Immunological Characteristics between αβ T DC and γδ T DC Cells in the Spleen of Breast Cancer-Induced Mice

Objective  To evaluate the antitumoral role of γδ T DC cells and αβ T DC cells in an experimental model of breast cancer. Methods  Thirty female Balb/c mice were divided into 2 groups: control group ( n  = 15) and induced-4T1 group ( n  = 15), in which the mice received 2 × 10 5 4T1 mammary tumor cell line. Following the 28-day experimental period, immune cells were collected from the spleen and analyzed by flow cytometry for comparison of αβ T DC (TCRαβ + CD11c + MHCII + ) and γδ T DC (TCRγδ + CD11c + MHCII + ) cells regarding surface markers (CD4 + and C8 + ) and cytokines (IFN-γ, TNF-α, IL-12 and IL-17). Results  A total of 26.53% of γδ T DC - control group ( p  < 0.0001) - the proportion of αβ T DC was lower in splenic cells than γδ T DC ; however, these 2 cell types were reduced in tumor conditions ( p  < 0.0001), and the proportion of IFN-γ, TNF-α, IL-12 and IL-17 cytokines produced by γδ T DC was higher than those produced by αβ T DC , but it decreased under conditions of tumor-related immune system response ( p  < 0.0001). Conclusion  Healthy mice engrafted with malignant cells 4T1 breast tumor presented T DC with γδ TCR repertoire. These cells express cytotoxic molecules of lymphocytes T, producing anti-tumor proinflammatory cytokines.


Introduction
A new type of immune cell has been described, and these new cells have characteristics of innate and acquired immunity. T DC cells were identified in mice and in humans as cells that express T cell receptors (TCRαβ) specific of T lymphocytes, and simultaneously express the CD11c markers and major histocompatibility complex class II (MHCII or HLA [Human Leukocyte Antigen] in humans), found in innate cells, mainly in dendritic cells. These molecules in the same cell confer unique characteristics and properties; they carry out functions of dendritic cells (DCs) that do not need to be activated by antigen-presenting cells (APCs). When stimulated by specific receptors, such as the family of Toll-like receptors, they can produce interleukin-2 (IL-12) cytokine, as well as process and present antigens. 1 T lymphocytes respond in a specific manner to pathogens and cancer cells by the recognition of specific antigens due to TCR (T-cell receptor) in their membrane, similar to the role of the immunoglobulins in B cells. The TCR consists of 2 polypeptide chains; $ between 90 and 99% of all T cells have the αβ TCR, but a minority has γd chains. 2,3 Both cells originate from common thymic precursors, but the biological roles and molecular understanding of these two subsets differ substantially. The T lymphocytes that express αβ TCR depend on the presentation of antigens in a defined HLA molecule to be activated, and usually are tolerant to selfpeptides. On the other hand, the γd T lymphocytes do not rely on the recognition of classic HLA molecules, and the identification of tumor antigen is made by ubiquitous changes observed across many individuals, which allows these cells to not undergo the rejection process, and, consequently, they can be transferred more easily between individuals. Unlike αβ T cells that have their biological role well-characterized in cancer immune surveillance, the protective role of γd cells during tumor development has only been increasingly reported over the past two decades.
The presence of tumor-infiltrating γd T lymphocytes has been associated with good prognosis in patients with mela-noma 4 and gastric cancer, 5 and high levels of these types of circulating lymphocytes have been associated with reduced cancer risk, increased 5-year-disease-free and increased survival after bone marrow transplant for acute leukemia. 6 The antitumoral ability of γd T lymphocytes is associated with their synthesis of interferon γ (IFN-γ), and of tumor necrosis factor-α (TNF-α), as well as their cytotoxic potential. Other studies have also reported the role of interleukin-17 (IL-17) produced by γd T cells, mainly when they act together with immunogenic cell death-inducing chemotherapeutic drugs. 7 To clarify whether T DC cells could also have the γd chains and the possible antitumor role of this new cell population, we investigated the T DC population comparing both αβ TCR and γd TCR T DC , as well as their cytokines in an experimental model of mice engrafted with malignant cells.

Methods Animals
Thirty 8-week-old female Balb/c mice, kept in the sectoral vivarium of the Oncology Research Institute (IPON, in the Portuguese acronym) of the Universidade Federal do Triângulo Mineiro (UFTM, in the Portuguese acronym), were used. During the 28-day experimental period, the animals were divided into a control group (healthy mice) and a tumor group (4T1 breast tumor cell-engrafted mice). Each group consisted of 15 animals, was housed in plastic cages under a 12-hour light/dark cycle at 21 AE 3°C, with food and water available ad libitum. After the experimental period, the animals were euthanized by overdosing with 50 mg/kg of ketamine and 15 mg/kg of xylazine, and their spleens were removed for analysis. The present study was approved by the Ethics Committee on Animal Use of the UFTM, under number 379/2016 -CEUA/UFTM.

Tumor
The animals were selected at random, and the tumor-induced group was engrafted with 4T1 inoculated with 2 Â 10 5 cells in

Characterization of Immune Cells by Flow Cytometry
The spleens of the control group and of the tumor group were disclosed, filtered, and washed with saline solution, and after counting in a Neubauer chamber, 1 Â 10 6 cells were placed in tubes suitable for the flow cytometry technique. The cells were then labeled with extracellular antiγd TCR-antibodies (T lymphocyte receptor), anti-CD11c (adhesion molecules), anti-IA (antigen-presenting molecule), anti-CD4 (helper T lymphocytes), and anti-CD8 (cytotoxic T lymphocytes) -all antibodies acquired from BD Biosciences. After the 30-minute incubation, the cells were washed and prepared to receive the intracellular antibody labels for anti-IFN-γ, anti-TNF-α, IL-12, and IL-17 proinflammatory cytokines. To block nonspecific binding, the antimouse IgG2b Immunoglobulin G2b) -(mouse) Rabbit Monoclonal Antibody (IgG2b), anti-rat IgG2a Immunoglobulin G2a -(mouse) Rabbit Monoclonal Antibody (IgG2a), and antirat IgG2b isotypes were used. The cells were read on the BD FACSCalibur (BD Biosciences, Franklin Lakes, NJ, USA) cytometer, and the data analyzed using Flowing software.
The gating strategy used was the delimitation by size and granularity (FSCxSSC) of the spleen cells of the control and tumor groups. Subsequently, the double-positive labeling of CD11c and IA (MHCII [Major Histocompatibility Complexclass II]) was limited and, thus, the γd TCR labeling traced the γd T DC cells. Within this population of γd T DC , we analyzed the phenotypic and cytokine markers of interest.

Statistical Analysis
Statistical analyzes and graphs were prepared using Graph-Pad Prism 5.0 (GraphPad Software, San Diego, CA, USA). The Kolmogorov-Smirnov tests were used to verify the normality of the variables. Non-normal samples were analyzed by the Mann-Whitney test, both for comparison between the control group and the tumor-induced group from both profiles and for the comparison of αβ T DC and γd T DC cell expression. The data obtained were represented with their corresponding median, minimum and maximum values. The difference found between the groups was considered statistically significant when p < 0.05.

Results
The flow cytometry profile shows the comparison of αβ T DC (TCRαβ þ CD11c þ MHCII þ ) and γd T DC (TCRγd þ CD11c þ MHCII þ ) cell infiltrates in the spleen of healthy mice engrafted by breast cancer 4T1 cells (►Figs. 1 a, b, c and d). When analyzing the frequency of the γd T DC cell profile (►Fig. 1a), a significant decrease was found in the tumor group, with a median of 18.11 (17.21-19.01) compared with the control group (26.53; 23.62-29.99) (p < 0.0001). The frequencies of both αβ T DC and γd T DC cells were compared, and a significance was found in the tumorinduced group of both cell profiles, that is, there was a higher amount of αβ T DC cells (47.74; 22.97-57.36) than of γd T DC cells (18.11; 17.21-19.01) in the spleen of the 4T1 tumorinduced mice group (p < 0.0001).
The mean fluorescence of auxiliary T lymphocyte (CD4) and cytotoxic T lymphocyte (CD8) markers present in the αβ T DC and γd T DC cells of both groups (►Fig. 1b) was analyzed, and it was observed that the CD8 αβ T DC cells showed a decrease in the tumor group (764. Finally, the profiles of αβ T DC and γd T DC cells were compared, and an increase in the IFN-γ γd T DC cells of the control group (5,972; 5,649-6,297) was found, when compared with IFN-γ αβ T DC cells (4,720; 4,488-6,120) (p ¼ 0.0005). An increase was also found in the TNF-α γd

Discussion
Kuka et al 1 described T DC cells (TCRαβ þ CD11c þ MHCII þ ) as a cell subtype with properties common to polyclonal T αβ cells and dendritic cells. These rare cells have a morphological similarity to dendritic cells that express intermediate levels of CD11c and present Major Histocompatibility Complex (MHC) class II antigenic molecules. Besides, these cells are also characterized by the expression of costimulatory molecules (CD80, CD86) and lymphocyte surface markers (CD3, CD4, and TCR α/β). 1 The frequency of αβ T DC cells described by Kuka et al 1 is of $ 0.04% in the spleen of healthy mice. In our study, it was identified an average of 34.64% in healthy mice and of 47.74% in the group of 4T1 breast tumor cell-engrafted mice. Kuka et al 1 identified and characterized T DC cells by flow cytometry by analyzing the total cells. Our study delimits an area (gate) referring to lymphocytes, size and granulation of this cell type. T DC cells have similar morphology and size to T lymphocytes. 1 The presence of the cell profile for γd T DC (TCRγd þ CD11c þ MHCII þ ) was verified in the same conditions, and the presence of a percentage of 26.53% of γd T DC in the control group and of 18.11% in mice with breast cancer (p < 0.0001) was found.
The present study reports that between 1 and 4% of all T cells present in the thymus, in secondary lymphoid organs, and in the lungs of adult mice are γd T lymphocytes. In mucous membranes, such as the intestinal membrane, there are 25 to 40% of this cell type, where the most significant amount is concentrated, 8 in addition to presenting subtypes as well as phenotypic and functional dieting properties. 9 In our studies, the effect of a systemic immune response under the influence of tumor cells, which decreased both αβ T DC and γd T DC cells, was observed. However, when comparing these two cell profiles -αβ T DC and γd T DC -there was a higher amount of αβ T DC in the 4T1 tumor-induced tumor group than γd T DC (p < 0.0001).
The αβ T cell repertoire is higher in T lymphocytes and, most of the time, they have protective antitumor activity, mainly related to human melanoma tumors. 10 . The results were analyzed by the Mann-Whitney test to compare the mean fluorescence intensity of subtypes αβ T DC and γd T DC cells (statistical differences represented by the dashed line). Differences were considered statistically significant at p < 0.05 (5%). Ã p < 0.05; ÃÃ p < 0.001; ÃÃÃ p < 0.0001.
A study with human blood samples from 38 patients diagnosed with breast cancer compared with healthy controls showed that the proportion of γd T cells in the circulating blood of healthy controls is 1.6 times greater than in breast cancer patients. 11 These data corroborate with our study, since γd T DC cells are present in more significant quantities in the control group (p < 0.0001).
Concerning γd T cells, in an antitumor immune response, pioneering studies on the immunoprotective role of these cells in mice were performed in murine models with skin cancer, which were chemically induced by carcinogens or by subcutaneous transfer of melanoma tumor lineage. From these studies, relevant roles of γd T in antitumor immunity have been described, with mechanisms mediated by the NKG2D C-type lectin-like receptor expressed on NK (NKG2D) receptor by dendritic epidermal T cells (DETCs) Vγ5 þ residing in tissues. 12,13 Studies comparing tumor progression in mice with deficient γd T cells (due to genetic inactivation of the γd TCR receptor) versus mice with sufficient γd T cells (wild) have firmly established the protective role of γd T cells 10 because it was found that γd T cells prevented the progression of chemically induced papilloma to cutaneous squamous cell carcinomas. In contrast, αβ cells seemed to favor tumor progression 14 ; the same happened with spontaneous B cell lymphomas, 15 prostate cancer 16 and in the transplantable model of melanoma B16-F0. 17 Besides, some studies show, in the context of infections by cytomegalovirus and malaria, that γd T cells can be activated later, in the form of direct cytotoxicity, by the action of granzyme B and through stimulating effects such as the secretion of cytokines IFN-γ and TNF-α, or by the direct presentation of antigen. 18 Most γd T cells, unlike αβ T lymphocytes, do not exhibit CD4 or CD8 coreceptors, so antigen recognition is not restricted to antigen-presenting molecules. 8 Thus, the expression of αβ T DC and γd T DC cells related to helper T lymphocyte (TCD4) and cytotoxic (TCD8) markers was compared, revealing that the proportion of CD8 γd T DC is less expressed in splenic cells than CD8 αβ T DC , but these two cell types are decreased in tumor conditions (p < 0.0001).
A recently conducted study comparing subsets of γd T lymphocytes in 40 patients with Chron disease demonstrated a significant decrease in this cell population, concluding that this condition can affect the immune responses against this disease. 19 We believe that, like cancer, the suppressive conditions provided by them can have the same result with αβ and γd T DC, leading to a deficiency of this mechanism.
The central cytokines produced by γd T DC presented a higher proportion of this cell type than those produced by αβ T DC . That is, there is a higher production of the IFN-γ, TNF-α, IL-12 cytokines in the control group, and a lower proportion of IL-17 γd T DC in the group of mice with breast cancer. However, this condition decreases when there is a systemic immune response related to tumors (p < 0.0001).
It is inferred that γd T DC cells are similar to the mechanisms exerted by γd T cells. Studies show that this cell type is an important precursor source of IFN-γ and TNF-α, which inhibits tumor growth and angiogenesis. Also, the study performed with the combination of concanavalin A (ConA) and interleukin-2 (IL-2) demonstrated the potential for polarization and plasticity of γd T DC cells, which induced the intense proliferation of these cells and the consequent production of interleukin-12 (IL-12) and interleukin-2 (IL-18). 20 In inflammatory conditions, a situation observed in some cancers and infections, they favor the polarization of γd T cells toward an IL-17 producing phenotype. 21 A recent study of transcriptome sequencing in $ 18,000 tumor masses in humans revealed that, among tumor-infiltrating leukocytes, γd T cells were strongly associated with a good prognosis. 22 In our study, it was observed that γd T DC in a systemic immune response is suppressed regarding αβ T DC cells in tumor conditions (p ¼ 0.0157).
A specific type of γd T cells (γd TCD27 þ ) from mice secrete the IFN-γ cytokine, responsible for inhibiting tumor angiogenesis and improving the expression of MHC class I by tumor cells, thus promoting efficiency in the responses of CD8þ T cells. 23 In our studies, it was observed that γd T DC cells expressed IFN-γ in more significant quantities in healthy mice (p < 0.0001). In the study with a model of adoptive transfer of γd T cells in mice against melanoma B16-F0, it was observed that a specific subtype of γd T Vγ4 þ (but not Vγ þ T cells) had the protective function dependent on its high eomesodermin expression and IFN-γ production. 24 Even though IFN-γ is the main cytokine produced by mouse γd T cells, IL-17 is involved in the protective responses of γd T cells in some cancer models. 25 Interleukin-17-producing γd T cells cooperated in mediating bladder cancer regression. 26 In another study, IL-17-producing γd T cells are associated with chemotherapeutic agents (such as doxorubicin) in various models of epithelial tumor transplantation and demonstrated a better antitumor response. 27 In our study, it was identified that IL-17-producing γd T DC is less frequent in the breast cancer-induced group compared with IL17-producing T DC αβ (p < 0.0001). Thus, it can be inferred that, in a systemic antitumor response, these cells may be suppressed by tumor escape mechanisms to antitumor immune responses.
Therefore, according to the data found in the present study, we can conclude that γd T DC has immunological characteristics shared with conventional effector αβ T DC cells. The healthy mice engrafted with 4T1 breast tumor presented T DC with γd TCR repertoire. These cells express T helper and cytotoxic T lymphocyte molecules, producing antitumor proinflammatory cytokines, suggesting that γd T DC could have an antitumor role, and even be used in the future in antitumor immunotherapy. However, new studies investigating its function in other tumor types are necessary.

Contributors
All authors were involved in the design and interpretation of the analyses, contributed with the writing of the manuscript, read, and approved the final manuscript.

Funding
The present research was supported by the National Council for Scientific and Technological Development Rev Bras Ginecol Obstet Vol. 43 No. 5/2021 © 2021. Federação Brasileira de Ginecologia e Obstetrícia. All rights reserved.