Abstract
Cytotoxic CD8+ T cell exhaustion is one of the mechanisms underlying the tumor immune escape. The paradigm-shifting immune checkpoint therapy can mitigate CD8+ T lymphocyte exhaustion, reinvigorate the anticancer immunity, and achieve durable tumor regression for some patients. Emerging evidence indicates that CD4+ T lymphocytes also have a critical role in anticancer immunity, either by directly applying cytotoxicity toward cancer cells or as a helper to augment CD8+ T cell cytotoxicity. Whether anticancer CD4+ T lymphocytes undergo exhaustion during immunotherapy of solid tumors remains unknown. Here we report that melanoma antigen TRP-1/gp75-specific CD4+ T lymphocytes exhibit an exhaustion phenotype after being adoptively transferred into mice bearing large subcutaneous melanoma. Exhaustion of these CD4+ T lymphocytes is accompanied with reduced cytokine release and increased expression of inhibitory receptors, resulting in loss of tumor control. Importantly, we demonstrate that PD-L1 immune checkpoint blockade can prevent exhaustion, induce proliferation of the CD4+ T lymphocytes, and consequently prevent tumor recurrence. Therefore, when encountering an excessive amount of tumor antigens, tumor-reactive CD4+ T lymphocytes also enter the exhaustion state, which can be prevented by immune checkpoint blockade. Our results highlight the importance of tumor-specific CD4+ T lymphocytes in antitumor immunity and suggest that the current immune checkpoint blockade therapy may achieve durable anticancer efficacy by rejuvenating both tumor antigen-specific CD8+ T lymphocytes and CD4+ T lymphocytes.
Keywords: CD4+ T cells, exhaustion, adoptive transfer therapy, immune checkpoint blockade
Introduction
T lymphocytes are critical in adoptive immunity against infection and cancer. Adult human body contains about 4 × 1011 T lymphocytes. Upon antigen exposure, a small subset of T lymphocytes, the antigen-specific naïve T lymphocytes, are clonally selected and activated. Each individual antigen-specific naïve T cell can proliferate and expand into thousands of effector T lymphocytes. Following the clearance of antigens, most of effector T lymphocytes die, whereas a minor portion of the effector T lymphocytes transition to memory T cells, which are prepared to react rapidly to the same antigen in the future [1,2]. However, during chronic viral infection or in the cancer microenvironment, T lymphocytes are exposed to persistent antigens, and become exhausted [3]. Exhausting T lymphocytes gradually lose cytokine expression, and express multiple inhibitory receptors including PD-1, TIM-3, TIGIT, and LAG-3 [4]. As a result, exhausted T lymphocytes have lost their ability to control infections or tumor growth [5].
The molecular and cellular mechanisms involved in T cell exhaustion have been extensively studied in order to design therapeutic interventions to prevent T cell exhaustion. The most commonly used model of CD8+ T cell exhaustion is LCMV-clone 13 infection. Chen Z et al performed single cell RNA-seq analysis of LCMV-specific P14 cells on day 8 following LCMV-clone 13 infection, compared with LCMV-Armstrong infection [6]. Naïve P14 CD8+ T lymphocytes express TCF1 (encoded by Tcf7 gene) and Ly108 (encoded by Slamf6 gene); LCMV-Armstrong specific effector P14 CD8+ T lymphocytes express abundant Granzyme B, but low levels of TCF1, TOX, or PD-1. By contrast, LCMV-Clone 13 infection induces the exhaustion state of P14 CD8+ T lymphocytes, which have lost TCF1, and instead express high amounts of TOX, PD-1 and low level of cytotoxic molecules. Philip et al studied T cell exhaustion against tumor antigen SV40 [7]. It is found that as early as on day 5 after CD8+ T cell transfer, SV40-specific CD8+ T lymphocytes in the liver have already started to express exhaustion markers PD-1 and Lag3, and could not produce cytokine IFN-γ or TNF-α.
Besides cytotoxic CD8+ T lymphocytes, helper CD4+ T lymphocytes have a critical role in adaptive immunity [8]. Particularly, emerging evidence indicates that CD4+ T lymphocytes have a crucial role in antitumor immunity. CD4+ helper T lymphocytes can aid CD8+ T lymphocytes to target tumors [9]. CD4+ T lymphocytes directly engage conventional type 1 dendritic cells (cDC1) via CD40 signaling and to license cDC1 to prime tumor antigen-specific CD8+ T lymphocytes [10]. In mice, tumor antigen-specific CD4+ T lymphocytes can acquire the cytolytic ability and eradicate the subcutaneous melanoma in lymphatic ablated recipient mice through an MHC-II-dependent manner [11-13]. In human bladder cancer, it is intratumoral cytotoxic CD4+ T lymphocytes, but not canonical CD8+ T lymphocytes, that mediate anticancer immunity [14].
Whether tumor-specific CD4+ T lymphocyte exhaustion exists in tumor microenvironment and whether checkpoint blockade can prevent effector CD4+ T cell from being exhausted have not been clearly established. Hence, in this study we have studied the function state transition of tumor antigen-specific CD4+ T lymphocytes after adoptively transferred into lymphopenic recipient mice bearing large subcutaneous melanoma and dissected the mechanism by which immune checkpoint blockade prevents the transition of CD4+ T lymphocytes to the exhaustion state.
Materials and methods
Mice
C57BL/6 (CD45.1), Rag1-/-, and Tyrp1B-wRAG-/- TRP-1-specific CD4+ TCR transgenic mice were obtained from the Jackson Laboratory (Bar Harbor, MA). Because RAG is a key enzyme involved in V(D)J recombination, RAG knockout mice contain no T cells, B cells, or NKT cells which all play important roles in anticancer immunity. TRP-1 TCR transgene is located on the Y chromosome. CD4+ T lymphocytes in Tyrp1B-wRAG-/- TRP-1 male mice (CD4+ Vβ14+) express a transgenic TCR that recognizes the melanoma differentiation antigen tyrosinase-related protein 1 (TRP-1). All mice used in this study were handled in accordance with guidelines of the Comparative Medicine Program at Houston Methodist Research Institute (HMRI).
Tumor cell line and in vitro cell culture
The mouse B16F10 melanoma cells (American Type Culture Collection) were maintained in Dulbecco’s Modified Eagle Medium (DMEM) (Thermo Fisher Scientific) containing 10% fetal bovine serum, 1% Penicillin/Streptomycin in 37°C incubator equilibrated with 5% CO2.
Ectopic tumor implantation and tumor volume measurement
The right flank of C57BL/6 (CD45.1) or Rag1-/- mouse was shaved and subcutaneously (sc) injected with 0.5 × 106 B16F10 cells on day 0. Tumor volume was measured in two dimensions (length and width) using a vernier caliper every two days. Tumor volume was determined using the following formula: volume (cm3) = 0.5 × length × (width)2. Mice were euthanized at the indicated time points, or when tumor volume reaches the endpoint (volume >2 cm3, diameter >2 cm), or when mouse develops signs of discomfort (head tilt, weakness, or severely dehydration), in accordance with recommendations approved by the 2000 Report of the American Veterinary Medical Association panel on euthanasia and the IACUC Committee at Houston Methodist Research Institute.
Adoptive transfer of antigen-specific Trp-1 CD4+ T lymphocytes and immune checkpoint blockade
10 to 12 days after implantation, tumor diameter reached 0.8 to 1 cm, and mice received sub-lethal (5 Gy) whole body radiation. Adult male Tyrp1B-wRAG-/- TRP-1 CD4+ TCR transgenic mice (CD45.2) were euthanized, and spleen and lymph node cells were collected and filtered through a 70 μm cell strainer. Trp-1 CD4+ T lymphocytes were purified with Dynabeads™ Mouse CD4 Kit (Thermo Fisher Scientific). Irradiated recipient mice bearing B16F10 melanoma tumors were grafted with 5 × 104 purified Trp-1 CD4+ T lymphocytes through intravenous (iv) injection. After Trp-1 CD4+ T lymphocyte graft, mice were administered with either PBS or anti-PD-L1 monoclonal antibody (10F9G2, Bio X Cell) by intraperitoneal (ip) injection for 3 times, on day 15, day 18 and day 21, at a dose of 10 µg/g of body weight.
Tumor digestion and single cell suspensions
Mice were euthanized at indicated time points. Tumors were excised, sliced into small pieces, enzymatically digested with collagenase I (SCR103 Sigma-Aldrich) and DNase I (AMPD1, Sigma-Aldrich) in RPMI1640 (R6504, Sigma-Aldrich) medium at 37°C for 30 minutes, and mashed through a 70 μm cell strainer to disperse cells. Tumor infiltrating lymphocytes (TILs) were purified using Ficoll density gradient (GE Healthcare). Cells between the interfaces of the liquid layers were harvested and washed with PBS twice prior to cell staining. Mouse splenocytes were filtered through a 70 μm cell strainer, washed with PBS twice, and ACK lysing buffer (Gibco) was added to selectively deplete red blood cells.
Flow cytometry analysis of cell surface markers and intracellular factors
The TILs and splenocytes isolated from the mice were stained with Zombie Aqua dye (Biolegend) for 15 minutes at room temperature to identify live cells. For cell surface marker staining, antibodies that recognize the following murine markers were purchased from Thermo Fisher Scientific, including CD45.1 (A20), CD45.2 (104), CD4 (RFT-4g), CD62L (MEL-14), CD160 (CNX46-3), CD44 (IM7), CXCR6 (DANID2), Slamf6 (13G3-19D), Tim-3 (RMT3-23), Lag-3 (C9B7W), PD-1 (J43). Anti-mouse Vβ14 antibody (MR12-3) was purchased from BD Biosciences.
Intracellular transcription factors were stained with Foxp3/Transcription Factor Staining Buffer Set (eBioscience) for flow cytometry analysis. Mouse-specific Ki67 (SolA15) was purchased from Thermo Fisher Scientific and Anti-TCF1 rabbit mAb (C63D9) was purchased from Cell Signal Technology. Cytokines were stained with Fixation/Permeabilization Solution Kit with BD GolgiPlug (BD Biosciences). Lymphocytes were stimulated with 500 ng/ml ionomycin (Sigma-Aldrich), 50 ng/ml PMA (Sigma-Aldrich), and GolgiPlug (BD Biosciences) for 4 hrs in RPMI 1640 medium before cytokine staining. Antibodies against mouse IFN-γ (XMG1.2) and TNF-α (MP6-XT22) were obtained from Thermo Fisher Scientific.
TILs and splenocytes stained with different antibodies were analyzed on BD LSR II or BD LSRFortessa Flow Cytometer (BD Biosciences) at the HMRI Flow Cytometry Core. The data were analyzed manually with FlowJo software (version10, Tree Star).
Statistical analysis
Mice were randomly assigned to control group and treatment group. The data were processed using GraphPad Prism8 and presented as mean ± SD. The means of the two sample groups were compared by using an unpaired 2-tailed Student’s t test. The p-value of animal survival was determined by the log-rank test. A p-value less than 0.05 is regarded as statistically significant.
Results
Tumor-infiltrating TRP-1-specific CD4+ T lymphocytes displayed an exhaustion phenotype, which was prevented by immune checkpoint blockade
We were interested in understanding how tumor-specific Trp-1 CD4+ T lymphocytes respond when they encounter an excessive amount of tumor antigens in mice. WT B6 (CD45.1+) mice were subcutaneously inoculated with 5 × 105 B16F10 murine melanoma cells on day 0. Twelve days after implantation, when tumor diameter reached to 0.8-1 cm, mice were treated with sublethal 5-Gy total body irradiation to remove homeostatic cytokine sinks [15]. Six hours later on the same day, 5 × 104 freshly isolated naïve Trp-1 cells from 8-week-old male Trp-1 CD4+ TCR transgenic mice were intravenously injected into the irradiated mice. In addition, to test the effect of immune checkpoint blockade on TRP-1 CD4+ T lymphocytes, mice were randomly divided into two groups and each group received αPD-L1 antibody or PBS via ip injection (Figure 1A). On day 24, mice were euthanized, tumors were excised, and tumor infiltrating lymphocytes (TILs) were purified.
Figure 1.
Anticancer activity of adoptively transferred Trp-1 CD4+ T lymphocytes in mice bearing subcutaneous melanoma and efficacy of co-treatment with immune checkpoint blockade. A. Schematic diagram showing the treatment regimen and the timeline for mice bearing melanoma. On day 24 post-tumor inoculation, the adoptively transferred Trp-1 CD4+ T lymphocytes isolated from irradiated WT B6 (CD45.1+) recipient mice were examined. All plots and histograms of flow cytometric analysis were gated on adoptively transferred CD4+ T lymphocytes among living splenocytes and TILs. B and C. Proportions of Trp-1 CD4+ T lymphocytes (CD45.2+Vβ14+) among gated total CD4+ T lymphocytes isolated from spleens and tumors on day 24. D and E. Representative flow and bar graphs of Tim-3 and Lag-3 expression on tumor infiltrating Trp-1 CD4+ T lymphocytes of each treatment group. F and G. PD-1 expression in tumor infiltrating Trp-1 CD4+ T lymphocytes of each treatment group. Experiments were repeated three times independently and data are presented as mean ± SD (n=4) from one representative experiment. Not significant = ns: P value >0.05; *: P<0.05; **: P<0.01; ***: P<0.001; ****: P<0.0001. Unpaired 2-tailed Student’s t test.
The gating strategy for detecting the Trp-1 CD4+ T lymphocytes is shown in Supplementary Figure 1A. Flow cytometry analyses indicate that, in comparison with PBS treatment group, mice treated with αPD-L1 possessed a significantly higher percentage of Trp-1 CD4+ T lymphocytes in the spleen, 79.9% vs 48.4% (Figure 1B, 1C). There was no difference in the percentage of tumor infiltrating Trp-1 CD4+ T lymphocytes between αPD-L1 and PBS treatment groups (Figure 1B, 1C). In comparison with cells purified from spleen, tumor infiltrating Trp-1 CD4+ T lymphocytes lost Slamf6 expression and expressed high level of CXCR6, with or without αPD-L1 treatment (Supplementary Figure 1B). Moreover, there was a higher percentage of CD62L-CD44+ Trp-1 cells in spleens in mice treated with Trp-1 CD4+ T lymphocytes/αPD-L1 compared with mice treated with the regimen of Trp-1 CD4+ T cells/PBS (Supplementary Figure 1C). Tumor infiltrating Trp-1 CD4+ T lymphocytes lost the expression of CD62L, and instead expressed CD44, and became terminal-effector like T lymphocytes (data not shown). Meanwhile, αPD-L1 treatment prevented the expression of exhaustion markers Tim-3 and Lag-3 in tumor infiltrating Trp-1 CD4+ T lymphocytes (Figure 1D, 1E). Additionally, αPD-L1 treatment decreased the PD-1 expression on Trp-1 CD4+ T lymphocytes compared with PBS treatment (Figure 1F, 1G). Hence, tumor-specific Trp-1 CD4+ T lymphocytes indeed displayed the exhaustion phenotype after infiltration into tumors, which was alleviated by αPD-L1 immune checkpoint blockade.
Antitumor efficacy of adoptively transferred Trp-1 CD4+ T lymphocytes on established melanoma in lymphopenic recipient mice
In a parallel experiment, the mice that were treated with Trp-1 CD4+ T lymphocytes and αPD-L1 or PBS, were monitored for tumor growth and animal survival. Some mice were sacrificed on day 65 for flow cytometric analysis (Figure 2A). For the melanoma-bearing mice that did not receive any treatment, their tumor diameters reached 2 cm within 3 weeks (Figure 2B, left panel), and mice were euthanized. For mice that received Trp-1 CD4+ T lymphocytes and αPD-L1 antibody or PBS, their tumors were rejected by day 30 to 40 (Figure 2B, right panel). However, in the Trp-1 CD4+ T cells/PBS treatment group, their tumors regrew after a temporary regression (Figure 2C). Conversely, in the Trp-1 CD4+ T cells/αPD-L1 treatment group, only one mouse had tumor relapse (Figure 2B, right panel, and 2C). The difference in survival between these two groups is statistically significant. Figure 2D shows a recurrent tumor in a representative mouse administered with Trp-1 CD4+ T cells/PBS on day 50 post-tumor cell inoculation. Figure 2E shows complete tumor regression and significant vitiligo in a representative mouse administered with Trp-1 CD4+ T cells/αPD-L1 on day 100 post-tumor cell inoculation. Most of these mice that received Trp-1 CD4 T cells/αPD-L1 did not have tumor recurrence, and actually became autoimmune vitiligo, indicating the persistent in vivo activity of Trp-1 CD4+ T lymphocytes in the presence of αPD-L1. Our findings indicate that adoptive transfer of Trp-1 CD4+ T lymphocytes alone into sub-lethally irradiated lymphopenic recipient mice can cause regression of large established melanoma tumors; however tumor relapse is inevitable. Importantly, co-treatment with immune checkpoint αPD-L1 antibody can achieve complete tumor regression without recurrence.
Figure 2.
Adoptively transferred CD4+ T lymphocytes induced temporary regression of large established tumors, and prevention of tumor relapse by immune checkpoint blockade. (A) Schematic diagram showing the treatment regimen and the timeline for mice bearing large melanoma. WT B6 (CD45.1+) mice bearing B16F10 mouse melanoma (tumor diameter ~0.8 cm) received sublethal 5 Gy whole body irradiation and iv injected with tumor-specific Trp-1 CD4+ T lymphocytes. On day 15, 18 and 21, αPD-L1 or PBS control was administered for three times. Tumor size was measured with caliper every two days. (B) The growth curves of subcutaneous B16F10 melanoma in mice receiving three different treatment regimens: no treatment, Trp-1 CD4+ T cells/PBS, and Trp-1 CD4+ T cells/αPD-L1. (C) Survival curves of mice receiving different treatment regimens based on the log-rank test. Data shown were combined from two independent experiments (n≥6 per group). **: P<0.01. The tumor pictures of representative recipient mice that received Trp-1 CD4+ T cells/PBS on day 50 (D) and Trp-1 CD4+ T cells/αPD-L1 on day 100 (E).
Checkpoint blockade induced proliferative response of Trp-1 CD4+ T lymphocytes and their differentiation into effector T cell state
To further investigate the antitumor ability of Trp-1 CD4+ T lymphocytes, we examined the adoptively transferred Trp-1 CD4+ T lymphocytes in spleens and tumors from mice sacrificed on day 65 (Figure 2A). At this time point, tumors in mice administered with Trp-1 CD4+ T cells/αPD-L1 completely regressed. Therefore, we only analyzed the tumors from mice administered with Trp-1 CD4+ T cells/PBS. The results show that in peripheral lymphoid organs, the Trp-1 CD4+ T cells/αPD-L1-treated animals had a higher percentage of Trp-1 CD4+ T lymphocytes than Trp-1 CD4+ T cells/PBS treatment group (Figure 3A, 3B). The percentage of Trp-1 CD4+ T lymphocytes in tumors was significantly higher than in peripheral lymphoid organs, indicating that antigen-specific Trp-1 CD4+ T lymphocytes were specifically recruited into tumors to exert tumor-killing function (Figure 3A, 3C). In mice administered with Trp-1 CD4+ T cells/αPD-L1 combination regimen, Trp-1 CD4+ T lymphocytes had a higher capability of developing into CD62L-CD44+ T lymphocytes in the spleens (Figure 3D, 3E), whereas the Trp-1 CD4+ T lymphocytes in the recurrence group displayed more CD62L+CD44+ central memory-like phenotype in the spleens (Figure 3D). In contrast, the tumor infiltrating Trp-1 CD4+ T lymphocytes in the tumor recurrence group lost CD62L expression, but expressed high level of CD44 (Figure 3D, 3F).
Figure 3.
Checkpoint blockade induced proliferative response and terminal effector phenotype in Trp-1 CD4+ T lymphocytes. WT B6 (CD45.1+) mice bearing B16F10 murine melanoma were adoptively transferred with 5 × 104 naïve Trp-1 CD4+ T lymphocytes. On day 65 post-tumor inoculation, Trp-1 CD4+ T lymphocytes were isolated from spleens and tumors and analyzed by flow cytometry. All plots and histograms were gated on transferred Trp-1 CD4+ T lymphocytes among living splenocytes and TILs. A-C. The proportions of Trp-1 CD4+ T lymphocytes (CD45.2+Vβ14+) among gated total CD4+ T lymphocytes in spleens (Trp-1 CD4+ T cells/αPD-L1) or in spleens and tumors (Trp-1 CD4+ T cells/PBS). D-F. The percentage of CD62L-CD44+ terminal effector T lymphocytes among the transferred Trp-1 CD4+ T lymphocytes. Data are presented as mean ± SD (n=4). **: P<0.01, ***: P<0.001. Unpaired 2-tailed Student’s t test.
In addition, we further analyzed the expression levels of Slamf6 and CXCR6 in Trp-1 CD4+ T lymphocytes. In comparison with PBS treatment group, Trp-1 CD4+ T lymphocytes from animals administered with αPD-L1 expressed higher level of CXCR6 in peripheral lymphoid organs (Figure 4A, 4B). In the PBS treatment group, the tumor infiltrating Trp-1 CD4+ T lymphocytes expressed higher levels of CXCR6 than those cells in peripheral lymphoid organs (Figure 4A, 4C). Because CXCR6 expression correlates with lymphocyte migration, in support of the observation that Trp-1 CD4+ T lymphocytes from animals treated with αPD-L1 had a higher capacity to infiltrate into tumors. Slamf6 is reported as a negative regulator of CD8+ T lymphocyte function and deficiency of Slamf6 increases anticancer activity of cytotoxic CD8+ T lymphocytes [16]. Our data show that, in peripheral lymphoid organs, Slamf6 expression level on Trp-1 CD4+ T lymphocytes from animals treated with αPD-L1 was significantly lower than those cells from mice treated with PBS (Figure 4D, 4E). Meanwhile, the expression level of Slamf6 on tumor infiltrating Trp-1 CD4+ T lymphocytes was lower than those cells in spleens (Figure 4D, 4F).
Figure 4.
Checkpoint blockade prevented tumor relapse and altered the expression of CXCR6 and Slamf6 in Trp-1 CD4+ T lymphocytes. WT B6 (CD45.1+) mice bearing B16F10 mouse melanoma were adoptively transferred with 5 × 104 naïve Trp-1 cells. On day 65 post tumor inoculation, adoptively transferred Trp-1 CD4+ T lymphocytes were isolated from spleens and tumors and analyzed by flow cytometry. All plots and histograms of flow cytometric analysis were gated on adoptively transferred CD4+ T lymphocytes among living splenocytes and TILs. A-C. The percentage of CXCR6+ effector T lymphocytes among the transferred CD45.2+Vβ14+ Trp-1 cells in spleens (Trp-1 CD4+ T cells/αPD-L1) or in spleens and tumors (Trp-1 CD4+ T cells/PBS). D-F. Representative contour plots and bar graphs display the percentage of Slamf6+ T lymphocytes among the transferred CD45.2+Vβ14+ Trp-1 cells in spleens only (Trp-1 CD4+ T cell/αPD-L1) or spleens and tumors (Trp-1 CD4+ T cells/PBS). Data are presented as mean ± SD (n=4). *: P<0.05, ***: P<0.001, ****: P<0.0001. Unpaired 2-tailed Student’s t test.
Taken together, αPD-L1 checkpoint blockade improved antitumor immunity of Trp-1 CD4+ T lymphocytes through inducing proliferative response and their differentiation toward effector T cell state in peripheral lymphoid organs.
Co-treatment with checkpoint blockade prevented tumor relapse by blocking Trp-1 CD4+ T cell transition to exhaustion state
To further dissect the mechanisms of checkpoint blockade in augmenting the antitumor efficacy of CD4+ T lymphocytes, we analyzed the expression levels of exhaustion markers on the adoptively transferred Trp-1 CD4+ T lymphocytes on day 65 post-tumor implantation. Compared with PBS treatment group, Trp-1 CD4+ T lymphocytes from animals treated with αPD-L1 expressed low levels of Tim-3, Lag-3 (Figure 5A, 5B, 5D), PD-1 and CD160 (Figure 6A, 6B, 6D, 6E) in peripheral lymphoid organs, indicating that αPD-L1 checkpoint blockade prevented the expression of inhibitory receptors on effector CD4+ T lymphocytes. By contrast, the expression levels of these exhaustion markers were significantly higher in tumor infiltrating Trp-1 CD4+ T lymphocytes likely caused by the immune suppression in the tumor microenvironment (Figures 5A, 5C, 5E, 6A, 6C, 6D and 6F). Hence, checkpoint blockade can prevent tumor recurrence by decreasing the expression of inhibitory receptors and avoiding CD4+ T cell exhaustion.
Figure 5.
Checkpoint blockade inhibited the expression of exhaustion markers Tim-3 and Lag-3 in Trp-1 CD4+ T lymphocytes. WT B6 (CD45.1+) mice bearing B16F10 mouse melanoma were adoptively transferred with 5 × 104 naïve Trp-1 cells. Adoptively transferred Trp-1 cells isolated from spleens and tumors on day 65 post tumor inoculation and were analyzed by flow cytometry. All plots and histograms of flow cytometric analysis were gated on adoptively transferred CD4+ T lymphocytes among living splenocytes and TILs. A. The percentage of Tim-3+ and Lag-3+ exhausted T lymphocytes among the transferred CD45.2+Vβ14+ Trp-1 cells in spleens only (Trp-1 CD4+ T cells/αPD-L1) or in both spleens and tumors (Trp-1 CD4+ T cells/PBS). B and C. The percentage of Tim-3+ exhausted T lymphocytes among the transferred CD45.2+Vβ14+ Trp-1 cells in spleens only (Trp-1 CD4+ T cells/αPD-L1) or both spleens and tumors (Trp-1 CD4+ T cells/PBS). D and E. Bar graphs display the percentage of Lag-3+ exhausted T lymphocytes among the transferred CD45.2+Vβ14+ Trp-1 cells in spleens only (Trp-1 CD4+ T cells/αPD-L1) or both spleens and tumors (Trp-1 CD4+ T cells/PBS). Data are presented as mean ± SD (n=4). *: P<0.05, ***: P<0.001, ****: P<0.0001. Unpaired 2-tailed Student’s t test.
Figure 6.
Checkpoint blockade inhibited the expression of exhaustion markers PD-1 and CD160 in Trp-1 CD4+ T lymphocytes. WT B6 (CD45.1+) mice bearing B16F10 murine melanoma were adoptively transferred with 5 × 104 naïve Trp-1 cells. Adoptively transferred Trp-1 cells isolated from spleens and tumors on day 65 post-tumor inoculation and analyzed by flow cytometry. All plots and histograms of flow cytometric analysis were gated on adoptively transferred CD4+ T lymphocytes among living splenocytes and TILs. A-C. The percentage of PD-1+ T lymphocytes among the transferred CD45.2+Vβ14+ Trp-1 cells in spleens only (Trp-1 CD4+ T cells/αPD-L1) or both spleens and tumors (Trp-1 CD4+ T cells/PBS). D-F. Representative contour plots and bar graphs display the percentage of CD160+ T lymphocytes among the transferred CD45.2+Vβ14+ Trp-1 cells in spleens only (Trp-1 CD4+ T cells/αPD-L1) or both spleens and tumors (Trp-1 CD4+ T cells/PBS). Data are presented as mean ± SD (n=4). ****: P<0.0001. Unpaired 2-tailed Student’s t test.
Checkpoint blockade did not affect the expression of Ki-67 while induce a progenitor-effector T cell phenotype in peripheral lymphoid organs
We next investigated the proliferative capacity of the adoptively transferred Trp-1 CD4+ T lymphocytes in WT B6 (CD45.1+) recipients on day 65 post-tumor inoculation. Shown in Figure 7A and 7B, there was no difference in Ki-67 expression levels between the Trp-1 CD4+ T lymphocytes treated with αPD-L1 and PBS. In addition, the percentage of Ki-67 positive Trp-1 CD4+ T lymphocytes was similar in spleens and tumors among the PBS-treated recipients (Figure 7A, 7C). The expression level of TCF-1, a key progenitor/naïve cell marker, was higher in Trp-1 CD4+ T lymphocytes isolated from the peripheral lymphoid organs of mice treated with αPD-L1 regimen (Figure 7D, 7E). TCF-1+ CD8+ T lymphocytes are recognized as precursor-effector cells which are able to self-renew and produce more TCF-1- terminal effector T lymphocytes to increase anticancer immunity [17-19]. By contrast, tumor infiltrating Trp-1 CD4+ T lymphocytes all showed low expression level of TCF-1 (not shown). Therefore, αPD-L1 checkpoint blockade did not affect the proliferative capacity of Trp-1 CD4+ T lymphocytes, and instead induced a precursor-effector T cell phenotype in peripheral lymphoid organs to expand antitumor immunity.
Figure 7.
Checkpoint blockade did not affect the expression of Ki-67 while induced a progenitor-effector T cell phenotype in peripheral lymphoid organs. WT B6 (CD45.1+) mice bearing B16F10 mouse melanoma were adoptively transferred with 5 × 104 naïve Trp-1 CD4+ T lymphocytes. On day 65 post-tumor inoculation, adoptively transferred Trp-1 CD4+ T lymphocytes were isolated from spleens and tumors and were analyzed by flow cytometry. All plots and histograms of flow cytometric analysis were gated on adoptively transferred CD4+ T lymphocytes among living splenocytes and TILs. A-C. The percentage of Ki-67+ T lymphocytes among the transferred CD45.2+Vβ14+ Trp-1 cells in spleens only (Trp-1 CD4+ T cells/αPD-L1) or both spleens and tumors (Trp-1 CD4+ T cells/PBS). D and E. Overlay histogram and bar graphs display the mean fluoresce intensity of TCF-1 among the transferred CD45.2+Vβ14+ Trp-1 cells in spleens (Trp-1 CD4+ T cells/αPD-L1 group and Trp-1 CD4+ T cells/PBS group). Data are presented as mean ± SD (n=4). ns: P>0.05. Unpaired 2-tailed Student’s t test.
Checkpoint blockade increased cytokine production by Trp-1 CD4+ T lymphocytes
To further investigate the mechanisms of checkpoint blockade in promoting anticancer immunity of Trp-1 CD4+ T lymphocytes, we examined the production of anticancer cytokines by the adoptively transferred tumor-specific CD4+ T lymphocytes in WT B6 (CD45.1+) recipient mice on day 65 post-tumor implantation. Shown in Figure 8A and 8B, Trp-1 CD4+ T lymphocytes isolated from spleens of animals treated with αPD-L1 expressed high amounts of IFN-γ and TNF-α, whereas Trp-1 CD4+ T lymphocytes isolated from spleens of mice treated with PBS barely expressed any of these cytotoxic molecules (Figure 8A, 8B). Meanwhile, among PBS-treated recipient mice, the tumor infiltrating Trp-1 CD4+ T lymphocytes expressed higher levels of IFN-γ and TNF-α in comparison with Trp-1 CD4+ T lymphocytes in spleens (Figure 8A, 8C). Therefore, checkpoint blockade helped Trp-1 CD4+ T lymphocytes produce more effector cytokines in peripheral lymphoid organs which prevented the recurrence of tumors.
Figure 8.
Checkpoint blockade increased Trp-1 CD4+ T lymphocyte production of anticancer cytokines. WT B6 (CD45.1+) mice bearing B16F10 mouse melanoma were adoptively transferred with 5 × 104 naïve Trp-1 CD4+ T lymphocytes. On day 65 post-tumor inoculation, adoptively transferred Trp-1 cells were isolated from spleens and tumors and analyzed by flow cytometry. All plots and histograms of flow cytometric analysis were gated on adoptively transferred CD4+ T cells among living splenocytes and TILs. A-C. The percentage of IFN-γ+ TNF-αhi T lymphocytes among the transferred CD45.2+Vβ14+ Trp-1 cells in spleens only (Trp-1 CD4+ T cells/αPD-L1) or both spleens and tumors (Trp-1 CD4+ T cells/PBS). Data are presented as mean ± SD (n=4). **: P<0.01, ****: P<0.0001. Unpaired 2-tailed Student’s t test.
Discussions
It is now well known that cytotoxic CD8+ T lymphocytes enter the state of exhaustion or dysfunction inside the immunosuppressive tumor microenvironment or encountering an excessive amount of tumor antigen. The immune checkpoint blockade therapies including anti-PD-1 and anti-PD-L1 monoclonal antibodies can mitigate CD8+ T cell exhaustion and rejuvenate anticancer immunity [17-20].
In this study, using a transgenic TCR Trp-1 CD4+ T lymphocytes as an in vivo model, we have provided evidence that adoptively transferred tumor-specific Trp-1 CD4+ T lymphocytes exhibited an exhaustion phenotype after infiltrating into solid tumors, resulting in tumor recurrence after a temporary regression. Similar to CD8+ T lymphocytes, exhausted Trp-1 CD4+ T lymphocytes also expressed typical inhibitory receptors including PD-1, Tim-3, Lag-3, and CD160. Importantly, the exhausted CD4+ T lymphocytes also lost the expression of stemness marker TCF-1, in support of the critical role of TCF-1 in maintenance of precursor-effector subset of CD4+ T lymphocytes. Tumor infiltrated Trp-1 CD4+ T lymphocytes also lost the expression of CD62L and Slamf6 [16,21], and instead expressed CD44 and CXCR6 [22].
However, this exhaustion can be prevented by co-treatment with anti-PD-L1 immune checkpoint blockade. When αPD-L1 antibody was co-administered with Trp-1 CD4+ T lymphocytes to the recipient mice, the tumors were completely rejected and most of them did not relapse. Further flow cytometry analyses indicate that the Trp-1 CD4+ T lymphocytes in peripheral lymphoid tissues showed a TCF1+ stem-like phenotype in mice co-treated with αPD-L1. Moreover, we found that in lymphopenic B6.Rag1-/- recipients, which lack B and T lymphocytes, the tumors underwent complete regression and mice developed vitiligo (depigmentation) after a combination regimen with Trp-1 CD4+ T lymphocytes and αPD-L1 (Supplementary Figure 2A, 2B), indicating the tumor rejection immunity was not contributed by endogenous B or T lymphocytes. Moreover, treatment with anti-PD-L1 antibody only did not have effect on the growth of B16F10 subcutaneous melanoma, or the functional states of endogenous CD4+ and CD8+ T lymphocytes (Supplementary Figures 3 and 4), indicating that the antitumor effect of anti-PD-L1 antibody was mostly mediated by adoptively transferred tumor-specific Trp-1 CD4+ T lymphocytes in this model.
In conclusion, after encountering an excessive amount of tumor antigen, tumor-specific CD4+ T lymphocytes enter an exhaustion state, which clearly dampen their antitumor immunity, resulting in loss of tumor control. Fortunately, co-treatment with immune checkpoint blockade αPD-L1 can prevent the exhaustion and induce proliferation response of tumor-specific CD4+ T lymphocytes. Our results suggest that the current commonly used immune checkpoint blockade therapies might achieve durable anticancer efficacy by rejuvenating tumorspecific CD4+ T lymphocytes, in addition to conventional cytotoxic tumor antigen-specific CD8+ T lymphocytes.
Acknowledgements
This work was supported by the start-up funding and the Career Cornerstone Award from Houston Methodist Research Institute to WC, and by National Institute of Health (NIH) National Cancer Institute R01CA211861 and Houston Methodist Research Institute start-up funding to BH.
Disclosure of conflict of interest
None.
Supporting Information
References
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