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TIGIT is upregulated by HIV-1 infection and marks a highly functional adaptive and mature subset of natural killer cells

Vendrame, Elenaa; Seiler, Christofb; Ranganath, Thanmayia; Zhao, Nancy Q.a,c; Vergara, Rosemarya; Alary, Micheld,e,f; Labbé, Annie-Claudeg,h; Guédou, Fernandi; Poudrier, Johannej,k; Holmes, Susanb; Roger, Michelj,k; Blish, Catherine A.a,c,l

Author Information
doi: 10.1097/QAD.0000000000002488



Natural killer (NK) cells are among the first responders to viral infections and can swiftly recognize and kill virus-infected cells [1]. These responses are traditionally thought to be nonspecific, as NK-cell function is mediated by the expression of germ-line-encoded receptors rather than antigen-specific receptors [2]. However, recent evidence has revealed that NK cells are capable of antigen-specific adaptive responses against viruses, such as cytomegalovirus (CMV), Epstein--Barr Virus, Varicella Zoster Virus, and influenza virus [1,3–9].

HIV infection profoundly alters the NK-cell compartment, with expansion of a CD56CD16+ subpopulation [10,11], downregulation of several activating NK-ell receptors, including FcRγ (FcεRIγ), NKp30, and NKp46 [12–14], and impairment of NK-cell function [15–18]. NK cells also play a critical role in the immune response against HIV [19,20]. NK cells may protect from HIV acquisition, as increased NK-cell activity is associated with lower risk of acquiring HIV in highly exposed individuals [21–23]. Consistent with this, genotypes of specific NK-cell receptors and human leukocyte antigens (HLA) are enriched in exposed seronegative individuals [21,24–26]. The expression of specific NK-cell receptor/HLA class I ligand pairs (KIR3DL1/KIR3DS1 with HLABw4-80I) is also associated with slower disease progression [27,28] and NK cells from long-term nonprogressors show increased function compared with HIV+ typical progressors [12,29]. Altogether, these data raise the possibility that specific NK receptor--ligand interactions may contribute to HIV control and could be used as targets to improve HIV-specific NK-cell function.

T cell immunoreceptor with Ig and ITIM domains (TIGIT) is an inhibitory receptor expressed on T cells and NK cells [30,31]. The TIGIT inhibitory signal is mediated by ligation with its high-affinity ligand, the poliovirus receptor (CD155 or PVR), and its low-affinity ligand CD112 (Nectin-2 or PVRL2) [30,32,33]. TIGIT has been associated with CD4+ T-cell, CD8+ T-cell, and NK-cell exhaustion both in the setting of chronic viral infections and malignancy [31,34–40]. Blockade of the TIGIT/CD155/CD112 pathway to improve the function of T cells and NK cells against solid cancers is currently under investigation [41,42].

In HIV-1 infection, TIGIT marks exhausted T cells, correlates with disease progression and is decreased on CD4+ T cells from elite controllers [34,43]. Early initiation of antiretroviral treatment (ART) in HIV-infected individuals does not return TIGIT/CD155 to normal levels on CD8+ T cells [37]. Additionally, co-expression of TIGIT with immune checkpoint inhibitor PD-1 marks CD4+ T cells harboring latent virus [44–46]. These data suggest that the TIGIT/CD155/CD112 pathway in T cells could contribute to HIV pathogenesis.

In NK cells, less is known regarding the role of TIGIT during HIV infection. A recent report showed that TIGIT is increased on NK cells from HIV-1-infected patients and that TIGIT blockade improves NK-cell responses to cytokines ex vivo[47], consistent with the idea that TIGIT may represent a marker of NK-cell exhaustion. Here, we used blood samples from Beninese women to study the effect of HIV infection on the NK-cell compartment, with a particular focus on TIGIT expression and function.


Study participants and sample processing

Cryopreserved peripheral blood mononuclear cells (PBMCs) were obtained from a study of commercial sex workers in Cotonou, Benin. HIV-1-infected women were enrolled from a sex-worker clinic and HIV-1-uninfected women were enrolled from a general health clinic, as described previously [48,49]. PBMCs were obtained from 20 untreated HIV-infected women, 20 HIV-infected women receiving ART and 10 healthy women (Table S1, Written informed consent was obtained from all participants. The study was approved by the Comité National Provisoire d’Éthique de la Recherche en Santé in Cotonou and the Centre Hospitalier de l’Université de Montréal (CHUM) Research Ethics Committees.

For in-vitro studies using healthy donors, leukoreduction system chambers were purchased from the Stanford Blood Bank. PBMCs were purified using Ficoll density gradient centrifugation and cryopreserved in fetal bovine serum (FBS) with 10% DMSO.

Cell isolation

For profiling PBMCs from Beninese women, 1 × 106 PBMCs were stained for mass cytometry with the ligand antibody panel (Table S2, NK cells were purified from PBMCs by magnetic-activated isolation via negative selection (Miltenyi, Bergisch Gladbach, Germany) and stained with the NK-cell antibody panel (Table S3,, Figure S2a,

For the in vitro co-cultures, cryopreserved PBMCs from healthy donors were used to purify CD4+ T cells and NK cells by magnetic-activated isolation via negative selection (Miltenyi).

Antibody conjugation, mass cytometry staining and data acquisition

Antibodies were conjugated using MaxPar ×8-labeling kits (Fluidigm, South San Francisco, California, USA). To ensure antibody stability over time, antibody panels were lyophilized into single-use pellets prior to use (Biolyph, Chaska, Minnesota, USA). Cells were stained for mass cytometry as described previously [50,51] and acquired on a Helios mass cytometer (Fluidigm).

In vitro HIV infection

Given that the majority of HIV strains circulating in Benin are clade A or G, the clade A early HIV-1 strain Q23-FL was chosen for in vitro infection. Replication competent Q23-FL was made by transfection and titrated as described [50]. CD4+ T cells were activated for 2 days with plate-bound anti-CD3 (clone OKT3; eBioscience, San Diego, California, USA; 10 μg/ml), soluble anti-CD28/CD49d (clones L293/L25; BD Biosciences, San Jose, California, USA; 1 μg/ml each) and phytohemagglutinin (eBioscience, 2.5 μg/ml). CD4+ T cells were infected overnight with Q23-FL via ViroMag R/L magnetofection (Oz Biosciences, San Diego, California, USA) at a multiplicity of infection of 20. HIV infection was as measured by intracellular p24.

TIGIT blockade assay

Cells were cultured in RPMI media containing 10% FBS (Thermo Fisher Scientific, Waltham, Massachusetts, USA) and 1% penicillin/streptomycin/amphotericin (Thermo Fisher Scientific) (RP10). NK cells were incubated in 24-well plates at a concentration of 2 × 106 cells/ml in for 3 days at 37 °C with 5% CO2, with addition of rhIL-2 (R&D Systems, Minneapolis, Minnesota, USA, 300 IU/ml). After 3 days, NK cells were washed with fresh media and plated in 96-well plates (80 000–100 000 cells/well). Blocking mIgG1κ antihTIGIT antibody (eBioscience, clone MBSA43, 10 μg/ml) or a mIgG1κ isotype control (eBioscience, clone P3., 10 μg/ml) were added. Cells were incubated at 4 °C for 20 min, washed with PBS + 2% FBS and resuspended in media.

CD112 and CD155 mRNA expression levels

RNA from resting, mock-infected, or HIV-infected CD4+ T cells was isolated using the RNeasy Mini Kit with QIAshredder columns (Qiagen, Hilden, Germany). cDNA was produced using the SuperScript III First Strand Synthesis Kit (Thermo Fisher Scientific). Quantitative PCR (qPCR) was achieved with Taqman Universal Master Mix (Applied Biosystems, Foster City, California, USA) with the following Taqman Assays, using FAM-MGB probes: Nectin-2/CD112 (Thermo Fisher Scientific), PVR/CD155 (Thermo Fisher Scientific), and Human GAPDH (Applied Biosystems). qPCR was run on the Applied Biosystems Step One Plus Real-Time PCR System. Transcription levels were normalized to GAPDH. mRNA fold change expression was calculated using the double delta Ct analysis method with resting CD4+ T cells as the control group [52].

Natural killer cell : CD4+ T-cell co-cultures, cytokine stimulation and determination of natural killer cell function

All cells were cultured in RP10. NK cells (80,000–100,000/well) were incubated with HIV-infected or mock-infected autologous CD4+ T cells (320,000/well at a 1 : 4 effector:target ratio), 0.4 μl/200 μl of cell stimulation cocktail (phorbol 12-myristate 13-acetate (PMA) and ionomycin, eBioscience, San Diego, CA, USA), a cocktail of IL-12, IL-15 and IL-18 (rhIL-12, R&D Systems, 5ng/ml; rhIL-15; Pepro-Tech, Rocky Hill, New Jersey, USA; 20ng/ml; rhIL-18, R&D Systems, 0.5 μg/ml), or with the cancer cell line K562 (ATCC, used at passage 2–10, 300 000 cells/well at a 1 : 3 effector : target ratio). Cells were incubated for 4 h at 37°C with 5% CO2, in the presence of brefeldin, monensin (eBioscience) and anti-CD107a-APC-H7 antibody (BD Biosciences, Clone H4A3). After incubation, cells were stained with LIVE/DEAD Fixable Yellow Staining Kit (Thermo Fisher Scientific) for 20 min at room temperature, then fixed (BD FACS Lyse), permeabilized (BD FACS Perm II) and stained with anti-IFN-γ-V450 (BD Biosciences, Clone B27), anti-TIGIT-APC (eBioscience, Clone MBSA43), anti-CD3-PerCP-Cy5.5 (Biolegend, San Diego, California, USA; Clone UCHT1), anti-HIVp24-FITC (Beckman Coulter, Brea, California, USA; Clone KC57) for 30 min at 4°C. Data was acquired on a MACSQuant Analyzer Flow Cytometer (Miltenyi, Bergisch Gladbach, Germany).

Data analysis

Bead normalization was performed before downstream analyses ( [53]. Data were visualized using FlowJo v10.5.3 (Ashland, Oregon, USA) and FAS-L, Ki-67 and CXCR6 were excluded from subsequent analyses because of poor staining. The original data is available at ImmPort ( under study accession SDY1535. Serial negative gating was performed to identify NK cells from any contaminating cells remaining after purification (Figure S1a, Samples with cell number less than 1000 were excluded from analyses. The open source statistical package R ( was used for all statistical analyses [54]. Normalized signal intensities were transformed using the asinh function with a cofactor of 5 prior to generalized linear model (GLM) analysis, multidimensional scaling (MDS) and Uniform Manifold Approximation and Projection (UMAP) visualization. For the GLM analysis and MDS projection, we used the custom-made package ‘CytoGLMM’ [55,56]. For comparison between groups, we used the Wilcoxon rank-sum test. Whenever appropriate, P values were adjusted for multiple comparisons using the Benjamini--Hochberg method. UMAP embeddings were calculated using the R package ‘uwot’, with n_neighbor = 5 and min_dist = 0.2 [57]. Embeddings were visualized in Cytobank (


TIGIT is upregulated on natural killer cells from untreated HIV-infected women

To investigate the effect of HIV-1 infection on natural killer cells, we used CyTOF to profile the NK cells of 20 untreated HIV-1-infected women, 20 HIV-1 infected women on ART and 10 healthy women (Fig. 1a). A generalized linear model with bootstrap resampling revealed that HIV-1 infection is associated with a profound alteration of the NK-cell phenotype, with higher levels of CD38 and lower levels of Siglec-7 predicting chronic HIV-1 infection (Fig. 1b), as previously described [58,59]. This analysis also confirmed that TIGIT is upregulated in untreated HIV-1-infected individuals [47]. Additionally, our data revealed that TIGIT is not elevated on NK cells from antiretroviral-treated HIV-1-infected women (Fig. 1b and c). We performed correlations with CD4+ T-cell counts and found that TIGIT levels did not correlate with CD4+ T-cell counts (Figure S3,

Fig. 1
Fig. 1:
TIGIT is upregulated on natural killer cells from untreated HIV-infected women.

TIGIT expression is increased in CD56dim and CD56 natural killer cells from untreated HIV-1+ women

To explore the nature of this increased TIGIT expression, we evaluated TIGIT expression within different NK-cell subpopulations. NK cells are classically divided into three distinct subpopulations: CD56bright, which secrete cytokines at high levels, CD56dim, a more cytotoxic subpopulation, and CD56, a subpopulation of NK cells that are thought to be functionally impaired and are expanded during chronic viral infections, including HIV [10,60–62]. Here we confirmed that CD56 NK cells are increased in HIV-infected individuals, regardless of treatment status (Fig. 2a and 2b). Additionally, TIGIT expression was higher on CD56 NK cells from untreated HIV-1 infected women compared with healthy controls, and on the CD56dim subpopulation in untreated HIV-1+ women compared with treated women (Fig. 2c).

Fig. 2
Fig. 2:
TIGIT expression is increased in CD56dim and CD56- natural killer cells of untreated HIV-1+ women.

TIGIT blockade does not alter natural killer cell response to HIV-infected cells

TIGIT blockade increases ex-vivo NK-cell responses to cytokines in HIV-1 infected men [47] and elicits improved antitumor responses in mouse models [35]. To explore the effect of TIGIT blockade on NK-cell responses against HIV in vitro, we treated NK cells from healthy donors with an anti-TIGIT monoclonal antibody and measured NK-cell responses (CD107a and IFN-γ) by flow cytometry after incubation with mock-infected or HIV-infected autologous CD4+ T cells (Fig. 3a). Blockade of TIGIT did not improve NK-cell responses, when compared with untreated NK cells or cells treated with an isotype control (Fig. 3b and 3c). This lack of effect was not because of inadequate blockade, as less than 10% of NK cells could bind the anti-TIGIT antibody at the end of co-culture, suggesting continued binding of the blocking antibody (Figure S4,

Fig. 3
Fig. 3:
TIGIT blockade does not alter HIV-specific natural killer cell responses to HIV and TIGIT ligands are not upregulated by during HIV infection.

TIGIT ligands are not substantially upregulated by HIV infection

The TIGIT inhibitory signal is mediated by ligation with CD155 (high affinity) or CD112 (low affinity) [30,32]. We, therefore, measured the expression of CD155 and CD112 on HIV-infected CD4+ T cells ex vivo and in vitro. CD155 and CD112 were both expressed at low levels on CD4+ T cells from HIV+ women and there was no significant difference in expression levels between healthy women, untreated HIV+ women and treated HIV+ women (Fig. 3d and S2b). CD155 was not significantly induced in CD4+ T cells infected with HIV in vitro (Fig. 3e and Fig. S2c, A median of 36% of cells expressed p24 in vitro and CD155 was not differentially expressed between mock-infected and p24+CD4+ T cells. Although CD112 expression was significantly higher in p24+CD4+ T cells, its overall expression level was extremely low with a median of only 1.6% of cells expressing it (Figure S5, Finally, CD112 and CD115 transcription levels did not differ between mock-infected and HIV-infected CD4+ T cells (Fig. 3f) with a median fold change of HIV relative to mock-infected CD4+ T cells 1.15 (interquartile range = 0.94–2.4) for CD155 and 1.07 (interquartile range = 0.93–3.13) for CD112.

TIGIT expression is increased in adaptive/mature natural killer cells

To characterize NK cells with increased TIGIT expression, we gated on TIGIT and compared TIGIT+ and TIGIT NK cells from our Beninese women (Fig. 4a). Using MDS, we found that TIGIT expression marks a distinct population of NK cells that was not explained by HIV infection or treatment status (Fig. 4b--d). We then focused our analysis on features distinguishing TIGIT+ and TIGIT NK cells. We found that TIGIT+ NK cells express higher levels of activating markers (DNAM-1, NTB-A, 2B4, CD2) and exhibit an adaptive/mature phenotype [CD57hi, NKG2Chi, LILRB1 (ILT2, CD85j)hi, FcRγlo, Syklo] (Fig. 4e and f). To follow-up on this finding, we used UMAP to visualize NK-cell subsets and found that adaptive/mature NK cells (CD57hi, NKG2Chi, LILRB1hi, NKG2Alo, NKp46lo, NKp30lo, FcRγlo, Syklo) were separated on the right side of the UMAP1 axis (Fig. 4g). Additionally, when we colored the UMAP plot by TIGIT expression, we found that TIGIT co-localized with markers of adaptive/mature NK cells (Fig. 4h).

Fig. 4
Fig. 4:
TIGIT expression is increased in adaptive/mature natural killer cells.

TIGIT expression is associated with increased natural killer cell responses

As mature NK cells are typically associated with higher functional activity, we sought to better characterize the function of TIGIT+ and TIGIT NK cells. We isolated NK cells from healthy donors and cultured them with mock-infected or HIV-infected autologous CD4+ T cells (Fig. 5a). We then measured the functional responses (CD107a and IFN-γ) of TIGIT+ and TIGIT- NK cells by flow cytometry (Fig. 5b--e). We found that TIGIT+ NK cells had increased responses to mock-infected and HIV-infected autologous CD4+ T cells (Fig. 5b and 5c). To better define whether this difference was specific to HIV, we measured the responses to PMA/ionomycin, a cocktail of IL-12, IL-15 and IL-18, and the leukemia cell line K562 (Fig. 5a, d and e). TIGIT+ NK cells also exhibited increased responses to PMA/ionomycin, cytokines and tumor cells (Fig. 5d and e).

Fig. 5
Fig. 5:
TIGIT expression is associated with increased natural killer cell responses.


NK cells undergo significant changes during chronic HIV infection [16,63], with expansion of a hypofunctional CD56CD16+ NK-cell subpopulation [10,11], downregulation of several activating NK-cell receptors [12–14] and adaptive reconfiguration [64]. Some of these changes are not observed after treatment or in the setting of natural HIV control [65], indicating that diminished NK-cell function could contribute to disease pathogenesis. To better understand the role of different NK-cell receptors in the pathogenesis of HIV, we profiled the NK cells from infected and uninfected HIV+ women. A particularly striking finding was that TIGIT is upregulated in untreated HIV infection, but not in the setting of ART. TIGIT is an inhibitory receptor that has been associated with hypofunctional/exhausted NK cells in cancer [31,35]. TIGIT is also associated with exhausted CD8+ T cells in hematologic malignancy and in HIV infection [36,39,43], making it an interesting potential target for immunomodulation in the setting of HIV infection. TIGIT upregulation on NK cells from HIV-infected men has been recently described [47]. Here, we demonstrate that TIGIT is upregulated in HIV-infected women and that this upregulation does not occur in women receiving ART. Further, TIGIT upregulation is more prominent on CD56/CD16+ NK cells, a subpopulation thought to be functionally exhausted [10,11].

Several studies have explored the use of TIGIT blockade to rescue T and NK-cell function. Blocking TIGIT is associated with improved antitumoral response in tumor-bearing mice and in patients with colorectal cancer [35], and with improved NK-cell responses to cytokine stimulation in HIV+ patients [47]. However, we found that TIGIT blockade does not improve NK-cell responses to HIV-infected CD4+ T cells in healthy donors. The lack of a benefit from TIGIT blockade may be explained by the fact that TIGIT is unlikely to be engaged by its ligands during NK-cell recognition of HIV-infected cells. We found that CD155 is not upregulated on CD4+ T cells actively infected in vitro, consistent with previous reports [66–68]. In our study, CD155 is also not upregulated on CD4+ T cells from HIV-infected women. This differs from the findings of Yin et al.[47], who reported elevated CD155 expression on CD4+ T cells from HIV-infected men, though with extremely low expression levels. Notably, Yin et al. did not observe improvement in NK-cell responses by blocking CD155, suggesting that its engagement was not contributing to NK-cell suppression. Although we showed that CD112 is upregulated on CD4+ T cells infected in vitro, only a median 1.6% of cells express this receptor and upregulation is not observed in vivo, calling into question the biological significance of this modest upregulation. Together, the data suggest that the TIGIT/CD155/CD112 pathway may not be directly involved in the regulation of NK-cell responses to HIV-infected cells.

Although TIGIT itself may not play a key role in immune response to HIV, we noted that TIGIT marks a distinct NK subpopulation, co-expressing several activation markers (DNAM-1, NTB-A, 2B4, CD2). Co-expression of D NAM-1 is of particular interest as it shares the ligands CD112 and CD155 with TIGIT [69] and is associated with NK-cell memory to murine CMV [70]. TIGIT outcompetes DNAM-1 for binding to CD155 [71]. However, this interaction is unlikely to be relevant in the context of HIV infection, as we show that HIV does not upregulate CD155 in CD4+ T cells. In our cohort, TIGIT+ NK cells also exhibit a more adaptive/mature phenotype (CD57hi, NKG2Chi, LILRB1hi, FcRγlo, Syklo) and adaptive NK cells demonstrated higher TIGIT expression in unsupervised clustering via UMAP. This is consistent with the notion that NK cells undergo adaptive reconfiguration during HIV infection [64]. Notably, Sarhan et al.[72] demonstrated that adaptive NK cells from healthy donors express TIGIT at lower levels. Our contrasting finding could reflect unique differentiation pathways driven by HIV infection or a differential downregulation of TIGIT in response to cytokine stimulation, given that Sarhan et al. evaluated cytokine-treated NK cells rather than PBMCs directly ex vivo. Importantly, NKG2C+CD57+ adaptive NK cells are typically expanded in response to human CMV infection, making it difficult to distinguish CMV-related from HIV-related NK phenotypic changes. Unfortunately, CMV status is not available from our study, but the CMV seroprevalence in Benin is extremely high [73]. Thus, although we cannot exclude an effect of CMV in our cohort, we can reasonably assume that most of our women are CMV+ and that TIGIT upregulation in untreated HIV+ women is thus due to the direct effect of HIV infection rather than CMV, which both HIV-infected and uninfected women are likely to have.

Adaptive NK cells show increased responses to FcγRIIIa triggering [74–76], are expanded in HIV-infected patients and robustly respond to HIV peptides [14]. Here we demonstrate that TIGIT+ NK cells have higher functional activity against mock-infected and HIV-infected CD4+ T cells, compared with TIGIT- NK cells. Further, higher functional responses also occur in the presence of non-HIV stimuli. This increased activity could be explained by the co-expression of activating receptors, which may overwhelm the inhibitory signal provided by TIGIT, or by changes in the activation threshold during adaptive reconfiguration. Alternatively, a recent report showed that TIGIT could contribute to NK-cell licensing [77], suggesting that the presence of TIGIT may be required during NK-cell maturation to ensure the development of optimal effector function.

Some limitations should be noted in our study. First, the limited sample availability only allowed us to profile NK cells from a small sample size of female subjects. To overcome this, functional experiments were done on both female and male subjects. Additionally, we did not have sufficient samples from viremic HIV+ patients, thus functional experiments were carried on healthy, CMV-negative, HIV-negative subjects. Healthy individuals have lower TIGIT levels, potentially hindering the effect of TIGIT blockade. However, NK cells were treated with IL-2, which increases TIGIT expression (Figure S6,, thus providing sufficient substrate for the blocking assays.

In conclusion, our work demonstrates that TIGIT is overexpressed on NK cells of untreated HIV+ women, but fully corrected by ART. We find that TIGIT does not directly participate in the NK-cell response to HIV-infected cells, but rather marks a mature NK-cell subpopulation with adaptive features that is enhanced in functional responses to virus-infected cells, tumors and cytokine stimulation. These results need to be confirmed in independent cohorts that evaluate both the direct functional contributions of TIGIT and the role of ART in regulating TIGIT expression.


We are grateful to the Beninese study participants. We are indebted to N. Geraldo, A. Gabin, C. Assogba and C. Agossa-Gbenafa for their clinical expertise, to M. Massinga-Loembe, G. Ahotin, L.Djossou and E. Goma for their technical assistance and to G. Batona and other field workers who helped with recruitment of commercial sex workers.

Grant support: This work was supported by: NIH Ruth L. Kirschstein Institutional National Research Service Award T32AI007502 and TL1TR001084 (E.V.), NIH/NIAID K08 K08AI138640 (E.V.), ITI/Bill & Melinda Gates Foundation Pilot Grant (C.A.B.), NIH/NIAID DP2 AI1219301 (C.A.B.), NIH/NIDA Avant Garde Award for HIV Research DP1 DA045089 (C.A.B.), Burroughs Wellcome Fund Investigators in the Pathogenesis of Infectious Diseases (C.A.B.), Grant # PJT-148529 from the Canadian Institutes of Health Research and by the R�seau SIDA from the Fonds de Recherche du Qu�bec en Sant� (M.R.).

The content of this work is solely the responsibility of the authors and does not necessarily represent the official views of the NIH.

Conflicts of interest

There are no conflicts of interest.


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adaptive; HIV; mature; natural killer cells; T cell immunoreceptor with Ig and ITIM domains (TIGIT)

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