Multifaceted role of BTLA in the control of CD8⁺ T cell fate after antigen encounter

Abstract

Purpose

Adoptive T-cell therapy using autologous tumor-infiltrating lymphocytes (TIL) has shown an overall clinical response rate 40–50% in metastatic melanoma patients. BTLA (B-and-T lymphocyte attenuator) expression on transferred CD8⁺ TIL was associated with better clinical outcome. The suppressive function of the ITIM and ITSM motifs of BTLA is well described. Here, we sought to determine the functional characteristics of the CD8⁺BTLA⁺TIL subset and define the contribution of the Grb2 motif of BTLA in T cell co-stimulation.

Experimental Design

We determined the functional role and downstream signal of BTLA in both human CD8+ TIL and mouse CD8+ T cells. Functional assays were used including single cell analysis, Reverse Phase Protein Array (RPPA), antigen-specific vaccination models with adoptively transferred TCR-transgenic T cells as well as Patient-Derived Xenograft (PDX) model using Immunodeficient NOD-scid IL2Rgammanull (NSG) tumor-bearing mice treated with autologous TIL.

Results

CD8⁺BTLA⁻ TIL could not control tumor growth in vivo as well as their BTLA⁺ counterpart and antigen-specific CD8⁺BTLA⁻ T cells had impaired recall response to a vaccine. However CD8⁺BTLA⁺ TIL displayed improved survival following the killing of a tumor target and heightened “serial killing” capacity. Using mutants of BTLA signaling motifs we uncovered a costimulatory function mediated by Grb2 through enhancing the secretion of IL-2 and the activation of Src after TCR stimulation.

Conclusions

Our data portrays BTLA as a molecule with the singular ability to provide both co-stimulatory and co-inhibitory signals to activated CD8⁺ T cells, resulting in extended survival, improved tumor control and the development of a functional recall response.

Introduction

Adoptive T cell therapy (ACT) using the body’s own expanded tumor-infiltrating lymphocytes (TIL) with prior lymphodepletion and followed by high dose IL-2 administration has demonstrated an overall response rate of 38–51% in multiple clinical trials for stage IIIC/IV metastatic melanoma (1–4). A comprehensive immunophenotyping of TIL infusion products has revealed an unexpected finding that a population of CD8⁺TIL expressing a molecule known to attenuate T cell response, the B and T lymphocyte attenuator (BTLA), was strongly associated with a positive clinical response (4).

BTLA is an inhibitory molecule expressed by T cells, B cells, dendritic cells, and NK cells (5). Herpes virus entry mediator (HVEM), a tumor necrosis factor receptor superfamily member 14 (TNFRSF14), is the known ligand for BTLA (6). The cytoplasmic domain of BTLA consists of three motifs; an immunoreceptor tyrosine-based inhibition motif (ITIM), an immunoreceptor tyrosine-based switch motif (ITSM), and a growth factor receptor-bound protein 2 motif (Grb2). Ligation of BTLA by HVEM has been shown to recruit Src homology 2 (SH2) domain-containing phosphatase 1 and 2 (SHP-1 and SHP-2) to the ITIM and ITSM motifs, resulting in suppression of T cell receptor (TCR) activation (5,7). Indeed, ITIM and ITSM motifs were required for the full function of BTLA to inhibit T cell proliferation and cytokine production including IFN-γ, IL-2, and IL-10 (8,9). A recent report indicated that PD-1 ligation, which also contains ITIM and ITSM motifs, selectively inhibits both the Akt and Ras-MEK-ERK pathways (10). So far, it remains inconclusive whether BTLA utilizes mechanism similar to PD-1 as ITIM and ITSM motifs are commonly shared between these two receptors.

Unlike PD-1, BTLA also harbors a Grb2 motif. Although the function of the Grb2 motif remains unclear, some evidence suggests that it may actually transmit a positive signal. In fact, an in vitro binding assay demonstrated the potential interaction of the Grb2 binding motif with the Grb2 adaptor protein and the p85 subunit of phosphatidylinositol 3-kinase (p85 PI3K) (7,11). Interestingly, a gene expression analysis of mouse CD4⁺T cells following activation by anti-CD3 and anti-BTLA demonstrated a highly overlapping transcription profile with that produced by anti-CD3 in combination with positive co-stimulators (CD28, ICOS, and CD80), but not with inhibitory molecules (PD-1 and CTLA-4) (12).

Emerging evidence also demonstrates that BTLA serves as a T cell differentiation marker in human T cells as BTLA expression is highly enriched in naïve T cells and central memory T cells (Tcm) and down-regulated upon T cell differentiation (13). Our recent work demonstrated that CD8⁺BTLA⁺TIL exhibited the molecular signature of less-differentiated T cells as compared to their CD8⁺BTLA⁻ counterpart and had increased persistence following adoptive transfer in treated patients (14). Several studies in both immunodeficient murine and non-human primate models also demonstrated that central memory derived effector CD8⁺ T cells established a pool of in vivo persistent memory T cells (15–18). In addition, adoptive transfer of memory T cell with stem cell properties (T_SCM) was shown to confer in vivo persistence and better tumor control due to enhanced survival and anti-tumor properties (15).

Thus far, it remains understudied whether the intrinsic properties of less-differentiated TIL highly enriched in BTLA expressing cells and/or BTLA signaling itself contribute to the favorable clinical outcome of TIL treated patients. In this study, we have uncovered a survival advantages of the BTLA⁺ subset that allows for serial killing of target tumor cells, which may explain our previous correlation between this subset and response to TIL ACT. In addition, our results unveiled a role for the BTLA-associated Grb2-binding motif in T cell proliferation and IL-2 production following TCR engagement that was independent of the inhibitory function of ITIM/ITSM motifs. The use of a pmel BTLA knockout system demonstrates a weaker priming of T cells in response to the cognate antigen and the absence of a recall response. Overall, this study has uncovered a previously unappreciated role of the Grb2 motif of BTLA in providing positive co-stimulatory signal to T cells and the ability of CD8⁺BTLA⁺TIL to function as serial killers.

Materials and Methods

Cell lines

Platinum-E retroviral packaging cell line, MEL 526 tumor line, and primary melanoma tumor cell line #2549, as well as B16F10, and B16OVA were maintained in Roswell Park Memorial Institute (RPMI) supplemented with 10% fetal bovine serum (FBS) (Gemini bio product), 10 mM HEPES (Gibco™), 10 mM Penicillin-streptomycin (Gibco™), and 10 mM Glutamine (Gibco™), selenium-transferrin-insulin (Gibco™), and 0.05 mM Beta-mercaptoethanol (Gibco™) as previously described (2, 34). Platinum-E retro packing cell line was purchased from Cellbiolabs. MEL 526 tumor line was obtained from Dr Steven A. Rosenberg at the National Cancer Institute. Autologous primary melanoma tumor cell line #2549 was generated at M.D. Anderson Cancer Center from a tumor sample of a patient enrolled on an ongoing adoptive T cell therapy study. The cell line #2549 was last authenticated on 03/24/2015 by STR DNA fingerprinting using the Promega 16 High Sensitivity STR Kit (Catalog #DC2100). The STR profiles were compared to online search databases (DSMZ/ATCC/JCRB/RIKEN) of 2455 known profiles; along with the MD Anderson Characterized Cell Line Core (CCLC) database of 2556 know profiles. The STR profiles matched known DNA fingerprint of patient’s PBMCs. No authentication was performed in all other cell lines.

Patient tumor sample acquisition

Tumor samples were obtained from patients with Stage IIIc and Stage IV melanoma undergoing surgery at The University of Texas MD Anderson Cancer Center according to an Institutional Review Board-approved protocol and with patient consent (IRB# 2004-0069, LAB06-0755, NCT00338377). This study was carried out in compliance with the protocol and Good Clinical Practice concerning medical research in humans, as described in the Declaration of Helsinki.

Generation of TIL

Fragments from melanoma tumors were cut into 1 to 2 mm³. Each fragment was placed into a single wells in 24-well-culture plates (Falcon) and maintained with RPMI supplemented with 10% heat inactivated Human AB serum (Gemini bio product), IL-2 6000 IU/ml (Proleukin, Novartis), 10 mM HEPES, 10 mM Penicillin-streptomycin (Gibco™), and 10 mM Glutamine (Gibco™) as previously described (19).

Retroviral constructs of BTLA wild-type and mutants

Murine BTLA was amplified from fully sequenced murine BTLA from Mammalian Gene Collection Clones (MGC, Open biosystem) by PCR with primers mBTLA-F and R (Supplementary Table S1). The PCR products were cloned into pRVKM retroviral vectors. To generate BTLA mutants, we substituted tyrosine for phenyl alanine in either Grb2 motifs or ITIM and ITSM motifs of BTLA cytoplasmic tails (Supplementary Table S2). These included murine ΔGrb2 mutants (Y245F), and murine Δ ITIM and ITSM mutants (Y274F and Y299F). Sequences of all constructs were validated by DNA sequencing.

Retroviral transduction of mouse BTLA-KO-T cells

pRVKM retroviral vectors and pEco plasmids were co-transfected into Plate-E cells using using PolyJet (Signagen Laboratories). The supernatants were harvested 60 h later and concentrated using Vivaspin-20 (Vivaproducts). Splenocytes from OT-1 BTLA KO mice were cultured with RPMI1640 with 10% FBS and hIL-2 at 300 IU/ml, and activated with anti-mouse CD3 at concentration of 0.3 ug/ml (Clone 145-2C11, eBioscience) for 24 h. The cells were then infected with a concentrated retrovirus and further expanded in RPMI1640 with 10% FBS and hIL-2 for 3 days. The cells were sorted based on the expression of GFP using a FACSAria (BD Bioscience) and propagated with hIL-2 at 300 IU/ml for 5 days.

Reverse phase protein array (RPPA)

Murine OT-1 BTLA KO cells overexpressing BTLA WT or mutants constructs were re-stimulated with either 10 ng/ml anti-mouse CD3 (Clone 145-2C11, BD Pharmingen™) alone or with recombinant mouse HVEM Fc (R&D systems) plate-bound for 8 h prior to harvest with the cell lysis buffer (kindly provided by RPPA core facility at The University of Texas M.D. Anderson Cancer Center). The cell lysates were centrifuged at 14,000 rpm for 10 minutes at 4°C. The protein supernatant was quantified using protein assay kit (Thermo scientific). RPPA was processed and normalized as previously described (20). Differential fold expression of protein was analyzed using Linear models and empirical Bayes methods(21). Volcano plots were generated using R system. For human TIL, four TIL lines were stained with anti-CD8 (clone RPA-T8, BD Pharmingen™), anti-BTLA (clone J168, BD Pharmingen™), and Sytox blue (Molecular Probe™) under aseptic condition. The cells were sorted based on expression of CD8⁺BTLA⁺ using FACSAria (BD Biosciences). On the next day, sorted TIL were re-stimulated with anti-human CD3 (clone OKT-3, eBioscience) with or without recombinant human HVEM-Fc (R&D systems) plate-bound for 8 h prior to harvest with the cell lysis buffer. The protein samples were processed, normalized, and analyzed as similar to the mouse experiment described above.

Intracellular cytokine staining

OT-1 BTLA KO T cells overexpressing BTLA WT or mutants were re-activated with either dendritic cells pulsed with OVA peptide (SIINFEKL) (American peptide) or dendritic cells alone at ratio of 1:40 in the presence of BD™ GolgiStop™ (according to the manufacturer’s instruction, BD Pharmingen™). After 4 h, cells were fixed and permeabilized using BD Cytofix/Cytoperm™ kits (according to the manufacturer’s instruction, BD Pharmingen™), and subsequently stained with anti-mouse IFN-γ (clone XMG1.2, BD Pharmingen™) and anti-mouse TNF-α (clone MP6-XT22, BD Pharmingen™).

Cytokine multiplex assays

Murine OT-1 BTLA KO cells overexpressing BTLA WT or mutants were re-stimulated with either 10 ng/ml anti-mouse CD3 (Clone 145-2C11, BD Pharmingen™) alone or with recombinant mouse HVEM-Fc (R&D systems) plate-bound for 24 h. Supernatants were collected to quantify the secreted cytokines using a MILLIPLEX MAP Mouse CD8⁺ T Cell Magnetic Bead Panel (Millipore).

Killing assays

T cells were co-cultured with tumor cells labeled with eFluor670® at ratio of 1:1, 1:3, and 1:10. After 3 h, the cells were fixed and permeabilized using BD Cytofix/Cytoperm (according to manufacturer protocol, BD Pharmingen™), then stained with anti-cleaved caspase-3 (clone CPP32, BD Pharmingen™), and analyzed by a BD FACSCanto II (BD Biosciences).

Cell proliferation assay

Murine OT-1 BTLA KO expressing BTLA WT or mutants were labeled with eFluor670® and re-stimulated with dendritic cells alone or dendritic cells pulsed with OVA peptide (SIINFEKL) (American peptide) at a ratio of 1:40 for 48 h prior to being analyzed using a BD FACSCanto II (BD Biosciences).

Nanowell array-based cytolytic assay

TIL and Tumor cells were labeled with 1 µmol/L of red fluorescence dye (PKH26, Sigma) and 1 µl of green fluorescence dye (PKH68, Sigma) respectively. The cells were loaded onto nanowell at concentration of 1×10⁶/mL. Target cell cytolysis mediated by TIL was monitored using a Carl Zeiss Axio Observer fitted with Hamamatsu EM-CCD camera using 10 × 0.3 NA objective. Apoptotic cells became green when stained with Annexin V conjugated with Alexa 647as previously described (22).

In vivo killing of melanoma tumors using an NSG ACT model

NOD-scid IL2Rgamma^null (NSG) mice were engrafted with either 5 × 10⁶ MEL 526 MEL tumor cells or 5 × 10⁶ autologous primary melanoma tumor cells. On day 12, either 10 ×10⁶ sorted CD8⁺BTLA⁺ or sorted CD8⁺BTLA⁻ were adoptively transferred into tumor-bearing mice (n= 5 to 8 per group). Recombinant human IL-2 (Proleukin, Prometheus) was administered intraperitoneally at a concentration of 6 ×10⁵ I.U. immediately after TIL transfer and daily for three days. Tumor size was measured every other day. Mice were sacrificed when tumors exceeded 15 mm diameter. Peripheral blood was collected every other day, lysed with ACK lysis buffer, then stained for AQUA (Invitrogen), anti-human CD45 (clone HI30, BD Pharmingen™), and anti-human CD8 (clone RPA-T8, BD Pharmingen™).

Vaccination model

C57BL/6J mice were intravenously administered 0.5 × 10⁶ naïve pmel-1 or OT-I T cells, and vaccinated with gp100 peptide or OVA peptide (100µg) together with anti-CD40 (50 µg) and imiquimod (50 mg). Recombinant human IL-2 at 1.2 ×10⁶ IU was administered once, and 6 × 10⁵ IU twice daily for the next 2 days (i.p.). Peripheral blood was collected every other day to determine the frequency of circulating pmel or OT-I T cells. Mice were boosted with gp100 or OVA peptide following the contraction phase and peripheral blood was collected every other day. Mice were sacrificed on day 120, and splenocytes were isolated to determine the presence of pmel T cells.

Animals

NOD-scid IL2Rgamma^null (NSG) mice were purchased from the Jackson Laboratory (Bar Harbor, ME). OT-1 BTLA KO C57BL/6 and Pmel BTLA KO C57BL/6 mice were kindly provided from Dr. Roza Nurieva. Pmel BTLA WT C57BL/6 mice were kindly provided by Dr. Willem Overwijk. C57BL/6 mice were purchased from Charles River Labs. All mice were housed in a specific pathogen-free facility at The University of Texas M.D. Anderson Cancer Center. Animal protocols were approved by The University of Texas M.D. Anderson Cancer Center (#00001229 (RN)).

Statistical analyses

Statistical analyses were performed using GraphPad Prism and the R system. For all survival curve analysis data, a long rank test was used to compare distribution of the two groups. We applied Linear models and empirical Bayes methods to compare the differential protein expression from RPPA data sets. Differences in tumor cell death of B16OVA and B16F10 and the percentage of positive virally transduced mouse T cells producing IFN-γ and TNF-α were analyzed using a two-way ANOVA, and a two-tailed Student’s t-test was used to determine statistical significance for all other analyses.

Results

High expression of CD8a and BTLA correlates with an improved survival of stage III metastatic melanoma patients

To investigate whether BTLA is associated with melanoma patient survival, we conducted a Kaplan-Meier survival analysis in stage III metastatic melanomas according to gene expression data in tumor tissues from TCGA (The Cancer Genome Atlas) (23). We found that patients expressing high transcript levels of either CD8a or BTLA had much better survival as compared with either CD8a low or BTLA low respectively (CD8a high vs CD8a low, P=0.0007, N=42; BTLA high vs BTLA low, P=0.001, N=42) (Fig. 1a and 1b). When both markers were analyzed together, the association of CD8a high and BTLA high conferred the greatest survival benefit as compared with other combinations (CD8a high BTLA high vs CD8a low BTLA low, P =0.0006, N =98) (Fig. 1c). BTLA can be expressed by immune cells other than T cells such as NK cells, B cells or dendritic cells, with the highest levels found in B cells (5). Interestingly, we did not observe improved survival in patients with high BTLA expression when associated with either high NK cells (identified by the expression of NCR1) or B cells (CD19) (Supplementary Fig. S1a and S1b). This data suggests that high BTLA expression in association with high CD8⁺ signal is associated with improved melanoma patient survival. The co-expression of CD8 and BTLA cannot be inferred by gene expression profiling from tumor tissue however the improved survival benefit measured in conjunction with BTLA and CD8 expression is not seen when BTLA is evaluated in conjunction with markers from other cell populations known to express BTLA. This data lends support to our hypothesis that CD8⁺BTLA⁺ TIL exert efficient anti-tumor control based on our previous observation that this T cell population correlated with better response to TIL ACT (4).

Figure 1. Correlation of BTLA and CD8a co-expression with overall survival in stage III melanoma.

Kaplan-Meier survival curves in stage III metastatic melanoma from The Cancer Genome Atlas (TCGA) consortium depicts; (a) CD8a expression; CD8a high versus CD8a low (b) BTLA expression; BTLA high versus BTLA low (c) Combined CD8 and BTLA expression; CD8a high BTLA high versus CD8a low BTLA high versus CD8a high BTLA low versus CD8a low BTLA low. Total number of patients=98, (high- above median, low-below median). Statistical significance was determined using a log-rank. (P <0.001 and P <0.0001).

CD8⁺BTLA⁺TIL subset exhibits greater in vivo tumor control

It has been noted that more differentiated T cells secrete more cytotoxic and cytolytic proteins compared to less-differentiated T cells (24). Thus, we conducted both in vitro and in vivo tumor control studies to compare the killing capacity of the BTLA⁺ and BTLA⁻ CD8⁺ TIL subsets. To determine CTL-mediated tumor killing and ensure an equal tumor-specific population of both BTLA subsets, MEL 526 melanoma tumor cells expressing the MART-1 antigen were co-cultured with either sorted MART-1 recognizing (tetramer positive) CD8⁺BTLA⁺ (CD8⁺MART-1⁺BTLA⁺) or CD8⁺BTLA⁻ (CD8⁺MART-1⁺BTLA⁻). We found comparable MART-1 antigen-specific tumor killing ability after a short term (3h) TIL and tumor co-incubation between the BTLA⁺TIL and BTLA⁻TIL subsets in two TIL lines (Fig. 2a left panel TIL#2559 and mid panel TIL #2765). In addition, comparable killing ability was observed using bulk non-antigen restricted, sorted BTLA subsets in a third TIL line (Fig. 2a, right panel TIL #2549). Since we did not observe a difference in in vitro killing between the two subsets, but have demonstrated a better clinical outcome in patients infused with more CD8⁺BTLA⁺ TIL, we next determined whether this subset could exhibit better in vivo tumor control. Immunodeficient NOD-scid IL2Rgamma^null mice (NSG) were engrafted with 5×10⁶ MEL 526 human melanoma tumor cells for 10 days prior to adoptive transfer of 10×10⁶ human CD8⁺MART⁺BTLA⁺TIL or CD8⁺ MART⁺BTLA⁻TIL. Tumor burden was measured every other day and transferred TIL were quantified from peripheral blood based on the expression of CD45 and CD8. We observed that the CD8⁺BTLA⁺ TIL subset exhibited significantly better tumor control on day 14 as compared with its BTLA⁻ counterpart in NSG mice treated with either MART-1 tumor antigen restricted TIL line (2559 MART⁺; P=0.002 (Fig. 2b, left panel and 2765 MART⁺; P=0.042 (Fig. 2b mid panel)) as well as in a non-tumor antigen restricted setting (2549; P=0.049) (Fig. 2b, right panel). In addition, we found that the level of the CD8⁺BTLA⁺ TIL subset in the peripheral blood was significantly higher than the CD8⁺BTLA⁻ subset early after therapy in mice treated with antigen-specific TIL lines (2559 MART⁺; P=0.039 on day 4, (Fig. 2c, left panel) 2765 MART⁺; P=0.0021 and 0.02 on day 2 and 4 respectively (Fig. 2c, mid panel)), but not in those treated with the non-restricted antigen-specific TIL line (2549; P=0.06 on day 2) (Fig. 2c, right panel). In summary, this data suggests that the CD8⁺BTLA⁺ and CD8⁺BTLA⁻ TIL subsets are equally able to directly kill tumor targets in a short term in vitro assay. However, the BTLA positive subset provides superior in vivo tumor control and tends to persist better following TIL transfer.

Figure 2. CD8⁺BTLA⁺ TIL demonstrate a superior anti-tumor effect in vivo.

(a) MART1-reactive CD8⁺ TIL (left and mid panel) and TIL 2549 (right panel) were sorted into BTLA⁺ and BTLA⁻ subsets. MEL 526 (melanoma tumor expressing MART-1 antigen, left and mid graph) or autologous tumor line 2549 (right graph) were stained with eFluor670® and co-cultured with TIL at the following TIL-to-tumor cell ratios (1:10, 1:3, and 1:1). Tumor cell death is measured by the percentage of caspase-3 positive cells. (b) Ten million sorted CD8⁺BTLA⁺ or sorted CD8⁺BTLA⁻ TIL were intravenously injected into tumor bearing mice previously subcutaneously implanted with either MEL 526 or autologous melanoma tumor line 2549. Tumor burden was measured using calipers and diameter graphed as mm². (c) Bar graph shows the percentage of CD45⁺CD8⁺ in the peripheral blood on days 2, 4, 6, and 8 post-adoptive transfer in the same experiment described in (b). N= 5–8 animals per group. *P <0.05, **P <0.001, ***P <0.0001. All values in figure are expressed as mean ± s.e.m. P-values were calculated using a two-tailed Student’s t-test.

Shorter target seeking but longer target killing time for CD8⁺BTLA⁺ TIL subset

As demonstrated above, the CD8⁺BTLA⁺ subset provides significantly better tumor control than its CD8⁺BTLA⁻ counterpart in vivo; however, this difference is not observed in an in vitro setting. Because our previous in vitro tumor killing experiments were conducted using population assays and for only 3 hours, this limited our ability to track T cell fate following tumor target cell interactions. To further understand the behavioral difference between the CD8⁺BTLA⁺ and CD8⁺BTLA⁻ subsets in mediating tumor killing, we utilized Timelapse Imaging Microscopy In Nanowell Grids (TIMING), to study the dynamic interactions between individual tumor targets and effector T cells in high-throughput (25,26). To this end, labeled tumor cells and TIL were loaded onto the nanowell chip, consisting of 28,224 wells regularly separated in 7×7 blocks, and the interactions were quantified at two separate effector-to-target cell ratios of 1:1 and 1:2. This assay allowed us to dissect the kinetics of T cell killing by calculating the following sequential parameters: 1) Time needed to establish conjugation between the T cell and tumor target (t_seek), 2) duration of the contact between the T cell and the tumor target (t_contact), and 3) time between the first T cell contact with the tumor cell and tumor cell apoptosis (t_death) (Fig. 3a). Interestingly, the CD8⁺BTLA⁺ subset was more efficient in tumor seeking at effector-to-target cell ratios of 1:1 (N = 3319) as the time utilized to make first contact with tumor cells was significantly less than that of the CD8⁺BTLA⁻ subset (P<0.0001) (Fig. 3b, left panel). This difference was not observed when 2 targets were present in the well (1:2, P=0.22) (Fig. 3b, left panel). It is possible that loading 2 targets in each nanowell rendered the distance to travel to meet each target too short to be able to measure a difference in the ability of T cell subsets to seek the target. However, in comparison to CD8⁺BTLA⁻ TIL individual CD8⁺BTLA⁺ TIL spent longer time in contact with a tumor cell target and induced apoptosis with slower kinetics at both E:T ratios studied (t_contact; 1:1 and 1:2, P<0.0001, t_death; 1:1 and 1:2, P<0.0001) (Fig. 3b, mid and right panels, respectively). Overall, individual CD8⁺BTLA⁺ TIL found their target faster, but required longer durations to complete tumor cell killing (Supplementary Movie 1 and 2).

Figure 3. CD8⁺BTLA⁺TIL subset mediates killing of multiple tumor targets through enhanced survival properties.

Nanowell array-based cytolysis assay was used to determine tumor killing capacity at single cell level. Effector cell and Tumor target are labeled in different colors and loaded into the nanowell at effector-to-target cell ratios of 1:1 and 1:2. Interaction between effector and target cells is monitored by automated time-lapse camera coupled with a fluorescence microscope. (a) Schematic diagram demonstrates the sequential events that effector cells seek (tseek), contact (tcontact), and mediate tumor cell death (tdeath). (b) Either CD8⁺MART⁺BTLA⁺ or CD8⁺MART⁺BTLA⁻ subset was co-incubated with MEL 526. Time (min) between each sequential event is evaluated. Dot plots depict tseek (left), tcontact (middle), tdeath (right) in comparison between CD8⁺BTLA⁺ (blue) and CD8⁺BTLA⁻ (red) subsets. All error bars depicts the mean ± s.e.m. All P-values were calculated using a two-tailed Student’s t-test. (N=497). (c) Donut charts demonstrate the frequency of tumor cell death following effector cell killing by either CD8⁺BTLA⁺ (left) or CD8⁺BTLA⁻ (right) subset. Inner circle and outer circle depict E:T ratio of 1:1 and 1:2 respectively. (d) Kaplan-Meier survival curves of T cell-contacted tumor target resulting in a killing event in the first 250 minutes in comparison between CD8⁺BTLA⁺ and CD8⁺BTLA⁻ subsets at effector-to-target cells ratios of 1:1 (left) and 1:2 (right). (e) Kaplan-Meier survival curves of T cell-contacted tumor target resulting in a killing event in the last 250 minutes minutes in comparison between CD8⁺BTLA⁺ and CD8⁺BTLA⁻ subset at effector-to-target cells ratios of 1:1 (left) and 1:2 (right). Statistical significance in (d) and (e) was determined using a log-rank. (N=3319). (***P <0.0001). (f) Kaplan-Meier survival curves of tumor-contacted effector cells following tumor cell death in comparison between CD8⁺BTLA⁺ and CD8⁺BTLA⁻ subsets at an effector-to-target ratio of 1:1. Statistical significance was determined using a log-rank. (N= 3319) (*P<0.05).

CD8⁺BTLA⁺ TIL subset is characterized by a heightened ability to kill multiple targets

We next examined whether one subset was more potent in its overall tumor killing capacity by evaluating tumor target cell survival over the 8 hour co-culture period. Overall, both T cell subsets killed 18% of the tumor targets at an E:T ratio of 1:1 (Fig. 3c, red inner circle). However, at the 1:2 ratio the CD8⁺BTLA⁺ subset was able to kill a total of 21% of the tumor targets upon contact as opposed to only 14% for the CD8⁺BTLA⁻ subset (Fig. 3c, red and green sections, outer circle). Secondly, when presented with two tumor targets, the CD8⁺BTLA⁺ subset was twice as likely to kill both targets (14% for the BTLA⁺ vs 7% for the BTLA, Fig. 3c).

Examination of the kinetics of tumor killing offered clues to understand the differences in the overall killing potential of the two subsets. The analysis of the full 500 minutes assay revealed a different killing pattern early after tumor encounter (first 250 minutes) in comparison to the second half of the incubation (last 250 minutes). A close look at the first 250 minutes demonstrated that the CD8⁺BTLA⁻ subset was more effective in tumor killing than the CD8⁺BTLA⁺ subset as the survival of the contacted tumor targets dropped significantly faster when co-incubated with CD8⁺BTLA⁻ TIL at effector-to-target cell ratios of 1:1 and 1:2 (left panel 1:1, P<0.03, right panel 1:2, P<0.0001) (Fig. 3d). However, the overall percentage of killed targets at both ratios at the 250 minutes mark is the same between both TIL subsets. Therefore, the killing potential of the two subsets is comparable in the first 250 minutes but their kinetics of killing are different.

Over the last 250 minutes of the assay the picture changes (Fig. 3e). The blue curve (BTLA⁺) now dips below the red curve (BTLA⁻), signifying that the BTLA⁺ subset is catching up with the BTLA⁻ subset in terms of the speed at which it kills (E:T ratio of 1:1, p=0.54), or takes a significant lead (E:T ratio of 1:2, p<0.0001). It is also worth noting that very limited killing occurs with the BTLA⁻ in the second half of the experiment at any ratio while the BTLA⁺ subset achieves higher killing when more targets are present in the well (1:2). We reasoned that this situation could be explained by a differential ability for serial killing, or the ability to kill a second target, which can only be detected if there is more than one target in the well therefore at a 1:2 ratio (Fig. 3e).

Improved survival of the CD8⁺BTLA⁺ TIL subset following killing of a target

After activation, T cells can be eliminated through the process of activation-induced cell death (AICD). The ability of the CD8⁺BTLA⁺ subset to kill multiple targets could be linked to an enhanced ability to survive AICD after completing its effector function. To test this hypothesis, we quantified effector T cell death after a tumor killing event. We found that in fact, CD8⁺BTLA⁻ are more susceptible to undergo apoptosis as compared with their CD8⁺BTLA⁺ counterpart (P<0.01) (Fig. 3f). This data demonstrates that the CD8⁺BTLA⁺ subset survives better after killing a tumor target and thus is able to sustain its cytotoxic functionality and eliminate additional tumor cells. In summary, these results argue that both subsets kill equally well in a short-term killing assay (250 minutes) but the differences emerge when the T cell and tumor co-incubation is prolonged to 8 hours wherein the CD8⁺BTLA⁺ TIL subset is found to have an increased propensity to survive a killing event and therefore function as serial killers, thus confirming that the CD8⁺BTLA⁺ TIL subset is qualitatively better consistent with the in vivo results (Supplementary Movie 3).

Memory recall response is defective in BTLA deficient T cells

Our data suggests a difference in survival of BTLA⁺ anti-tumor CD8⁺ T cells rather than a change in killing potential. We next sought to evaluate the long-term fate of CD8⁺BTLA⁺ antigen-specific T cells after in vivo antigenic challenge in an immunocompetent animal. Our study of the expression of BTLA in human and mouse CD8⁺ T cells showed a different expression pattern (Supplementary Fig. 2 and 3). Murine naïve CD8 (CD44lowCD62Lhigh) are negative for BTLA but expression of BTLA is acquired as the cells differentiate to memory (CD44high) while human naïve CD8 (CCR7⁺, CD45RA⁺) are positive for BTLA and expression is progressively lost as the CD8 differentiate to terminal effector cells (EMRA, CCR7⁻, CD45RA⁺). However memory stages of differentiation are positive for BTLA in both mouse and human. Based on this we reasoned that a vaccination model would be the most appropriate in vivo antigen trigger to measure the role of BTLA on the function of CD8. We aimed to measure the impact of BTLA expression on newly generated memory CD8⁺ T cells on their capacity to respond to a second antigen exposure (recall). In this study, we sought to determine the role of BTLA in the persistence of antigen-specific CD8⁺ T cells in vivo after antigenic stimulation. We used TCR transgenic CD8⁺ T cells that are either WT or KO for BTLA and study their response to immunization and memory-recall response using a mouse vaccination model as previously described (27). Splenocytes (5e⁵) from either Pmel-1 WT or Pmel-1 BTLA KO (recognizing gp100 peptide) were adoptively transferred into C57BL/6 recipient mice. On the following day, mice were vaccinated with gp100 peptide together with anti-mouse CD40. Imiquimod cream 5% was also applied on the vaccination site to boost the innate immune response. Additionally, IL-2 was also provided to support in vivo T cell proliferation following vaccination (Fig. 4a and b). The frequency of Pmel-1 T cells was tracked in peripheral blood after vaccination (Fig. 4c). On day 20 following priming, we observed a significantly higher frequency of Pmel-1 WT in peripheral blood when compared with Pmel-1 BTLA KO. The frequency of both Pmel-1 WT and Pmel-1 BTLA KO contracted to pre-priming frequency by day 30. Boost vaccination was performed on day 60. We observed that Pmel-1 WT T cells had a robust recall response that was absent in the Pmel-1 BTLA KO group (Fig. 4c). To determine the impact on the generation of a long-term memory response, we assessed the frequency of Pmel-1 T cells in the spleen on day 120. We found a significantly higher percentage of Pmel-1 WT T cells in the spleen as compared to Pmel-1 BTLA KO T cells (Fig. 4d, p=0.038). Similar data was obtained with the transfer of BTLA WT or KO OT-1 T cells and vaccination with OVA peptide SIINFEKL (Supplementary Fig S4). In this context, the use of BTLA KO OT-1 T cells led to a marked attenuation of the recall response but not the complete lack observed with the Pmel-1 T cells. Nonetheless these results suggests that BTLA is critical for the generation of a recall memory response.

Figure 4. Defective memory recall response of BTLA deficient T cells.

Schematic diagram depicting experimental design of mouse model for vaccination. 0.5 × 10⁶ (a) Pmel-1 Thy 1.1. wild type splenocytes or (b) Pmel-1 BTLA KO splenocytes were adoptively transferred into C57BL/6 mouse recipients (i.v.) (n=5 mice per group). On the following day, the recipients were vaccinated with gp100 peptide (100µg) together with anti-CD40 (50 µg) and imiquimod (50 mg). Recombinant human IL-2 at 1.2 ×10⁶ IU was administered once, and 6 × 10⁵ IU twice daily for the next 2 days (i.p.). Peripheral blood was collected every other day to determine the frequency of circulating Pmel-1 Thy 1.1 T cells. On day 60, the mic e were vaccinated with gp100 peptide, and peripheral blood was collected every other day until Pmel-1 T cells were no longer detected. On day 120, the mice were euthanized, and spleens were collected to determine the presence of Pmel-1 Thy 1.1 T cells. (c) Plot graph depicts the percentage of CD3⁺CD8⁺Pmel⁺ T cells in the peripheral blood following the priming (first peak from day 15 to day 30) and boosting (second peak from day 60 to 70). The frequency of CD3⁺CD8⁺Pmel⁺T cells was significantly higher in C57BL/6 mouse recipients receiving Pmel-1 Thy 1.1 wild type splenocytes as compared to those receiving Pmel-1 Thy 1.1 BTLA KO splenocytes. (d) Comparison of the percentage of CD3⁺CD8⁺Pmel⁺ T cell in spleen on day 120 of mice receiving either Pmel-1 Thy 1.1 wild type splenocytes or Pmel-1 Thy 1.1 BTLA KO splenocytes. Pmel-1 Thy 1.1 BTLA KO splenocytes failed to develop memory recall response following vaccination and boosting.

Dichotomous BTLA signaling: ITIM/ITSM dampens effector cytokine secretion while Grb2 enhances IL-2 production

Our in vivo experiments with human TIL and single cell killing analyses have suggested that the CD8⁺BTLA⁺ subset is endowed with a superior pro-survival function leading to better anti-tumor activity. Conceivably, the superiority of this subset could result from either 1) properties of less-differentiated T cells marked by BTLA expression or 2) signaling through the BTLA molecule itself. Our vaccination study suggests a direct involvement of the BTLA molecule. However, to further test the contribution of the BTLA signaling, we performed functional studies of BTLA by overexpressing wild-type BTLA (WT BTLA) or variants of BTLA’s intracellular motifs in OT-1 T cells from OT-1 BTLA KO mice. To dissect the signaling contributions from the ITIM/ITSM and the Grb2 motifs independently, retroviral constructs were generated with point mutations by substitution of tyrosine for phenylalanine in either the Grb2 binding motif (ΔGrb2) or the ITIM and ITSM motifs (ΔITSM) (Fig. 5a). OT-1 BTLA KO T cells were transduced with constructs containing WT BTLA, ΔGrb2, and ΔITSM as well as an empty vector control (EM). Virally transduced OT-1 T cells were co-cultured with either B16F10 (negative for OVA antigen) or B16OVA tumor cells at an effector to target ratio of 1:10, 1:3, and 1:1. HVEM expression on both tumor targets was confirmed by flow cytometry (Supplementary Fig. S5). Indeed, the tumor killing capacity of the TIL in this short term in vitro assay was comparable regardless of the presence of WT BTLA or BTLA mutants at all ratios tested (ratio 1:1, P=0.67; ratio 1:3, P=0.46; ratio 1:10, P=0.29) (Fig. 5b). We did not observe differences in the number of T cells producing IFN-γ or TNF-α among these different groups (IFN-γ, P=0.11; TNF-α, P=0.59) (Fig. 5c, left panel). However, the amount of effector cytokines being made by the WT BTLA transduced T cells was significantly less than that made by those transduced with the empty vector alone following re-stimulation with dendritic cells pulsed with OVA peptide (IFN-γ MFI; EM vs WT, P=0.04, TNF-α MFI; EM vs WT, P=0.0045) (Fig. 5c, right panel). The BTLA-mediated inhibition of IFN-γ and TNF-α production was relieved by the disruption of the ITIM/ITSM motifs; however, it was not significantly impacted by the disruption of the Grb2 motif, suggesting that ITIM and ITSM motifs are mainly involved in regulating the amount of effector cytokines produced following TCR triggering.

Figure 5. BTLA signaling motifs show no effect on tumor killing, while Grb2 motif augments IL-2 production and T cell proliferation.

(a) Schematic diagram depicts the structure of BTLA WT (left), BTLAΔGrb2 (middle), and BTLAΔITSM (right). Signaling motifs with modified Tyrosine to Phenylalanine are indicated by a dotted pattern over a black background. A table summarizing the phenotype observed with the expression of BTLA or of the different BTLA constructs is presented on the right panel. (b) B16 OVA (mouse melanoma tumor positive for OVA) or B16F10 (mouse melanoma tumor negative for OVA) were stained with eFluor670® and co-cultured with OT-1 BTLA KO T cells overexpressing WT BTLA or BTLA mutants at the following T cell-to-tumor cell ratios (1:10, 1:3, and 1:1). Tumor cell death is depicted by the percentage of caspase-3 positive cells. N=3 independent experiments (c) OT-1 BTLA KO T cells overexpressing WT BTLA or its variants were re-stimulated with dendritic cells pulsed with OVA peptide. TNF-α and IFN-γ production by virally transduced OT-1 BTLA KO T cells was evaluated by intracellular staining. Bar graph depicts the percentage of positive cells (left panel) and mean fluorescence intensity (MFI) (right panel). Each bar represents three independent experiments. (Two-way ANOVA; *P<0.05). (d) OT-1 BTLA KO T cells overexpressing WT BTLA or its variants were labeled with eFluor670® and re-stimulated with dendritic cells pulsed with OVA peptide. Cell proliferation was determined by the dilution of eFluor670®. Histogram plots of eFluor670® demonstrate proliferation of OT-1 BTLA KO T cells overexpressing WT BTLA or its variants. Bar graph depicts MFI of virally transduced T cells in the same experiment shown in the left panel. N=3 *P <0.05, **P <0.001, ***P<0.0001. All error bars depicts the mean ± s.e.m. All P-values were calculated using a two-tailed Student’s t-test. (e) Virally transduced OT-1 BTLA KO T cells were stimulated with plate-bound anti-mouse CD3 and HVEM Fc. Supernatants were assessed for IL-2 production using by MILLIPLEX MAP Mouse CD8⁺ T Cell Magnetic Bead Panel Assays. Each bar graph represents two independent experiments. *P <0.05, **P <0.001. All error bars depicts the mean ± s.e.m. All P-value were calculated using a two-tailed Student’s t-test.

We have previously demonstrated that the CD8⁺BTLA⁺TIL subset had an improved proliferative capacity in response to IL-2 (14). Thus, we sought to determine whether BTLA signaling motifs could play a role in T cell proliferation. OT-1 BTLA KO T cells overexpressing BTLA WT or mutants were labeled with the cell proliferation dye eFluor670® and re-stimulated with dendritic cells pulsed with OVA peptide for two days. T cell proliferation was comparable between control and WT BTLA groups. (MFI: WT versus EM; P=0.30) (Fig. 5d, histogram). On the other hand, subtle differences are appreciable between T cells expressing the different BTLA constructs. We observed that OT-1 BTLA KO T cells expressing ΔITSM had a lower mean fluorescence intensity (MFI) of the eFluor670® dye as compared with the empty vector control which is indicative of a more robust proliferation (MFI: ΔITSM versus EM; P=0.012, ΔITSM versus WT; P=0.0057) (Fig. 5d). On the contrary, attenuation of T cell proliferation was observed in T cells expressing a disrupted Grb2 motif (ΔGrb2), (MFI: ΔGrb2 versus ΔITSM; P=0.0003, ΔGrb2 versus EM; P=0.0008, ΔGrb2 versus WT; P= 0.0009) (Fig. 5d). These data suggest that the T cell proliferation post TCR triggering is regulated positively by the Grb2 motif and negatively by ITIM/ITSM motifs.

Our previous report demonstrated that CD8⁺BTLA⁺ human TIL produce more IL-2 upon activation (14). Thus, we further investigated whether BTLA signaling could be responsible for IL-2 secretion. OT-1 BTLA KO T cells overexpressing WT BTLA or its mutants were re-stimulated with anti-CD3 in the presence of HVEM-Fc fusion protein to engage the BTLA molecule, and IL-2 secretion was assessed. Indeed, we found that IL-2 production was significantly increased in activated T cells transduced with the BTLA WT and ΔITSM constructs, but not in ΔGrb2 and the empty vector. This suggests that the BTLA-dependent IL-2 production was mediated through Grb2 motif, independently of the ITIM/ITSM motifs (WT versus EM; P=0.02, ΔITSM versus EM; P=0.005, WT versus ΔGrb2; P=0.02, ΔITSM versus ΔGrb2; P=0.01, EM versus Grb2; P=0.79, WT versus ΔITSM; P=0.32) (Fig. 5e). This data highlights that Grb2 signaling contributes to IL-2 production following BTLA ligation during T cell activation. A summary of the phenotypes seen with the expression of BTLA or its variants is presented in Fig. 5a (right panel).

Signaling downstream of mouse BTLA

To identify proteins activated downstream of BTLA, we employed Reverse Phase Protein Array (RPPA), a high-throughput method developed for functional proteomic studies. OT-1 BTLA KO mouse T cells overexpressing WT BTLA or BTLA mutants were stimulated with anti-CD3 with or without HVEM-FC for 8 hours. The full list of tested proteins can be found in Supplementary Table S3. The protein expression levels found in activated T cells transduced with BTLA WT was used as a reference to compare the differential protein changes in ΔGrb2 and ΔITSM. The protein expression profile of the T cells expressing the BTLA variants were also compared to that obtained with the empty vector. (Supplementary Table S4). We observed that the phosphorylation of Akt at T308 as well as the phosphorylation of one of its substrates, pPRAS40 at T246, were significantly attenuated in ΔGrb2 in comparison to BTLA WT (pAkt T308; P=0.045, pPRAS40 T246; P=0.007) (Fig. 6a, left panel and Supplementary Table S4) (28). As expected, the disruption of ITIM/ITSM motif resulted in a remarkable enhancement of the phosphorylation of several proteins such as Src, Chk1, Chk2, as well as members of the Wnt pathway GSK-3b, and of Beta-Catenin at T41 and S45 (pSrc at S527; P=0.005, pA-Raf at S299; P=0.01, and pC-Raf at S338; P=0.02, pChk1 at S286; P=0.01, pChk2 at T68; P=0.02, GSK-3b at S9; P=0.04, Beta-Catenin at T41 and S45; P=0.04) (Fig. 6a, right panel and Supplementary Table S4).

Figure 6. BTLA-HVEM signaling axis suppresses MAPK, Akt, and NF-κB pathways, but selectively augments the Src pathway.

(a) OT-1 BTLA KO T cells overexpressing WT BTLA or its variants were re-stimulated with plate-bound anti-CD3 and HVEM-Fc for 8 h prior to harvest. Cells were lysed and the protein supernatant was collected to perform RPPA. Volcano plots depict fold change of proteins with the following comparisons: ΔGrb2 versus WT (left), ΔITSM versus WT (middle), and ΔITSM versus EM (right). Data shown represent two independent experiments. P <0.05. P-values were calculated using Linear models and empirical Bayes methods. (b and c) Sorted CD8⁺BTLA⁺ TIL were stimulated with plate-bound anti-CD3 (0, 10, 30, 100, 300, and 1000 ng/ml) alone or with HVEM-Fc for 8 h prior to harvest. Cells were lysed and the protein supernatant was collected to perform RPPA. (b) Volcano plot depicts fold change of proteins in CD8⁺BTLA⁺TIL upon T cell activation with anti-CD3 alone (left), anti-CD3 + HVEM (middle), and anti-CD3 in comparison with anti-CD3 + HVEM (right). (c) Bar graph demonstrates proteins that significantly change in comparison between anti-CD3 activation alone (red) and anti-CD3 + HVEM ligation (blue). (d) Signaling network from the proteins that significantly change in (c) were clustered by Ingenuity Pathway Analysis. N=5, P <0.05. P-values were calculated using Linear models and empirical Bayes methods.

BTLA-HVEM axis in human TIL selectively suppresses Akt and NF-kB pathways but enhances Src pathway

To exclude the bias of overexpression of WT BTLA and genetically modified BTLA in BTLA knockout mouse, we further investigated BTLA signaling pathway in human TIL, which is more physiologically and clinically relevant to our observation in our adoptive T cell therapy clinical trial. Sorted CD8⁺BTLA⁺ human TIL were stimulated with plate bound anti-human CD3 at concentrations of 10, 30, 100, 300, and 1000 ng/mL with or without HVEM-Fc for 8h and proteins extracted from cell lysates were used to perform RPPA in 5 separate patient TIL samples. We observed a general decrease in differentially phosphorylated protein changes in several signaling pathways when T cell activation happened in the presence of HVEM. These included the MAPK kinase pathway (pP38 at T180; P=2.08 × 10⁻⁹, pP90RSK at T573; P=3.26 × 10⁻⁴, pS6 at S235; P= 1.68×10⁻²), NF-κB pathway (pNF-κB p65 at S536; P=3.15 × 10⁻²), the Akt pathway (pAKT at S473; P=9.6×10⁻³), and the Beta-catenin pathway (GSK-3a–b pS21; P=9.6×10⁻³) (Fig. 6b and c and Supplementary Table S5). Inhibition of these positive signaling pathways clearly support the role of BTLA as a co-inhibitory molecule. Interestingly, we found a significant elevation of Src phosphorylation at S416 (pSrc at S416; P= 3.36 × 10⁻⁶) when the T cell activation happened in the presence of HVEM. Our results suggest that the TCR signaling pathway is not completely suppressed by BTLA, but is specifically attenuated in certain pathways as indicated above and specifically potentiated in very select pathway(s). Unexpectedly, we found that phosphorylation of HER2 at tyrosine 1248 was prominently increased in an anti-CD3 dose-dependent manner regardless of the presence of HVEM. This strengthens the notion that the BTLA/HVEM axis acts on specific targets. To further comprehensively understand the downstream signaling pathway of human BTLA, we generated the signaling network of proteins significantly changed during HVEM ligation using Ingenuity pathway analysis (IPA). We observed that Src signaling node was exclusively clustered and separated from Akt, NF-kB, and Beta-catenin signaling nodes. This suggests that the Src signaling pathway is likely not or minimally interfered by SHP1/2 (Fig. 6d).

Discussion

Our recent report demonstrated that the CD8⁺BTLA⁺ TIL are less differentiated, respond better to IL-2 and persist longer in the patient post infusion (14). In the current study, we shed light on the dichotomy of the signaling downstream of BTLA and highlight a role for BTLA in the development of CD8⁺ T cell memory-recall and the survival of effector memory CD8⁺ T cells sustaining their anti-tumor function.

We observed that the CD8⁺ BTLA⁺ and CD8⁺ BTLA⁻ subsets had comparable in vitro killing, which did not explain the superiority of the CD8⁺ BTLA⁺ subset in controlling disease in patients. The use of a single cell nanowell-based cytolytic assay helped elucidate fundamental differences between the two subsets. Importantly, the results demonstrated that both subsets efficiently kill a first target but the CD8⁺BTLA⁺ subset is more likely to survive after this killing event and kill another target, thus providing a rationale for the improved in vivo efficacy of the CD8⁺BTLA⁺ TIL.

The defective recall response seen in BTLA KO antigen-specific T cell in vivo models used contrasts with previous literature. A publication by Krieg et al evaluates the response of the CD8⁺ BTLA-KO OT-1 T cells in a vaccination model and claims that there is more efficient memory formation in the BTLA-KO OT-1 T cells than their WT counterpart (29). This claim is based on the fact that there are twice as many CD8⁺ OT-1 BTLA-KO T cells than WT counterpart in the spleen 30 days after the primary stimulation even though the BTLA-KO OT-1 subset peaked at lower frequency in the early days of the primary response. The BTLA-KO OT-1 also had a slightly lower fold expansion early (day 4) during the secondary expansion (7 fold versus 10 fold) following boosting with peptide-pulsed DC. This is in contrast with the data obtained when boosting with an infectious agent expressing OVA, LM-OVA, where the BTLA-KO OT-1 expand more than the BTLA WT OT-1 at day 5 (91 fold versus 191 fold). Unfortunately the experiment was not carried out longer. The authors conclude that BTLA KO OT-1 can form memory, and that the amplitude of the recall response may depend on the type of antigenic challenge used. Interestingly, another study utilizing BTLA KO animals and LM-OVA infection concluded that HVEM expression on T cells transduces a survival signal through interaction with BTLA expressed on other cells in the host and that interaction was required for CD8⁺ T cell memory generation(30).

There are a few key differences between our vaccination model and the reported studies. First of all, the adoptive cell transfer in the paper by Krieg et al is performed to allow competition between the cell subsets in the host. The authors actually transfer both BTLA WT OT-1 and BTLA KO OT-1 T-cells at a 1:1 ratio to BTLA WT hosts and vaccinate the animals one day later with peptide-pulsed dendritic cells. While these experiments were designed to test BTLA’s function, the known expression of BTLA’s receptor, HVEM, on activated T cells, and its ability to transduce a survival signal by interactions in trans may become a confounding factor. In this system it is possible that BTLA expressed on BTLA WT OT-1 may have interacted with HVEM on both transferred subsets which could have delivered a survival signal potent enough to limit the contraction phase and enhance memory formation for both subsets. The amplitude of the memory generation in this system could be controlled by the strength of TCR signal which clearly will be stronger in BTLA KO OT-1 since all studies are in agreement that BTLA suppresses TCR signaling.

Our experiments were designed to study the response of the two subsets separately, in different animals. Our data rather shows BTLA-mediated survival of T cells following effector function, probably due to dampening of TCR signaling and prevention of AICD. We believe that BTLA acts as a rheostat reducing TCR signaling during strong or chronic antigenic stimulation while also promoting cell survival due to the presence of both activating and inhibitory signaling motifs in its intracellular signaling domain. Our vaccination was administered by injection of the cognate peptide and anti-CD40 in saline subcutaneously, with topical application of the TLR 7 agonist imiquimod on the injection site and IP IL-2. This vaccination strategy, optimized by our collaborator Drs. Hailemichael and Overwijk, results in a powerful activation of the antigen-specific T-cell response (27). This stimulation produces a robust recall response in WT OT-1 and Pmel T cells however in our hands the BTLA KO OT-1 cells have a reduced ability to generate a recall response and Pmel BTLA KO cannot recall at all. We reasoned that BTLA expression may be essential to suppress overstimulation conducive to AICD and allow the survival of cells to form a memory pool. The method employed to vaccinate in the study by Krieg et al, the use of peptide-pulsed DC, may also not cause as much AICD as the stimulation used in our system, thus not requiring dampening of TCR stimulation by BTLA for survival.

We have demonstrated previously that resting CD8⁺BTLA⁺TIL manifested enhanced mitochondrial function and spare respiratory capacity (SRC) as compared to their CD8⁺BTLA⁻ TIL counterpart (31). High SRC has been reported to be a feature of memory CD8⁺ T cells distinguishing them from naïve and effector subsets, and was found to play a role in survival of the memory CD8⁺ subset conducive to the establishment of a long-lived memory pool (32). Our findings of the better survival of the CD8⁺BTLA⁺ TIL subset after a killing event are consistent with a pro-survival advantage. A recent study indicated that increased SRC was strongly associated with a superior bioenergetic capacity, which helped improve T cell survival and motility under hypoxic conditions (33). The intrinsic properties of this subset provide an intriguing possibility for why infusion of large numbers of CD8⁺BTLA⁺TIL positively correlates with clinical response to TIL ACT in metastatic melanoma patients.

We have found that a BTLA-dependent potentiation of IL-2 secretion entirely depends on Grb2 motif. Disruption of the ITIM and ITSM motifs did not alter the levels of IL-2 induced by BTLA, suggesting a signaling pathway independent of the pro-inhibitory influence of ITIM and ITSM. Our findings are consistent with previous reports that have shown that Grb2-linked SLP-76 and Vav interaction are involved in IL-2 production, and that recruitment of Grb2 is essential for CD28-induced IL-2 production (34–36). Our current study reveals that BTLA engagement in the context of TCR stimulation results in IL-2 production in a manner similar to CD28 which positions BTLA as both a co-inhibitory and co-stimulatory molecule. The in vivo relevance of BTLA-dependent IL-2 production in the context of T cell activation warrants further study. Autocrine IL-2 production by CD8⁺ T cells has been shown to be essential for secondary expansion of CD8⁺ memory T cells which could potentially explain the lack of secondary expansion seen in BTLA KO cells after boosting with the vaccinating antigen (37). The pro-survival properties conferred by BTLA on CD8⁺ T cells may come from the interaction of BTLA with HVEM on activated T cells as it was demonstrated for CD4⁺ murine T cells (38,39) However the HVEM interaction with its alternative ligand LIGHT rather than BTLA has been implicated in furthering CD4⁺ T cell activation and survival(39). In our experiments, the absence of BTLA may render HVEM available to bind to its alternative ligand LIGHT to provide co-stimulatory signaling to T cells however this mechanism was not sufficient to rescue the establishment of memory cells able to respond to a recall antigen stimulation.

Conversely we did not observe any major differences in in vitro killing capacity and cytotoxic cytokine production between BTLA KO T cells versus BTLA KO T cells overexpressing BTLA WT or its mutants. This suggests that BTLA signaling does not affect tumor killing capability. Our findings are consistent with the previous report showing that the BTLA blockade in γδT cells had no effect on the tumor lysis (40).

We did not observe a significant impact of WT BTLA when the molecule was reintroduced in BTLA KO T cells. However, malfunction of ITIM and ITSM motifs significantly enhanced T cell proliferation while Grb2 alteration reduced the proliferation. A lack of impact of BTLA on CD4⁺ T cell proliferative response has been reported previously (12). Previous studies in both human and mouse settings indicated that CD8⁺ T cells were intrinsically less susceptible to BTLA-mediated inhibition as compared to CD4⁺ T cells (9,41). This might explain the minimal inhibition of WT as compared to EM in CD8⁺ T cells in terms of proliferation.

We noticed that the Akt signaling pathway was remarkably attenuated in BTLA ΔGrb2, while the phosphorylation of Src was enhanced in BTLA ΔITSM. This finding is consistent with a previous report demonstrating that ITIM and ITSM motifs of PD-1 inhibited signals through Akt and MAPK (10). However, in the presence of co-stimulation mediated through the BTLA Grb2 motif, as in the case of ΔITIM/ITSM, the phosphorylation of Akt was unaltered, suggesting that Grb2 is not directly involved in maintaining or augmenting Akt phosphorylation. Taken together, this data suggests that the Akt pathway in BTLA was likely targeted by SHP1/2, similar to what was found in PD-1.

Overexpression of Grb2 in osteoclasts was shown to promote phosphorylation of Src at Y416 and the opposite result was obtained when Grb2 expression was disrupted (42). From our data, it is clear that the phosphorylation of Src increased when Grb2 was providing BTLA signaling in the absence of functional ITIM and ITSM motifs. This could result from either the direct effect of Grb2 signal transduction or the lack of dephosphorylation normally caused by SHP1/2 via ITIM and ITSM motifs. Since the phosphorylation of Src is not significantly influenced by the ITIM/ITSM co-signaling (ΔGrb2), we conclude that it is likely to be Grb2 mediated Src activation.

Our data of BTLA signaling in mouse T cells was found to be consistent with changes observed when activated human CD8⁺BTLA⁺TIL were co-stimulated by HVEM. Indeed, we observed an attenuation of MAPK, NF-κB, and Akt signaling proteins, but phosphorylation of Src was enhanced when human TIL were stimulated with anti-CD3 and HVEM as compared to CD3 alone. This suggested that the downstream signaling pathway of BTLA in mice and humans possibly share similar downstream signaling targets.

Overall, our study sheds light on a dual role of BTLA as both a co-stimulatory and co-inhibitory molecule. This observation is supported by a study reporting that the gene expression profile of CD4⁺ T cells activated with CD3 and BTLA engagement mimics the profile induced by co-stimulatory molecules (12). In our hands, the integration of the positive and negative signals transduced by BTLA promotes IL-2 secretion while reducing effector cytokine production and proliferation in certain contexts. In addition, the inherent properties of the less-differentiated T cells expressing BTLA also display enhanced resistance to apoptosis and an efficient bioenergetic profile providing a survival advantage following tumor killing. These findings support the concept that the intrinsic attribute of the less-differentiated CD8⁺BTLA⁺ human TIL subset together with balanced signals transduced by the engagement of HVEM in the tumor microenvironment on melanoma cells could provide a costimulatory signal to CD8⁺BTLA⁺ TIL promoting IL-2 secretion, T cell survival and anti-tumor function.

Translational Relevance.

BTLA (B-and-T lymphocyte attenuator) is a negative T cell co-signaling molecule. Its expression on CD8⁺ Tumor-Infiltrating Lymphocytes (TIL) unexpectedly associated with better clinical outcome in metastatic melanoma patients treated with adoptive T-cell therapy using TIL. We sought to determine whether BTLA signaling could positively impact T cells. Our results demonstrate that the CD8⁺BTLA⁺ TIL subset has a survival advantage following the killing of a tumor target in comparison to its BTLA⁻ counterpart, which may explain the superior in vivo persistence of this subset after TIL infusion. Interestingly, the Grb2 motif of BTLA was found to promote IL-2 production following BTLA engagement in the context of a TCR stimulation, and the presence of BTLA on CD8⁺ T cells was required to develop a robust recall response. Therefore, developing a strategy to selectively expand and infuse CD8⁺BTLA⁺ TIL may enhance TIL persistence post-infusion and result in superior clinical outcome.