Infiltrating CD4+ T cells attenuate chemotherapy sensitivity in prostate cancer via CCL5 signaling

Abstract Background Chemotherapy with Docetaxel (Doc) is efficient in a subset of prostate cancer (PCa) cases; however, most patients ultimately develop resistance to Docetaxel. The tumor immune microenvironment and secreted cytokines play a substantial role in development of resistance to chemotherapy. Our previous study has demonstrated that CD4+ T cells in prostate tumor microenvironment contribute to PCa progression; meanwhile, we found increased CD4+ T‐cell infiltration in tumor area after Doc treatment; however, their effects on PCa chemosensitivity remain unclear. Here, we aim to explore the role and mechanisms of CD4+ T cells in PCa chemotherapy sensitivity. Methods CD4+ T‐cell infiltration in Doc‐treated paraffin‐embedded specimens from transurethral resection of prostate, radical prostatectomy, or bone metastasis was detected by immunohistochemistry. The castration‐resistant PCa cell lines—C4‐2 and CWR22RV1, and CD4+ T‐cell lines—HH and Molt‐3 were used in the coculture system. After coculture with the lymphocytes, PCa cell chemosensitivity was detected by cell counting kit‐8, terminal deoxynucleotidyl transferase dUTP nick‐end labeling assays, and Western blot analysis. Various cell cytokines were determined by cytokine arrays and reverse‐transcription polymerase chain reaction. The recombinant human C‐C motif chemokine ligand 5 (CCL5) was added to PCa cells for further confirming its effects and anti‐CCL5 antibody was used for neutralization. S3I‐201, a signal transducer and activator of transcription 3 (STAT3) inhibitor, was added to the coculture system to detect STAT3 role in chemosensitivity. Tumor xenografts in nude mice were used for confirming effects of CD4+ T cells in vivo study. Results We found more infiltrated CD4+ T cells in human PCa lesions than in the adjacent noncancerous tissues after Doc treatment. In vitro cell line study confirmed that CD4+ T cells increase the PCa Doc resistance. Quantative polymerase chain reaction and cytokine arrays indicated that after coculture with PCa, CD4+ T cells could secrete large amounts of CCL5. Moreover, CCL5 stimulation enhanced PCa resistance to Doc, and anti‐CCL5 antibody could partly reverse this process. We found that CD4+ T cells could activate P‐STAT3 signaling via secreting CCL5 and adding a STAT3 inhibitor can reverse the chemoresistance. In vivo mouse model with xenografted 22RV1 cells and CD4+ T cells also confirmed the in vitro results. Conclusions Together, our results indicate that infiltrating CD4+ T cells could promote PCa chemotherapy resistance via modulation of the CCL5/STAT3 signaling pathway.

could secrete large amounts of CCL5. Moreover, CCL5 stimulation enhanced PCa resistance to Doc, and anti-CCL5 antibody could partly reverse this process.
We found that CD4+ T cells could activate P-STAT3 signaling via secreting CCL5 and adding a STAT3 inhibitor can reverse the chemoresistance. In vivo mouse model with xenografted 22RV1 cells and CD4+ T cells also confirmed the in vitro results.

Conclusions: Together, our results indicate that infiltrating CD4+ T cells could promote
PCa chemotherapy resistance via modulation of the CCL5/STAT3 signaling pathway. half of all patients do not respond to Doc and those who do eventually develop resistance to Doc within 24 months of initial exposure. 3,4 Resistance to Doc is poorly understood and may be caused by a number of mechanisms. These mechanisms may include androgen receptor (AR) signaling, activation of prosurvival pathways, and the acquisition of a cancer stem cell morphology. [5][6][7][8] Further, tumor immune microenvironment and overexpression of inflammation-associated molecules have an important role in the development of Doc resistance. 7,8 Among infiltrating immune cells, innate and adaptive immune cells were shown to significantly correlate with PCa aggressiveness. [9][10][11] Moreover, mast cells could enhance PCa resistance to chemotherapy and radiotherapy via activation of p38/p53/p21 and ATM protein kinase signals. 12 Similarly, cytokines from immune cells also affect chemotherapy resistance, such as interleukin 6 (IL6), IL8, CCL2, and transforming growth factor-β1. 8,13 T cells, especially CD4+ T cells, are an important part of the tumor immune inflammatory microenvironment. Accumulating evidence suggests that CD4+ T cells could contribute to a tumor immune evasion and tumor progression. 14,15 Our previous study has shown that CD4+ T cells in the prostate tumor microenvironment contribute to PCa progression, 10 and we found increased CD4+ T-cell infiltration in PCa tissue after Doc treatment. However, their effects on PCa chemosensitivity remain unclear. Here, we studied the role of infiltrating CD4+ T cells in PCa chemotherapy sensitivity.

| Patients
We recruited 15 patients whose prostate biopsies showed clinical evidence of PCa, and who received Doc treatment. These paraffinembedded specimens from radical prostatectomy, transurethral resection of prostate (TURP), or bone metastasis. Patients with CRPC received Doc treatment often show local progression and then suffer from urinary obstruction due to tumor growth. In these patients, transurethral resection of the tumor often helps them to regain normal voiding function. In our study, TURP specimens were also selected. Pathologically confirmed prostate carcinoma bone metastasis specimens also were obtained from patients that had undergone Doc CD4+ T-lymphocytic cell lines HH and Molt-3 were acquired from the American Type Culture Collection (Rockville, MD) and maintained in 10% heat-inactivated FBS, RPMI media with 1% pen/strep. All cell lines were cultured in a 5% CO 2 humidified incubator at 37℃.

| Western blot analysis
The expressions of specific genes were determined by Western blot analysis according to a previous study. 10   were processed according to the manufacturer's protocols and evaluated using the chemiluminescence system (Thermo Fisher Scientific).

| Immunohistochemistry
The prostate tumor samples from patients and mice were fixed in 4% neutral buffered paraformaldehyde and embedded in paraffin.

| Isolation of CD4+ T cells and flow cytometry
Human peripheral blood mononuclear cells (PBMCs) were isolated from the whole blood of healthy patients, using Ficoll-Paque

| In vivo subcutaneous implantation and Doc administration studies
Male nude mice (6-to 8-week-old) were used to create the animal model. In brief, 2 × 10 6 CWR22Rv1 cells with 2 × 10 5 HH cells mixed with Matrigel were injected subcutaneously in the right axillary region  F I G U R E 3 CCL5 is highly secreted from CD4+ T cells after coculture with PCa and plays a pivotal role in inducing PCa chemoresistance. A, Cytokine chip was used to screen high expression and specific cytokines; CCL5 was increased in cocultured conditioned medium compared to control medium. B, RT-PCR analysis was used to detect related cytokines, and the results showed that CCL5 was highly expressed in cocultured CD4+ T cells. Data are presented as mean ± SD, n = 3. *P < 0.05 vs control. CCL5, C-C motif chemokine ligand 5; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; PCa, prostate cancer; RT-PCR, real-time polymerase chain reaction [Color figure can be viewed at wileyonlinelibrary.com] F I G U R E 4 CCL5 is highly secreted from CD4+ T cells after coculture with PCa and plays a pivotal role in inducing PCa chemoresistance. A, B, C4-2 and 22RV1 cells were pretreated with recombination human CCL5 (100 ng/mL) 24 hours and then treated with 3 nM Doc for 48 hours. Cell viability was detected using CCK8, Data are presented as mean ± SD, n = 3. *P < 0.05 vs control. Western blot analysis was used to detect apoptosis-related proteins levels. Data are also presented as mean ± SD, n = 3. *P < 0.05 vs control. CCL5 stimulation enhances PCa Doc resistance. C-E, C4-2 and 22RV1 cells were pretreated with anti-CCL5 (1 μg/mL) and HH/Molt-3 cells 24 hours and then treated with 3 nM Doc for 48 hours. Cell viability was detected using CCK8, Data are presented as mean ± SD, n = 3. *P < 0.05 vs control. Tunel assay analysis was used to detected cell apoptosis. Western blot analysis was used to detect apoptosis-related proteins levels. Data are also presented as mean ± SD, n = 3. *P < 0.05 vs control. Anti-CCL5 could reverse the effects of CD4+ T cells on PCa chemoresistance. CCK8, cell counting kit-8; CCL5, C-C motif chemokine ligand 5; Doc, Docetaxel; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; PCa, prostate cancer; Tunel, terminal deoxynucleotidyl transferase dUTP nick-end labeling [Color figure can be viewed at wileyonlinelibrary.com] F I G U R E 5 CD4+ T cells induced chemoresistance of PCa via CCL5/P-STAT3 signaling. A, C4-2 and 22RV1 cells were treated with CCL5 for 48 hours. Western blot analysis shows that phosphorylation of STAT3 (Tyr705) is increased. Similarly, after coculture with HH cell for 48 hours, phosphorylation of STAT3 (Tyr705) was also increased. Data are also presented as mean ± SD, n = 3. *P < 0.05 vs control. B, Effect of P-STAT3 inhibitor treatment. C4-2 and 22Rv1 cells were pretreated with S3i-201(P-STAT3 inhibitor) for 24 hours and subsequently coculture with CD4+ T cells for an additional 24 hours, and then treated with 3 nM Docetaxel for 48 hours. Finally, the apoptosis-related protein levels were detected by Western blot analysis. The P-STAT3 inhibitor treatment can reverse CD4+ T-cell-induced chemoresistance of PCa. Data are also presented as mean ± SD, n = 3. *P < 0.05 vs control. And ns is no statistical difference. CCL5, C-C motif chemokine ligand 5; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; PCa, prostate cancer; STAT3, signal transducer and activator of transcription 3 deviation. Differences in mean values between each group were analyzed by the two-tailed Student test, and the mean values of more than two groups were compared by one-way analysis of variance.
P < 0.05 was considered statistically significant. All experiments were repeated three times.

| CD4+ T-cell infiltration in PCa patients with Doc treatment and altered PCa chemotherapy sensitivity
To examine the potential impact of CD4+ T cells on chemosensitivity on the neighboring PCa, we first compared CD4+ T-cell infiltration in PCa clinical specimens after chemotherapy using IHC staining. The results showed increased CD4+ T-cell infiltration in the tumor area than in adjacent noncancerous tissues in Doc-treated paraffinembedded specimens from PCa patients ( Figure 1A).
To further study the effect of CD4+ T cells on the chemosensitivity of neighboring PCa cells, we applied the coculture system.

Results showed that after coculture with CD4+ T cells (HH cells),
C4-2 and 22Rv1 cells became more resistant to Doc treatment in a dose-dependent manner ranging from 1 nM to 8 nM by CCK8 ( Figure 1B). The level of cleaved PARP, another indicator of apoptosis, was also decreased when coculture with HH cells ( Figure   1C). Furthermore, we indicated that CD4+ T cells inhibit Doc-induced apoptosis in C4-2 and CWR22RV1 cells through the Tunel assay ( Figure 1D). Similar results were also obtained when HH cells were replaced with another CD4+ T cell-Molt-3 cells (Figure 2A-C). Using CD4+ T cells, which from peripheral blood mononuclear cells also increase PCa cell chemotherapy resistance ( Figure S1).
Together, these results suggest that infiltrating CD4+ T cells could decrease PCa chemotherapy sensitivity to Doc.

| CCL5 is secreted from CD4+ T cells after coculture with PCa and plays a pivotal role in inducing PCa chemoresistance
To determine which factors influence acquired Doc resistance after coculture with PCa, we compared the expression of some cytokines related to chemoresistant cancer. Cytokine/chemokine assay was performed and found that CCL5 was significantly increased after F I G U R E 6 CD4+ T cells induced chemoresistance of PCa via CCL5/P-STAT3 signaling. A, B, Effect of P-STAT3 inhibitor treatment. Cell viability was detected using CCK8, Data are presented as mean ± SD, n = 3. *P < 0.05 vs control. and cell apoptosis was detected by Tunel assay. | 1027 coculture with CD4+ T cells compared with the control medium ( Figure 3A). Further, we found that CCL5 expression was increased in CD4+ T cells after coculture with PCa by qPCR ( Figure 3B).
To verify whether CCL5 is one of the key factors released from CD4+ T cells to increase neighboring PCa chemoresistance, we directly added CCL5 recombinant proteins into C4-2 and CWR22RV1 cells. C4-2 and CWR22RV1 cells were more resistant to Doc after CCL5 treatment compared with the control groups ( Figure 4A).
Furthermore, the level of cleaved PARP also decreased ( Figure 4B).
Moreover, a neutralizing antibody against CCL5 was added into the coculture system. The CCK8 assay, Tunel, and Western blot analysis indicated that neutralizing against CCL5 antibody could reverse the effects of CD4+ T cells on PCa chemoresistance ( Figure 4C-E). These results support that CCL5 stimulation enhances Doc resistance.
F I G U R E 7 CD4+ T cells enhance PCa cell chemotherapy resistance in an in vivo mouse study. Ten male nude mice were injected subcutaneously with 2 × 10 6 CWR22RV1 cells or 2 × 10 6 CWR22RV1 cocultured with 2 × 10 5 HH cells. After 2 weeks, the mice were treated with Docetaxel for three weeks and then killed. The tumor weight was assessed after Doc treatment for 4 weeks.
After Doc administration, CWR22Rv1 tumor volumes were two-fold larger in mice bearing CWR22Rv1 and HH cells than those in mice bearing CWR22Rv1 tumors alone ( Figure 7A-C).
To assess the role of CD4+ T cell-induced chemoresistance of PCa tissues, we detected the expression of molecules examined in vitro through IHC, such as Ki67, P-STAT3, cleaved PARP, and CCL5 in the xenograft tumors, and results were consistent with the findings in vitro ( Figure 7D). Moreover, similar results were obtained when we replace CWR22RV1 cells with C4-2 cells in vivo mouse study ( Figure S2). We also found the key cytokine-CCL5, also known as RANTES which is the most secreted factor by CD4+ T cells after cocultured with PCa cells. It is one of the members of the CC chemokine family of proteins, and upregulation of CCL5 can increase the aggressive potential of PCa cells and the size of PCa stem cell populations. [30][31][32][33] Moreover, it has also been verified that overexpression of CCL5 facilitates tumor progression 19,21 and can activate PI3K/Akt and STAT3 signaling pathway, which plays a vital role in tumor progression, adhesion, and drug resistance in breast and ovarian cancers. 17,18,20 CCL5, as previously shown, 32 directly increased PCa cell migration.

| DISCUSSION
To further study the mechanism of CD4+ T cell and the secreted CCL5 promoting PCa cells chemotherapy-resistant, we then focused on the STAT3 signaling pathway, a member of the STAT transcription factor family. In PCa, STAT3 activation correlates with Gleason score and pathological stage, 34,35 promotes tumor invasion and metastasis, 36 is involved in resistance to enzalutamide 37 and Doc. 38 Here, we found that CD4+ T cells might affect PCa chemosensitivity through CCL5 activation of STAT3 via upregulation of STAT3 phosphorylation.

| CONCLUSIONS
The most striking finding of the present study was that infiltrating CD4+ T cells could promote PCa chemotherapy resistance via modulation of the CCL5/STAT3 signaling pathway ( Figure 8). Therefore, STAT3