CD8+ T-Cell Number and Function are Altered by Shkbp1 Knockout Mediated Suppression of Tumor Growth in Mice

Xiaolan Guo Institute of Basic Medical Sciences, Guangdong Pharmaceutical University, China Haobin Li Institute of Basic Medical Sciences, Guangdong Pharmaceutical University, China Xiuqiong Meng Institute of Basic Medical Sciences, Guangdong Pharmaceutical University, China ZhiBin Zhao Guangdong Academy of Medical Sciences Rongxin Zhang Guangdong Pharmaceutical University Lijing Wang Institute of Basic Medical Sciences, Guangdong Pharmaceutical University, China Jiangchao Li (  lijiangchao@gdpu.edu.cn ) Guangdong Pharmaceutical University


Introduction
In recent years, many genes have been successfully used as targets in cancer treatment. Targeted therapy is an effective way to signi cantly improve the survival rate of certain cancer patients. Vascular endothelial growth factor (VEGF ) and epidermal growth factor receptor (EGFR ) promote tumor growth by unregulated vascular and tumor cell cycle respectively. Inhibition of VEGF and EGFR can effectively inhibit the growth of tumors, thereby improving the survival rate of patients, and has been successfully applied to clinical targeted therapy [1][2][3].
Tumor immunotherapy is another application of targeted therapy methods that target genes on tumor cells or immune cells, enhancing the immune response to the body. Moreover, immune cells and effectors enhance the host immune system, and the immune system is coordinated to kill tumor cells and inhibit tumor growth. T lymphocytes e ciently scan nearly all parts of the body, looking for unwanted or foreign bodies; thus, childhood and effector T cells are highly skilled cells, critical for the development of immune surveillance, and infection and cancer. The differentiation and phenotype of T cells are strictly regulated by transcription factors, cytokines, chemokines, integrins, and metabolic signals. Immune checkpoint inhibitors are designed to block dysfunctional T cells (including CD8 + T cells) by transferring T cells that use CD8 + genetically engineered receptors, blocking inhibitory immune receptor revitalization. Chimeric antigen receptor (CAR) -modi ed T-cell adoptive immunotherapy involves injection of T cells with speci c antitumor activity into tumor patients with low immune function and exerts an antitumor effect in vivo to achieve tumor treatment [4]. Therapeutic tumor vaccines stimulate immune responses after inoculation of patients with tumor speci c antigens [5]. Overall, the ultimate goal of treatment is to improve the killing capacity of CD8 + cells [6].
Cytotoxic CD8 + T cells of the adaptive immune system are the most potent effectors of the anticancer immune response and form the backbone of cancer immunotherapy [6]. CD8 + T cells are derived from bone marrow stem cells. Mature CD8 + T cells develop and migrate to peripheral lymphoid tissues after negative T-cell selection and positive T-cell selection in the thymus. During this process, due to noncontact antigen stimulation, CD8 + T cells are in a relatively quiescent state [7].The spleen is the largest peripheral immune organ. Naive CD8 + T cells reside in the central PALS of WP and await antigen presentation by APCs [8]. Once activated, CD8 + T cells respond to splenic infection with a well-de ned target range [9]. Upon initiation, activated cytotoxic T lymphocytes (CTLs) exit the MZ and WP in the RP through the sternal canal (BC) and ultimately clear the spleen of "peripheral" infection (RP). Subsequently, some CD8 + memory T cells from the WP return to the PALS, and some CD8 + memory T cells remain in the RP [10] − [11].
There are two ways for CD8 + T cells to exert antitumor immunity [12]. First, CTL-mediated direct killing requires cell-to-cell contact, usually caused by the release of cytolytic enzymes such as granzyme B.
Perforin released by CTLs generates holes in the target cell membrane, causing passive inward diffusion of granzyme B, and then inducing apoptosis of target cells. Second, in addition to the direct killing mechanism, CTLs indirectly induce the death of "next-door" tumor cells by secreting cytokines that act at a distance. For example, TNF-α secretion can induce apoptosis in adjacent tumor cells expressing TNF receptors. However, the inhibitory molecules PD-1, CTLA4, TIM-3, and LAG-3 on the surface of the CTL membrane stimulated by continuous antigen in tumor microenvironment gradually increase, and the production of cytokines promoting immune activity by TNF-α and IFN-γ is inhibited.
Knockout of one gene in mice, changes the CD8 + T-cell number or CD8 + function, which can identify speci c tumor antigens and kill tumors [13].
In our study, we observed the high expression of Shkbp1(Sh3kbp1 binding protein 1) in immune organs and immune cells. Shkbp1 belongs to the KCTD3 family [14], which widely exists in various species. The KCTD gene is involved in transcriptional repression, cytoskeletal regulation and other biological processes. These proteins play an important role in proliferation, differentiation, apoptosis and metabolism [15]. Shkbp1, also known as CIN85 binding protein, and SETA binding protein, has two proline-rich domains, namely PXXXPR, and is a signal adapter protein. The common structure of adaptor proteins includes three SH3 (Src homology 3) domains, one proline rich domain and one coiled-coil domain. Studies in osteosarcoma and glioma have shown that Shkbp1 can directly or indirectly participate in the development of tumors and inhibit the development of tumors [16,17]. Studies have also shown that Shkbp1 can inhibit degradation after activating EGFR, and increase the transcriptional level of downstream transcription factors after activating EGFR, thereby enhancing EGFR pathway activity [18,19].
In our study, we did not focus on Shkbp1 itself but on the relationship among Shkbp1 knockout, CD8 + cells and tumors. Using Shkbp1 knockout mice we explored the in uence of Shkbp1 on CD8 + immune function and tumor immunity changes in mice, which suppress tumor growth. Our study may provide new therapeutic targets for cancer immunotherapy. Knockout of this gene increased the antitumor function of CD8 + cells in tumor growth.

Cell culture
Mouse cutaneous melanoma cells (B16-F10 cells) were purchased from the Shanghai Institute of Cell Biology, Chinese Academy of Sciences. Cell lines were cultured in DMEM containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin. Cells were passaged before reaching 90% con uence by harvesting with 0.25% trypsin containing 1mM EDTA for 5 minutes at 37°C. Cell lines were con rmed to have no mycoplasma contamination by PCR.

Hematoxylin and eosin (H&E) staining
Spleen sections (4-6-µm thick) were dewaxed in xylene and rehydrated in 100%, 90%, 80%, and 70% ethanol and then in PBS. After hematoxylin staining, and rinsing with running water for half an hour, cell nuclei were stained blue. The sections were stained with eosin and washed with water to remove multiple dyes on glass slides. Dehydration of dyed slices was performed natural air drying and neutral gum sealing, and the slides were observed under a microscope.

Quantitative real-time RT-PCR (qPCR)
Total RNA was extracted from cells and spleen tissues using AG RNA ex Pro RNA (AG21101, Accurate Biology). Potential genomic DNA contaminants of RNA samples were eliminated by digesting with DNase I before the reverse transcription step.
Total RNA samples were reverse transcribed for complementary DNA (cDNA) with the Evo M-MLV Mix Kit with gDNA Clean for qPCR(AG11728, Accurate Biology). Real time PCR was performed using an SYBR Green Premix Pro Taq HS qPCR kit (AG11701, Accurate Biology) on a LightCycler 96 (Real-Time PCR Detection System, Roche). The data were normalized to the endogenous control GAPDH, and the fold change was calculated using relative quanti cation (2^-∆∆Ct).

Flow Cytometry
Single-cell suspensions of spleen cells and peripheral blood mononuclear cells (PBMCs) from mice were prepared. Sample cells were concentrated on centrifugation at 250 g for 5 min at 4°C and incubated in 2 ml of erythrocyte lysates (1:10, BD555899, Biolegend). The cells were washed in phosphate buffered saline (PBS) prior to staining for ow cytometry. The cells were then labeled with dead cells (Live/Dead kit, Invitrogen) to gate out the dead cells and stained with antibodies speci c to anti-CD45 antibody (103108, Biolegend), PerCP/Cy5.5 anti-mouse CD3 antibody (100218, Biolegend), PE anti-mouse CD4 antibody (100408, Biolegend), BV421 anti-mouse CD8a antibody (100738, Biolegend). The samples were incubated for 30 min at room temperature. Cells were xed in 4% formaldehyde and washed twice with PBS and acquired via ow cytometry within 24 hours. All ow cytometry experiments were performed on an LSRII ow cytometer (BD Biosciences).

Western Blot
A total of 60-80 mg spleen tissues was lysed with 500 µL of RIPA lysate containing 1% PMSF protease inhibitor, and placed on ice. The lysates were clari ed by centrifugation at 10,000 × g for 10 min at 4°C and taken the supernatant as a protein sample. Equal amounts of protein were separated by 8% SDS-PAGE and transferred to PVDF membranes. The membranes were cut to the size of the protein of interest and blocked in PBS, 0.1% Tween 20 with 5% nonfat dry milk for 1 hour at room temperature on a shaker and then incubated with primary antibodies in 5% BSA overnight at 4°C with rocking. Antibodies against Shkbp1 (sc169308, Santa Cruz), CD4 (ab183685, Abcam), CD8a (ab203035, Abcam), GAPDH (sc-32233, Santa Cruz) were used.

Lentiviral Infection
The lentiviruses used in this study were purchased from Gima Gene Company. Three Shkbp1 sequences targeting mice were designed and constructed, and the lentivirus sequences are shown in the Supplemental Table. The effectiveness of the virus was veri ed and the virus infected mice with the best interference e ciency were selected. B16-F10 cells in good condition were plated in six-well plates with 1x10 6 cells per well. Viruses were added to each well according to the ratio of virus: nal volume = 1: 50. Three days after infection, the status of viral interference was observed under a microscope, RNA was extracted from infected cells, and its knockdown e ciency was detected.

ELISA for Perforin and Granzyme B
The reagent and sample were restored to room temperature before detection. Standard sample wells had different concentrations of standard (50 µl), blank wells did not have sample added, and samples were added to the sample wells (50 µl). HRP-labeled antibody (100 µl) was added to the blank wells. After 60 minutes at 37°C, the plate was washed, and100 µl of substrate mixture was added. After incubation at 37°C for 15min, 50 µl of termination liquid was added to the plate, and the absorbance value (OD) was read on a microplate reader.

Xenograft model
Six-week-old Shkbp1 −/− mice and C57BL/6J mice (Shkbp1 +/+ mice) were used; the tumor site was depilated in advance to facilitate the injection of the tumor cell suspension and subsequent tumor observation and measurement. B16-F10 cells were predigested, counted and diluted with sterile 1x PBS to a single-cell suspension at a concentration of 5x10 5 cells/ml and then injected subcutaneously with 200 µL of cell suspension. The body weight of each mouse was recorded by Vernier calipers every day, and tumor volume was obtained by the following formula: V = 4/3Π*a*b 2 , where "a" is the longest diameter of tumor and "b" is the shortest diameter.

Statistical analysis
Unpaired t tests were performed on the statistical data using GraphPad Prism 9 software, p value of less than 0.05 was considered statistically signi cant.

The proportion of white pulp in the spleen of Shkbp1 knockout mice is changed
To determine the role of the Shkbp1 gene in tumor progression and development, we used a tumor database, which showed that the expression of Shkbp1 in various types of tumor tissues was signi cantly higher than that in adjacent normal tissues (Supplemental Fig. 1A), our previously published data showed that Shkbp1 affected not only tumor cells but also CD8 + T cell [19]. Shkbp1 was expressed in immune organs and cells (Supplemental Fig. 1B-C). Shkbp1 was highly expressed at RNA and protein levels in the spleen and lymph nodes (Supplemental Fig. 1D) and expressed in myeloid cells and αβT cells (Supplemental Fig. 1E). Compared with the wild type mice, the Shkbp1 knockout mice showed a normal weight and normal offspring. The spleen is the largest secondary lymphoid organ and an important organ for initiating systemic immune responses to pathogens [20]. In the Shkbp1 −/− mice and the Shkbp1 +/+ mice, there was no signi cant difference in the size and length of the spleen, and the spleen weight and index (the ratio between the weight of the spleen in milligrams relative to the total body weight in grams [21]) signi cantly increased (*P < 0.05, Fig. 1A). The ratio of splenic weight to body weight was reported to remain fairly constant regardless of age and, in rats, is typically approximately 0.2% [22]. Next, we used histological methods to analyze spleen tissue sections with hematoxylin and eosin (H&E) staining and viewed them under an optical microscope. Compared with that of the Shkbp1 +/+ mice, the spleen tissue structure of the Shkbp1 −/− mice was dramatically changed. The white pulp area was irregular in shape and size, and the distribution was scattered and irregular, with white pulp fusion (Fig. 1B). The ratio of white pulp to red pulp, the percentage of white pulp and the percentage of red pulp were quanti ed using ImageJ software. The ratio of white pulp and red pulp and the percentage of splenic white pulp in total area of the Shkbp1 −/− mice signi cantly increased, while the percentage of red pulp decreased (*P < 0.05) (Fig. 1C-E).
We also performed routine blood tests of the Shkbp1 −/− mice and the Shkbp1 +/+ mice to observe the effect of Shkbp1 knockout on all mouse blood parameters. The results showed that the number of eosinophils (EOs) in peripheral blood of the Shkbp1 −/− mice was signi cantly increased (****P < 0.0001), and further study of EO was not performed here. There were no signi cant changes in the number of white blood cells (WBCs), neutrophil (NEUTs), lymphocytes (LYMPHs) and monocyte (MONOs) in the Shkbp1 −/− mice (Supplemental Fig. 2A).

Knockout of Shkbp1 causes an unbalance of T-cell subtypes
The above results showed that Shkbp1 knockout signi cantly affected the structure of the mouse spleen. The spleen as the largest peripheral immune organ, is an important site for immune response. Therefore, we performed mRNA transcriptome sequencing in the spleens of the Shkbp1 −/− mice and the Shkbp1 +/+ mice ( Fig. 2A). Sequencing data were subjected to GO enrichment analysis and KEGG pathway analysis. Shkbp1 knockout affected many genes related to the immune system and cell cycle (Fig. 2B). T cells are divided into αβ T cells and γ δ T cells according to their TCR expression [23].Bioinformatics analysis showed that Shkbp1 was highly expressed in αβT cells. αβT cells are mainly CD3 + T cells, which can be divided into CD4 + T cells and CD8 + T cells. The spleen mRNA sequence analysis of the Shkbp1 −/− mice and the Shkbp1 +/+ mice showed that the mRNA levels of CD3, CD4 and CD8 increased after Shkbp1 knockout (Fig. 2C). When activated by antigen-presenting cells (APCs), naive CD4 + T cells can differentiate into functionally distinct T-cell lineages, including Th1, Th2, Th17, and regulatory T (Treg) cells [24]. Therefore, the corresponding factors of T-cell subsets were analyzed, and the results showed that the secreted factor IL-21 in the spleen of the Shkbp1 −/− mice increased signi cantly (Fig. 2D-H). In addition, cell surface molecules and effectors corresponding to CD8 + T-cell subsets were analyzed, and the mRNA expression of CD8 + T-cell-related genes in the spleen of the Shkbp1 −/− mice was increased, among them, the chemokines CCL3 and CCL4 were increased signi cantly (Fig. 2I). The T-cell subtype biomarkers of central memory, effector memory, and effector related genes were all upregulated (Fig. 2J).
According to the GO enrichment analysis and KEGG pathway analysis of the spleen mRNA sequences of mice, Shkbp1 knockout affected the cell cycle and immune system. The killer cell secretory factor was upregulated (Fig. 2K), the expression of the CD8 + T-cell failure-related indicators PD1 and LAG3 was upregulated (Fig. 2L), and the expression of CD69 and CD25, which are related to CD8 + T-cell activation was also upregulated (Fig. 2M). TCR signaling downstream factors (Fig. 2N), including RCAN1, RCAN2, PPP3CA, and PPP3CB, were upregulated. Genes related to CD8 + T-cell proliferation and activation (Fig.  2O), such as ARG2, SH3RF1, and FADD, were also upregulated.

The number of CD8 + T-cells increased in the Shkbp1 knockout mice
The above results suggested that Shkbp1 knockout affects T-cell subsets in which the mRNA expression of related genes is altered. In all T-cell subtypes, CD8 + T cells play a key protective role in antitumor immune response [25]. Therefore, we further investigated whether Shkbp1 knockout affects CD8 + T cells. We detected the expression of CD4 + T cells and CD8 + T cells in spleen by immunohistochemical staining. The results showed that the numbers of CD4 + T cells and CD8 + T cells with positive expression in the spleen of the Shkbp1 −/− mice were increased signi cantly (*P < 0.05) (Fig. 3A). In addition, the number of CD8 + T cells with positive expression in the spleen of the Shkbp1 −/− mice was increased in both white pulp and red pulp (Fig. 3B). The expression of CD8 + T-cell proteins was detected by Western Blotting (Fig. 3C). As expected, the expression of CD8 proteins in the spleen of the Shkbp1 −/− mice increased (*P < 0.05).

Shkbp1 knockout increases the effector function of CD8 + T cells in mice with tumors
Cytotoxic T lymphocytes (CTLs), usually called CD8 + T cells, are the key components of the adaptive immune system, a key target of immunotherapeutic approaches and important mediators for protection against viral infection and immunity to malignant tumors [26]. Naive CD8 + T cells differentiate and become activated into effector CD8 + T cells after antigen-recognition and antigen-presenting cell (APC) co-stimulated [27]. Activated CD8 + T cells rapidly express effector molecules, such as perforin, granzyme B,IFN-γ and TNF-α[28].
To explore whether Shkbp1 knockout affects the function of CD8 + T cells, we tested spleen RNA from the Shkbp1 +/+ mice and Shkbp1 −/− mice, and spleen RNA from the Shkbp1 +/+ mice and Shkbp1 −/− mice by RT-qPCR. The results showed that perforin, granzyme B, TNF-α were increased signi cantly (*P < 0.05) under physiological conditions (Fig. 4A). There was a trend of increasing IFN-γ expression in Shkbp1 knockout mouse spleens. In mice with subcutaneous tumor, the RNA expression levels of caspase3, granzyme B, perforin and INF-γ in the spleen of the Shkbp1 knockout mice were increased signi cantly (*P < 0.05). During antigen stimulation and activation of CD8 + T cells, T-cell depletion may occur, manifested as upregulation of the expression of coinhibitory molecules (such as programmed cell death protein 1 (PD-1), cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4), T-cell immunoglobulin domain and mucin domain-3(TIM-3) and lymphocyte activating gene-3(LAG-3). Here, the RNA levels of the PD-1, CTLA-4 and TIM3 were elevated in the spleens of the Shkbp1 knockout mice with subcutaneous tumors (Fig. 4B).
For further exploration, the serum of the Shkbp1 +/+ mice, Shkbp1 −/− mice, Shkbp1 +/+ tumor-bearing mice and Shkbp1 −/− tumor-bearing mice was collected from the four groups. ELISAs were performed to detect the levels of perforin and granzyme B in the serum of each group. Compared with those of the Shkbp1 +/+ mice, the levels of perforin (*P < 0.05) and granzyme B (*P < 0.05) in the serum of the Shkbp1 −/ − mice were signi cantly increased (Fig. 4C). In subcutaneous tumor models, the levels of perforin and granzyme B (**P < 0.01) the serum of the tumor-bearing Shkbp1 −/− mice were signi cantly increased.

Shkbp1 knockout would inhibits tumor growth
The above results suggested that Shkbp1 knockout can signi cantly increase the number and function of CD8 + T cells in the mouse spleen. We further explored whether Shkbp1 knockout regulates T-cell function and is involved in the regulation of tumor immunity. Shkbp1 +/+ mice and Shkbp1 −/− mice bearing tumors were established with subcutaneous xenograft models. Compared with that of th Shkbp1 +/+ mice, the tumor volume of the Shkbp1 −/− mice was signi cantly reduced( Fig. 5A-B) (**P < 0.01) and the ratio of tumor weight to body weight was signi cantly reduced( Fig. 5A) (*P < 0.05).The Shkbp1 −/− mice subcutaneous xenograft model showed that Shkbp1 knockout can inhibit the growth of B16-F10 melanoma, suggesting that Shkbp1 knockout had involved in the regulation of tumor immunity and inhibits tumor growth by regulating T-cell function.
To investigate whether Shkbp1 knockout affected the in ltration of T cells into melanoma tumors, we performed immunohistochemical staining on para n sections of tumor and spleen tissues. We assessed the spleen, tumor periphery and tumor interior, and compared with that of the Shkbp1 +/+ tumor-bearing mice, the expression of CD8 + cells in the Shkbp1 −/− mice were signi cantly increased (Fig. 5D). Moreover, the ow cytometry results showed that the proportion of CD4 + T cells and CD8 + T cells in spleens of the Shkbp1 −/− tumor-bearing mice increased signi cantly (Fig. 5C).

Lentiviral interference with Shkbp1 suppresses the growth of melanoma cells
The above results showed that Shkbp1 knockout inhibited tumor growth and CD8 + number in tumor tissue. We wanted to obtain Shkbp1 inhibitor, but there was no Shkbp1 inhibitor available for purchase.
We investigated whether in vitro lentivirus interference could inhibit tumor growth. Further development of drugs targeting Shkbp1 is important. Therefore, we designed and synthesized three kinds of interference lentiviruses targeting mouse Shkbp1. The e ciency of lentivirus infection was veri ed by RT-qPCR, and the results showed that the SH153 lentivirus had the best e ciency (Fig. 6A). The SH153 lentivirus was subsequently used to treat tumor-bearing mice. Shkbp1 +/+ mice aged 6-8 weeks were selected. The activity status, weight change and tumor volume of the mice were observed and recorded every day after tumor loading. From the sixth day after tumor injection, Shkbp1 lentivirus (SH-153) at 1x10 7 titer/100 µl was injected intraperitoneally every two days, and the control group was injected with 1x PBS every two days. Sampling was performed after 12 days of treatment.
Compared with those of the control group, the general size of the tumor (Fig. 6B) and the tumor volume of the group injected with SH153 were signi cantly reduced (Fig. 6C). Similarly, tumor volume and weight were signi cantly reduced (Fig. 6D-F) (*P < 0.05). However, compared with the PBS-treated group, the lentivirus treatment group showed a signi cant increase in spleen weight (Fig. 6G-I) (**P < 0.01).
Further analysis by immunohistochemical staining, showed that compared with those of the control group, CD8 + T cells in the spleen and tumor interior were signi cantly increased (Fig. 6J) (*P < 0.05) in the lentivirus treatment group. The percentage of CD8 + T cells increased in the lentivirus-treated group by ow analysis (Fig. 6K).

Discussion
Our previously published data demonstrated that Shkbp1 expressed in tumor cells and that Shkbp1 knockout in mice can affect CD8 + cells in tumor tissues. Shkbp1 has been proven to play a role in promoting tumor development by protecting EGFR from degradation in the process of tumor development [16,29],which was con rmed in our previously published paper [19]. However, we further con rmed that Shkbp1 was highly expressed in tumor tissues and that immune cells in the tumor microenvironment were also related with Shkbp1. Shkbp1 knockout affects immune cell function in tumors, CD8 + number and antitumor function.
The expression of Shkbp1 in the spleen is very high, and the spleen is the main reaction site of the immune response. Moreover, Shkbp1 was the most highly expressed in αβT cells. αβT cells, which account for more than 95% of the total T cells, are the main mediator of cellular immunity in adaptive immunity, and show speci city, diversity, and memory. The above results indicate that this gene is highly likely to affect immune system development. The expression of Shkbp1 on αβT cells is higher than that of other immune cells, suggesting that there may be an intrinsic mechanism between Shkbp1 and immune cells, which may change immune function by affecting immune cells or in immune cell development.
Interestingly, eosinophils (EOs) are increased and are mostly elevated in diseases such as parasitic infections [30,31]. Generally, EOs are mainly concentrated in the intestine, thymus, bone marrow and adipose tissue. Shkbp1 knockout leads to a signi cant increase in EOs, which may be because Shkbp1 can inhibit the synthesis of EOs, or hinder the transfer of EOs from the bone marrow circulation pool to blood. More work should be performed to investigate this issue in the future The spleen consists of three different interrelated areas: the red pulp, the white pulp, and the marginal zone, each of which performs different functions [32].The WP consists of a periarterial lymphatic sheath (PALS), a column consisting of a circular network (CFR) and a central artery extending along its axis, and lymphoid follicles (LFs). The PALS consists of T lymphocytes, macrophages, and interdigital cells. This zone contains a dense population of T cells called the T-cell zone (TCZ). When an immune response occurs, the region splits and proliferates, and T cells proliferate. The LF, also called the spleen node, is located next to the PALS. The top of the lid faces toward the B-cell-rich red pith, hence the name B-cell zone (BCZ) [33]. Compared with that of the Shkbp1 +/+ mice, the white pulp of the Shkbp1 −/− mice was uneven in shape and size, had an unclear boundary, and was accompanied by two or more white pulp fusions. Compared with the Shkbp1 +/+ mice, the Shkbp1 −/− mice had an increased proportion of total white pulp area. The results suggested that the proportion of white pulp in the spleen of the Shkbp1 −/− mice increased, the number of T cells increased, and their antitumor immune function was enhanced, but further studies are needed to explore this mechanism.
Sequencing data showed that the expression levels of CD3, CD4 and CD8 were increased after the Shkbp1 knockout, suggesting that Shkbp1 can regulate the expression of CD3, CD4 and CD8 in T cells. Then, we analyzed ve subsets of CD4 + T cells, namely Th1, Th2, Th17, Tfh and Treg cells [34], and three different states of CD8 + T cells, namely central memory, effector memory, effector. The difference between the expression of IL-21 and secreted IL-5 was the most signi cant. The expression of IL-5 in the spleen decreased signi cantly. IL-5 is an important stimulating factor of eosinophils, which can promote the transfer of mature eosinophils from bone marrow to blood. Eosinophils in the blood are signi cantly increased. Therefore, we hypothesize that Shkbp1 is inevitably related to IL-5. The sequencing analysis showed that IL-5 is signi cantly decreased, suggesting that Shkbp1 may affect the distribution of IL-5, or that Shkbp1 can directly inhibit the synthesis of EOs. When synthesis of Shkbp1 knockout Eos increased signi cantly, the total content also increased. IL-21 is usually produced by CD4 + T cells, and is involved in immune processes and in ammatory reactions, which promote the proliferation of T cells. More importantly, this molecule can promote the ampli cation and activation of CD8 + T cells. IL-21 can also regulate the proliferation, differentiation, and apoptosis of B cells. The increase of IL-21 in the spleen may promote the proliferation of T cells, activate CD8 + T cells and strengthen the immune ability of the body [35]. Some genes were upregulated and some downregulated, while almost all the indicators of CD8 + T cells were upregulated. We concluded that Shkbp1 is closely related to CD8 + T cells, among which CCL3, CCL4 and CCL5 showed obvious changes. These small molecules can induce cells to move speci c sites, induce the homing of lymphocytes, in ltrate swollen immune cells and play an immune role [36]. Increased levels of IFN-γ and TNF-α can activate T-cell immunity and kill tumor cells, suggesting that Shkbp1 knockout enhances the immune function of CD8 + T cells [37].
Recently, popular immunosuppressive checkpoints were shown to inhibit tumor growth and improve patient survival after screening of effective immunosuppressive targets and administration of appropriate drug. The common immunosuppressive targets are PD-1, CTLA4, LAG3 and TIM3 [38]. The results showed that the expression levels of these four genes were all increased, suggesting that CD8 + T cells might be in a state of functional exhaustion after Shkbp1 knockout. Similarly, the more inhibitory targets for CD8 + T cells, the easier it is to bind to the matched inhibitory receptors on tumor cells during tumor immunity, achieving immune escape. We also detected important molecules downstream of T-cell antigen receptor (TCR) signal, such as RCAN1, RCAN2, PPP3CA, PPP3CB. After Shkbp1 knockout, most of the downstream molecules were upregulated, indicating that Shkbp1 knockout can strengthen the reactivity of TCRs and increase TCR molecules downstream. thereby strengthening the immune response and enhancing the immune ability [39,40].The expression changes of genes related to proliferation and activation of multiple T cells, such as ARG2, SH3RF1 and FADD. most of them were signi cantly increased, and the increase in the expression of activated genes indicated that T cells were more sensitive to immune responses [41].The above results suggested that Shkbp1 knockout affected the expression of immune system-related genes in the spleen, and could increase CD3, CD4 and CD8 in the spleen, improve the expression of TCR downstream molecules and strengthen the proliferation and activation of T cells. T cells showed a more rapid response to the immune response.
In normal mouse spleens, CD4 expression was low and was mainly concentrated in the white pulp. We found that CD4 + T cells were mainly concentrated on the lymphatic sheath around the artery under microscope observation. Compared with the Shkbp1 +/+ mice, the Shkbp1 −/− mice expressed more CD4 + T cells in spleen, which was consistent with sequencing results. Naive CD8 + T cells remain in the WP central PALS, awaiting antigen submission of APCs[8].Upon initiation, activated cytotoxic T lymphocytes (CTLs) leave the WP through the sternum canal (BC) to reach the MZ and RP. This process helps to eliminate infection [9].Compared with that of the Shkbp1 +/+ mice, the expression of CD8 + T cells in spleen of the Shkbp1 −/− mice increased both in white pulp and red pulp. Then, Western blotting was used to con rm whether Shkbp1 knockout increased the CD4 + T cells and CD8 + T cells, thereby enhancing immunity. The results were as expected.
We further investigated whether Shkbp1 can affect tumor immunity, and whether Shkbp1 knockout can successfully inhibit tumor growth. We constructed B16-F10 subcutaneous melanoma mouse model. The results showed that the tumor volume of the Shkbp1 −/− mice was smaller, the tumor weight was lighter, and the Shkbp1 knockout inhibited tumor growth. IHC was used to visually observe the distribution and location of the target protein, while ow cytometry was used to count the number of cells that showed no difference in CD8+. More experiments are needed to con rm our ndings.
Therefore, further experiments were needed to verify whether Shkbp1 can affect the proliferation of T cells. PD1 expression did not differ between the Shkbp1 KO and WT groups, but under tumor growth conditions, the expression of PD1 in the spleen increased, suggesting that CD8 + T cells are in a state of depletion [42].
GZMB is the main secreted factor by which CD8 + T cells exert their killing ability. Both CTLs and NK cells can be secreted and expressed, and both functional cells that can directly contact antigens and have a killing function. GZMB generally acts together with perforin [43]. qPCR data showed that the expression of GZMB increased after Shkbp1 knockout, and its ability to kill tumor cells was improved. In addition, IHC revealed CD8 + T cells in tumor tissue. The in ltration of CD8 + T cells in Shkbp1 +/+ mice was increased in tumor peripheral tissues. The Shkbp1 −/− mice had a higher degree of in ltration of and CD8 + T cells.
Shkbp1 −/− mice had a smaller tumor size, lighter tumor weight, and higher T-cell expression and in ltration within the tumor, all of which suggest that enhanced immunity present in Shkbp1 knockout mice. Therefore, to further clarify the function of tumor-in ltrating T cells when Shkbp1 knocked out, we need to design more experiments. According to all of the above results, Shkbp1 knockout can enhance the tumor immunity of the body Finally, we further explored drugs targeting Shkbp1, using SH153 adenovirus as a therapeutic agent to treat tumor-bearing mice. The spleen weight of the virus group was signi cantly decreased, and RNA detection was performed on the spleen. The results showed that SH153 successfully reduced the expression of Shkbp1 in the spleen. Whether it was suitable for tumor immunity was still an unknown.
However, the potential immune regulation CD8 + T cell is increased in the Shkbp1 knockout mice, although the mechanism remains unknown. This study shown that Shkbp1 affects CD8 + T cell number and function, which increases antitumor immunity, and suppresses tumor growth.
In conclusion, Shkbp1 knockout increased the number and function of CD4 + T cells and CD8 + T cells, thereby suggesting that Shkbp1 may be an effective target for increasing CD8 + antitumor activity. Figure 1 The proportion of white pulp in spleen of Shkbp1 knockout mice was changed A: The spleen weight and the spleen index (the ratio between the weight of the spleen in milligram relative to the total body weight in grams) B:To observe the structure of spleen, the spleen tissues were stained with H&E staining. H&E staining results ( 4X, 10X) C-E: Proportion of white pulp and red pulp , Ratio of white pulp and red pulp