference was detected between the two groups (Fig. \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA).The diversity of TCR \u0026delta; CDR3 repertoires in the two groups was evaluated using three metrics, namely the D50 index(Fig. \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB), Hill index(Fig. \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC), and Gini-Simpson index(Fig. \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). The results consistently demonstrated that the TCR \u0026delta; CDR3 repertoire diversity of the cattle group was higher than that of the buffalo group. Specifically, the higher D50 index of the cattle group indicated a more uniform clonotype distribution, while the Hill index and Gini-Simpson index further verified that the cattle group had a more even clonotype distribution and richer diversity.\u003c/p\u003e\n \u003cp\u003eSubsequent analyses were performed to characterize the proportion of rare clonotypes and top clonotypes in the two groups. For rare clonotypes, both the cattle and buffalo groups exhibited a low proportion of low-frequency clonotypes, with clonotypes present in fewer than 30 copies accounting for less than 10% of the total in each group(Fig. \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE). Moreover, the two groups shared a highly similar distribution pattern of clonotypes across different abundance levels (Fig. \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF). Regarding top clonotypes, the top 10 most abundant clonotypes accounted for a higher proportion in the buffalo group than in the cattle group(Fig. \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG), and the cumulative proportion of the top 100 clonotypes exceeded 50% in both groups (Fig. \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eH). These findings indicated that both buffalo and cattle repertoires were dominated by medium-to-high frequency clonotypes, with the buffalo group relying more heavily on a small number of highly abundant clonotypes. The result was consistent with the aforementioned conclusion, further confirming that the cattle group had a more uniform clonotype distribution and higher repertoire diversity.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\n \u003ch2\u003eV and J gene usage and correlation analysis of TCR \u0026delta; CDR3 repertoires\u003c/h2\u003e\n \u003cp\u003eIMGT has documented 55 TRDV genes in cattle, among which 52 were detected in this experiment. Meanwhile, 57 V genes were identified in this study out of the 65 annotated V genes in buffalo. Analysis of V gene usage frequencies showed that most V genes were utilized at low frequencies. In the buffalo group, TRDV1-13 exhibited the highest usage frequency (nearing 10%), which was significantly higher than that in the cattle group. In contrast, the cattle group preferentially used TRDV1-1, TRDV1-15 and TRDV1-33 at high frequencies, with TRDV1-33 having the highest frequency (approximately 9%); the usage frequencies of these three genes were all significantly higher than those in the buffalo group. Several V genes, such as TRDV1-10, TRDV1-12, TRDV1-18 and TRDV1-2, were expressed at low frequencies in both groups, yet significant inter-group differences were still observed (Fig. \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Collectively, V gene usage displayed distinct species-specific preferences between the two groups. Correlation analysis of V gene usage indicated a strong positive correlation among samples within each group, whereas the inter-group correlation was weak. These results demonstrated that V gene usage differed between groups and possessed species-specific characteristics (Fig. \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB).\u003c/p\u003e\n \u003cp\u003eFour TRDJ genes of cattle have been recorded in IMGT, and our research team annotated four TRDJ genes in buffalo. The same four TRDJ genes were detected in both cattle and buffalo, and all of them were expressed across all samples. Analysis of J gene usage frequencies revealed that both groups predominantly utilized TRDJ1 and TRDJ4 at high frequencies. Among these, TRDJ1 was the most frequently used (close to 60%), and no significant difference was found between the two groups. TRDJ2 and TRDJ3 were expressed at low frequencies in both groups but still showed significant inter-group differences (Fig. \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). Correlation analysis of J gene usage showed a strong positive correlation both within and between the two groups, suggesting that J gene usage was not species-specific and was highly conserved across species (Fig. \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\n \u003ch2\u003eLength distribution, K-mers and motif sequence analysis of TCR \u0026delta; CDR3 repertoires\u003c/h2\u003e\n \u003cp\u003eAnalysis revealed that the CDR3 length distribution of both groups exhibited a highly consistent bell-shaped pattern, with lengths mainly concentrated between 15 and 30 amino acids and a peak at 23 amino acids for each group. No significant difference was observed in clonotype abundance across different lengths between the two groups (Fig. \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). These results indicated that the CDR3 length distribution was conserved between the two species.\u003c/p\u003e\n \u003cp\u003eTo further analyze the K-mers of the TCR \u0026delta; CDR3 repertoires, the top 10 high-frequency 5-mer sequences were selected for frequency analysis. The results showed that the buffalo and cattle groups shared some K-mers while also displaying species-specific preferences. Among all 12 samples, DKLIF and TDKLI were the most frequently utilized K-mers. In the analysis of the top 10 high-frequency K-mers, QYPLI and YPLIF were predominantly present in the buffalo group, with both sequences accounting for over 30% of the total in buffalo5; by contrast, NPLIF and QNPLI were mainly detected in the cattle group, with each sequence representing more than 30% of the total in cattle4. The remaining K-mers showed minimal differences between the two groups (Fig. \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB).\u003c/p\u003e\n \u003cp\u003eSubsequently, motif sequence analysis was performed based on amino acid composition. It was found that polar amino acids and hydrophobic amino acids exhibited relatively high frequencies in the core regions, which constituted the basic amino acid composition characteristics of CDR3 sequences. Amino acids including glycine (G), arginine (R), leucine (L) and valine (V)-which belong to polar, basic and hydrophobic categories-were detected in all samples, with G showing the highest frequency. In addition, the proportion of V at position 2 was higher in the buffalo group than in the cattle group, and individual differences were observed in the proportion of L at position 3 within the buffalo group (Fig. \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). These findings suggested that the order and arrangement of amino acid sequences differed significantly between species and among individuals.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\n \u003ch2\u003eConsensus amino acid sequences and Overlap analysis of TCR \u0026delta; CDR3 repertoire\u003c/h2\u003e\n \u003cp\u003eAnalysis of conserved amino acid sequences in the TCR \u0026delta; CDR3 repertoires of the two groups showed that 1,168 unique sequences were identified in the cattle group and 615 in the buffalo group, with 1,194 sequences shared between the two groups (Fig. \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). Analysis of conserved clonotype overlap revealed that the extent of clonotype sharing among individuals within the cattle group was higher than that within the buffalo group. For instance, the overlap values between cattle4 and cattle6, as well as between cattle5 and cattle6, exceeded 2,000. By contrast, only a few individual pairs, such as buffalo4 and buffalo1, exhibited relatively high overlap in the buffalo group. When comparing between the two groups, the overall overlap values were low, but several cross-group pairs, including buffalo1 and cattle6, buffalo4 and cattle6, and buffalo4 and cattle4, showed higher overlap values than those observed within either group (Fig. \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB).\u003c/p\u003e\n \u003cp\u003eAnalysis of shared amino acid sequences across samples demonstrated that 18,259 sequences were shared by 2 out of the 12 samples, whereas only 4 sequences were conserved across 11 samples (Fig. \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). Within the buffalo group, 2,894 sequences were shared by 2 samples, while merely 13 sequences were present in all 5 samples (Fig. \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD). In the cattle group, 8,779 sequences were shared by 2 samples, and only 24 sequences were detected across all 7 samples (Fig. \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE).\u003c/p\u003e\n \u003cp\u003eTracking of the top five most frequently shared amino acid sequences across all samples indicated that buffalo1, buffalo4, cattle4 and cattle6 had relatively high numbers of shared sequences, with CATHLHGEQSGKLIF being the most prevalent shared sequence (Fig. \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF). When focusing on the top five most frequently shared sequences within the buffalo group, buffalo1, buffalo4, cattle4 and cattle6 were again found to harbor abundant shared sequences, and CATHLHGEQSGKLIF remained the most dominant shared sequence (Fig. \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eG). For the cattle group-specific top five most frequently shared sequences, cattle1, cattle2 and buffalo4 showed high levels of sequence sharing, with CATMDHSRARGTQTDKVIF identified as the most commonly shared sequence (Fig. \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eH). These results suggested that highly abundant shared clonotypes were enriched only in a small subset of individuals.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eγδ T cells account for an extremely high proportion in ruminants, a feature that is significantly different from that observed in other γδ low species such as humans and mice. Early studies have indicated that γδ T cells in ruminants including sheep and cattle represent the dominant subsets in both peripheral blood and tissues, a characteristic that may reflect the unique surveillance requirements of the ruminant immune system in response to external stimuli[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. In addition, γδ TCRs are capable of recognizing non-classical antigens and acting as a bridge between innate and adaptive immunity, endowing these cells with a critical role in the rapid response to pathogenic challenges[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. This study systematically compared the characteristics of TCRδ CDR3 immune repertoires between two bovine species, cattle and buffalo. Multiple dimensions including clonal diversity, V(D)J gene usage, CDR3 structural features and shared clonotypes were investigated, which revealed remarkable differences between the two species against the backdrop of their highly similar γδ T cell populations. Overall, although both species exhibited a TCR δ CDR3 repertoire architecture dominated by medium-to-high frequency clonotypes, the clonotype distribution in cattle was more homogeneous, with an overall diversity level higher than that in buffalo. These observations suggest that γδ T cells of different bovine species may adopt distinct immune strategies to combat external antigen stimulation, even when they occupy similar immune niches.\u003c/p\u003e \u003cp\u003eThe basic structure and composition of the TRD locus are the root causes of the complexity of the TRD repertoire. In bovids, the TRD gene pool is extremely large, containing a large number of TRDV1 subgroup genes and multiple D, J genes, a phenomenon of amplification that is prevalent in γδ high species[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Specifically, the TRD locus exhibits abundant TRAV/TRDV members, including different subgroups such as TRDV1, TRDV2, TRDV3 and TRDV4, in the annotation of multiple genome versions, along with multiple TRDD and TRDJ segments. These provide a rich genetic basis for high CDR3 diversity[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Such gene amplification is not only prominent in cattle but also shows a similar pattern in other ruminants like sheep, indicating an evolutionary commonality in the topological expansion of TRDV1 among ruminant species[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. In this study, we observed that the cattle group used a total of 52 V genes, among which 47 belonged to the V1 family, accounting for 90.4%. In contrast, 57 V genes were detected in the buffalo group, with 53 from the V1 family, representing a proportion of 93.0%. This result further confirms the amplification characteristic of the TRDV1 subgroup in bovids. Notably, both the total number of TRDV genes used and the proportion of the V1 family in buffalo were slightly higher than those in cattle, suggesting that the TRD repertoire in buffalo may have richer diversity. Meanwhile, the V1 family genes dominated in both species, highlighting their important role in γδ T cell receptor assembly and conforming to the evolutionary common feature of TRDV1 topological expansion in ruminants. Additionally, studies have found that the expansion of TRDV1\u0026thinsp;+\u0026thinsp;γδ T cells in ruminants may be attributed to the unique surveillance requirements of their immune systems [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn terms of V gene usage patterns, cattle and buffalo exhibited distinct species-specific preferences. Correlation analysis of TRDV usage between cattle and buffalo showed a high degree of consistency within each group but low correlation between the two groups, suggesting that TRDV gene usage is driven by species-specific regulatory mechanisms. Although both buffalo and cattle retain a large TRDV1 gene pool[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e], the frequency and usage preference of their specific gene members are not completely consistent between the two species. This may reflect the different selective pressures experienced by the two species during long-term independent evolution and ecological adaptation. In addition, different V gene usage patterns may also be associated with their immune responses to the same pathogens and the outcomes of infection. In a Schistosoma japonicum infection model, after infection, cattle (Bos taurus) are more prone to developing hepatic white granulomas and significant inflammatory cell infiltration, whereas buffalo (Bubalus bubalis) exhibit relatively milder inflammatory responses[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Similarly, during Mycobacterium bovis infection, although both species can produce antigen-specific IFN‑γ and TNF‑α cytokine responses, differences in cytokine expression patterns have been observed between buffalo and cattle, indicating interspecific variations in immune responses[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn contrast to the species-specific differences observed in V gene usage, TRDJ genes exhibited higher conservation between cattle and buffalo. Both groups predominantly used TRDJ1 and TRDJ4, with an extremely high correlation across the two species. Such conservation in J gene usage also shows similar cross-species stability in the β chain locus of αβ TCR. Previous immune repertoire and comparative genomics analyses have demonstrated that TRBJ segments in TCR β repertoires of multiple mammalian species, including bank voles, mice and humans, display significant sequence conservation and play a critical role in shaping the terminal structure of CDR3[\u003cspan additionalcitationids=\"CR21\" citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. This conservation may be attributed to the functional constraints of J genes in forming the structural framework of the CDR3 terminal region after V(D)J recombination. Specifically, J segments provide the structural backbone of the CDR3 region, enabling the variable region of the entire receptor to stably bind to D/V segments and maintain the overall three-dimensional conformation. This functional characteristic is supported by comparative analyses of gene loci across various species[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe TCR δ CDR3 length distribution in both cattle and buffalo showed a typical bell-shaped pattern, with lengths concentrated in the range of 15\u0026ndash;30 amino acids. This range is wider than that of the TCR β chain CDR3 in αβ high species such as humans and mice, which usually falls between 12 and 15 amino acids[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. This phenomenon may be attributed to the fact that the TRD gene pool of ruminants including cattle and sheep contains a large number of TRDV1 subgroups and multiple D/J genes, which provides a genetic basis for generating CDR3 regions with diverse and variable lengths[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. For example, in sheep, the usage of different numbers of TRDD genes directly leads to a linear increase in CDR3 length, with the longest CDR3 even exceeding approximately 26 amino acids[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. This mechanism may also apply to cattle and buffalo, enabling them to generate longer and more diverse CDR3 repertoires that can better adapt to the immune task of broad-spectrum antigen recognition.\u003c/p\u003e \u003cp\u003eIn this study, the number of shared amino acid sequences between the TRD CDR3 repertoires of cattle and buffalo was relatively small, and highly abundant shared clonotypes were only enriched among a small number of individuals. This phenomenon not only reflects the immunological essence of the γδ TCR repertoire but may also be associated with the differences accumulated between the two species during long-term evolution and genomic divergence. Previous studies have demonstrated that the human γδ T cell receptor repertoire exhibits a prominent private clonotype dominance among different individuals. In contrast, public clonotypes shared across individuals are mostly associated with the relatively conserved rearrangement structures in Vδ2\u0026thinsp;+\u0026thinsp;γδ T cells, and their occurrence tends to be related to generation probability, V(D)J recombination preferences and expansion status rather than driven by a single antigen. For example, deep sequencing data have indicated that multiple public clonotypes exist in adult Vγ9Vδ2\u0026thinsp;+\u0026thinsp;repertoires, whereas other γδ subsets such as Vδ1\u0026thinsp;+\u0026thinsp;are dominated by private clonotypes[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. In addition, cattle (Bos taurus) and buffalo (Bubalus bubalis) both belong to the tribe Bovini, yet their genomes exhibit significant differences in phylogenetic history and chromosomal structure. Comparative genomics studies have shown that the two species have undergone multiple structural chromosomal rearrangement events since diverging from their common ancestor. These genomic changes help to explain the potential differences in rearrangement preferences near specific gene loci[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Finally, the divergence time between cattle and buffalo can be traced back to millions of years ago. The two species have experienced distinct natural and artificial selection pressures under their respective ecological and domestication backgrounds, which may fundamentally affect the rearrangement tendency and generation probability distribution of the TRD locus[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e].\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eIn summary, the present study comprehensively characterized the species-specific differences in the TRD immune repertoires of cattle and buffalo from multiple perspectives, including repertoire architecture, clonotype distribution, diversity indices, V/J gene usage and sequence features. Moreover, an interpretive framework was provided by integrating existing studies on the TRD genome and its functions in ruminants. These findings not only enrich the understanding of the mechanisms underlying immune diversity in γδ high species but also lay a foundation for further investigations into the roles of γδ T cells in pathogen recognition, vaccine responses and species-specific immune adaptability.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eCDR3 \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Complementarity-determining region 3\u003c/p\u003e\n\u003cp\u003eTRD \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;T cell receptor delta chain\u003c/p\u003e\n\u003cp\u003eTCR \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;T cell receptor\u003c/p\u003e\n\u003cp\u003eMHC \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Major histocompatibility complex\u003c/p\u003e\n\u003cp\u003eHTS \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;High-throughput sequencing\u003c/p\u003e\n\u003cp\u003eTRG \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;TCR \u0026gamma; chain\u003c/p\u003e\n\u003cp\u003ePCR \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; Polymerase Chain Reaction\u0026nbsp;\u003c/p\u003e"},{"header":"Declarations","content":" \u003ch2\u003eEthics approval and consent to participate\u003c/h2\u003e \u003cp\u003eThis study was approved by the Ethics Committee of Zunyi Medical University (Approval No. ZMU21-2203-111). All procedures involving animals were performed in accordance with institutional and national guidelines.\u003c/p\u003e \u003cp\u003e \u003cstrong\u003eConsent for publication\u003c/strong\u003e \u003cp\u003eNot applicable\u003c/p\u003e \u003ch2\u003eCompeting Interests\u003c/h2\u003e \u003cp\u003eAuthors declare that they have no competing interests.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThe Science and Technology Program of Guizhou Province (ZK[2023]) and Zunyi Medical University Doctoral Initiation Fund ([F950]) funded this study.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eY.Z. and F.W. contributed equally to this work and share first authorship. J.L. and X.Y. designed the study, Y.Z., F.W.,performed the experiments, and analyzed the data. L.M. assisted with data analysis and figure preparation. J.L. supervised the study, provided resources, and wrote the manuscript. All authors read and approved the final manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eWe thank to IMGT for sharing the full VDJC annotation for cattle.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eThe data supporting the findings of this study have been deposited in Zenodo and are openly available at https://zenodo.org/records/18346729. The raw sequencing data have been uploaded to the NCBI with BioProject accession number: PRJNA1417106.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eYirsaw A, Baldwin CL. Goat gammadelta T cells. Dev Comp Immunol. 2021;114:103809.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGuzman E, Price S, Poulsom H, Hope J. Bovine gammadelta T cells: cells with multiple functions and important roles in immunity. 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BMC Vet Res. 2025;21(1):202.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"γδ T cells, TCR CDR3 repertoire, V(D)J recombination, cattle, water buffalo","lastPublishedDoi":"10.21203/rs.3.rs-8686230/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8686230/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eγδ T cells constitute a substantial proportion of lymphocytes in ruminants, and the diversity of their immune receptors is critical for understanding species-specific immune functions. The complementarity-determining region 3 (CDR3) of the T cell receptor delta chain (TRD) is a key structural determinant of γδ T cell antigen recognition; however, systematic comparative analyses of TRD immune repertoire characteristics across different bovine species remain limited. In this study, high-throughput sequencing and comprehensive analysis of the TRD CDR3 immune repertoire were performed in 7 cattle (Bos taurus) and 5 water buffalo (Bubalus bubalis).\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eThe results demonstrated good consistency in sequencing depth and data quality between the two groups. Diversity analysis revealed that the cattle TCRδ CDR3 repertoire exhibited higher clonal evenness and overall diversity than that of buffalo. Clonotype composition analysis showed that both species were dominated by medium- to high-frequency clonotypes, whereas buffalo relied more heavily on a limited number of highly expanded clonotypes. V(D)J gene usage analysis identified pronounced species-specific preferences in TRDV gene usage, with high intra-group consistency but low inter-species correlation; in contrast, TRDJ gene usage was highly conserved between the two species. The CDR3 length distributions in both groups displayed similar bell-shaped patterns, suggesting structural constraints during evolution, while K-mer and motif analyses revealed differences in CDR3 microstructural features between species. Furthermore, shared clonotype analysis indicated a limited number of public CDR3 amino acid sequences between cattle and buffalo, with highly abundant shared clonotypes enriched only in a small subset of individuals.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eCollectively, this study provides a systematic characterization of the similarities and differences in the TRD CDR3 immune repertoires of cattle and water buffalo, offering fundamental data and a comparative perspective for understanding the mechanisms shaping γδ TCR diversity and their potential immunological functions in high γδ T cell species.\u003c/p\u003e","manuscriptTitle":"High-throughput profiling of the T cell receptor delta CDR3 repertoire reveals species-specific patterns in cattle (Bos taurus) and water buffalo(Bubalus bubalis)","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-04-07 18:29:33","doi":"10.21203/rs.3.rs-8686230/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"6bafe35f-3b5c-4d9b-864a-a00aa12b33a4","owner":[],"postedDate":"April 7th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-04-15T10:23:11+00:00","versionOfRecord":[],"versionCreatedAt":"2026-04-07 18:29:33","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8686230","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8686230","identity":"rs-8686230","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}