Belatacept-related cytomegalovirus infection: Advocacy for tailored immunosuppression based on individual assessment of immune fitness.

Belatacept, a fusion protein combining cytotoxic T-lymphocyte antigen-4 (CTLA-4) and the Fc region of human IgG1, is increasingly used as a calcineurin inhibitor-sparing regimen in patients with chronic graft dysfunction. Older kidney transplant recipients, particularly from expanded criteria donors, may be switched to belatacept due to poor renal recovery. However, late-onset cytomegalovirus (CMV) reactivation is increasingly reported with this treatment, especially in older patients with graft dysfunction. This suggests a progressive loss of CMV-specific T cell response, potentially driven by T cell exhaustion. Contributing factors include preexisting T cell dysfunction, increased viral antigen exposure, and interference in the PD-L1/PD-1 pathway by belatacept. mTOR inhibitors have shown efficacy in preventing CMV reactivation by reinvigorating CMV-specific T cells. These findings support combining belatacept with mTOR inhibitors in high-risk CMV-seropositive recipients and emphasize the need for personalized immune assessments to guide immunosuppressive strategies.

Cancer immunotherapy by γδ T cells.

The premise of cancer immunotherapy is that cancers are specifically visible to an immune system tolerized to healthy self. The promise of cancer immunotherapy is that immune effector mechanisms and immunological memory can jointly eradicate cancers and inoperable metastases and de facto vaccinate against recurrence. For some patients with hitherto incurable diseases, including metastatic melanoma, this promise is being realized by game-changing immunotherapies based on αß T cells. Today's challenges are to bring benefit to greater numbers of patients of diverse ethnicities, target more cancer types, and achieve a cure while incurring fewer adverse events. In meeting those challenges, specific benefits may be offered by γδ T cells, which compose a second T cell lineage with distinct recognition capabilities and functional traits that bridge innate and adaptive immunity. γδ T cell-based clinical trials, including off-the-shelf adoptive cell therapy and agonist antibodies, are yielding promising results, although identifiable problems remain. In addressing those problems, we advocate that immunotherapies be guided by the distinctive biology of γδ T cells, as elucidated by ongoing research.

Long-lived central memory γδ T cells confer protection against murine cytomegalovirus reinfection.

The involvement of γδ TCR-bearing lymphocytes in immunological memory has gained increasing interest due to their functional duality between adaptive and innate immunity. γδ T effector memory (TEM) and central memory (TCM) subsets have been identified, but their respective roles in memory responses are poorly understood. In the present study, we used subsequent mouse cytomegalovirus (MCMV) infections of αß T cell deficient mice in order to analyze the memory potential of γδ T cells. As for CMV-specific αß T cells, MCMV induced the accumulation of cytolytic, KLRG1+CX3CR1+ γδ TEM that principally localized in infected organ vasculature. Typifying T cell memory, γδ T cell expansion in organs and blood was higher after secondary viral challenge than after primary infection. Viral control upon MCMV reinfection was prevented when masking γδ T-cell receptor, and was associated with a preferential amplification of private and unfocused TCR δ chain repertoire composed of a combination of clonotypes expanded post-primary infection and, more unexpectedly, of novel expanded clonotypes. Finally, long-term-primed γδ TCM cells, but not γδ TEM cells, protected T cell-deficient hosts against MCMV-induced death upon adoptive transfer, probably through their ability to survive and to generate TEM in the recipient host. This better survival potential of TCM cells was confirmed by a detailed scRNASeq analysis of the two γδ T cell memory subsets which also revealed their similarity to classically adaptive αß CD8 T cells. Overall, our study uncovered memory properties of long-lived TCM γδ T cells that confer protection in a chronic infection, highlighting the interest of this T cell subset in vaccination approaches.

Human NLRC4 expression promotes cancer survival and associates with type I interferon signaling and immune infiltration.

The immune system can control cancer progression. However, even though some innate immune sensors of cellular stress are expressed intrinsically in epithelial cells, their potential role in cancer aggressiveness and subsequent overall survival in humans is mainly unknown. Here, we show that nucleotide-binding oligomerization domain-like receptor (NLR) family CARD domain-containing 4 (NLRC4) is downregulated in epithelial tumor cells of patients with colorectal cancer (CRC) by using spatial tissue imaging. Strikingly, only the loss of tumor NLRC4, but not stromal NLRC4, was associated with poor immune infiltration (mainly DCs and CD4+ and CD8+ T cells) and accurately predicted progression to metastatic stage IV and decrease in overall survival. By combining multiomics approaches, we show that restoring NLRC4 expression in human CRC cells triggered a broad inflammasome-independent immune reprogramming consisting of type I interferon (IFN) signaling genes and the release of chemokines and myeloid growth factors involved in the tumor infiltration and activation of DCs and T cells. Consistently, such reprogramming in cancer cells was sufficient to directly induce maturation of human DCs toward a Th1 antitumor immune response through IL-12 production in vitro. In multiple human carcinomas (colorectal, lung, and skin), we confirmed that NLRC4 expression in patient tumors was strongly associated with type I IFN genes, immune infiltrates, and high microsatellite instability. Thus, we shed light on the epithelial innate immune sensor NLRC4 as a therapeutic target to promote an efficient antitumor immune response against the aggressiveness of various carcinomas.

Mechanisms of systemic low-grade inflammation in HIV patients on long-term suppressive antiretroviral therapy: the inflammasome hypothesis.

OBJECTIVE: We aimed to determine the contribution of inflammasome activation in chronic low-grade systemic inflammation observed in patients with HIV (PWH) on long-term suppressive antiretroviral therapy (ART) and to explore mechanisms of such activation. DESIGN: Forty-two PWH on long-term suppressive ART (HIV-RNA < 40 copies/ml) were compared with 10 HIV-negative healthy controls (HC). METHODS: Inflammasome activation was measured by dosing mature interleukin (IL)-1ß and IL-18 cytokines in patient serum. We explored inflammasome pathways through ex vivo stimulation of PWH primary monocytes with inflammasome activators; expression of inflammasome components by transcriptomic analysis; and metabolomics analysis of patient sera. RESULTS: Median (Q1; Q3) age, ART and viral suppression duration in PWH were 54 (48; 60), 15 (9; 20) and 7.5 (5; 12) years, respectively. Higher serum IL-18 was measured in PWH than in HC (61 (42; 77) vs. 36 (27-48 pg/ml), P = 0.009); IL-1ß was detected in 10/42 PWH (0.5 (0.34; 0.80) pg/ml) but not in HC. Monocytes from PWH did not produce more inflammatory cytokines in vitro , but secretion of IL-1ß in response to NOD like receptor family, pyrin domain containing 3 (NLRP3) inflammasome stimulation was higher than in HC. This was not explained at the transcriptional level. We found an oxidative stress molecular profile in PWH sera. CONCLUSION: HIV infection with long-term effective ART is associated with a serum inflammatory signature, including markers of inflammasome activation, and an increased activation of monocytes upon inflammasome stimulation. Other cells should be investigated as sources of inflammatory cytokines in PWH. Oxidative stress might contribute to this chronic low-grade inflammation.

WARS1, TYMP and GBP1 display a distinctive microcirculation pattern by immunohistochemistry during antibody-mediated rejection in kidney transplantation.

Antibody-mediated rejection (ABMR) is the leading cause of allograft failure in kidney transplantation. Defined by the Banff classification, its gold standard diagnosis remains a challenge, with limited inter-observer reproducibility of the histological scores and efficient immunomarker availability. We performed an immunohistochemical analysis of 3 interferon-related proteins, WARS1, TYMP and GBP1 in a cohort of kidney allograft biopsies including 17 ABMR cases and 37 other common graft injuries. Slides were interpreted, for an ABMR diagnosis, by four blinded nephropathologists and by a deep learning framework using convolutional neural networks. Pathologists identified a distinctive microcirculation staining pattern in ABMR with all three antibodies, displaying promising diagnostic performances and a substantial reproducibility. The deep learning analysis supported the microcirculation staining pattern and achieved similar diagnostic performance from internal validation, with a mean area under the receiver operating characteristic curve of 0.89 (± 0.02) for WARS1, 0.80 (± 0.04) for TYMP and 0.89 (± 0.04) for GBP1. The glomerulitis and peritubular capillaritis scores, the hallmarks of histological ABMR, were the most highly correlated Banff scores with the deep learning output, whatever the C4d status. These novel immunomarkers combined with a CNN framework could help mitigate current challenges in ABMR diagnosis and should be assessed in larger cohorts.

Does Inflammation Contribute to Cancer Incidence and Mortality during Aging? A Conceptual Review.

Aging is associated with chronic low-grade inflammation, cancer incidence and mortality. As inflammation contributes to cancer initiation and progression, one could hypothesize that age-associated chronic low-grade inflammation contributes to the increase in cancer incidence and/or mortality observed during aging. Here, we review the evidence supporting this hypothesis: (1) epidemiological associations between biomarkers of systemic inflammation and cancer incidence and mortality in older people, (2) therapeutic clues suggesting that targeting inflammation could reduce cancer incidence and mortality and (3) experimental evidence from animal models highlighting inflammation as a link between various mechanisms of aging and cancer initiation and progression. Despite a large body of literature linking aging, inflammation and cancer, convincing evidence for the clear implication of specific inflammatory pathways explaining cancer incidence or mortality during aging is still lacking. Further dedicated research is needed to fill these gaps in evidence and pave the way for the development of applications in clinical care.

The Proteome of Antibody-Mediated Rejection: From Glomerulitis to Transplant Glomerulopathy.

Antibody-mediated rejection (ABMR) is the leading cause of allograft failure in kidney transplantation. Its histological hallmark is represented by lesions of glomerulitis i.e., inflammatory cells within glomeruli. Current therapies for ABMR fail to prevent chronic allograft damage i.e., transplant glomerulopathy, leading to allograft loss. We used laser microdissection of glomeruli from formalin-fixed allograft biopsies combined with mass spectrometry-based proteomics to describe the proteome modification of 11 active and 10 chronic active ABMR cases compared to 8 stable graft controls. Of 1335 detected proteins, 77 were deregulated in glomerulitis compared to stable grafts, particularly involved in cellular stress mediated by interferons type I and II, leukocyte activation and microcirculation remodeling. Three proteins extracted from this protein profile, TYMP, WARS1 and GBP1, showed a consistent overexpression by immunohistochemistry in glomerular endothelial cells that may represent relevant markers of endothelial stress during active ABMR. In transplant glomerulopathy, 137 proteins were deregulated, which favor a complement-mediated mechanism, wound healing processes through coagulation activation and ultimately a remodeling of the glomerular extracellular matrix, as observed by light microscopy. This study brings novel information on glomerular proteomics of ABMR in kidney transplantation, and highlights potential targets of diagnostic and therapeutic interest.

Incidence of cytomegalovirus infection in seropositive kidney transplant recipients treated with everolimus: A randomized, open-label, multicenter phase 4 trial.

Cytomegalovirus (CMV) persists as the most frequent opportunistic infection among solid organ transplant recipients. This multicenter trial aimed to test whether treatment with everolimus (EVR) could decrease the incidence of CMV DNAemia and disease. We randomized 186 CMV seropositive kidney transplant recipients in a 1:1 ratio to receive EVR or mycophenolic acid (MPA) in association with basiliximab, cyclosporin, and steroids and 87 in each group were analyzed. No universal prophylaxis was administered to either group. The composite primary endpoint was the presence of CMV DNAemia, CMV treatment, graft loss, death, and discontinuation of the study at 6 months posttransplant. In the modified intent-to-treat analysis, 42 (48.3%) and 70 (80.5%) patients in the EVR and MPA groups reached the primary endpoint (OR = 0.21, 95% CI: 0.11-0.43, p < .0001). Fewer patients of the EVR group received treatment for CMV (21.8% vs. 47.1%, p = .0007). EVR was discontinued in 31 (35.6%) patients. Among the 56 patients with ongoing EVR treatment, only 7.4% received treatment for CMV. In conclusion, EVR prevents CMV DNAemia requiring treatment in seropositive recipients as long as it is tolerated and maintained.

mTOR Inhibitors Prevent CMV Infection through the Restoration of Functional αß and γδ T cells in Kidney Transplantation.

BACKGROUND: The reported association of mTOR-inhibitor (mTORi) treatment with a lower incidence of cytomegalovirus (CMV) infection in kidney transplant recipients (KTR) who are CMV seropositive (R+) remains unexplained. METHODS: The incidence of CMV infection and T-cell profile was compared between KTRs treated with mTORis and mycophenolic acid (MPA), and in vitro mTORi effects on T-cell phenotype and functions were analyzed. RESULTS: In KTRs who were R+ and treated with MPA, both αß and γδ T cells displayed a more dysfunctional phenotype (PD-1+, CD85j+) at day 0 of transplantation in the 16 KTRs with severe CMV infection, as compared with the 17 KTRs without or with spontaneously resolving CMV infection. In patients treated with mTORis (n=27), the proportion of PD-1+ and CD85j+ αß and γδ T cells decreased, when compared with patients treated with MPA (n=44), as did the frequency and severity of CMV infections. mTORi treatment also led to higher proportions of late-differentiated and cytotoxic γδ T cells and IFNγ-producing and cytotoxic αß T cells. In vitro, mTORis increased proliferation, viability, and CMV-induced IFNγ production of T cells and decreased PD-1 and CD85j expression in T cells, which shifted the T cells to a more efficient EOMESlow Hobithigh profile. In γδ T cells, the mTORi effect was related to increased TCR signaling. CONCLUSION: Severe CMV replication is associated with a dysfunctional T-cell profile and mTORis improve T-cell fitness along with better control of CMV. A dysfunctional T-cell phenotype could serve as a new biomarker to predict post-transplantation infection and to stratify patients who should benefit from mTORi treatment. CLINICAL TRIAL REGISTRY NAME AND REGISTRATION NUMBER: Proportion of CMV Seropositive Kidney Transplant Recipients Who Will Develop a CMV Infection When Treated With an Immunosuppressive Regimen Including Everolimus and Reduced Dose of Cyclosporine Versus an Immunosuppressive Regimen With Mycophenolic Acid and Standard Dose of Cyclosporine A (EVERCMV), NCT02328963.

Human γδ T cell sensing of AMPK-dependent metabolic tumor reprogramming through TCR recognition of EphA2.

Human γδ T cells contribute to tissue homeostasis and participate in epithelial stress surveillance through mechanisms that are not well understood. Here, we identified ephrin type-A receptor 2 (EphA2) as a stress antigen recognized by a human Vγ9Vδ1 TCR. EphA2 is recognized coordinately by ephrin A to enable γδ TCR activation. We identified a putative TCR binding site on the ligand-binding domain of EphA2 that was distinct from the ephrin A binding site. Expression of EphA2 was up-regulated upon AMP-activated protein kinase (AMPK)-dependent metabolic reprogramming of cancer cells, and coexpression of EphA2 and active AMPK in tumors was associated with higher CD3 T cell infiltration in human colorectal cancer tissue. These results highlight the potential of the human γδ TCR to cooperate with a co-receptor to recognize non-MHC-encoded proteins as signals of cellular dysregulation, potentially allowing γδ T cells to sense metabolic energy changes associated with either viral infection or cancer.

Innate (and Innate-like) Lymphoid Cells: Emerging Immune Subsets With Multiple Roles Along Transplant Life.

Transplant immunology is currently largely focused on conventional adaptive immunity, particularly T and B lymphocytes, which have long been considered as the only cells capable of allorecognition. In this vision, except for the initial phase of ischemia/reperfusion, during which the role of innate immune effectors is well established, the latter are largely considered as "passive" players, recruited secondarily to amplify graft destruction processes during rejection. Challenging this prevalent dogma, the recent progresses in basic immunology have unraveled the complexity of the innate immune system and identified different subsets of innate (and innate-like) lymphoid cells. As most of these cells are tissue-resident, they are overrepresented among passenger leukocytes. Beyond their role in ischemia/reperfusion, some of these subsets have been shown to be capable of allorecognition and/or of regulating alloreactive adaptive responses, suggesting that these emerging immune players are actively involved in most of the life phases of the grafts and their recipients. Drawing upon the inventory of the literature, this review synthesizes the current state of knowledge of the role of the different innate (and innate-like) lymphoid cell subsets during ischemia/reperfusion, allorecognition, and graft rejection. How these subsets also contribute to graft tolerance and the protection of chronically immunosuppressed patients against infectious and cancerous complications is also examined.

Characterization of a Unique γδ T-Cell Subset as a Specific Marker of Cytomegalovirus Infection Severity.

Cytomegalovirus (CMV) is a major infectious cause of death and disease after transplantation. We have previously demonstrated that the tissue-associated adaptive Vδ2neg γδ T cells are key effectors responding to CMV and associated with recovery, contrasting with their innatelike circulating counterparts, the Vγ9posVδ2pos T cells that respond to phosphoantigens but not to CMV. A third Vγ9negVδ2pos subgroup with adaptive functions has been described in adults. In the current study, we demonstrate that these Vγ9negVδ2pos T cells are also components of the CMV immune response while presenting with distinct characteristics from Vδ2neg γδ T cells. In a cohort of kidney transplant recipients, CMV seropositivity was the unique clinical parameter associated with Vγ9negVδ2pos T-cell expansion and differentiation. Extensive phenotyping demonstrated their substantial cytotoxic potential and activation during acute CMV primary infection or reinfection. In vitro, Vγ9negVδ2pos T cells responded specifically to CMV-infected cells in a T-cell receptor-dependent manner and through strong interferon γ production. Finally, Vγ9negVδ2pos T cells were the only γδ T-cell subset in which expansion was tightly correlated with the severity of CMV disease. To conclude, our results identify a new player in the immune response against CMV and open interesting clinical perspectives for using Vγ9negVδ2pos T cells as an immune marker for CMV disease severity in immunocompromised patients.

Restoration of T-cell Effector Function, Depletion of Tregs, and Direct Killing of Tumor Cells: The Multiple Mechanisms of Action of a-TIGIT Antagonist Antibodies.

TIGIT is an immune checkpoint inhibitor expressed by effector CD4+ and CD8+ T cells, NK cells, and regulatory T cells (Tregs). Inhibition of TIGIT-ligand binding using antagonistic anti-TIGIT mAbs has shown in vitro potential to restore T-cell function and therapeutic efficacy in murine tumor models when combined with an anti-PD(L)-1 antibody. In the current work, we demonstrate broader TIGIT expression than previously reported in healthy donors and patients with cancer with expression on γδ T cells, particularly in CMV-seropositive donors, and on tumor cells from hematologic malignancies. Quantification of TIGIT density revealed tumor-infiltrating Tregs as the population expressing the highest receptor density. Consequently, the therapeutic potential of anti-TIGIT mAbs might be wider than the previously described anti-PD(L)-1-like restoration of αß T-cell function. CD155 also mediated inhibition of γδ T cells, an immune population not previously described to be sensitive to TIGIT inhibition, which could be fully prevented via use of an antagonistic anti-TIGIT mAb (EOS-448). In PBMCs from patients with cancer, as well as in tumor-infiltrating lymphocytes from mice, the higher TIGIT expression in Tregs correlated with strong antibody-dependent killing and preferential depletion of this highly immunosuppressive population. Accordingly, the ADCC/ADCP-enabling format of the anti-TIGIT mAb had superior antitumor activity, which was dependent upon Fcγ receptor engagement. In addition, the anti-TIGIT mAb was able to induce direct killing of TIGIT-expressing tumor cells both in human patient material and in animal models, providing strong rationale for therapeutic intervention in hematologic malignancies. These findings reveal multiple therapeutic opportunities for anti-TIGIT mAbs in cancer therapeutics.

Prognostic Role of Inflammasome Components in Human Colorectal Cancer.

(1) We wanted to assess the prognostic impact of inflammasomes involved in gut epithelial homeostasis and the development of human colorectal cancer (CRC). (2) We investigated the expression of inflammasome components in colonic epithelial cells at the protein level in patient tissues, through an immunofluorescence assay. (3) In a cohort of 104 patients, we found that all inflammasome components were downregulated in CRC. Loss of epithelial (but not stromal) expression of NLRP6, caspase-1 and IL-18 was associated with an increased mortality of 72%, 58% and 68% respectively and to disease progression into metastasis. The loss of epithelial and stromal IL-18 but not NLRP6, was associated to lower tumor immune infiltrates in the lymphoid compartment and higher Programmed cell Death receptor 1 (PD-1) expression. Finally, we found that combined downregulation of IL-18 and NLRP6 was associated with a worse outcome. Indeed, 5-year survival rates were 26% for the NLRP6low/IL-18low tumors, compared to 64.4% for the entire cohort. This downregulation was associated with a more advanced disease (p < 0.0001) and a trend to lower lymphoid cell infiltration. (4) We identified critical inflammasome markers that may help in better stratifying patients for prognosis in CRC and could help clinicians to determine which patients may benefit from immunotherapies.

Understanding human γδ T cell biology toward a better management of cytomegalovirus infection.

Cytomegalovirus (CMV) infection is responsible for significant morbidity and mortality in immunocompromised patients, namely solid organ and hematopoietic cell transplant recipients, and can induce congenital infection in neonates. There is currently an unmet need for new management and treatment strategies. Establishment of an anti-CMV immune response is critical in order to control CMV infection. The two main human T cells involved in HCMV-specific response are αß and non-Vγ9Vδ2 T cells that belong to γδ T cell compartment. CMV-induced non-Vγ9Vδ2 T cells harbor a specific clonal expansion and a phenotypic signature, and display effector functions against CMV. So far, only two main molecular mechanisms underlying CMV sensing have been identified. Non-Vγ9Vδ2 T cells can be activated either by stress-induced surface expression of the γδT cell receptor (TCR) ligand annexin A2, or by a multimolecular stress signature composed of the γδTCR ligand endothelial protein C receptor and co-stimulatory signals such as the ICAM-1-LFA-1 axis. All this basic knowledge can be harnessed to improve the clinical management of CMV infection in at-risk patients. In particular, non-Vγ9Vδ2 T cell monitoring could help better stratify the risk of infection and move forward a personalized medicine. Moreover, recent advances in cell therapy protocols open the way for a non-Vγ9Vδ2 T cell therapy in immunocompromised patients.

Effect of mTOR inhibitors during CMV disease in kidney transplant recipients: Results of a pilot retrospective study.

mTOR inhibitors exert a preventive effect on cytomegalovirus (CMV) disease in CMV seropositive (R+) kidney transplant recipients, but their impact during the curative treatment of CMV disease in high-risk kidney transplant recipients has not been investigated. We aimed to evaluate the efficacy and tolerance of mTOR inhibitors compared with mycophenolic acid in 63 consecutive kidney transplant recipients (80% of D+R-) suffering from CMV disease with a persistent or a recurrent CMV DNAemia. In this monocentric retrospective study, 16 had their treatment converted to mTOR inhibitors and 47 did not. The Kaplan-Meier curves did not show any significant differences in CMV DNAemia eradication (77% vs. 88% respectively; hazard ratio (HR), 1.648 [95% confidence interval (CI), 0.913-2.973]; log-rank test, P = .132), DNAemia recurrence (36% vs. 47%; HR, 1.517 [95% CI, 0.574-4.007]; log-rank test, P = .448) and CMV clinical recurrence (17% vs. 27%; HR, 1.375 [95% CI, 0.340-5.552]; log-rank test, P = .677) between patients who received mTOR inhibitors and those who did not. These results were confirmed in uni- and multivariate time-dependent Cox regressions. In summary, conversion from mycophenolic acid to mTOR inhibitors seems inadequate for improving CMV clearance or in better preventing CMV recurrences during severe or persistent CMV disease.

Uncovering the Anticancer Potential of Murine Cytomegalovirus against Human Colon Cancer Cells.

Human cytomegalovirus (HCMV) components are often found in tumors, but the precise relationship between HCMV and cancer remains a matter of debate. Pro-tumor functions of HCMV were described in several studies, but an association between HCMV seropositivity and reduced cancer risk was also evidenced, presumably relying on recognition and killing of cancer cells by HCMV-induced lymphocytes. This study aimed at deciphering whether CMV influences cancer development in an immune-independent manner. Using immunodeficient mice, we showed that systemic infection with murine CMV (MCMV) inhibited the growth of murine carcinomas. Surprisingly, MCMV, but not HCMV, also reduced human colon carcinoma development in vivo. In vitro, both viruses infected human cancer cells. Expression of human interferon-ß (IFN-ß) and nuclear domain (ND10) were induced in MCMV-infected, but not in HCMV-infected human colon cancer cells. These results suggest a decreased capacity of MCMV to counteract intrinsic defenses in the human cellular host. Finally, immunodeficient mice receiving peri-tumoral MCMV therapy showed a reduction of human colon cancer cell growth, albeit no clinical sign of systemic virus dissemination was evidenced. Our study, which describes a selective advantage of MCMV over HCMV to control human colon cancer, could pave the way for the development of CMV-based therapies against cancer.

Butyrophilin-2A1 Directly Binds Germline-Encoded Regions of the Vγ9Vδ2 TCR and Is Essential for Phosphoantigen Sensing.

Vγ9Vδ2 T cells respond in a TCR-dependent fashion to both microbial and host-derived pyrophosphate compounds (phosphoantigens, or P-Ag). Butyrophilin-3A1 (BTN3A1), a protein structurally related to the B7 family of costimulatory molecules, is necessary but insufficient for this process. We performed radiation hybrid screens to uncover direct TCR ligands and cofactors that potentiate BTN3A1's P-Ag sensing function. These experiments identified butyrophilin-2A1 (BTN2A1) as essential to Vγ9Vδ2 T cell recognition. BTN2A1 synergised with BTN3A1 in sensitizing P-Ag-exposed cells for Vγ9Vδ2 TCR-mediated responses. Surface plasmon resonance experiments established Vγ9Vδ2 TCRs used germline-encoded Vγ9 regions to directly bind the BTN2A1 CFG-IgV domain surface. Notably, somatically recombined CDR3 loops implicated in P-Ag recognition were uninvolved. Immunoprecipitations demonstrated close cell-surface BTN2A1-BTN3A1 association independent of P-Ag stimulation. Thus, BTN2A1 is a BTN3A1-linked co-factor critical to Vγ9Vδ2 TCR recognition. Furthermore, these results suggest a composite-ligand model of P-Ag sensing wherein the Vγ9Vδ2 TCR directly interacts with both BTN2A1 and an additional ligand recognized in a CDR3-dependent manner.