Preferential HLA-B27 Allorecognition Displayed by Multiple Cross-Reactive Antiviral CD8+ T Cell Receptors

T cells provide essential immunosurveillance to combat and eliminate infection from pathogens, yet these cells can also induce unwanted immune responses via T cell receptor (TCR) cross-reactivity, also known as heterologous immunity. Indeed, pathogen-induced TCR cross-reactivity has shown to be a common, robust, and functionally potent mechanism that can trigger a spectrum of human immunopathologies associated with either transplant rejection, drug allergy, and autoimmunity. Here, we report that several virus-specific CD8+ T cells directed against peptides derived from chronic viruses (EBV, CMV, and HIV-1) presented by high frequency HLA-A and -B allomorphs differentially cross-react toward HLA-B27 allotypes in a highly focused and hierarchical manner. Given the commonality of cross-reactive T cells and their potential contribution to adverse outcomes in allogeneic transplants, our study demonstrates that multiple antiviral T cells recognizing the same HLA allomorph could pose an extra layer of complexity for organ matching.


INTRODUCTION
A hallmark of human antiviral T cells is their ability to recognize viral peptide antigen bound to a self-human leukocyte antigen (HLA) on the surface of infected cells. Whilst this recognition often displays exquisite specificity, it is not uncommon for some of these T cells to cross-react with closely related peptide-HLA (pHLA) complexes, such as a peptide from a different viral strain (1). Given that T cells are inherently cross-reactive, by nature of thymic selection (i.e., recognition of self) and their interaction with foreign antigen in the periphery, cross-strain reactivity is a beneficial property affording protection to mutant viral strains and preventing immune escape. More remarkably, some T cells are also capable of recognizing apparently distinct pHLA including non-self or allogeneic pHLA (2)(3)(4), self-pHLA that have undergone some form of perturbation resulting in an altered self-peptide repertoire (5), and self-pHLA expressed in different tissues (6). These forms of heterologous immunity, otherwise known as T cell cross-reactivity, are not beneficial to the host and can lead to transplant rejection, drug hypersensitivity and autoimmunity, respectively. Moreover, these potentially hazardous T cell responses are the price paid to maintain immune potential to combat the vast array of pathogenic challenges during a lifetime. Hence, cross-reactivity is an intrinsic feature of T cells, necessitated by the limited availability of unique human T cell receptor (TCR) clonotypes (<10 8 distinct TCRs) to maintain immunity against tremendous pathogenic diversity (>10 15 pHLA combinations) (7).
Childhood exposure to common viruses results in the induction of a robust immune response that controls the infection and generates long lasting immune memory. A small proportion of some viruses (e.g., herpesviruses including Epstein-Barr virus [EBV] and cytomegalovirus [CMV]) are able to evade the immune response by entering into a latent state inside the host cells. In fact, for these common herpesviruses up to 90% of individuals maintain viral latency by adulthood (8). The persistence of a memory pool of T cells against the virus generally controls outbreaks of viral reactivation. Recurrent reactivation episodes maintain these memory T cells at high frequency, facilitating rapid deployment and activation. Virally triggered cross-reactive T cells have predominantly been explored in infections where there is a high likelihood of their relevance after transplantation. This is particularly so for EBV and CMV, which establish latency in the host following naturally acquired or vaccine-induced immunity. These viruses have been directly implicated as risk factors associated with allograft rejection and graft vs. host disease (8), with studies demonstrating that high frequencies of herpesvirus-derived cross-reactive T cells (up to 85%) or clones (up to 45%) co-recognize alternate HLA allotypes (3,(9)(10)(11). Whilst there is a high likelihood that cross-reactive T cells are involved in clinical rejection (11)(12)(13), this has yet to be formally proven.

Study Participants and Peripheral Blood Mononuclear Cells Isolation
Participant HLA typing is shown in Supplementary Table 1. Peripheral blood mononuclear cells (PBMC) were isolated by standard Ficoll-Paque (GE Healthcare, Uppsala, Sweden) density gradient centrifugation and cryopreserved at −196 • C until required.

αβTCR Identification
Virus-specific CD8 + T cells lines were incubated with 1 µM peptide or relevant peptide-pulsed C1R transfected cells for 2 h before detection of cytokine secretion using an anti-IFNγ antibody (IFNγ Secretion Assay Detection Kit APC; Miltenyi Biotec, Auburn, CA) as previously described (28). CD8 + T cells were single-cell sorted directly into semi-skirted 96-well plates (Bio-Rad Laboratories Inc., USA) based on tetramer specificity and ± IFNγ production (FACSAria I, BD Biosciences operated by FlowCore, Monash University). Sorted plates were immediately stored at −80 • C until required. TCR analysis of paired complementarity determining region (CDR)3α and β loops were carried out using multiplex nested RT-PCR and sequencing of α and β gene products as previously described (29). For virus-specific CD8 + T cell clones, αβTCR usage was determined by DNA Sanger sequencing using either TCR-specific PCR for HD9G6 (30) or nextgeneration sequencing using published primer sequences (31) for A16 and 457.

TCR Expression in SKW3.hCD8αβ Cells
Full-length human TCRα and TCRβ cDNA was cloned into a self-cleaving 2A peptide-based pMIG vector as described previously (32). HEK293T packaging cells were incubated with 4 mg pEQ-pam3(-E) and 2 mg pVSV-G packaging vectors, in the presence of 4 mg pMIG vector each containing a specific TCR transgene using Lipofectamine 3000 (Life Technologies). HEK293T cell culture supernatant containing virus particles carrying the TCR transgene was then used to retrovirally transduce GFP-tagged SKW3.hCD8αβ cells or GFP-tagged SKW3.hCD8αβ.CD3 [for LTR5 TCR only (28)], which are negative for endogenous TCRαβ but contain CD3 and signaling components, as previously described (28). SKW3.hCD8αβ.TCR (hereafter referred to as SKW3) cell lines were maintained in RF10. Routine monitoring of TCR cell surface expression on SKW3 transduced cells was performed using anti-CD3 PE-Cy7

Generation of Virus-Specific CD8 + T Cell Lines and Clones
Virus-specific CD8 + T cells can be generated following stimulation with viral cognate peptide-pulsed autologous PBMCs. To demonstrate both specificity and functionality, in vitro expanded T cell lines or clones were co-stained with anti-CD8 and tetramer phenotypic markers for identification of virus-specific T cells. As expected, variations in the frequency of expanded tetramer + CD8 + T cell lines were observed for both EBV and CMV (Figures 1A,B, middle panels), ranging between 32.6-90.7% (n = 6) and 6.15-74.0% (n = 2) of the total CD8 + T cell population for B7 RPP and A2 NLV , respectively. In addition, CD8 + T cell clones raised against the HIV-1 B57 TW10 epitope in patients A16 and 457 showed FIGURE 1 | Characterization of virus-specific CD8 + T cells. Virus-specific CD8 + T cell lines and clones for (A) EBV, (B) CMV, and (C) HIV-1 were examined for specificity following either re-stimulation with HLA-restricted APCs pulsed with cognate viral peptide or bulk PBMC sorting, using both anti-CD8 and specific tetramer. The functionality of virus-specific CD8 + tetramer + T cells was assessed either using IFNγ production for T cell lines or via the CD137 activation marker for HIV-1 T cell clones. Cells were gated on FSC vs. SSC, single cells, CD8 + , CD8 + tetramer + , CD8 + IFNγ + cells. Representative plots are shown.
very high frequencies following tetramer-specific PBMC bulk sorting ( Figure 1C, middle panels), which were similar to the high frequencies observed against the EBV-B7 RPP epitope (i.e., HD9G6). To assess the functionality of the EBV-or CMV-specific CD8 + T cell lines to produce the pro-inflammatory cytokine IFNγ, cells were restimulated with HLA-restricted APCs pulsed with cognate viral peptide. The frequency of IFNγ production ranged from 17.2 to 67.0% and 38.4 to 68.2% of the CD8 + tetramer + T cell population for B7 RPP and A2 NLV , respectively (Figures 1A,B, lower panels). For HD9G6, the functionality of this B7 RPP -specific CD8 + T cell clone is published elsewhere (21). For B57 TW10 -specific CD8 + T cell clones A16 and 457, the activation marker CD137, which induces downstream effects of proliferation and cytolytic activity, was used to assess functionality when stimulated with cognate TW10 peptide ( Figure 1C), with data for 457 reported elsewhere (20).
Increased Sensitivity for TCR Cross-Reactivity Detection Using SKW3 Reporter Cells We have previously reported that CMV-specific CD8 + T cells raised against A2 NLV were differentially cross-reactive toward three HLA-B27 allotypes (B * 27:07 > B * 27:09 > B * 27:05). These T cells were also shown to remain relatively stable following lung transplantation, but increased significantly in response to CMV reactivation (4,11). Further characterization of the crossreactive A2 NLV -specific TCR repertoires from two unrelated individuals showed a striking similarity for the cross-reactive TCR clonotype. Additionally, this study also demonstrated that expression of cross-reactive TCRs in SKW3 cells was a robust system that maintains specificity without the need for continuous in vitro expansion of T cell lines or clones for further functional immunoassays (28). In this study, we extended the HLA-B27 allotype panel (B * 27:01-B * 27:10) to map the immunogenic FIGURE 2 | Activation of SKW3.A2 NLV TCR cells by HLA-B27-expressing APCs. SKW3.TCR activation was measured using cell surface CD69 upregulation after 16-20 h stimulation with C1R.A*02:01 ± cognate NLV peptide and a panel of C1R.B27 transfectants. CD69 MFI values were calculated after gating on FSC vs. SSC, single cells, GFP + cells, live cells, CD3 + CD8 + cells then CD69 + cells. Mean ± SEM are shown (a single experiment with triplicate data is shown from independent biological replicates performed at least twice). Statistical significance denoted by *p < 0.05 and **p < 0. 01 was determined by repeated measures non-parametric ANOVA (Kruskal-Wallis test) with post-hoc Dunn's multiple comparison test.  (Figure 2). Given the utility of SKW3 reporter cells for profiling TCR cross-reactivity, we adopted this approach to further explore HLA-B27 allorecognition patterns by immunodominant HLA-restricted virus-specific T cells. Here, several cognate peptide-specific CD8 + T cells identified using either the ICS immunoassay (i.e., T cell lines) or tetramer sorting (i.e., T cell clones) were sequenced for paired TCR α and β chains. The highest frequency αβTCR was then selected for retrovirus transduction into SKW3 cells ( Table 1). Following transduction, extremely high levels of clonality of >90% were easily achieved and maintained by sorting the top 10% of GFP + CD3 + cells if TCR expression decreased during long-term sub-culturing (Figure 3).
A recent report highlighted that HIV-1-specific memory T cells generated from Gag B57 TW10 epitope can mediate abacavir-induced hypersensitivity reactions through molecular mimicry (20). Therefore, we explored the alloreactive potential of B57 TW10 -specific CD8 + T cells toward HLA-B27. TCRs from two T cell clones (A16 and 457) raised against the Gag B57 TW10 epitope were expressed in SKW3 cells for functional evaluation. Sequencing of the A16 T cell clone revealed two α-chains with different junction regions and CDR3 loops, therefore both TCRs were independently expressed in SKW3 cells (  Figure 5, Table 2). Furthermore, we examined whether immunodominant IAV A2 GIL -specific CD8 + T cells, from an alternate RNA virus that induces acute viral infection, could also alloreact toward HLA-B27 allotypes. Here, a total of six healthy donors were screened, and interestingly no significant allorecognition was observed above background levels (Supplementary Figure 5).

DISCUSSION
In this study, we examined the cross-reactive potential of CD8 + T cells specific for immunodominant epitopes derived from three different chronic viruses (i.e., CMV, EBV, and HIV-1), presented by commonly expressed HLA (i.e., A2, B7, and B57). We demonstrated that these virus-specific CD8 + TCRs were capable of vigorous cross-reactivity toward specific HLA-B27 allotypes, and that the immune responses were hierarchical and varied considerably across the three chronic viruses.
Whilst, we previously reported a defined pattern of strong HLA-B27 T cell cross-reactivity (B * 27:07 > 09 > 05) by CMV A2 NLV CD8 + TCRs for both LTR5 and HC5 (11,28), this study extended the number of B27 subtypes examined and revealed additional cross-reactivity toward B * 27:10 > 03 > 02 for HC5. Interestingly, despite subtle sequence differences in the CDR3 regions of both the α-and β-chains (2 and 1 amino acids, respectively) between LTR5 and HC5, the fine specificity of strong TCR interactions with B * 27:07/09 allotypes were maintained. The data suggests that the composition of the allopeptide(s) presented by each HLA-B27 allomorph are similar or alternatively, of high affinity and that molecular flexibility of the CDR3 loops aids promotion of TCR engagement (33,34). In contrast, weaker responses toward B * 27:02/03/10 show delineation in TCR interaction, with LTR5 not demonstrating recognition of these allotypes, which may be due to weak TCR interactions below the assay sensitivity threshold. This suggests that the allopeptide contribution required to form the ternary complex is impacted by the variability observed in the CDR3 regions, which is supported by structural studies of the murine 2C TCR demonstrating that variations in the CDR3α loop dictated TCR affinity and cross-reactivity between distinct ligands (35). Indeed, the importance of the TCR variable domains in promoting high affinity interactions with pHLA complexes was also shown with the human HLA-A2-restricted cancer antigen MART-1 (36). Further investigations are required to decipher the allopeptide(s) presented by these HLA-B27 allotypes and determine their exact role in conferring cross-reactivity.
We next examined the magnitude of cross-reactivity exhibited by EBV-specific B7 RPP CD8 + T cells toward HLA-B27 allotypes. In the five HLA-B7 + individuals, including a healthy donor and immunosuppressed patients, allorecognition resulted in production of proinflammatory Th1 cytokines (IFNγ and TNFα) mainly toward either B * 27:02 or B * 27:08. Although, it should be noted that an additional screen of four healthy donors showed no HLA-B27 cross-reactivity, suggesting that allorecognition is driven by private TCR usage. The B7 RPP CD8 + TCR repertoires were sequenced for three of these individuals to determine their clonotypic profiles. Interestingly, only two clonotypes were observed for LTR54 (i.e., LTR54.1 and LTR54.2), which differed in the CDR3 and J regions of both TCRα-and β-chains. Both TCRs were expressed in SKW3 cells for further functional validation. Additionally, comparison of the B7 RPP CD8 + TCR clonotypes showed a high degree of similarity between LTR54.1 and LTR117, with differences only noted in the CDR3α-and β-loops. Whilst, LTR119 and the previously reported B7 RPP CD8 + T cell clone, HD9G6 (21), are vastly different from the other TCRs in this cohort. Interestingly, the strongest TCR cross-reactivity was relatively restricted to B * 27:08 (LTR54.1, LTR54.2, HD9G6) and B * 27:02 (LTR117), although there was a degree of allorecognition toward other subtypes for most TCRs. These observations highlight that both private (i.e., LTR119 and HD9G6) and shared (i.e., LTR54.1 and LTR117) TCR specificities contribute to cross-reactivity, and that the cross-reactive pattern diversity is dependent on the Vβ region (2,15,16). Furthermore, Amir et al. (2) also reported that T cell clones with identical Vβ regions from the same individual held private specificities and generated different alloreactions. For example, in donor BDV a T cell clone raised against CMV B7 RPHERNGFTVL with TCR Vβ7.2 recognized DRB1 * 08:01, whilst another T cell clone from same individual with the identical Vβ did not. Additionally, in donor FKR an influenza A2 GIL T cell clone with Vβ17 recognized allogeneic HLA-B * 64:01 but another T cell clone with the identical Vβ failed. These T cell clones had private differences in TCR sequence, which effectively abrogated alloreactivity.
For the herpesvirus TCRs, the allorecognition hierarchy remained relatively static for the strongest responses, but this was not observed in the case of HIV-1 B57 TW10 CD8 + TCRs in that 457 and A16 TCRs were completely focused toward different HLA-B27 allotypes. Here, we show that 457 TCR cross-reacted strongly toward B * 27:02 > 01, with two TCRs derived from A16 strongly recognizing B * 27:05/07 for A16.1 and B * 27:05 for A16.2.
Comparison of their TCRs revealed that their signatures were completely different, supporting that the B57 TW10 specificity is driven by private TCR usage in these two individuals. Particularly of interest was the dual expression of two different TCRαchains from the A16 T cell clone, which when independently expressed in SKW3 reporter cells, showed reactivity differences not only toward the B27 subtypes but also importantly against the cognate antigen. We observed that the A16.2 TCR was geared toward cognate antigen recognition, with the A16.1 TCR being more alloreactive. Up to 30% of human peripheral T cells naturally express dual TCRα-chains (37), with multiple studies demonstrating that the allelic inclusion facilitates a heightened immune response by providing an additional chance for antigen recognition and engagement [extensively reviewed in (38)].
So, what drives the preferential HLA-B27 allorecognition displayed by these virus-specific TCRs? Undoubtedly, the polymorphic nature of the B27 molecule itself greatly influences the peptide cargo being displayed to surveying T cells (Figure 7). In our study, the A2 NLV CD8 + TCRs preferentially bind to B * 27:07/09/05, which differ by 1 (B * 27:09) and 5 (B * 27:07) amino acids compared to the consensus B * 27:05 allotype. These polymorphisms directly impact the D/E (position 114, peptide contacts P5-P7) and F (position 116, peptide contact P9) peptidebinding pockets, which are known immunological hot spots for non-permissive HLA mismatches in transplantation (39)(40)(41)(42)(43). Given that the public A2 NLV CD8 + TCR co-recognizes these three molecules and their relative impact on the peptide-binding pockets D/E and F, suggests that each may be presenting an alternate allopeptide with affinity above a threshold to promote TCR engagement. This is supported by the prototypic HLA-B8restricted LC13 TCR which is capable of engaging with HLA-B * 44:05 presenting either an allotype or mimotope (18). In addition, we cannot exclude that the same allopeptide may also be presented by all HLA-B27 molecules, with allorecognition being impacted by differences in conformational flexibility. Indeed, a study by Loll et al. demonstrated that a HLA-B27-derived self-peptide derived from vasoactive intestinal peptide receptor type 1 (epitope; RRKWRRWHL) is differentially presented by AS-associated B * 27:04 and B * 27:05 compared to the non-ASassociated B * 27:06 and B * 27:09 due to structural variations in molecular dynamics (44). For B7 RPP CD8 + TCRs, recognition was focused toward B * 27:08/02/01, with B * 27:01 and B * 27:02 differing by a single amino acid (position 80) and both differing from B * 27:08 by 5 amino acids (positions 77, 80-83), all of which also influence the F peptide-binding pocket (Figure 7). Finally, for the B57 TW10 CD8 + TCRs we observed completely divergent recognition of B27 allotypes by 457 (B * 27:01/02) and A16 (B * 27:05/07). However, a common feature is involvement of the F pocket at positions 80 and 116, respectively. Interestingly, the F pocket not only determines the carboxy terminal motif of HLA-I peptides (45), but in other HLA-B27 allotypes has also been shown to affect anchoring sites (i.e., B * 27:06; P3, P -2, and P ) (46). Moreover, positions 114 and 116 are important for the chaperone tapasin, involved in loading of optimal peptides on HLA-I molecules (47,48). Whilst, the identification of allopeptides has been a major limiting factor hampering translational impact in clinical studies, further investigations are warranted to assess the true impact of T cell cross-reactivity.