CD4+ and CD8+ T cells have opposing roles in breast cancer progression and outcome

The Cancer Immunoediting concept has provided critical insights suggesting dual functions of immune system during the cancer initiation and development. However, the dynamics and roles of CD4⁺ and CD8⁺ T cells in the pathogenesis of breast cancer remain unclear. Here we utilized two murine breast cancer models (4T1 and E0771) and demonstrated that both CD4⁺ and CD8⁺ T cells were increased and involved in immune responses, but with distinct dynamic trends in breast cancer development. In addition to cell number increases, CD4⁺ T cells changed their dominant subsets from Th1 in the early stages to Treg and Th17 cells in the late stages of the cancer progression. We also analyzed CD4⁺ and CD8⁺ T cell infiltration in primary breast cancer tissues from cancer patients. We observed that CD8⁺ T cells are the key effector cell population mediating effective anti-tumor immunity resulting in better clinical outcomes. In contrast, intra-tumoral CD4⁺ T cells have negative prognostic effects on breast cancer patient outcomes. These studies indicate that CD4⁺ and CD8⁺ T cells have opposing roles in breast cancer progression and outcomes, which provides new insights relevant for the development of effective cancer immunotherapeutic approaches.     

Levels of circulating regulatory CD4+CD25+ T cells are decreased in breast cancer patients after vaccination with a HER2/neu peptide (E75) and GM-CSF vaccine

SummaryPurpose We are conducting clinical trials in breast cancer (BrCa) patients to test the HER2/neu peptide vaccine (E75). We have investigated the impact of this vaccine on circulating levels of regulatory T cells (T_reg) and the resulting effects on antitumor responses.Experimental design Twenty-two blood samples from healthy individuals and from 22 BrCa patients including pre- and post-vaccination samples from seven vaccinated HLA-A2⁺ patients were stained for CD4, CD25, and CD69 as well as CD8 and E75:HLA-A2 Ig dimer and quantified by flow cytometry. Cytotoxic activity against HER2/neu ⁺ tumors was measured by ⁵¹Cr-release. Serum from BrCa patients and normal subjects were analyzed for TGF-β levels.Results BrCa patients have a greater percentage of circulating T_reg (CD4⁺CD25⁺, 4.45% versus 2.96%; p = 0.007) than normal subjects. HLA-A2⁺ BrCa patients had more T_reg compared to the HLA-A2^– BrCa patients (CD4⁺CD25⁺, 5.63% versus 3.28%; p = 0.001). E75 vaccination increased circulating activated CD4⁺ T cells post-vaccination (CD4⁺CD69⁺, 1.23 versus 3.81%; p = 0.03). However, T_reg were significantly reduced after vaccination (CD4⁺CD25⁺, 5.31–1.81%; p < 0.0001). Furthermore, activated T_reg also decreased (CD4⁺CD25⁺CD69⁺, 0.23% versus 0.08%; p = 0.06). Importantly, post-vaccination decreases in T_reg were temporally associated with increased E75 vaccine-specific CD8⁺ T cells and corresponding HER2/neu ⁺ tumor cytotoxicity. Serum TGF-β levels were significantly elevated in BrCa patients compared to normals (3548 pg/ml versus 1007 pg/ml; p = 0.007). Four of seven vaccinated patients showed decreased serum TGF-β levels post-vaccination.Conclusions T_reg, are increased in BrCa patients along with serum levels of TGF-β. E75 vaccination resulted in CD4⁺ recruitment but was associated with a significant decrease in circulating T_reg and TGF-β levels in the majority of the vaccinated patients. Successful cancer vaccination strategies may require the alteration of complex immune interactions.The opinions or assertions contained herein are the private views of the authors and are not to be construed as official or reflecting the views of the Department of the Army or the Department of Defense     

Mobilized CD34+ cells as a biomarker candidate for the efficacy of combined maximal tolerance dose and continuous infusional chemotherapy and G-CSF surge in gastric cancer

We investigated whether the level of bone marrow-derived progenitor cells and mature endothelial cells could be used as predictors of clinical outcome in patients receiving taxotere-based chemotherapy for advanced gastric cancer. Peripheral blood mononuclear cells were obtained from 49 gastric cancer patients who received taxotere combined with 5-FU and leucovorin and prophylactic G-CSF treatment. To categorize the cells, the cell markers CD34, vWF, P1H12, and CD31 were stained. Changes in these cells were examined before and after chemotherapy, and the clinical significance of these changes to response prediction and prognosis were investigated. Before the second cycle of chemotherapy, the number of CD34⁺/vWF⁺ and CD34⁺ cells was higher in non-responders as compared to the responders. Patients with 6.2 CD34⁺/vWF⁺ cells/ml had a shorter progression free survival (3.7 months) as against patients with <6.2 CD34⁺/vWF⁺/ml (6.0 months, p = 0.076). Patients with 5.8 CD34⁺ cells/ml had shorter progression free survival (4.0 months) than patients with <5.8 CD34⁺ cells/ml (6.1 months, p = 0.046). In an ex vivo pharmacokinetic study, the maximum inhibition (I_max) for HUVEC and YCC3 cells was 13.0 ± 6.6% and 74.0 ± 2.0%, respectively. The time to reach I_max (T_max) was 72 h in all HUVEC cells and 0.5 hours in YCC3 cells. We suggested that CD34⁺/vWF⁺ and CD34⁺ cells can be used as a biomarker for prediction and CD34⁺ cells for prognosis.     

Expression of caspase-3 in cord blood CD34<Superscript>+</Superscript> cells during culture in vitro

Objective: To investigate the expression and significance of caspase-3 protein in CD34⁺ cells from cord blood (CB) during culturein vitro with different growth factors.Methods: RT-PCR, Western blot and flow cytometry techniques were used to detect the expression of caspase-3 in CD34⁺ CB cells during culturein vitro. Results: Caspase-3 mRNA was constitutively expressed at a low level in freshly isolated CD34⁺ cells. The expression of caspase-3 mRNA and protein was upregulated when these cells were first expanded in suspension culture with growth factors for 3 days. However, only the 32 kDa inactive caspase-3 proenzyme was detected in the freshly isolated CD34⁺ cells as well as during the first 3 days expansion with cytokines. With longer culture timein vitro, especially in the presence of the combination of IL-3, IL-6 and GM-CSF, caspase-3 was activated and a cleavage product of 20 kDa became detectable.Conclusion: Caspase-3 is involved in apoptosis of primitive CB CD34⁺ cells during expansionin vitro. The study was supported by a grant from the National Natural Science Foundation of China (No. 39928010)     

Expansion of CD3+CD8+PD1+ T lymphocytes and TCR repertoire diversity predict clinical responses to adoptive cell therapy in advanced gastric cancer

The adoptive cell therapy (ACT) and delivery of ex vivo activated cellular products, such as dendritic cells (DCs), NK cells, and T cells, have shown promise for the treatment of gastric cancer (GC). However, it is unknown which cells can improve patient survival. This study was focused on the antitumour activity of a subset of these cellular products and their relationships with clinical outcomes. Nineteen patients were enrolled at the Capital Medical University Cancer Center, Beijing Shijitan Hospital, from June 1, 2013, to May 30, 2016. CD8⁺PD1⁺ T-cell sorting was carried out using flow cytometry, and the T-cell receptor (TCR) repertoire during ex vivo expansion for 15 days was analyzed by next-generation sequencing. After 15 days of culture, the number of CD8⁺ T cells had increased significantly, and the number of CD4⁺ T cells had increased correspondingly. After ex vivo expansion, CD8⁺ T cells exhibited significantly enhanced expression of PD-1, LAG-3, and TIM-3 but not 4-1BB. Survival analysis showed that patients with a pro/pre value of CD8⁺PD-1⁺ T cells >2.4 had significantly favorable overall survival (OS) (median OS time, 248 days versus 96 days, P=0.02) and progression-free survival (PFS) (median PFS time, 183 days vs. 77 days, P=0.002). The sorted CD8⁺PD-1⁺ T cells displayed enhanced antitumor activity and increased IFN-γ secretion after coculture with autologous tumor cell lines. TCR repertoire diversity was decreased after ex vivo expansion, which decreased the Shannon index and increased the clonality value. The prognosis of patients was significantly improved and was associated with the extent of CD8⁺PD-1⁺ T-cell expansion. In summary, this study showed that after ex vivo expansion for 15 days, CD8⁺PD-1⁺ T cells could be identified as tumor-reactive cells in patients treated for GC. Changing TCR species can predict the extent of CD3⁺CD8⁺PD1⁺ T-cell growth and the effect of ACT treatment.     

Effect of Homoharringtonine on Bone Morrow CD34^＋CD7^＋ Cells in Chronic Granulocytic Leukemia

Objective： To explore the effect of homoharringtonine （HHT） on bone morrow CD34^＋CD7^＋ cells in chronic granulocytic leukemia （CGL）. Methods： The changes of bone morrow CD34^＋CD7^＋ cells were observed after the treatment of HHT in 23 cases with CGL. The proliferation and apoptosis of CD34^＋CD7^＋ cells treated with HHT in vitro were studied. Results： The proportion of CD34^＋CD7^＋ cells in CGL （0.145±0.021） was higher than that of normal control （0.052±0.013）. The proportion of CD34^＋CD7^＋ cells in patients who got cytogenetic responses to HHT （0.072±0.020） decreased remarkably, but not in those patients who did not got cytogenetic responses to HHT, （0.137±0.023）. the proliferation of CD34^＋ cells was inhibited and the proportion of CD34^＋CD7^＋ cells decreased after cultured with HHT （0.134 in 24 h, 0.126 in 48 h and 0.102 in 72）. The apoptosis rate of CD34^＋CD7^＋ cells was higher than that in CD34^＋CDT cells （35.39%±4.39% versus 24.57%±4.01%, P〈0.05） 72 h after culture with HHT. Conclusion： The proportion of CD34^＋CD7^＋ cells in CGL was higher than that of normal control and HHT may inhibit the proliferation and induce apoptosis of bone marrow CD34^＋CD7^＋ cells.     

The prognostic value of peripheral CD4+CD25+ T lymphocytes among early stage and triple negative breast cancer patients receiving dendritic cells-cytokine induced killer cells infusion

Objective

This study aimed to assess the prognostic value of CD4⁺CD25⁺ T lymphocyte in peripheral blood among breast cancer patients treated with adoptive T lymphocytes immunotherapy.

Methods

217 patients participated in the follow-up study. CD4⁺CD25⁺ proportion was measured by flow cytometry in peripheral T cells. The median survival was estimated by Kaplan-Meier curve, Log-rank test and Cox hazard proportion regression model, between groups of CD4⁺CD25⁺ proportion more than 5% and less than or equal to 5% in peripheral T cells.

Results

Peripheral CD4⁺CD25⁺ T lymphocytes had not a relationship with progression-free survival. It was featured that above 5% peripheral CD4⁺CD25⁺ proportion of T cells was related with the median overall survival by a shorten of 51 months (p < 0.05) with the HR 1.65 (95%CI 1.04, 2.62). Above 5% CD4⁺CD25⁺proportion of T cells produced the HR to be 1.76 (95%CI 1.07, 2.87) In stage 0-II patients, and 3.59 (95%CI 1.05, 12.29) in triple negative breast cancer patients.

Conclusion

Cellular immunity restoration recovered by adoptive T cell infusions which resulted in less proportion of peripheral CD4⁺CD25⁺T lymphocytes could be a potential prognostic indicator among early stage and triple negative patients.     

Anti-4-1BB antibody-based combination therapy augments antitumor immunity by enhancing CD11c+CD8+ T cells in renal cell carcinoma

To improve the potential treatment strategies of incurable renal cell carcinoma (RCC), which is highly resistant to chemotherapy and radiotherapy, the present study established a combination therapy with immunostimulatory factor (ISTF) and anti-4-1BB monoclonal antibodies (mAbs) to augment the antitumor response in a murine RCC model. ISTF isolated from Actinobacillus actinomycetemcomitans stimulates macrophages, dendritic cells and B cells to produce IL-6, TNF-α, nitric oxide and major histocompatibility complex class II expression. 4-1BB (CD137) is expressed in activated immune cells, including activated T cells, and is a promising target for cancer immunotherapy. The administration of anti-4-1BB mAbs promoted antitumor immunity via enhancing CD11c⁺CD8⁺ T cells. The CD11c⁺CD8⁺ T cells were characterized by high killing activity and IFN-γ-producing ability, representing a phenotype of active effector cytotoxic T lymphocytes. The present study showed that combination therapy with ISTF and anti-4-1BB mAbs promoted partial tumor regression with established RCC, but monotherapy with ISTF or anti-4-1BB mAbs did not. These effects were speculated to be caused by the increase in CD11c⁺CD8⁺ T cells in the spleen and tumor, and IFN-γ production. These insights into the effector mechanisms of the combination of ISTF and anti-4-1BB mAbs may be useful for targeting incurable RCC.     

Transgenic expression of IL-33 activates CD8 T cells and NK cells and inhibits tumor growth and metastasis in mice

IL-33 is a multifunctional cytokine in immune regulation that activates Th1 cells, Th2 cells, CD8⁺ T cells and NK cells. Our study showed that transgenic expression of IL-33 attenuated tumor metastasis in the B16 melanoma and Lewis lung carcinoma (LLC) metastatic models. The percentages and cytotoxicity of CD8⁺ T cells and NK cells and their infiltration into the tumor tissues were significantly increased by the transgenic expression of IL-33 in tumor-bearing mice. Treatment with recombinant IL-33 could also increase the cytotoxicity of CD8⁺ T cells and NK cells in vitro. In addition, depletion of CD8⁺ T cells and NK cells using anti-CD8 or anti-asialo GM1 antibody abolished the pulmonary metastasis inhibition mediated by IL-33. Furthermore, IL-33 stimulated the activation of NF-κB and increased CD69 expression, which is a marker of the activated form of the two cell subsets, in CD8⁺ T cells and NK cells. Our results suggest that IL-33 stimulated NF-κB signaling and promoted the proliferation, activation and infiltration of CD8⁺ T cells and NK cells, which resulted in the inhibition of pulmonary metastasis in B16 melanoma and LLC mice models.     

Amphiregulin activates regulatory T lymphocytes and suppresses CD8+ T cell-mediated anti-tumor response in hepatocellular carcinoma cells

CD8⁺ T cell-mediated immune response plays an important role in inhibiting progression of hepatocellular carcinoma (HCC). For strategic immunotherapy, it is critical to understand why some of the tumor cells escape from this immune attack. In this study, we investigated how HCC cells alter endogenous anti-tumor immunity and their related signaling pathways. We found that HCC cells, both in vitro and in vivo, substantially secret and express amphiregulin (AR). AR in turn activates immunosuppressive function of intratumoral CD4⁺Foxp3⁺ regulatory T cells (Tregs), a major inhibitor of CD8⁺ T cells. Using either lentiviral siRNA, or AR neutralizing antibody, we blocked the expression and function of AR to test the specificity of AR mediated activation of Tregs, Biochemical and cell biology studies were followed and confirmed that blocking of AR inhibited Tregs activation. In addition, we found that AR can trigger the activation of rapamycin complex 1(mTORC1) signaling in Tregs. The mTORC1 inhibitor rapamycin treatment led to compromise Treg function and resulted in enhancing anti-tumor function of CD8⁺ T cells. Blocking AR/EGFR signaling in Tregs with Gefitinib also enhanced anti-tumor immunity and decreased tumor size in a mouse xenograft tumor model. Taken together, our study suggested a novel mechanism of functional interaction between HCC and Tregs for regulating anti-tumor function of CD8⁺ T cells.     

CD15<Superscript>+</Superscript>/CD16<Superscript>low</Superscript> human granulocytes from terminal cancer patients: granulocytic myeloid-derived suppressor cells that have suppressive function

Myeloid-derived suppressor cells (MDSCs) are a subpopulation of myeloid cells with immunosuppressive function whose numbers are increased in conditions such as chronic infection, trauma, and cancer. Unlike murine MDSCs defined as CD11b⁺/Gr-1⁺, there are no specific markers for human MDSCs. The goal of this study was to delineate a specific human MDSCs subpopulation in granulocytes from terminal cancer patients and investigate its clinical implications. Here, we show that the CD15⁺/CD16^low subset was increased in terminal cancer patients compared with healthy donors (P = 0.009). Phorbol 12-myristate 13-acetate-activated granulocytes (CD16^low/CD66b⁺⁺/CD15⁺) that have a phenotype similar to MDSCs from cancer patients, effectively suppressed both proliferation and cytotoxicity of normal T cells. Among cancer patients, T-cell proliferation was highly suppressed by granulocytes isolated from terminal cancer patients with a high proportion of CD15⁺/CD16^low cells. Patients with low peripheral blood levels of CD15⁺/CD16^low cells had significantly longer survival than those with high levels (P = 0.0011). Patients with higher levels of CD15⁺/CD16^low also tended to have poor performance status (P = 0.05). These data suggest that CD15⁺/CD16^low granulocytes found in terminal cancer patients may play a role in the progression of cancer by inhibiting tumor immunity.     

Numbers and cytotoxicities of CD3+CD56+ T lymphocytes in peripheral blood of patients with acute myeloid leukemia and acute lymphocytic leukemia

Recent reports have highlighted the role of cellular immunity in anti-tumor defenses. T lymphocytes are known to play important part in anti-cancer immunity. The number and function of T lymphocytes are altered in chronic leukemia patients. CD3⁺CD56⁺ T lymphocytes have also been found to be abnormal in cancer patients. We therefore investigated changes in the number and cytotoxicity of CD3⁺CD56⁺ T lymphocytes in the peripheral blood of acute leukemia (AL) patients (excluding acute promyelocytic leukemia), to improve our understanding of the role of this T lymphocyte subset. We analyzed CD3⁺CD56⁺ T lymphocyte numbers and cytotoxicities in healthy controls, AL patients, and AL patients with complete remission. Lymphocyte counts were performed in peripheral blood and flow cytometry was used to determine cell numbers and cytotoxicities. The absolute number of CD3⁺CD56⁺ T lymphocytes was increased in AL patients (including acute myeloid [AML] and acute lymphocytic leukemia [ALL]) compared with healthy controls (P < 0.05), but their functioning was significantly reduced (P < 0.05). The number of CD3⁺CD56⁺ T lymphocytes in AML and ALL patients who achieved remission following chemotherapy was close to healthy controls (P > 0.05), but their functioning was still significantly reduced (P < 0.05). In addition, the number of CD3⁺CD56⁺ T lymphocytes increased significantly in AML patients with increased peripheral blood white blood cell (WBC) counts, and in ALL patients without increased WBCs. These results suggest that cellular immunity may respond to AML and ALL, but that lymphocyte cytotoxicity remains impaired. Dysfunction of CD3⁺CD56⁺ T lymphocytes in AML and ALL patients may contribute to the failure of the host immune response against leukemic blasts.     

An evaluation of the clinical significance of FOXP3<Superscript>+</Superscript> infiltrating cells in human breast cancer

Studies in mice have shown that thymic-derived CD4+ CD25+ regulatory T cells (T reg; FOXP3⁺ lymphocytes) inhibit an antitumour immune response. Additional studies have also reported that the T reg population increases in peripheral blood and tumour tissues from patients with cancer. However, the relationship between the T reg population and the patient prognosis remains controversial. Our aim was to determine the prognostic value of T reg cell density in breast cancer using immunohistochemical assessment of FOXP3, which has been shown to be the optimal marker for T regs. Tissue microarrays were used, and the density of FOXP3⁺ cells was determined in a series of 1445 cases of well-characterised primary invasive breast carcinoma cases with long-term follow up. FOXP3⁺ cell numbers were counted in tumour nests, in tumour-adjacent stroma, and in distant stroma. The total number of FOXP3⁺ cells significantly correlated with higher tumour grade (r _s = 0.37, P < 0.001) and ER negativity (Mann–Whitney U test, P < 0.001). In addition, FOXP3 infiltration positively correlated with HER2 expression and basal phenotype subclass. On univariate analysis, FOXP3⁺ cells were associated with a worse prognosis (P = 0.012, log rank = 6.36). This association was found for intratumoural FOXP3⁺ and for tumour-adjacent stromal FOXP3⁺-cells (tumour-cell associated FOXP3, P = 0.001 and log rank 10.35). However, the number of FOXP3⁺ cells was not found to be an independent prognostic factor in multivariate analysis. We therefore conclude that FOXP3⁺ infiltrating cells do not have a dominant role in breast cancer prognosis and suggest that other inflammatory cell subsets may be more critical variables.     

Induction of anti-mammary cancer immunity by engaging the OX-40 receptor in vivo

The OX-40 receptor (OX-40R) is a member of the tumor necrosis factor receptor (TNF-R) superfamily that is expressed on activated CD4⁺ T cells. The OX-40R is a costimulatory molecule that induces CD4⁺ T cell activation when engaged by its ligand (OX-40L; found on antigen presenting cells). In human and murine tumors, we have shown upregulation of the OX-40R on CD4⁺ T cells from tumor-infiltrating lymphocytes (TIL) and tumor-draining lymph node cells (TDLNC) but not on systemic CD4⁺ T cells, such as peripheral blood lymphocytes (PBL) or splenocytes. In order to examine potentially heightened anti-tumor immunity through enhanced costimulation when engaging OX-40R in vivo, we inoculated mice with a murine mammary cancer cell line (SM1) and then treated with a soluble form of the OX-40L. Mice injected with a lethal inoculum of SM1 cells were given two intraperitoneal injections (days 3 and 7 post-inoculation) of 100g soluble OX-40L. Seven of 28 treated mice survived the lethal tumor inoculum, as compared to one of 28 control mice, demonstrating a significant survival benefit with treatment (p=0.0136, log rank analysis). Mice that did not develop tumor by day 90 were rechallenged; all remained tumor-free. Mice were also injected with a second mammary tumor line (4T1) and treated with OX-40L:Ig with similar therapeutic results. Activation of OX-40R⁺ CD4⁺ T cells during mammary cancer priming stimulated an anti-tumor immune response resulting in enhanced survival and protective anti-tumor immunity. These results should have practical applications for treatment modalities for patients with breast cancer.     

CD4+ T cell exhaustion leads to adoptive transfer therapy failure which can be prevented by immune checkpoint blockade

Cytotoxic CD8⁺ T cell exhaustion is one of the mechanisms underlying the tumor immune escape. The paradigm-shifting immune checkpoint therapy can mitigate CD8⁺ T lymphocyte exhaustion, reinvigorate the anticancer immunity, and achieve durable tumor regression for some patients. Emerging evidence indicates that CD4⁺ T lymphocytes also have a critical role in anticancer immunity, either by directly applying cytotoxicity toward cancer cells or as a helper to augment CD8⁺ T cell cytotoxicity. Whether anticancer CD4⁺ T lymphocytes undergo exhaustion during immunotherapy of solid tumors remains unknown. Here we report that melanoma antigen TRP-1/gp75-specific CD4⁺ T lymphocytes exhibit an exhaustion phenotype after being adoptively transferred into mice bearing large subcutaneous melanoma. Exhaustion of these CD4⁺ T lymphocytes is accompanied with reduced cytokine release and increased expression of inhibitory receptors, resulting in loss of tumor control. Importantly, we demonstrate that PD-L1 immune checkpoint blockade can prevent exhaustion, induce proliferation of the CD4⁺ T lymphocytes, and consequently prevent tumor recurrence. Therefore, when encountering an excessive amount of tumor antigens, tumor-reactive CD4⁺ T lymphocytes also enter the exhaustion state, which can be prevented by immune checkpoint blockade. Our results highlight the importance of tumor-specific CD4⁺ T lymphocytes in antitumor immunity and suggest that the current immune checkpoint blockade therapy may achieve durable anticancer efficacy by rejuvenating both tumor antigen-specific CD8⁺ T lymphocytes and CD4⁺ T lymphocytes.     

Formosanin C promotes the curative efficacy of ultrasound-guided radiofrequency ablation in a mouse model of breast cancer

Breast cancer is the leading cause of tumor-associated death among women worldwide, and new therapeutic strategies are required to improve the post-surgery prognosis and quality of life of patients. Radiofrequency ablation (RFA) is a less invasive approach compared with traditional surgical resection to treat malignancies, and the combination of RFA and chemotherapeutic agents, including formosanin C (FC), can synergistically improve the curative effects against breast carcinoma. However, the detailed mechanisms remain unclear. In the present study, nude mice were used to identify the influence of FC on the therapeutic efficacy of RFA for breast cancer. Flow cytometry was performed to demonstrate the proportional alteration of CD8⁺ and CD45⁺ T cells with different biomarkers, including CD107a, IFNγ and TNFα. It was demonstrated that FC enhanced the therapeutic efficacy of RFA in breast cancer, while RFA combined with FC improved the proportion of IFNγ⁺ and TNFα⁺ CD8⁺ T cells and CD107a⁺ CD8⁺ T cells in tumor-infiltrating lymphocytes, thus increasing the immune responses caused by surgery and chemotherapy. The present study indicated that FC may promote the curative efficacy of ultrasound-guided RFA against breast tumor by regulating adaptive immune responses.     

Immune landscape of vulvar cancer patients treated with surgery and adjuvant radiotherapy revealed restricted T cell functionality and increased IL-17 expression associated with cancer relapse

For vulvar cancers, radiotherapy is targeting cancer cells, but also affects the host immune system. As this may affect treatment outcome, in this prospective study, we characterized the individual T cell immune milieu induced by surgery and adjuvant radio +/− chemotherapy (aRT) systemically in the blood of vulvar cancer patients and found increased frequencies of Interleukin (IL)-17-producing CD4⁺ and CD8⁺ T cells after aRT while frequencies of Th1 and perforin-producing CD8⁺ killer cells were strongly diminished. Phenotypic characterization revealed enhanced expression of the ectonucleotidase CD39 on Th17 and Tc17 cells as well as CD8⁺ perforin⁺ cells after aRT. Furthermore, the aRT cohort exhibited increased proportions of Programmed Cell Death Protein (PD-1) expressing cells among Th1 and CD8⁺ perforin⁺ cells, but not among Th17 and Tc17 cells. High post-therapeutic levels of Th17 and Tc17 cells and low proportions of Th1 and CD8⁺ perforin⁺ cells expressing PD-1 was associated with reduced recurrence free survival on follow-up. In conclusion, our study defines individual therapy-induced changes in the cellular immune milieu of patients and their association with cancer relapse. Our results may help to explain differences in the individual courses of disease of vulvar cancer patients and suggest PD-1 and IL-17 as targets for immunotherapy in vulvar cancer.     

Low miR-16 expression induces regulatory CD4+NKG2D+ T cells involved in colorectal cancer progression

MiR-15a/16 is a member of the miRNA cluster that exhibits tumor suppression and immune modulation via targeting multiple genes. Decreased miR-15a/16 expression is involved in many cancer cells. Here, miR-16 had decreased expression in NK1.1^-CD4⁺NKG2D⁺ T cells and bound with the 3’-UTR of NKG2D gene. MiR-15a/16-deficient mice had many CD4⁺NKG2D⁺ T cells, which produced TGF-β1 and IL-10 and inhibited the IFN-γ production of CD8⁺ T cells. Adoptive transfer of NK1.1^-CD4⁺NKG2D⁺ T cells from miR-15a/16-deficient mice promoted tumor growth in vivo. However, no changes for NK1.1^-CD4⁺NKG2D⁺ T cells were found in the miR-15a/16-transgenic mice. Although the miR-15a/16 transgenic mice transplanted with B16BL6 or MC38 cells exhibited rapid growth, these tumor-bearing mice did not show changes in NK1.1^-CD4⁺NKG2D⁺ T cell distributions in either spleens or tumors. When NK1.1^-CD4⁺ T cells were stimulated by α-CD3/sRAE-1 ex vivo, the NKG2D expression was difficult to induce in the T cells of miR-15a/16-transgenic mice. Finally, increased frequencies of regulatory CD4⁺NKG2D⁺ T cells with low miR-16 levels were observed in patients with late-stage colorectal cancer (Duke’s C, D). Thus, miR-16 modulates NK1.1^-CD4⁺NKG2D⁺ T cell functions via targeting NKG2D. Low miR-16 expression in CD4⁺ T cells induces the regulatory CD4⁺NKG2D⁺ T subpopulation, which promotes tumor evasion via the secretion of immune-suppressive molecules.     

Increased lactate in AML blasts upregulates TOX expression,leading to exhaustion of CD8+ cytolytic T cells

Recently, the role of lactate as merely an end product of cancer cell metabolism has been reassessed. Lactate has been implicated in more biological processes than previously understood and drives tumor progression. Here, we demonstrated that the bone marrow lactate concentrations in acute myeloid leukemia (AML) patients were substantially higher than those in their healthy control counterparts. Moreover, AML blasts from bone marrow expressed significantly higher lactate dehydrogenase-A (LDHA) levels. Further studies revealed that LDHA expression was regulated through the HIF1α pathway. Elevated lactate levels were indicative of alterations in CD8⁺ T cell cytolytic phenotype and activity. An in vitro study showed that the lactate treatment group had significantly higher percentages of CD8⁺ TEM and CD8⁺ TEMRA cells as well as higher PD-1 expression in these cells than the control group. Lactate induced the loss of the effector function of CD8⁺ T cells by altering lytic granule exocytosis. T cell dysfunction is characterized by an increase in terminally differentiated phenotypes, sustained expression of PD-1, and accelerated decline of cytolytic competence. Moreover, the TOX gene was found to be correlated with lactate production and implicated in CD8⁺ T cell dysfunction. AML patients in complete remission after chemotherapy had markedly lower lactate concentrations, reduced CD8⁺ TEM and CD8⁺ TEMRA cells and PD-1 expression, and increased perforin and granzyme B. However, no difference was found in the relapsed patients. The study presented here has established lactate as a predictive biomarker for patient response to antitumor therapies and demonstrated that targeting this gene in AML patients could be a meaningful precision therapeutic strategy.     

Clonal expansion of bone marrow CD8+ T cells in acute myeloid leukemia patients at new diagnosis and after chemotherapy

CD8⁺ T cells are crucial adaptive immune effectors and express receptors (T cell receptors, TCRs) that specifically recognize and eradicate tumor cells. The diversity of the TCR repertoire is generated by specialized genetic diversification mechanisms, which lead to an extremely variable TCR repertoire that is capable of recognizing a wide range of antigens. However, the variations in CD8⁺ TCR diversity and their clinical implications in acute myeloid leukemia (AML) patients remain unknown. CD8⁺ T cells were enriched from 10 healthy donors and 31 AML patients at diagnosis and after chemotherapy, and TCRβ deep sequencing was performed to analyze CD8⁺ T cell clonal expansion and TCR repertoire diversity. Diminished TCR repertoire diversity and increased T cell clone expansion were noted in the bone marrow of AML patients. In relapsed patients, T cells were found to be more clonally expanded after chemotherapy than at new diagnosis. Moreover, significantly more expanded TCRβ clonotypes were noted in CD8⁺ PD-1⁺ T cells than in CD8⁺ PD-1^- T cells regardless of the time of examination. Our systematic T cell repertoire analysis may help better characterize CD8⁺ T cells before and after chemotherapy in AML, which may provide insights into therapeutic strategies for hematological malignancies.