Tumor immunology has been studied extensively. Tumor immunology–based cancer immunotherapy has become one of the most promising approaches for cancer treatment. However, one of the fundamental aspects of tumor immunology—the initiation of antitumor immunity—is not fully understood. Compared to that of CD8+ T cells, the effect of CD4+ T cells on antitumor immunity has not been fully appreciated. Using a gene knockout mouse model, the mice of which are deficient in the TCRα repertoire, specifically lacking invariant NKT and mucosal-associated invariant T cells, we found that the deficiency in TCRα repertoire diversity did not affect the antitumor immunity, at least to B16BL6 melanoma and EO771 breast cancer. However, after acquiring thymocytes or splenocytes from wild-type mice, these knockout mice exhibited greatly enhanced and long-lasting antitumor immunity. This enhanced antitumor immunity depended on CD4+ T cells, especially CD4+ tissue-resident memory T (TRM) cells, but not invariant NKT or CD8+ T cells. We also present evidence that CD4+ TRM cells initiate antitumor immunity through IFN-γ, and the process is dependent on NK cells. The CD4+ TRM/NK axis appears to control tumor formation and development by eliminating tumor cells and modulating the tumor microenvironment. Taken together, our results demonstrated that CD4+ TRM cells play a dominant role in the initiation of antitumor immunity.

Visual Abstract

Tumor immunoediting theory has clearly outlined the interaction between the immune system and tumor cells during tumor formation and development (1). Most clinically visible tumors are in the third phase of tumor immunoediting, that is, escape. Tumor cells evade the immune system by decreasing immune cell recognition, inducing immune cell dysfunction, or inhibiting immune cell infiltration into the tumor. Most preclinical and clinical tumor immunology studies are focused on immune response in the third phase of immunoediting, specifically on CD8+ T cells, because CD8+ T cells are the primary killer cells to eliminate tumor cells, and the accumulation of tumor-infiltrating CD8+ T cells is associated with a favorable prognosis (2). Indeed, CD8+ T cell–based cancer immunotherapies have achieved unprecedented clinical success in cancer treatment (3). However, the low number of tumor-infiltrating CD8+ T cells and CD8+ T cell dysfunction in the tumor microenvironment (TME) lead to a low response rate to cancer immunotherapy (4). Type 1 conventional dendritic cells (cDC1s) play an essential role in CD8+ T cell infiltration and sustaining CD8+ T cell function in the TME (5, 6). NK cells are crucial for cDC1 migration and maturation in the TME (7, 8). The mechanism of regulating NK cell function in the TME is not fully understood.

Compared to the extensive study on CD8+ T cell antitumor immunity, the CD4+ T cell antitumor function has not been fully appreciated. Most research has focused on investigating CD4+ T cell helper function in antitumor immunity, although new studies have indicated that cytotoxic CD4 T cells exist in the immune system and play an important role in antitumor immunity (4, 9, 10). Some preclinical and clinical studies indicate that CD4+ T cells can effectively control tumor development and progression independent of CD8+ T cells (1113). The CD4+ T cell antitumor function has shown to be even more effective than CD8+ T cells (14, 15). Importantly, note that these studies were performed by transferring transgenic or in vitro–expanded tumor Ag-specific CD4+ T cells into CD4+ T cell–depleted mice or patients. The underlying mechanism remains unclear.

Although it is crucial to study the antitumor immune response in clinically visible tumors, which allows us to develop a therapeutic strategy for cancer treatment, it is equally important to study the mechanism of the initiation of antitumor immunity. Most cancer patients die of metastasis rather than the primary tumor (16). Understanding the initiation of the antitumor immune response would allow for developing a therapeutic strategy to prevent tumor metastasis. It is generally accepted that the immune response, such as antiviral immune response, is triggered by activation of innate immune cells, and the activated innate immune cells activate the adaptive immune system (17). Recent research in antiviral immunity has indicated that tissue-resident memory T (TRM) cells, which are bona fide adaptive immune cells, play potent roles in activating innate immune cells and initiating an antiviral immune response (18).

TRM cells are a lineage of memory T cells that reside in the tissues, especially in nonlymphoid tissues, such as epithelial mucosa and skin, without recirculating (19). One of the most critical functions of TRM cells is their quick response to viral infection. CD8+ TRM cells can get activated within 6 h after viral infection (18). Activated CD8+ TRM cells produce IFN-γ, IL-2, and TNF-α cytokines further to activate NK cells and DCs (18). Therefore, CD8+ TRM cells act as sentinels to initiate an antiviral immune response (20). Although the studies on CD4+ TRM cells are not as extensive as the studies on CD8+ TRM cells due to some of the CD4+ TRM cells lacking the typical TRM cell markers such as CD103, many elegant studies indicate that CD4+ TRM cells play the same important role as, or even more dominant role than, CD8+ TRM cells in antiviral immunity (21, 22).

Only recently, the antitumor function of TRM cells has drawn attention in preclinical and clinical studies (23, 24). TRM cells have been found in many types of tumors, and more TRM cells in tumors are correlated with a more favorable prognostic outcome (2527). Most of these studies have focused on CD8+ TRM cells and found that CD8+ TRM cells cannot only mediate durable anti-melanoma immunity but also amplify antitumor immunity by triggering Ag spreading (28, 29). How CD4+ TRM cells regulate antitumor immunity has not been well defined.

Using Ja281 knockout (KO) mice, we found that the deficiency in invariant NKT (iNKT) cells and TCRα repertoire diversity did not cause an apparent difference in antitumor responses compared with wild-type (WT) mice, at least for B16BL6 melanoma and E0771 breast cancer. However, when transferring thymocytes or splenocytes to the Ja281 KO mice and their WT counterparts, only the cell-transferred Ja281 mice acquired potent antitumor immunity. We further found that this enhanced antitumor immunity is mediated by CD4+ T but not iNKT or CD8+ T cells. More specifically, the enhanced antitumor immunity is dominated by CD4+ TRM cells and depends on NK cells. CD4+ TRM cells can initiate an antitumor immune response. The CD4+ TRM/NK cell axis orchestrates the formation of a TME in favor of antitumor immunity.

C57BL/6N mice at the age of 6–7 wk were purchased from the National Institutes of Health/Charles River Laboratories (Wilmington, MA) or Envigo (Indianapolis, IN). Ja281−/− iNKT KO (Ja281 KO) mice with a C57BL/6 background were obtained from the National Institutes of Health and bred at the Washington State University Wegner Hall Vivarium in Pullman, WA and the PBS Vivarium in Spokane, WA. Traj18 KO mice (B6(Cg)-Traj18tm1.1Kro/J), CD45.1 mice (B6.SJL-Ptprca Pepcb/BoyJ), IFN-γ KO mice (B6. 129S7-Ifngtm1Ts/J), IL-4 KO mice (C57BL/6-Il4tm1Nnt/J), CXCR6 KO mice (B6.129P2-Cxcr6tm1Litt/J), and IL-17A KO mice (Il17atm1.1(icre)Stck/J) were purchased from The Jackson Laboratory (Bar Harbor, ME). IFN-γ/iNKT double KO mice were created at the Washington State University Wegner Hall Vivarium through crossing Ja281 KO mice with IFN-γ KO mice. Mice were housed in a specific pathogen–free room, in plastic cages with microfilter tops and CareFresh beddings, and were allowed free access to Purina 5001 rodent laboratory chow and sterilized Milli-Q water. The animal protocols used in this study were approved by the Institutional Animal Care and Use Committee at Washington State University.

The following FITC-, PE-, allophycocyanin-, PE-Cy5.5–, PE-Cy7–, biotin-, PE-eFluor 610–labeled anti-mouse Abs were purchased from eBioscience (San Diego, CA) or BioLegend (San Diego, CA): anti-CD3 (145-2C11), anti-CD4 (GK1.5), anti-CD8 (53-6.7), anti-CD11c (N418), anti-CD11b (M1/7), anti–Gr-1 (RB6-8C5), anti-NK1.1 (PK136), anti-CXCR6 (SA051D1), anti–PD-1 (29F.1A12), anti–Tim-3 (B8.2C12), anti-CD19 (6D5), anti-CD16/32 (clone 93), anti–IFN-γ (XMG1.2), anti–TNF-α (MP6-XT22), anti–IL-13 (eBio13A), anti–IL-10 (JES5-16E3), and anti–TGF-β1 (TW7-16B4). The following Abs were purchased from Santa Cruz Biotechnology (Dallas, TX): anti-arginase (sc-271430), anti-NOS2 (sc-7271), and anti-actin (sc-47778). MojoSort streptavidin nanobeads, a MojoSort mouse CD4 T cell isolation kit (catalog no. 480033), and a MojoSort mouse CD8 T cell isolation kit (catalog no. 480035) were purchased from BioLegend (San Diego, CA). FTY720 was purchased from Sigma-Aldrich (St. Louis, MO).

Thymocytes or splenocytes were isolated from the thymus or spleen of the indicated donor mice following our previously published methods (30, 31). Cells were suspended in sterilized PBS at 1.6 × 108 cells/ml for thymocytes and 1 × 108 cells/ml for splenocytes. Cells were transferred into recipient mice via tail vein injection at 8 × 107 cells/mouse for thymocytes or 5 × 107 cells/mouse for splenocytes. CD4+ T and CD8+ T cells were isolated from splenocytes using a MojoSort mouse CD4 T cell isolation kit and a MojoSort Mouse CD8 T cell isolation kit. CD4+CXCR6+ and CD4+CXCR6 cells were purified from splenic CD4+ T cells by incubating with biotin-conjugated anti-mouse CXCR6 Ab and isolated with MojoSort streptavidin nanobeads. Purified CD4+, CD8+, CD4+CXCR6+, and CD4+CXCR6 cells were transferred into recipient mice via tail vein injection at 2–5 × 106 cells/mouse.

FTY720 was dissolved in sterilized Milli-Q water at 5 mg/ml to make a stock solution. The mouse was injected i.p. with 25 μg of FTY720 in 100 μl of sterilized Milli-Q water daily for 5 consecutive days, starting 2 d before tumor inoculation.

Two weeks after cell transfer and 1 d before tumor inoculation, Ultra-LEAF purified anti–asialo-GM1 Ab (BioLegend, San Diego, CA) was injected i.p. into mice at 250 μg/mouse in 200 μl of PBS to deplete NK cells. For CD4+ and CD8+ T cell depletion, 250 μg of anti-mouse CD4 (GK1.5, BioLegend, San Diego, CA) or anti-mouse CD8 (53-6.7, BioLegend, San Diego, CA) Ab was injected i.p. into mice 2 wk after cell transfer 5 d before tumor inoculation.

Two weeks after CD45.1+ cell transfer, anti-CD45.1 Ab (clone A20, BioLegend, San Diego, CA) was injected i.p. into mice at 10 μg/mouse. Tumor cells were inoculated 2 d after anti-CD45.1 Ab injection.

B16BL6 and EO771 cells were cultured in DMEM supplemented with 10% FBS, 1% ampicillin, and streptomycin in an incubator at 37°C and 5% CO2. Cells were harvested when they reached 70% confluence and suspended in sterilized PBS. For B16BL6 melanoma s.c. injection, tumor cells were inoculated in the right hip area at 2 × 105 cells in 200 μl of PBS. For EO771 breast cancer cell inoculation, 1 × 106 tumor cells in 100 μl of PBS were injected into the fourth mammary gland fat pad. Tumor size was measured by a caliper every other day, 1 wk after tumor inoculation when the tumor was palpable and calculated by the formula v = (a × b2)/2, where v is the volume of the tumor, a is the length of the tumor, and b is the width of the tumor. For lung melanoma growth, B16BL6 tumor cells were injected via the tail vein at 5 × 104 cells in 200 μl of PBS. Mice were euthanized 3 wk after tumor inoculation. The lungs were fixed with Fekete’s solution. The tumor colonies on five lobes of the lung were counted using a stereomicroscope. Without specific indication, all of the tumor cells were inoculated 2 wk after cell transfer.

Leukocytes from the thymus, spleen, blood, and lymph nodes were isolated following our previously published methods (31). The protocol for tumor-infiltrating leukocyte isolation was modified from a method for immune cell isolation developed by Blom et al. (32). Briefly, tumor tissue was rinsed with ice-cold PBS and crushed with a plastic syringe plunger to pass through a stainless wire mesh strainer. Cells were suspended in 50 ml of PBS. The cell suspension was centrifuged at 60 × g for 1 min. The supernatant was transferred into a fresh tube and centrifuged at 480 × g for 10 min. The cell pellet was resuspended in 10 ml of 37.5% Percoll in PBS and centrifuged at 850 × g for 30 min. The cell pellet was resuspended in 5 ml of RBC lysis buffer for 5 min. Cells were washed with PBS + 0.1% BSA and collected by centrifugation. Cell phenotype was analyzed by flow cytometry following our previously published methods (33). Fluorescence minus one controls, including isotype controls, were used to set the gate on the interested cells. For tumor samples, anti-mouse CD45 was used to gate on leukocytes for further analysis. A Beckman Coulter Gallios flow cytometer and Kaluza acquisition and analysis software (Beckman Coulter, Miami, FL) were used to collect and analyze the cell phenotype data.

Lymphocytes were activated by 50 ng/ml PMA and 500 ng/ml ionomycin in RPMI 1640 medium supplemented with 10% FBS and 5 μg/ml brefeldin A at 37°C for 4 h. Cytokine-producing cells were determined by flow cytometry–based intracellular staining following our previously published method (30). The fluorescence minus one gate strategy, a Beckman Coulter Gallios flow cytometer, and Kaluza acquisition and analysis software (Beckman Coulter, Miami, FL) were used to collect and analyze the cell phenotype data.

Western blotting was employed to study protein expression in the tumor tissue. The method was the same as we reported previously (34).

TRIzol reagent was used to extract total RNA from tumor tissue following the manufacturer’s instructions. The RNA concentration was determined by NanoDrop. cDNA was synthesized by using Moloney murine leukemia virus reverse transcriptase (Promega, Madison, WI). SsoAdvanced universal SYBR Green supermix (Bio-Rad, Hercules, CA) was used to run real-time PCR with an Analytik Jena qTOWER3 real-time PCR thermal cycler. Actin and GAPDH were used as internal controls.

Data were analyzed by Microsoft Excel and GraphPad Prism 7. A normality test was performed. A power analysis on the number of animals to be used in each experiment was performed. The confidence level was set to 95% and the type I error was 0.05. An unpaired, two-tailed Student t test or one-way ANOVA with Tukey’s test was used to determine the significance of the difference between or among groups where appropriate. Kaplan–Meier survival analysis and a log-rank (Mantel–Cox) test (conservative) were used to analyze survival. The difference was considered significant when p < 0.05.

Ja281 KO mice were originally developed to target Va14Ja18 iNKT cells and have been used to study iNKT cell function, including antitumor immunity (3537). A later study found that the PGK (phosphoglycerate kinase)-neo cassette that was used to replace the Ja18 gene fragment in the Ja281 KO mice severely affected the transcription of the Ja genes upstream of Ja18, which led to an ∼60% loss of the diversity of TCRα repertoire, including a deficiency in iNKT and mucosal-associated invariant T (MAIT) cells (3840). Because both iNKT and MAIT cells play critical roles in antitumor immunity (35, 41), we wanted to know how the deficiency in iNKT, MAIT, and TCRα diversity in Ja281 mice and reconstitution of these T cells by transferring WT thymocytes or splenocytes into Ja281 KO mice affect antitumor immunity. We found that neither s.c. B16BL6 melanoma development and growth nor survival of the tumor-bearing Ja281 KO mice was affected compared with their WT counterparts (Fig. 1A, 1B). Surprisingly, the cell transfer significantly inhibited tumor development and growth in Ja281 KO mice (Fig. 1A). The cell transfer also greatly extended the survival of the tumor-bearing Ja281 KO mice (Fig. 1B). We also transferred the same amount of WT thymocytes or splenocytes into WT mice. The tumor development, growth, and survival of the tumor-bearing WT mice were not significantly affected by the cell transfer (Fig. 1A, 1B). These results suggest that the transfer of WT thymocytes or splenocytes greatly enhanced antitumor immunity in the Ja281 KO mice, but not the WT mice.

FIGURE 1.

Transferring thymocytes or splenocytes into Ja281 KO mice significantly increases the antitumor immune response to B16BL6 melanoma and EO771 breast cancer. (A) Tumor growth of B16BL6 melanoma in female WT C57BL/6 or Ja281 KO mice transferred with or without thymocytes or splenocytes. a is different from b (p < 0.01). (B) Survival of B16BL6 melanoma-bearing female WT C57BL/6 or Ja281 KO mice transferred with or without thymocytes or splenocytes. Numbers in the parentheses are the days of median survival time. a is different from b (p < 0.001). (C) Lung colonies of B16BL6 melanoma in female Ja281 KO mice transferred with or without thymocytes or splenocytes. *p < 0.05, ***p < 0.001. (D) Survival of EO771 tumor-bearing female WT C57BL/6 and Ja281 KO mice transferred with or without thymocytes. Numbers in the parentheses are the days of median survival time. In each independent experiment, each group contained 8–10 mice. Experiments were repeated at least once with similar results. C57BL/6_Spl, C57BL/6 mice transferred with WT splenocytes; C57BL/6_Thy, C57BL/6 mice transferred with WT thymocytes; C57BL/6 (WT), WT C57BL/6 mice; J281 KO, Ja281 KO mice; Ja281 KO_Spl, Ja281 KO mice transferred with WT splenocytes; Ja281 KO_Thy, Ja281 KO mice transferred with WT thymocytes.

FIGURE 1.

Transferring thymocytes or splenocytes into Ja281 KO mice significantly increases the antitumor immune response to B16BL6 melanoma and EO771 breast cancer. (A) Tumor growth of B16BL6 melanoma in female WT C57BL/6 or Ja281 KO mice transferred with or without thymocytes or splenocytes. a is different from b (p < 0.01). (B) Survival of B16BL6 melanoma-bearing female WT C57BL/6 or Ja281 KO mice transferred with or without thymocytes or splenocytes. Numbers in the parentheses are the days of median survival time. a is different from b (p < 0.001). (C) Lung colonies of B16BL6 melanoma in female Ja281 KO mice transferred with or without thymocytes or splenocytes. *p < 0.05, ***p < 0.001. (D) Survival of EO771 tumor-bearing female WT C57BL/6 and Ja281 KO mice transferred with or without thymocytes. Numbers in the parentheses are the days of median survival time. In each independent experiment, each group contained 8–10 mice. Experiments were repeated at least once with similar results. C57BL/6_Spl, C57BL/6 mice transferred with WT splenocytes; C57BL/6_Thy, C57BL/6 mice transferred with WT thymocytes; C57BL/6 (WT), WT C57BL/6 mice; J281 KO, Ja281 KO mice; Ja281 KO_Spl, Ja281 KO mice transferred with WT splenocytes; Ja281 KO_Thy, Ja281 KO mice transferred with WT thymocytes.

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We used the i.v. injection of B16BL6 melanoma to determine whether the cell transfer–enhanced antitumor immunity in the Ja281 KO mice is only limited to a s.c. tumor or may also affect tumor growth in the lung. Results indicated that both thymocyte and splenocyte transfer significantly inhibited the formation of melanoma colonies in the lung in the Ja281 KO mice compared with the WT mice (Fig. 1C). However, the effect of inhibition on tumor formation induced by thymocyte transfer was more significant than by splenocyte transfer (Fig. 1C). To further determine whether the cell transfer–induced antitumor immunity in Ja281 KO mice was melanoma specific, we tested it in the EO771 breast cancer model. Results indicated that there was no difference in survival of non–cell-transferred, tumor-bearing mice between the Ja281 KO mice and the WT mice (Fig. 1D). However, thymocyte transfer dramatically suppressed EO771 breast cancer development in Ja281 KO mice. In the group of 10 thymocyte-transferred Ja281 KO mice, only one mouse developed a tumor within the period of the experiment; the survival time of this mouse was significantly longer than any of the WT or Ja281 KO counterparts without cell transfer (Fig. 1D). Taken together, these results indicated that the deficiency of iNKT, MAIT, and TCR repertoire diversity in Ja281 KO mice did not significantly affect antitumor immunity to melanoma and breast cancer. However, transferring thymocytes or splenocytes into Ja281 KO mice dramatically enhanced antitumor immunity compared with the WT mice that possessed an intact T cell immune system, which implies that the loss of TCR repertoires in Ja281 KO mice facilitates the formation of acquired antitumor immunity by reconstitution of T cells.

The Ja281 KO mice are deficient not only in iNKT and MAIT cells but also in 60% of TCRα repertoire diversity (38). We next determined whether the cell transfer–induced antitumor tumor immunity is mediated by iNKT cells. Traj18 KO mice were developed in M. Kronenberg’s laboratory by depleting only the Ja18 gene (39). The usage of all Ja genes, except for Ja18, in Traj18 KO mice is comparable to that of WT mice. Therefore, the Traj18 KO mice are deficient only in iNKT cells but have a normal repertoire of other TCRαs (39). We transferred WT thymocytes into respective Ja281 and Traj18 KO mice and transferred Traj18 KO thymocytes into Ja281 KO mice. Two weeks after the cell transfer, the mice were inoculated s.c. with B16BL6 cells. Results indicated that transferring WT thymocytes into Ja281 KO mice significantly inhibited tumor progression and increased the survival of the tumor-bearing Ja281 KO mice compared with the WT control (Fig. 2A, 2B). Instead of enhancing antitumor immunity, transferring WT thymocytes into Traj18 KO mice enhanced the tumor growth and decreased the survival of the tumor-bearing mice (Fig. 2A, 2B). Transferring Traj18 KO thymocytes, which lack iNKT cells, into Ja281 KO mice generated the same effective antitumor immunity as transferring WT thymocytes into Ja281 KO mice (Fig. 2A, 2B). These results indicated that cell transfer–induced antitumor immunity in Ja281 KO mice was not mediated by iNKT cells.

FIGURE 2.

Cell transfer–induced antitumor immunity in Ja281 KO mice is independent of iNKT or CD8+ T cells. (A) Tumor growth of B16BL6 melanoma in female WT C57BL/6 and Ja281 KO mice transferred with WT or iNKT KO thymocytes, or in female Traj18 KO mice transferred WT thymocytes. a is different from b (p < 0.05) and c (p < 0.001); b is different from c (p < 0.01). (B) Survival of B16BL6 melanoma-bearing female WT C57BL/6 and Ja281 KO mice transferred with WT or iNKT KO thymocytes, and Traj18 KO mice transferred with WT thymocytes. Numbers in the parentheses are the days of median survival time. *p < 0.05, **p < 0.01, ***p < 0.001; ns, not significant. (C) Survival of B16BL/6 melanoma-bearing female WT C57BL/6 and Ja281 KO mice transferred with CD4+ T or CD8+ T cells. Numbers in the parentheses are the days of median survival time. *p < 0.05, ***p < 0.001. (D) Survival of B16BL/6 melanoma-bearing male WT C57BL/6 and Ja281 KO mice transferred with CD4+ T or CD8+ T cells. Numbers in the parentheses are the days of median survival time. *p < 0.05, **p < 0.01. In each independent experiment, each group contained 8–10 mice. Experiments were repeated once with similar results. C57BL/6, WT C57BL/6 mice; F_C57BL/6, female WT C57BL/6 mice; F_J281 KO_CD4, female Ja281 KO mice transferred with WT CD4+ T cells from female C57BL/6 mice; F_Ja281 KO_CD8, female Ja281 KO mice transferred with WT CD8+ T cells from female C57BL/6 mice; Ja281 KO Traj18_Thy, Ja281 KO mice transferred with iNKT KO thymocytes from Traj18 KO mice; Ja281 KO WT_Thy, Ja281 KO mice transferred with WT thymocytes; M_C57BL/6, male WT C57BL/6 mice; M_J281 KO_CD4, male Ja281 KO mice transferred with WT CD4+ T cells from male C57BL/6 mice; M_Ja281 KO_CD8, male Ja281 KO mice transferred with WT CD8+ T cells from male C57BL/6 mice; Traj18 KO WT_Thy, Traj18 KO mice transferred with WT thymocytes.

FIGURE 2.

Cell transfer–induced antitumor immunity in Ja281 KO mice is independent of iNKT or CD8+ T cells. (A) Tumor growth of B16BL6 melanoma in female WT C57BL/6 and Ja281 KO mice transferred with WT or iNKT KO thymocytes, or in female Traj18 KO mice transferred WT thymocytes. a is different from b (p < 0.05) and c (p < 0.001); b is different from c (p < 0.01). (B) Survival of B16BL6 melanoma-bearing female WT C57BL/6 and Ja281 KO mice transferred with WT or iNKT KO thymocytes, and Traj18 KO mice transferred with WT thymocytes. Numbers in the parentheses are the days of median survival time. *p < 0.05, **p < 0.01, ***p < 0.001; ns, not significant. (C) Survival of B16BL/6 melanoma-bearing female WT C57BL/6 and Ja281 KO mice transferred with CD4+ T or CD8+ T cells. Numbers in the parentheses are the days of median survival time. *p < 0.05, ***p < 0.001. (D) Survival of B16BL/6 melanoma-bearing male WT C57BL/6 and Ja281 KO mice transferred with CD4+ T or CD8+ T cells. Numbers in the parentheses are the days of median survival time. *p < 0.05, **p < 0.01. In each independent experiment, each group contained 8–10 mice. Experiments were repeated once with similar results. C57BL/6, WT C57BL/6 mice; F_C57BL/6, female WT C57BL/6 mice; F_J281 KO_CD4, female Ja281 KO mice transferred with WT CD4+ T cells from female C57BL/6 mice; F_Ja281 KO_CD8, female Ja281 KO mice transferred with WT CD8+ T cells from female C57BL/6 mice; Ja281 KO Traj18_Thy, Ja281 KO mice transferred with iNKT KO thymocytes from Traj18 KO mice; Ja281 KO WT_Thy, Ja281 KO mice transferred with WT thymocytes; M_C57BL/6, male WT C57BL/6 mice; M_J281 KO_CD4, male Ja281 KO mice transferred with WT CD4+ T cells from male C57BL/6 mice; M_Ja281 KO_CD8, male Ja281 KO mice transferred with WT CD8+ T cells from male C57BL/6 mice; Traj18 KO WT_Thy, Traj18 KO mice transferred with WT thymocytes.

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Thymocytes primarily include CD4+ T cells and CD8+ T cells. NK cells in thymocytes are negligible. To further determine whether CD4+ or CD8+ T cells mediate the cell transfer–induced antitumor immunity in Ja281 KO mice, we isolated CD4+ and CD8+ T cells from the spleen of WT mice and separately transferred them into Ja281 KO mice. Two weeks after the cell transfer, B16B16 melanoma cells were inoculated s.c. The survival of tumor-bearing mice was observed. This experiment was conducted in both male and female mice. Results indicated that only CD4+ T cell transfer significantly increased the survival of tumor-bearing mice in both female and male mouse experiments (Fig. 2C, 2D). CD8+ T cell transfer did not affect survival tumor-bearing mice compared with their WT counterparts (Fig. 2C, 2D).

To further verify that CD4+ T cells but not CD8+ T cells mediated cell transfer–induced antitumor immunity, we used anti-CD4 and anti-CD8 Abs to deplete CD4+ and CD8+ T cells, respectively, in the Ja281 KO mice transferred with thymocytes and determined the tumor growth and survival of the B16BL6 tumor-bearing mice. Results indicated that depletion of CD8+ T cells did not affect cell transfer–induced antitumor immunity in Ja281 mice. Depletion of CD4+ cells partially compromised cell transfer–induced antitumor immunity in Ja281 KO mice (Supplemental Fig. 1).

These results indicated that cell transfer–induced antitumor immunity in Ja281 KO mice is mediated by CD4+ T cells, but not iNKT or CD8+ T cells.

We next determined whether the cell transfer–induced antitumor immunity acquired by Ja281 KO mice was transient or long-lasting. We inoculated three groups of Ja281 KO mice with B16BL6 melanoma at 1 wk, 3 wk, or 3 mo, respectively, after they had received thymocyte transfer. Ja281 KO mice without thymocyte transfer were used as the control. Results indicated no difference in tumor growth and survival between Ja281 KO mice that had received no thymocyte transfer and those that had received the transfer 1 wk before (Fig. 3A, 3B). However, the tumor growth was significantly slower in the mice that had received thymocyte transfer 3 wk or 1 mo before than those that had received no thymocyte transfer (Fig. 3A). Consistent with tumor growth, the survival of the tumor-bearing mice that had received the thymocyte transfer 3 wk or 1 mo before was significantly longer than those that received no cell transfer (Fig. 3B). Because >85% of the transferred thymocytes were immature T cells, they may need time for further maturation after transferring into the recipient mice. We used the fully matured splenic CD4+ T cells to test this possibility. B16BL6 cells were inoculated s.c. into male Ja281 KO mice after 1 d, 1 wk, or 2 wk of splenic CD4+ T cell transfer. WT C57BL/6 mice were used as the control. Results indicated no difference in tumor growth among the control, 1 d, and 1 wk cell-transferred groups. However, the tumor progression was significantly inhibited in the 2 wk cell-transferred group compared with the other three groups (Fig. 3C). These results indicated that cell transfer–induced antitumor immunity in Ja281 KO mice needs time to develop.

FIGURE 3.

Cell transfer–induced antitumor immunity in Ja281 KO mice is time-dependent and long-lasting. (A) Tumor growth of B16L6 melanoma in female Ja281 KO mice without cell transfer (Ja281 KO) and in those with the tumor cells inoculated 1 wk (Ja281 KO Thy-1w), 3 w (Ja281 KO Thy-3w), or 3 mo (Ja281 KO Thy-3m) after WT thymocyte transfer. (B) Survival of B16BL6 tumor-bearing mice shown in (A). Numbers in parentheses are the days of median survival time. *p < 0.05, **p < 0.01. (C) Tumor growth of B16BL6 melanoma in male WT C57BL/6 mice without cell transfer and in male Ja281 KO mice with tumor cells inoculated at 1 d (Ja281 KO CD4_1d), 1 wk (Ja281 KO CD4_1w), or 2 wk (Ja281 KO CD4_2w) after WT splenic CD4+ T cell transfer. a is different from b (p < 0.01). (D) Survival of female Ja281 KO mice inoculated with B16BL6 melanoma without (Ja281 KO) or with prior WT thymocyte transfer (2 wk before the melanoma inoculation). One year after the first tumor inoculation, the mice without tumor development were inoculated with B16BL6 cells for the second time (Ja281 KO Thy 2° tumor). The control mice were 2 mo of age at the time of tumor inoculation. The numbers in the parentheses are the days of median survival. Each group contained 6–10 mice. ***p < 0.001.

FIGURE 3.

Cell transfer–induced antitumor immunity in Ja281 KO mice is time-dependent and long-lasting. (A) Tumor growth of B16L6 melanoma in female Ja281 KO mice without cell transfer (Ja281 KO) and in those with the tumor cells inoculated 1 wk (Ja281 KO Thy-1w), 3 w (Ja281 KO Thy-3w), or 3 mo (Ja281 KO Thy-3m) after WT thymocyte transfer. (B) Survival of B16BL6 tumor-bearing mice shown in (A). Numbers in parentheses are the days of median survival time. *p < 0.05, **p < 0.01. (C) Tumor growth of B16BL6 melanoma in male WT C57BL/6 mice without cell transfer and in male Ja281 KO mice with tumor cells inoculated at 1 d (Ja281 KO CD4_1d), 1 wk (Ja281 KO CD4_1w), or 2 wk (Ja281 KO CD4_2w) after WT splenic CD4+ T cell transfer. a is different from b (p < 0.01). (D) Survival of female Ja281 KO mice inoculated with B16BL6 melanoma without (Ja281 KO) or with prior WT thymocyte transfer (2 wk before the melanoma inoculation). One year after the first tumor inoculation, the mice without tumor development were inoculated with B16BL6 cells for the second time (Ja281 KO Thy 2° tumor). The control mice were 2 mo of age at the time of tumor inoculation. The numbers in the parentheses are the days of median survival. Each group contained 6–10 mice. ***p < 0.001.

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The Ja281 KO mice are deficient in not only iNKT cells, but also TCRα repertoires. This might decrease T cell cellularity to induce the transferred T cell homeostatic proliferation. T cell homeostatic proliferation could enhance antitumor immunity (42). To test this possibility, we determined the T cell numbers in the different organs of Ja281 KO mice and WT mice. Results indicated that the numbers of CD4+ T and CD8+ T cells in the spleen, lymph nodes, and liver were comparable between Ja281 KO mice and their WT control (Supplemental Fig. 2).

Note that ∼20% of mice with a 2 wk or longer time of cell transfer did not develop a tumor after B16BL6 melanoma inoculation. We used these mice a second time and conducted a tumor inoculation 1 y after the first tumor inoculation. The tumor growth was significantly slower and the survival time was significantly longer than for the control mice, although the mice with the second time of tumor injection were much older than the control group (Fig. 3D).

Collectively, these results indicated that the transferred thymocytes or splenocytes need at least 2 wk to develop and acquire antitumor immunity in Ja281 KO mice. The acquired antitumor immunity is long-lasting.

The CD4+ cell transfer–induced antitumor immunity in Ja281 KO mice has shown the following features: 1) partial resistance to Ab depletion (Supplemental Fig. 1), 2) needing at least 2 wk to develop (Fig. 3A–C), and 3) a long-lasting effect (Fig. 3D). These features are the hallmarks of TRM cells (43). Therefore, we used FTY720-mediated lymphocyte egress blockade and low-dose Ab depletion of circulating lymphocytes to verify that the cell transfer–induced antitumor immunity in Ja281 KO mice is mediated by tissue-resident CD4+ T cells.

FTY720 is an antagonist of S1PR1 (44). FTY720 treatment blocks lymphocyte egress from lymph nodes to circulation (44). We treated the cell-transferred mice with FTY720 (Fig. 4A) in the EO771 tumor model. Results indicated that all WT C57BL/6 mice developed a tumor; cell-transferred Ja281 KO mice inoculated with 100 μl of water as a control did not develop a tumor; and two-thirds of cell-transferred Ja281 KO mice injected with FTY720 developed a tumor. However, the tumor growth was significantly slower than that in C57BL/6 mice (Fig. 4B). FTY720 treatment inhibits T cell IFN-γ and granzyme B production (45). Compared to the untreated cell-transferred mice, the development of tumors in FTY720-treated mice could be associated with impaired CD4+ T cell function induced by FTY720 treatment. These results suggest that blocking circulating lymphocyte egress from lymphoid organs compromises the cell transfer–induced antitumor immunity.

FIGURE 4.

Effects of FTY720 treatment or Ab depletion of circulating transferred CD4+ T cells on cell transfer–induced antitumor immunity in Ja281 KO mice. (A) Scheme shows the timeline of cell transfer, FTY720 injection, and tumor inoculation. (B) Growth of EO771 breast tumor in WT C57BL/6 mice (C57BL/6), Ja281 KO mice transferred with WT thymocytes (Ja281 KO_Thy), or Ja281 KO mice transferred with WT thymocytes and treated with FTY720 (Ja281 KO _Thy_FTY720). (C) Scheme shows the timeline of CD45.1+ thymocyte transfer, anti-CD45.1 mAb injection, and tumor inoculation. (D) Growth of EO771 breast tumor in female C57BL/6 WT mice and Ja281 KO mice transferred with CD45.1+ thymocytes and treated with anti-CD45.1 mAb (10 μg/mouse) (Ja281 KO_CD45.1+Thy_aCD45.1). Each line stands for one mouse. Four out of five cell-transferred mice treated with anti-CD45.1 mAb did not develop a tumor (the green squares on the x-axis are the mice without tumor).

FIGURE 4.

Effects of FTY720 treatment or Ab depletion of circulating transferred CD4+ T cells on cell transfer–induced antitumor immunity in Ja281 KO mice. (A) Scheme shows the timeline of cell transfer, FTY720 injection, and tumor inoculation. (B) Growth of EO771 breast tumor in WT C57BL/6 mice (C57BL/6), Ja281 KO mice transferred with WT thymocytes (Ja281 KO_Thy), or Ja281 KO mice transferred with WT thymocytes and treated with FTY720 (Ja281 KO _Thy_FTY720). (C) Scheme shows the timeline of CD45.1+ thymocyte transfer, anti-CD45.1 mAb injection, and tumor inoculation. (D) Growth of EO771 breast tumor in female C57BL/6 WT mice and Ja281 KO mice transferred with CD45.1+ thymocytes and treated with anti-CD45.1 mAb (10 μg/mouse) (Ja281 KO_CD45.1+Thy_aCD45.1). Each line stands for one mouse. Four out of five cell-transferred mice treated with anti-CD45.1 mAb did not develop a tumor (the green squares on the x-axis are the mice without tumor).

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To further verify that cell transfer–induced antitumor immunity in Ja281 KO mice is mediated by CD4+ TRM cells, we used a low dose of Ab to deplete circulating lymphocytes. TRM cells are resistant to low-dose Ab depletion (46). We transferred CD45.1+ thymocytes to the Ja281 KO mice. Two weeks after cell transfer, 10 μg of anti-CD45.1 Ab was injected into the cell-transferred Ja281 KO mice (Fig. 4C). EO771 tumor cells were inoculated 2 d after Ab administration. Results indicated that circulating CD45.1 cells were successfully depleted by the Ab (Supplemental Fig. 3), and only one-fifth of the Ab-treated mice grew EO771 breast tumor, whereas all of the control mice grew a tumor (Fig. 4D). These results further support that the cell transfer–induced antitumor immunity was not from the circulating portion of the transferred cells.

Taken together, these results indicated that cell transfer–induced antitumor immunity in Ja281 KO mice was mediated by CD4+ TRM cells.

TRM cells initiate an immune response through producing cytokines (18). CD4 T cells are a complicated T cell family. Based on cytokine production, CD4+ cells can at least be divided into Th1, Th2, and Th17 cells that produce their representative cytokines, that is, IFN-γ, IL-4, and IL-17A, respectively. We next determined whether these cytokines were critical for cell transfer–induced antitumor immunity in Ja281 KO mice. We transferred thymocytes from these specific cytokine KO mice into Ja281 KO mice to study B16BL6 tumor growth and the survival of tumor-bearing mice. Results indicated that transferring IFN-γ KO thymocytes to Ja281 KO mice abrogated cell transfer–induced antitumor immunity in terms of tumor growth and host survival (Fig. 5A, 5B). Transferring IL-4 KO thymocytes into Ja281 KO mice did not significantly affect tumor growth compared with the WT thymocyte-transferred group but impaired the cell transfer–induced host survival (Fig. 5A, 5B). Transferring IL-17A KO thymocytes did not affect cell transfer–induced inhibition on tumor growth but even enhanced the cell transfer–induced host survival (Fig. 5C, 5D). These results indicated that IFN-γ produced by the transferred cells was essential for cell transfer–induced antitumor immunity.

FIGURE 5.

IFN-γ and CXCR6 in donor cells are essential for cell transfer–induced antitumor immunity in Ja281 KO mice. (A) Tumor growth of B16BL6 melanoma in female Ja281 KO mice without cell transfer (Ja281 KO), transferred with WT thymocytes (Ja281 KO_Thy), IFN-γ KO thymocytes (Ja281 KO_IFN-γ KO-Thy), or IL-4 KO thymocytes (Ja281 KO_IL-4 KO-Thy). a is different from b (p < 0.05). (B) Survival of female Ja281 KO mice with or without cell transfer shown in (A). The numbers in the parentheses are the days of median survival time. **p < 0.01. (C) Growth of B16BL6 melanoma in female Ja281 KO mice without cell transfer (Ja281 KO), transferred with WT thymocytes (Ja281 KO_Thy) or IL-17A KO thymocytes (Ja281 KO_Thy-IL-17A). a is different from b (p < 0.001). (D) Survival of B16BL6 melanoma-bearing mice shown in (C). *p < 0.05, **p < 0.01. (E) Growth of B16BL6 melanoma in male WT C57BL/6 control (C57BL/6) mice and in Ja281 KO mice transferred with male WT thymocytes (Ja281 KO_Thy), WT splenic CD4+CXCR6+ cells (Ja281 KO_ CD4+CXCR6+), or WT splenic CD4+CXCR6 cells (Ja281 KO_CD4+CXCR6). a is different from b (p < 0.05).

FIGURE 5.

IFN-γ and CXCR6 in donor cells are essential for cell transfer–induced antitumor immunity in Ja281 KO mice. (A) Tumor growth of B16BL6 melanoma in female Ja281 KO mice without cell transfer (Ja281 KO), transferred with WT thymocytes (Ja281 KO_Thy), IFN-γ KO thymocytes (Ja281 KO_IFN-γ KO-Thy), or IL-4 KO thymocytes (Ja281 KO_IL-4 KO-Thy). a is different from b (p < 0.05). (B) Survival of female Ja281 KO mice with or without cell transfer shown in (A). The numbers in the parentheses are the days of median survival time. **p < 0.01. (C) Growth of B16BL6 melanoma in female Ja281 KO mice without cell transfer (Ja281 KO), transferred with WT thymocytes (Ja281 KO_Thy) or IL-17A KO thymocytes (Ja281 KO_Thy-IL-17A). a is different from b (p < 0.001). (D) Survival of B16BL6 melanoma-bearing mice shown in (C). *p < 0.05, **p < 0.01. (E) Growth of B16BL6 melanoma in male WT C57BL/6 control (C57BL/6) mice and in Ja281 KO mice transferred with male WT thymocytes (Ja281 KO_Thy), WT splenic CD4+CXCR6+ cells (Ja281 KO_ CD4+CXCR6+), or WT splenic CD4+CXCR6 cells (Ja281 KO_CD4+CXCR6). a is different from b (p < 0.05).

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CXCR6 is an important cell surface marker governing TRM cell retention and localization (47, 48). We sought to determine whether CXCR6 on donor cells was required for cell transfer–induced antitumor immunity in Ja281 mice. We isolated CD4+CXCR6+ and CD4+CXCR6 splenic cells from male WT C57BL/6 mice, transferred them into male Ja281 KO mice, and studied the antitumor immunity. Results indicated that transferring CD4+CXCR6+ cells but not CD4+CXCR6 cells, into Ja281 KO mice significantly inhibited tumor growth (Fig. 5E).

Taken together, these results indicated that the expression of IFN-γ and CXCR6 in donor cells is essential for cell transfer–induced antitumor immunity in Ja281 KO mice.

IFN-γ plays an essential role in antitumor immunity. We next determined whether the endogenous IFN-γ produced by recipient Ja281 KO mice plays a role in cell transfer–induced antitumor immunity. To this end, we created a strain of Ja281/IFN-γ double KO mice by crossing Ja281 KO mice and IFN-γ KO mice. We transferred WT thymocytes into Ja281/IFN-γ double KO mice and Ja281 KO mice, respectively. Nontransferred Ja281/IFN-γ KO and Ja281 KO mice were used as controls. The tumor growth and survival of B16BL6 tumor-bearing mice were determined. Results indicated that the tumor growth in Ja281/IFN-γ KO mice was significantly faster than in Ja281 KO mice, and the survival of tumor-bearing Ja281/IFN-γ KO mice was significantly decreased as opposed to that of Ja281 KO mice (Fig. 6). Transferring thymocytes into Ja281/IFN-γ KO mice did not affect antitumor immunity compared with the nontransferred Ja281/IFN-γ KO mice and Ja281 KO mice (Fig. 6). In line with the results shown above, transferring thymocytes into Ja281 KO mice significantly inhibited tumor growth and increased the survival of the tumor-bearing mice (Fig. 6). These results indicated that IFN-γ in the recipient Ja281 KO mice was required for the induction of cell transfer–induced antitumor immunity.

FIGURE 6.

Cell transfer–induced antitumor immunity is host IFN-γ– and NK cell–dependent. (A) Tumor growth of B16BL6 melanoma in female Ja281 KO mice without cell transfer (Ja281 KO), or with WT thymocyte transfer (Ja281 KO_Thy), thymocyte transfer, and NK cell depletion by anti–asialo-GM1 Ab (Ja281 KO_Thy-Anti-G), or female Ja281 and IFN-γ double KO mice without cell transfer (Ja281/IFN-γ KO), or with WT thymocyte transfer (Ja281/IFN-γ KO_Thy). a is different from b and c (p < 0.001). b is different from c (p < 0.05). (B) Survival of B16BL6 tumor-bearing mice shown in (A). The numbers in the parentheses are the days of median survival. a is different from b (p < 0.001) and c (p < 0.001); b is different c (p < 0.01).

FIGURE 6.

Cell transfer–induced antitumor immunity is host IFN-γ– and NK cell–dependent. (A) Tumor growth of B16BL6 melanoma in female Ja281 KO mice without cell transfer (Ja281 KO), or with WT thymocyte transfer (Ja281 KO_Thy), thymocyte transfer, and NK cell depletion by anti–asialo-GM1 Ab (Ja281 KO_Thy-Anti-G), or female Ja281 and IFN-γ double KO mice without cell transfer (Ja281/IFN-γ KO), or with WT thymocyte transfer (Ja281/IFN-γ KO_Thy). a is different from b and c (p < 0.001). b is different from c (p < 0.05). (B) Survival of B16BL6 tumor-bearing mice shown in (A). The numbers in the parentheses are the days of median survival. a is different from b (p < 0.001) and c (p < 0.001); b is different c (p < 0.01).

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In antiviral immunity, TRM cells usually act as a sentinel to initiate an antiviral immune response and need to activate other effector cells to complete the antiviral immune response. Because iNKT and CD8+ T cells were dispensable (Fig. 2) in cell transfer–induced antitumor immunity in Ja281 KO mice, we sought to determine whether NK cells were required in this cell transfer–induced antitumor immunity. We first used the anti–asialo-GM1 Ab to deplete NK cells from the cell-transferred Ja281 KO mice and then studied the B16BL6 melanoma growth. Results indicated that the depletion of NK cells abrogated cell transfer–induced antitumor immunity in Ja281 KO mice (Fig. 6).

These results indicated that the endogenous IFN-γ and NK cells are required for cell transfer–induced antitumor immunity in the Ja281 KO mice.

We next investigated how cell transfer affected the TME in Ja281 KO mice. Two weeks after cell transfer, mice were inoculated with B16BL6 melanoma. Tumor samples were collected 3 wk after tumor inoculation. Tumor-infiltrating lymphocytes (TILs) and myeloid cells were characterized by flow cytometry. Results indicated that the number of TILs per gram of tumor tissue in the cell-transferred Ja281 KO mice was 5-fold more than that in the nontransferred Ja281 KO mice (Fig. 7A). The increased TILs in cell-transferred Ja281 KO mice was mainly composed of CD4+ T, CD8+ T, and NK cells (Fig. 7B). The number of total CD19+ B cells per gram of tumor did not alter (Fig. 7B). However, the frequency of B cells in the CD45+ leukocyte population in the tumor was significantly lower in the cell-transferred mice than in the nontransferred mice (Supplemental Fig. 4A, 4B). The percentage of IFN-γ–producing T cells and NK cells was significantly higher in the tumor of cell-transferred mice than that of the nontransferred mice (Fig. 7C). Cell-transferred mice had fewer TNF-α–producing cells than did the non–cell-transferred control (Supplemental Fig. 4C). Therefore, the ratio of IFN-γ–producing cells to TNF-α–producing cells was significantly higher in the tumor of cell-transferred mice than that in the tumor of non–cell-transferred mice (Supplemental Fig. 4D). TNFR1 expression was significantly higher in the tumor of non–cell-transferred Ja281 KO mice compared with the cell-transferred Ja281 KO mice (Supplemental Fig. 4E). The frequencies of Foxp3+ CD4+ T (regulatory T [Treg]) and T-bet+CD4+ T cells (Th1 cells) in the cell-transferred mice were significantly higher than those in the non–cell-transferred control mice (Fig. 7D, 7E). The frequencies of CD69+CD103+ (TRM) cells in both CD8+ and CD4+ T cells were much higher in the cell-transferred mice than in the nontransferred control mice (Fig. 7F). Cell transfer significantly enhanced the expression of inhibitory receptors PD-1 and Tim3 on CD8+ T cells (Fig. 7G). Cell transfer significantly decreased CD11b+Gr1+ myeloid-derived suppressor cells (MDSCs) and increased tumor-associated macrophages (TAMs) (Fig. 7J), specifically CD11c+ type I TAMs in the tumor (Fig. 7H, 7I). The frequency of DCs in the tumor of cell-transferred mice was also significantly increased compared with their non–cell-transferred counterparts (Supplemental Fig. 4F).

FIGURE 7.

Cell transfer significantly alters the contents, phenotype, and function of the lymphocytes and myeloid cells in the tumor. Female Ja281 KO mice were transferred with thymocytes and inoculated with B16BL6 2 wk after the transfer. Leukocytes were isolated from the tumor and analyzed by flow cytometry 3 wk after tumor inoculation. (A) The number of TILs per gram of tumor tissue. (B) The number of the indicated lymphocytes per gram of tumor tissue. (C) Percentage of IFN-γ–producing cells in CD8+, CD4+, or NK cells. Cells were stimulated with PMA + ionomycin for 4 h, and the cytokine-producing cells were determined by intracellular staining and flow cytometry. (D) Percentage of Foxp3+ Treg cells in CD4+ T cells. (E) Percentage of T-bet+ cells in CD4+ T cells. (F) Percentage of CD69+CD103+ cells in CD8+ or CD4+ T cells. (G) PD1+ or Tim3+ cells in CD8+ T cells. (H) Dot plots show the gate strategy of CD45+ leukocytes (gates M, Q), Gr1+CD11b+ MDSCs (gate N), F4/80+Gr1 tumor-associated macrophages (TAMs: gate P), and CD11c+ TAMs. (I) Percentage of MDSCs in myeloid cells (gate M). (J) Percentage of TAM in myeloid cells. (K) Percentage of CD11c+ TAMs. Each group contained 9–13 mice. Data are shown as mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

FIGURE 7.

Cell transfer significantly alters the contents, phenotype, and function of the lymphocytes and myeloid cells in the tumor. Female Ja281 KO mice were transferred with thymocytes and inoculated with B16BL6 2 wk after the transfer. Leukocytes were isolated from the tumor and analyzed by flow cytometry 3 wk after tumor inoculation. (A) The number of TILs per gram of tumor tissue. (B) The number of the indicated lymphocytes per gram of tumor tissue. (C) Percentage of IFN-γ–producing cells in CD8+, CD4+, or NK cells. Cells were stimulated with PMA + ionomycin for 4 h, and the cytokine-producing cells were determined by intracellular staining and flow cytometry. (D) Percentage of Foxp3+ Treg cells in CD4+ T cells. (E) Percentage of T-bet+ cells in CD4+ T cells. (F) Percentage of CD69+CD103+ cells in CD8+ or CD4+ T cells. (G) PD1+ or Tim3+ cells in CD8+ T cells. (H) Dot plots show the gate strategy of CD45+ leukocytes (gates M, Q), Gr1+CD11b+ MDSCs (gate N), F4/80+Gr1 tumor-associated macrophages (TAMs: gate P), and CD11c+ TAMs. (I) Percentage of MDSCs in myeloid cells (gate M). (J) Percentage of TAM in myeloid cells. (K) Percentage of CD11c+ TAMs. Each group contained 9–13 mice. Data are shown as mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Close modal

Effector molecules, such as enzymes, cytokines, and chemokines, in the TME greatly affect TIL function (49). Arginase and inducible NO synthase 2 are produced by MDSCs and other cell types and inhibit T cell and NK cell function (50). We found that the expression of arginase and inducible NO synthase 2 in the tumor of cell-transferred Ja281 KO mice was significantly lower than that in the tumor of nontransferred Ja281 KO mice (Supplemental Fig. 4G). TGF-β, IL-10, and IL-13 are the inhibitory cytokines that inhibit TIL antitumor immunity (51). The expression of these cytokines was significantly downregulated in the tumor of the cell-transferred Ja281 KO mice (Supplemental Fig. 4H). XCL1 and FLT3L are important growth factors for cDC1 development and maturation (7, 8). CCL4 and CCL5 play a key role in attracting cDC1 migration to the tumor (7). CXCL9 and CXCL10 are critical chemoattractants for T cell migration into the tumor (6). The expression of CXL1, FLT3L, CCL4, CCL5, CXCL9, and CXCL10 was significantly upregulated in the tumor of the cell-transferred Ja281 KO mice compared with their non–cell-transferred counterparts (Supplemental Fig. 4I).

These results indicated that cell transfer in Ja281 KO mice had altered the TME by inducing a TME favoring antitumor immunity.

Using a TCRα repertoire deficient mouse model, we found that CD4+ TRM cells could initiate a potent antitumor immune response, significantly inhibiting B16BL6 melanoma progression in the s.c. tissue and in the lung. The antitumor immune response was even more pronounced in the triple-negative EO771 breast tumor model. This CD4+ TRM cell–initiated antitumor immunity was dependent on NK cells and IFN-γ but not iNKT and CD8+ T cells. In addition, the CD4+ TRM/NK cell axis could orchestrate the formation of TME that favors antitumor immunity.

The Ja281 KO mice were created to study iNKT cell function, including iNKT cell antitumor immunity (35). Using this strain of mice combined with iNKT cell transfer, several groups showed that iNKT cells play important roles in antitumor immunity by regulating DC and NK cell functions (52, 53). Because Ja281 KO mice are deficient in iNKT, MAIT, and other T cells, we used thymocytes, which contain all types and developmental stages of T cells, and splenocytes, which contain well-differentiated T cells, to reconstitute T cells in Ja281 KO mice to study antitumor immunity. Indeed, both splenocyte and thymocyte transfer could induce potent antitumor immunity. However, the results from Traj18 KO mice, which are only deficient in iNKT cells, as donors or recipients, unequivocally indicated that cell transfer–induced antitumor immunity in Ja281 KO mice was not mediated by iNKT cells. Ab depletion and purified CD4+ and CD8+ T cell transfer experiments further confirmed that cell transfer–induced antitumor immunity in Ja281 KO mice was mediated by CD4+ T but not CD8+ T cells.

Several elegant studies have demonstrated that CD4+ T cells can orchestrate potent antitumor immunity, which may be even more effective than CD8+ T cell–mediated antitumor immunity (14, 15, 54). Because these CD4+ T cell–based antitumor studies were performed in CD4+ T cell–deficient mice transferred with tumor-specific CD4+ transgenic cells or TILs, lymphopenia and depletion of Treg cells might play a key role in the observed antitumor immunities. The Ja281 KO mice we used in this study are deficient in the TCRα repertoire (38) but have no CD4+ T cell lymphopenia, as their CD4+ T cell number is comparable to that of WT mice (Supplemental Fig. 2). Ja281 KO mice with or without cell transfer had a higher level of Treg cells (Fig. 7). Thus, this CD4+ cell–mediated antitumor immunity in the cell-transferred Ja281 KO mice unlikely resulted from lymphopenia-induced CD4+ T cell activation or the lack of Treg cell inhibition. FTY720 treatment and low-dose Ab depletion experiments showed that the cell transfer–induced antitumor immunity in Ja281 KO mice was mediated by CD4+ TRM cells.

One crucial question is why transferring the same type of donor cells could only generate antitumor immunity in Ja281 KO but not in WT mice. We speculate that there is a population of T cells that can regulate TRM cell function, and this population of T cells is absent from the immune system of Ja281 KO mice because of the deficiency in the TCR repertoire. Besides iNKT cells, MAIT cells are also missing in Ja281 KO mice (40). How the lack of MAIT cells in the Ja281 KO mice affects the outcome of antitumor immunity is unknown. It is reported that MAIT cells facilitate tumor initiation, growth, and metastasis through MR1 on tumor cells (41). The deficiency of MAIT cells in the Ja281 KO mice should enhance the antitumor immunity of these mice. However, our results indicated that Ja281 KO mice exhibited comparable antitumor immunity to WT mice. This suggests that Ja281 KO mice may lack not only protumoral but also antitumoral cells. As we mentioned above, these antitumoral cells are likely the population of cells regulating TRM cell function. Cell transfer might only recover the antitumoral partitions.

CXCR6 is an important chemokine receptor governing TRM cell retention and localization (47, 55). Our results indicated that the expression of CXCR6 on splenic CD4+ T cells is essential for cell transfer–induced antitumor immunity in Ja281 KO mice.

It is noteworthy that both male and female Ja281 KO mice exhibited cell transfer–induced antitumor immunity. However, the magnitude of the response was different. Female mice showed a stronger response compared with male mice. This phenomenon is consistent with the antimelanoma response in WT mice (56).

In both antitumor and antiviral immune responses, CD8+ cells usually play the dominant role in eliminating tumor or virally infected cells. CD4+ T cells play a regulatory role to help CD8+ T cell function. CD8+ TRM cells can effectively control viral infection or tumor progression (57, 58). In this Ja281 KO mouse model, CD4+ TRM cells play a decisive role in controlling antitumor immunity, and this CD4+ TRM cell–mediated antitumor immunity is independent of CD8+ T cells. This provides us with a new strategy to develop tumor immunotherapy. CD4+ T cells regulate the immune response mainly through producing cytokines to modulate other effector cells. Indeed, IFN-γ and NK cells are essential for CD4+ TRM cell–mediated antitumor immunity. Interestingly, transferring IFN-γ KO thymocytes significantly, but not completely, abrogated cell transfer–induced antitumor immunity in Ja281 KO mice. This suggested that other cytokines produced by the transferred CD4+ T cells may also play a role in this CD4+ TRM cell–mediated antitumor immunity. In antiviral immunity, besides IFN-γ, TRM cell–produced IL-2 and TNFα also play a key role in activating NK cells and DCs (18). The transferred CD4+ T cells initiate the immune response by activating NK cells. Depletion of host NK cells and IFN-γ completely abrogated CD4+ T cell transfer–induced antitumor immunity, suggesting that the CD4+ TRM/NK cell axis can effectively fulfill antitumor immunity.

Cell transfer dramatically altered the TME of B16BL6 tumor in Ja281 KO mice. It significantly increased TILs, specifically IFN-γ–producing CD8+, CD4+, and NK cells, decreased MDSCs, and downregulated immunoinhibitory cytokine and enzyme expression. It was reported that the NK cell/DC axis defines the TME in melanoma (8). Our results support that the CD4+ TRM/NK cell axis orchestrates formation of the TME. Indeed, the expression of XCL1 and Flt3L—cytokines governing DC1 proliferation and maturation—was significantly upregulated in the tumors of cell-transferred Ja281 KO mice. Interestingly, cell transfer in Ja281 KO mice increased TILs, Treg cells, and the expression of inhibitory receptors PD-1 and Tim3 on CD8+ T cells. Therefore, combining cell transfer with immune checkpoint blockade might significantly enhance the efficacy of cancer immunotherapy.

In summary, using the Ja281 KO mouse model, we demonstrate that CD4+ TRM cells can initiate antitumor immunity. The CD4+ TRM/NK cell axis can effectively control tumor progression and orchestrate antitumor immunity by modulating TME formation. These results also imply that a population of T cells in the immune system controls CD4+ TRM cell formation and function. These findings shed light on how CD4+ TRM cells initiate and dominate antitumor immunity.

We thank Dr. Faya Zhang, Dr. Yuanfei Li, and Jasmine Nguyen for technical assistance, as well as Dr. Shisheng Li for valuable discussion and critical reading of the manuscript.

This work was supported by National Institutes of Health/National Institute on Alcohol Abuse and Alcoholism Grant AA022098 and by Washington State University College of Pharmacy and Pharmaceutical Sciences start-up funds to H.Z.

H.Z. designed and supervised the study, performed the experiments, analyzed and interpreted the data, and wrote the manuscript. Z.Z., S.M., and A.L. performed the experiments and analyzed the data.

The online version of this article contains supplemental material.

Abbreviations used in this article

cDC1

type 1 conventional dendritic cell

iNKT

invariant NKT

KO

knockout

MAIT

mucosal-associated invariant T

MDSC

myeloid-derived suppressor cell

TAM

tumor-associated macrophage

TIL

tumor-infiltrating lymphocyte

TME

tumor microenvironment

Treg

regulatory T

TRM

tissue-resident memory T

WT

wild-type

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Supplementary data