Anti PD1 therapy activates tumoricidic properties of NKT cells and contributes to overall deceleration of tumor progression in a model of murine mammary carcinoma

Introduction: Immune checkpoint therapy is well- established therapeutic
 approach in treatment of malignant disease and is thought to be mostly based
 on facilitating adaptive immune responses. However, cells of innate immune
 response, such as NKT cells, might also be important for successful
 anti-programmed cell death protein 1 therapy, as they initiate anti-tumor
 immune response. Materials and methods: For tumor induction, 4T1 cells
 syngenic to BALB/c background were used after which mice underwent
 anti-programmed cell death protein 1 treatment.After the mice were
 sacrificed, NKT cells, dendritic cells and macrophages derived from spleen
 and primary tumor tissue were analyzed using flow cytometry. Results:
 Anti-programmed cell death protein 1 therapy significantly decelerates tumor
 growth and enhanced expression of activating molecules CD69, NKp46, NKG2D in
 NKT cells of tumor and spleen. Anti-programmed cell death protein 1 therapy
 activates pro-tumoricidic changes in dendritic cells and macrophages of
 primary tumor tissue. Conclusion: Anti-programmed cell death protein 1
 therapy activates NKT cell directly, and indirectly via DCs. Activated NKT
 cells provide tumoricidic properties directly, by secreting perforin, and
 indirectly by stimulating M1 macrophages polarization. Since anti-programmed
 cell death protein 1 therapy induces significant changes in NKT cells,
 dendritic cells and macrophages, efficacy of overall anti-programmed cell
 death protein 1 therapy is increased, contributing to more efficient
 anti-tumor immune response.


Introduction
Immunotherapy is an emerging approach to treatment of many cancers nowadays 1 . Since the discovery of immune checkpoint inhibitors i.e. anti-programmed cell death protein 1 (PD-1), cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) antbodies, immune checkpoint inhibitors have been utilized in various diseases, such as autoimmune or even infectious diseases, but are predominantly used in malignant, with evolving strategies in management of the disease 2 .
Underlying mechanism in anti PD1 therapy is blockage of PDL/PD1 axis. Under physiological circumstances, programmed death ligand (PDL) is found on many epithelial, endothelilal and immune cells, such as dendritic cells and macrophages 3 . The main role of this ligand is to limit over-reactive immune response, therefore restricting tissue damage due to unrestrained immune response, since activation PDL/PD1 axis potently hinders Tcell receptor activation 4,5 .However, during a malignant disease, PDL is often found on cancer cells. PD1 molecule is mainly expressed on effector immune cells, such as T lymphocytes, NK cells and NKT cells 6 . Given the expression on cancer cells and effector cells of immune response, activation of PDL/PD1 axis in these terms subsequently leads to deteriorating of immune response to malignant disease 7 . Having in mind these assets of a PDL/PD1 axis, it is clear that its inhibitionis important for treating many diseases, especially cancers. Until now, anti-PD1 therapy is approved for many types of solid cancers: metastatic melanoma, non-small cell lung cancer, renal cell carcinoma, bladder cancer and triple negative breast cancer with high PDL expression 8,9,10 .
Although therapeutic PDL/PD1 blockage is thought to be mainly carried through blockage on T lymphocytes, there is emerging evidence that other effector cells, such as NK and NKT cells, take part in beneficial effects of PDL/PD1 axis blockage 11 . Until now, it is well known that in some malignant diseases PD-1 molecule is more expressed in NK cells, which suggests damaged NK cell function 12 . Since it is well known that anti-PD1 therapy increases cytokine production, especially in T lymphocytes, it remains unclear whether anti-PD1 therapy acts directly on NK cells or indirectly, via secretion of activating molecules, such as IFNγ 13 . Data are very modest when it comes to NKT cells and anti-PD1 therapy. These cells play an important role in interplay between innate and acquired immune response 14 . Also, it is known that NKT cells produce cytokines that can activate macrophages and dendritic cells and therefore coordinate immune response 15 . However, the effect of anti-PD1 therapy on NKT cells is yet to be elucidated. Our data imply that NKT cells might be also important for more effective anti-PD1 therapy in malignancies, and might contribute to overall effective immune response to mammary carcinoma, as anti-PD1 therapy induces phenotype changes in NKT cells. wild type anti-PD1 treated (WT anti-PD1 treated), that were treated with anti-PD1 antibody on the third, the sixth, the ninth and the eleventh day after induction of tumor.

Administration of anti-PD1 antibody
Murine anti-PD1 antibody was purchased from BioXcell. Antibody was administered intraperitoneally to mice on the third, the sixth, the ninth and the eleventh day, beginning from the day of tumor induction, at 150 μg per mice of anti-PD1 antibody dissolved in 150 μl of PBS, as previously described by Qun et al and Shmizu et al 16,17 . WT mice that didn't receive anti-PD1 therapy were injected with 150 μl of PBS only, on the same days, according to the model from the study of Vo et al 18 .

Evaluation of tumor growth
Appearance of primary tumor was monitored daily after induction of tumor, by palpation.
After tumor appearance, diameter of primary tumor was measured three times per week using caliper. On the 40 th day after tumor induction, mice were sacrificed; the primary tumor and spleen were surgically removed.

Flow cytometric analysis of splenocytes and tumor-infiltrating leukocytes
We analyzed spleen for assessment of systemic anti-tumor immune response, as our previous results illustrate that phenotype changes in splenocytes are more likely to resemble to phenotype changes occurring in tumor-infiltrating leukocytes 19 . Single-cell suspensions of spleen were obtained by mechanical dispersion, while single-cell suspensions of primary tumors were obtained by enzymatic digestion. Primary 4T1 tumors were minced and placed in 5 mL of DMEM containing 1 mg/mL collagenase I, 1 mM EDTA and 2% FBS (all from Sigma-Aldrich) for enzymatic digestion. After incubation for 2 hr at 37°C, 10 mL of 0.25% trypsin was added and incubated for 3 min followed by

Statistical analysis
The data were analyzed using commercially available software (SPSS version 23.0). All results were analyzed using the Student's t test, Mann-Whitney U test, ANOVA or Kruskal-Wallis test where appropriate. Data are presented as mean ± SEM. Statistical significance was set at p < 0.05.

Anti-PD1 therapy activates splenic NKT cells and skews its phenotype towards more tumoricidic one
Administration of anti-PD1 therapy significantly decelerated tumor growth, in comparison to untreated WT mice.The significant difference between tumor diameter was detected on 14 th day after tumor induction (WT vs. WT + anti-PD1: 1,57 mm vs. 0,50 mm; p<0.05) and remained until 40 th day when mice were sacrificed (WT vs. WT + anti-PD1: 11,93 mm vs. 9,37 mm; p<0.05). Further, we analyzed NKT cells in spleen of tumor bearing WT mice and WT mice treated with anti PD1 antibody. There was no difference in percentage of CD3 + CD49b + NKT cells between experimental and control group (Fig 1A). Expression of activation marker CD69 was significantly elevated in WT anti-PD1 treated mice in comparison to WT untreated mice (p<0.05; Fig 1B). The percentage of IFNγ + and perforin + CD3 + CD49b + NKT cells was significantly higher, while percentage of FoxP3 + CD3 + CD49b + NKT cells was significantly lower in WT anti-PD1 treated mice in comparison to WT untreated mice (p<0.05; Fig 1C-E). There were no significant changes in percentage and phenotype changes in macrophages and dendritic cells in spleen (data not shown).

Enhanced accumulation and alteration toward tumoricidal phenotype of NKT cells in tumor microenvironment
Within primary tumor tissue, the percentage of CD3 + CD49b + NKT cells was significantly higher in anti-PD1 treated mice in comparison to untreated group (p<0.05; Fig 2A).

Anti-PD1 therapy facilitates accumulation and polarisation of macrophages in tumor microenvironment
Anti-PD1 treatment significantly increased the percentage of F4/80 + cells within primary tumor tissue in comparison to WT untreated mice (p<0.05; Fig 3A). Also, expression of TNF-α in F4/80 + cells was significantly higher in anti-PD1 treated mice in comparison to untreated mice (p<0.05; Fig 3B).

Anti PD1 therapy diminishes expression of immunosuppressive molecules in dendritic cells within primary tumor
There was no statistical difference in the percentage of CD11c + cells in tumor microenvironment between groups (Fig 4A), however percentage of IL-10 producing CD11c + cells was significantly lower in tumor microenvironment of anti-PD1 treated mice (p<0.05; Fig 4B).

Discussion
As it is well known, checkpoint inhibitors are currently taking an important role in management of malignant diseases 20,21 . More specifically, anti PD1 antibody has been and is yet to be investigated in numerous oncological diseases, such as melanoma, lung, head and neck and genitourinary cancers 11,22 . As it prolongs half-life of effector immune cells, anti-PD1 therapy efficiently modulates and stimulates more efficient immune response 23 .
Many studies have shown beneficial effects on T lymphocytes. It has been shown that anti-PD1 therapy efficiently increases the percentage of cytotoxic T within tumor tissue. Also, there is some evidence that anti-PD1 therapy elevates the percentage of CD4 + cells in peripheral blood of patients undergoing anti-PD1 therapy [24][25][26][27][28] . Even though T lymphocytes are rather important for compliant anti-PD1 therapy, other cells, such as NK and NKT cells might contribute to more potent effects of anti-PD1 therapy. Until now, the anti-PD1 therapy has been thoroughly studied in terms of adaptive immune response 25,27 , but is yet to be studied in innate immunity during antitumor immune response, especially in regard to NKT cells. It is of great significance to elucidate effects of immune checkpoint therapy on NK and NKT cells, as these cells might be the key of initiating successful anti-PD1 therapy when function of T lymphocytes is impaired 29,30 . NKT cells have important role in anti-  (Fig 1A), phenotype of CD3 + CD49 + NKT cells is remolded towards a more active one. There was significantly higher percentage of CD69 + CD3 + CD49 + cellsin anti-PD1 treated mice, which implies that anti-PD1 therapy might enforce activation of CD3 + CD49 + cells in spleen (Fig 1B). In line with this finding, it has been shown that in highly immunosuppressive tumors, such as head and neck carcinomas, ligands for PD1 in tumor tissue potentially inhibit expression of CD69, and consequently dampen down activation of immune cells 36 . Anti-PD1 therapy also significantly raised the percentage of IFNγ + CD3 + CD49 + cells in spleen (Fig 2C). Available data suggests that anti-PD1 therapy increases expression of IFNγ and inhibits progression of aggressive tumors such as NK/T lymohomas 37 . Also, it has been shown that augmented IFNγ production in NKT cells stimulates cytotoxic T lymphocyte (CTL) mediated antitumor immunity in a model of highly immunogenic T cell lymphoma 38 . On the other hand, anti-PD1 therapy also enhanced production of perforin in CD3 + CD49 + cells, suggesting that anti-PD1 therapy can directly enhance cytotoxic potential of NKT cells (Fig 2D). In addition, expression of immunosuppressive marker FoxP3 was significantly lower in CD3 + CD49 + cells of anti-PD1 treated mice (Fig 2E). This indicates that anti-PD1 therapy, besides directly enhancing IFNγ production, simultaneously weakens immunosuppressive assets of NKT cells, therefore contributing to more tumoricidic phenotype of NKT cell altogether.
When it comes to tumor microenvironment, the percentage of CD3 + CD49 + cells was significantly higher in anti-PD1 treated mice (Fig 2A), implicating intensive accumulation of NKT cells in primary tumor tissue due to anti-PD1 therapy. As it is already known, the presence of NKT cells within primary tumor tissue modifies tumor microenvironment by secreting IFNγ, that activates effector cells, and suppresses immunosuppressive populations, therefore enabling more fluent antitumor immune response 39,40,41 . Our results imply that anti-PD1 therapy might stimulate these beneficial properties of NKT cells. Also, the percentage of NKp46 + and NKG2D + cells was also significantly increased in anti-PD1 treated mice (Fig 2 B, C), which reflects a more dexterous phenotype of NKT cell in tumor microenvironment. Similarly as in spleen, anti-PD1 therapy also raised the percentage of IFNγ-producing CD3 + CD49 + cells within primary tumor tissue (Fig 2D). Furthermore, the expression of FoxP3 and KLRG-1 markers in CD3 + CD49 + NKT cells was significantly diminished in tumor microenvironment, which is indicative of NKT cell phenotype that is less prone to anergy 39 .
As NKT cells are known to interact with many immune cells, such as T cells, dendritic cells and macrophages 42,43 , and the fact that 4T1 mammary carcinoma presents low immunogenic tumor, we further analyzed dendritic cells and macrophages within the primary tumor. Tumor-associated macrophages (TAMs) are one of the most abundant cells within primary tissue of the tumor 44 . Unfortunately, TAMs that reside in tumor microenvironment are mostly those of an immunosuppressive M2 phenotype, thus allowing immune escape of the tumor 45 . Given the vast range of macrophage immunomodulatory properties, facilitating these cells might be of a great significance when it comes to revealing more potent therapeutic strategies in a malignancy. As it is known, TAMs might stimulate anti tumor immune response, by secreting TNFα, and also suppress anti-tumor immune response, by secreting IL-10 that induces overall immunospuppression 46,47 . Our results showed that anti-PD1 therapy significantly enhanced the percentage of F4/80 + macrophages within tumor microenvironment (Fig 3A), and, in addition, significantly increased production of TNF-α in F4/80 + macrophages (Fig 3B), which is a hallmark of a M1 phenotype 48 . When it comes to dendritic cells, anti-PD1 therapy didn't alter the percentage of resident CD11c + dendriticcells within tumor microenvironment (Fig 4A).
Yet, the percentage of IL-10 producing CD11c + dendritic cells was significantly decreased in anti-PD1 treated group (Fig 4B). Dendritic cells, as professional antigen-presenting cells, are constantly circulating throughout tumor microenvironment where are continuously exposed to immunosuppressive molecules produced by cancer cells 49   Mann-Whitney Rank Sum test or Student's unpaired t-test, where appropriate.

Effects of anti-PD1 therapy on NKT, dendritic cells and macrophages.
Anti PD1 therapy acts directly on NKT cell by facilitating its pro-tumoricidic phenotype, which further polarize macrophages towards M1 phenotype via augmented IFNγ secretion. In addition, anti-PD1 therapy lowers IL-10 production in dendritic cells, making them less tolerogenic and more efficient in activating NKT cells.