A tumor lysate is an effective vaccine antigen for the stimulation of CD4+ T-cell function and subsequent induction of antitumor immunity mediated by CD8+ T cells

To develop a potent cancer vaccine, it is important to study how to prepare highly immunogenic antigens and to identify the most appropriate adjuvants for the antigens. Here we show that a tumor lysate works as an effective antigen to prime CD4⁺ T-cell help when baculovirus is employed as an adjuvant. When immunized intradermally with the combination (BLP) of baculovirus, a CT26 tumor lysate, and a cytotoxic T-cell epitope peptide before a tumor challenge, 60% of mice rejected tumors. In contrast, all mice vaccinated with baculovirus plus a tumor lysate (BL) developed tumors. In addition, flow cytometry showed that tumor-specific, interferon γ-producing CD8⁺ cytotoxic T lymphocytes (CTLs) were robustly activated by intradermal immunization with BLP. When BLP was administered therapeutically to tumor-bearing mice, antitumor efficacy was better compared to BL. The established tumor was completely eradicated in 50–60% of BLP-treated mice, and induction of tumor-specific CTLs was observed, suggesting that the antitumor efficacy of BLP is mediated by CD8⁺ T cells. Numerous CD4⁺ T cells infiltrated the tumors of BLP-treated mice, whereas the antitumor effect of BLP almost disappeared after removal of the tumor lysate from BLP or after depletion of BLP-immunized mice of CD4⁺ T cells. Thus, the combination of a peptide, lysate, and baculovirus provides stronger antitumor immunity than does a peptide plus baculovirus or a lysate plus baculovirus; effectiveness of BLP is determined by functioning of CD4⁺ T cells stimulated with a tumor lysate.     

Antigen spreading-induced CD8+T cells confer protection against the lethal challenge of wild-type malignant mesothelioma by eliminating myeloid-derived suppressor cells

A key focus in cancer immunotherapy is to investigate the mechanism of efficacious vaccine responses. Using HIV-1 GAG-p24 in a model PD1-based DNA vaccine, we recently reported that vaccine-elicited CD8⁺ T cells conferred complete prevention and therapeutic cure of AB1-GAG malignant mesothelioma in immunocompetent BALB/c mice. Here, we further investigated the efficacy and correlation of protection on the model vaccine-mediated antigen spreading against wild-type AB1 (WT-AB1) mesothelioma. We found that this vaccine was able to protect mice completely from three consecutive lethal challenges of AB1-GAG mesothelioma. Through antigen spreading these animals also developed tumor-specific cytotoxic CD8⁺ T cells, but neither CD4⁺ T cells nor antibodies, rejecting WT-AB1 mesothelioma. A majority of these protected mice (90%) were also completely protected against the lethal WT-AB1 challenge. Adoptive cell transfer experiments further demonstrated that antigen spreading-induced CD8⁺ T cells conferred efficacious therapeutic effects against established WT-AB1 mesothelioma and prevented the increase of exhausted PD-1⁺ and Tim-3⁺ CD8⁺ T cells. A significant inverse correlation was found between the frequency of functional PD1⁻Tim3⁻ CD8⁺ T cells and that of MDSCs or tumor mass in vivo. Mechanistically, we found that WT-AB1 mesothelioma induced predominantly polymorphonuclear (PMN) MDSCs in vivo. In co-cultures with efficacious CD8⁺ T cells, a significant number of PMN-MDSCs underwent apoptosis in a dose-dependent way. Our findings indicate that efficacious CD8⁺ T cells capable of eliminating both tumor cells and MDSCs are likely necessary for fighting wild-type malignant mesothelioma.     

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.     

CCL3 and CCL20-recruited dendritic cells modified by melanoma antigen gene-1 induce anti-tumor immunity against gastric cancer ex vivo and in vivo

Background

To investigate whether dendritic cell (DC) precursors, recruited by injection of chemokine ligand 3 (CCL3) and CCL20, induce anti-tumor immunity against gastric cancer induced by a DC vaccine expressing melanoma antigen gene-1 (MAGE-1) ex vivo and in vivo.

Methods

B6 mice were injected with CCL3 and CCL20 via the tail vein. Freshly isolated F4/80^-B220^-CD11c⁺ cells cultured with cytokines were analyzed by phenotype analysis and mixed lymphocyte reaction (MLR). For adenoviral (Ad)-mediated gene transduction, cultured F4/80^-B220^-CD11c⁺ cells were incubated with Ad-MAGE-1. Vaccination of stimulated DC induced T lymphocytes. The killing effect of these T cells against gastric carcinoma cells was assayed by MTT. INF-γ production was determined with an INF-γ ELISA kit. In the solid tumor and metastases model, DC-based vaccines were used for immunization after challenge with MFC cells. Tumor size, survival of mice, and number of pulmonary metastatic foci were used to assess the therapeutic effect of DC vaccines.

Results

F4/80^-B220^-CD11c⁺ cell numbers increased after CCL3 and CCL20 injection. Freshly isolated F4/80^-B220^-CD11c⁺ cells cultured with cytokines were phenotyically identical to typical DC and gained the capacity to stimulate allogeneic T cells. These DCs were transduced with Ad-MAGE-1, which were prepared for DC vaccines expressing tumor antigen. T lymphocytes stimulated by DCs transduced with Ad-MAGE-1 exhibited specific killing effects on gastric carcinoma cells and produced high levels of INF-γ ex vivo. In vivo, tumor sizes of the experimental group were much smaller than both the positive control group and the negative control groups (P < 0.05). Kaplan-Meier survival curves showed that survival of the experimental group mice was significantly longer than the control groups (P < 0.05). In addition, MAGE-1-transduced DCs were also a therapeutic benefit on an established metastatic tumor, resulting in a tremendous decrease in the number of pulmonary metastatic foci.

Conclusions

CCL3 and CCL20-recruited DCs modified by adenovirus-trasnsduced, tumor-associated antigen, MAGE-1, can stimulate anti-tumor immunity specific to gastric cancer ex vivo and in vivo. This system may prove to be an efficient strategy for anti-tumor immunotherapy.     

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.     

Targeting of the non-mutated tumor antigen HER2/neu to mature dendritic cells induces an integrated immune response that protects against breast cancer in mice

Introduction

Given their relative simplicity of manufacture and ability to be injected repeatedly, vaccines in a protein format are attractive for breast and other cancers. However, soluble human epidermal growth factor receptor (HER2)/neu protein as a vaccine has not been immunogenic. When protein is directly targeted to antigen uptake receptors, such as DEC205 (DEC), efficient processing and presentation of antigen take place. The aim of this study was to determine the immunogenicity of a HER2 protein vaccine that directly targets to DEC⁺ dendritic cells (DCs) in a mouse breast cancer model.

Methods

We genetically engineered the HER2 extracellular domain into a monoclonal antibody specific for DEC (DEC-HER2). Mice of various genetic backgrounds were immunized with DEC-HER2 in combination with DC maturation stimuli (poly IC ± CD40 Ab). Vaccine-induced T cell immunity was determined by analyzing the ability of CD4⁺/CD8⁺ T cell to produce interferon (IFN)-gamma and proliferate upon antigen rechallenge. Sera were assessed for the presence of antigen specific antibody (Ab). For vaccine efficacy, FVB/N mice were immunized with DEC-HER2 in combination with poly IC and protection against neu-expressing mammary tumors was assessed. Protection mechanisms and tumor-specific T cell responses were also evaluated.

Results

We demonstrate that DEC-HER2 fusion mAb, but not Ctrl Ig-HER2, elicits strong, broad and multifunctional CD4⁺ T cell immunity, CD8⁺ T cell responses, and humoral immunity specific for HER2 antigen. Cross-reactivity to rat neu protein was also observed. Importantly, mice xeno-primed with DEC-HER2 were protected from a neu-expressing mammary tumor challenge. Both CD4⁺ and CD8⁺ T cells mediated the tumor protection. Robust anti-tumor T cell immunity was detected in tumor protected mice.

Conclusions

Immunization of mice with HER2 protein vaccine targeting DEC⁺ DCs in vivo induced high levels of T- and B-cell immunity. Non-targeted HER2 protein was poorly immunogenic for CD4⁺ and CD8⁺ T cells. This vaccination approach provided long-term survival benefit for mice challenged with neu-expressing tumor following as little as 2.7 ??g of HER2 protein incorporated in the vaccine. Vaccine-induced CD4⁺ and CD8⁺ T cells were both essential for tumor protection. This immunization strategy demonstrates great potential towards the development of vaccines for breast cancer patients.     

slan/M-DC8+ cells constitute a distinct subset of dendritic cells in human tonsils

Human blood dendritic cells (DCs) include three main distinct subsets, namely the CD1c⁺ and CD141⁺ myeloid DCs (mDCs) and the CD303⁺ plasmacytoid DCs (pDCs). More recently, a population of slan/M-DC8⁺ cells, also known as “slanDCs”, has been described in blood and detected even in inflamed secondary lymphoid organs and non-lymphoid tissues. Nevertheless, hallmarks of slan/M-DC8⁺ cells in tissues are poorly defined. Herein, we report a detailed characterization of the phenotype and function of slan/M-DC8⁺ cells present in human tonsils. We found that tonsil slan/M-DC8⁺ cells represent a unique DC cell population, distinct from their circulating counterpart and also from all other tonsil DC and monocyte/macrophage subsets. Phenotypically, slan/M-DC8⁺ cells in tonsils display a CD11c⁺HLA-DR⁺CD14⁺CD11b^dim/negCD16^dim/negCX3CR1^dim/neg marker repertoire, while functionally they exhibit an efficient antigen presentation capacity and a constitutive secretion of TNFα. Notably, such DC phenotype and functions are substantially reproduced by culturing blood slan/M-DC8⁺ cells in tonsil-derived conditioned medium (TDCM), further supporting the hypothesis of a full DC-like differentiation program occurring within the tonsil microenvironment. Taken together, our data suggest that blood slan/M-DC8⁺ cells are immediate precursors of a previously unrecognizedcompetent DC subset in tonsils, and pave the way for further characterization of slan/M-DC8⁺ cells in other tissues.     

Stimulation of CD8+ T cell responses to MAGE-3 and Melan A/MART-1 by immunization to a polyvalent melanoma vaccine

A critical requirement for cancer vaccines is that they stimulate CD8⁺ T cell responses. In this study, we tested the ability of a polyvalent melanoma vaccine to induce CD8⁺ T cell responses to the melanoma associated antigens MAGE-3 and Melan A/MART-1. Fifteen HLA-A2⁺ patients with resected malignant melanoma were immunized with the vaccine s.c. every 2–3 weeks. CD8⁺ T cells in peripheral blood reacting to HLA-A2 restricted epitopes on MAGE-3 (FLWGPRALV) and Melan A/MART-1/(AAGIGILTV) were quantitated using a filter spot assay at baseline and following 4 immunizations. Vaccine immunization induced CD8⁺ T cells reacting to one or both of these peptides in 9 of the 15 (60%) patients. These cells were CD8⁺ and HLA-A2 restricted, as reactivity was abrogated by monoclonal antibodies (MAbs) to CD8 and class I HLA, but not by anti-CD4. All responding patients remained recurrence-free for at least 12 months (median 15 months, range 12 to >21 months), whereas melanoma recurred within 3–5 months in non-responders. The differences in outcome were unrelated to differences in disease severity or overall immunological competence between responders and non-responders. Our results demonstrate directly that MAGE-3 and Melan A/MART-1 can stimulate CD8⁺ T cell responses in humans, and suggest that these responses are protective and surrogate markers of vaccine efficacy. Int. J. Cancer 72:972–976, 1997. © 1997 Wiley-Liss, Inc.     

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.     

Genetically driven CD39 expression shapes human tumor-infiltrating CD8+ T-cell functions

In our study, we investigated the role of CD39 on tumor-infiltrating CD8⁺ T lymphocytes (CD8⁺ TILs) in colorectal, head and neck and pancreatic cancers. Partially confirming recent observations correlating the CD39 expression with T-cell exhaustion, we demonstrated a divergent functional activity in CD39⁺CD8⁺ TILs. On the one hand, CD39⁺CD8⁺ TILs (as compared to their CD39⁻ counterparts) produced significantly lower IFN-γ and IL-2 amounts, expressed higher PD-1, and inversely correlated with perforin and granzyme B expression. On the other, they displayed a significantly higher proliferative capacity ex vivo that was inversely correlated with the PD-1 expression. Therefore, CD39⁺CD8⁺ TILs, including those co-expressing the CD103 (a marker of T resident memory [TRM] cells), were defined as partially dysfunctional T cells that correlate with tumor patients with initial progression stages. Interestingly, our results identified for the first time a single nucleotide polymorphism (SNP rs10748643 A>G), as a genetic factor associated with CD39 expression in CD8⁺ TILs. Finally, we demonstrated that compounds inhibiting CD39-related ATPases improved CD39⁺CD8⁺ T-cell effector function ex vivo, and that CD39⁺CD8⁺ TILs displayed effective suppression function in vitro. Overall these data suggest that the SNP analysis may represent a suitable predictor of CD39⁺CD8⁺ T-cell expression in cancer patients, and propose the modulation of CD39 as a new strategy to restore partially exhausted CD8⁺ TILs.     

Lenvatinib enhances antitumor immunity by promoting the infiltration of TCF1+ CD8+ T cells in HCC via blocking VEGFR2

Lenvatinib is the favorable treatment for advanced hepatocellular carcinoma (HCC), and it is currently undergoing phase III clinical trials. However, the specific effects of lenvatinib on PD1⁺ CD8⁺ T cells in HCC microenvironment have not been systematically studied. Here, we established an orthotopic hepa1-6 mouse model treated with lenvatinib to investigate CD8⁺ T cells’ role in the tumor and spleen. We found an increasing proportion of TCF-1⁺ in PD1⁺ CD8⁺ T cells and proliferation of PD1⁺ CD8⁺ T cells after lenvatinib treatment. Meanwhile, lenvatinib treatment upregulated the expression of granzyme B on PD1⁺ CD8⁺ T cells both in vitro and in vivo. Lenvatinib activated the endogenous mTOR pathway of exhausted CD8⁺ T cells, and mTOR pathway blockade eliminated the antitumor effect of lenvatinib and function of PD1⁺ CD8⁺ T cells. The effects of the mTOR pathway on PD1⁺ CD8⁺ T cells after lenvatinib treatment were mediated by VEGFR2 inhibition. Overall, our work provides insight into the mechanism of lenvatinib's antitumor efficacy through exhausted CD8⁺ T cells in HCC treatment.     

Adenovirus vector‐based prime‐boost vaccination via heterologous routes induces cervicovaginal CD8+ T cell responses against HPV16 oncoproteins

Recent advances in immunotherapy against cancer underscore the importance of T lymphocytes and tumor microenvironment, but few vaccines targeting cancer have been approved likely due in part to the dearth of common tumor antigens, insufficient immunogenicity and the evolution of immune evasion mechanisms during the progression to malignancy. Human papillomaviruses (HPVs) are the primary etiologic agents of cervical cancer and progression from persistent HPV‐infection to cervical intraepithelial lesions and eventually cancer requires persistent expression of the oncoproteins E6 and E7. This offers the opportunity to specifically target these virus‐specific antigens for vaccine‐induced clearance of infected cells before cancers develop. Here we have evaluated the immunogenicity of Adenovirus Types 26 and 35 derived vectors expressing a fusion of HPV16 E6 and E7 oncoproteins after intramuscular (IM) and/or intravaginal (Ivag) immunization in mice. The adenovirus vectors were shown to transduce an intact cervicovaginal epithelium. IM prime followed by Ivag boost maximized the induction and trafficking of HPV‐specific CD8⁺ T cells producing IFN‐γ and TNF‐α to the cervicovaginal tract. Importantly, the cervicovaginal CD8⁺ T cells expressed CD69 and CD103; hallmarks of intraepithelial tissue‐resident memory CD8⁺ T cells. This prime‐boost strategy targeting heterologous locations also induced circulating HPV‐specific CD8⁺ T cell responses. Our study prompts further evaluation of Ivag immunization with adenoviral vectors expressing modified E6 and E7 antigens for therapeutic vaccination against persistent HPV infection and cervical intraepithelial neoplasia.     

Blockade of TGF‐β enhances tumor vaccine efficacy mediated by CD8+ T cells

Though TGF‐β inhibition enhances antitumor immunity mediated by CD8⁺ T cells in several tumor models, it is not always sufficient for rejection of tumors. In this study, to maximize the antitumor effect of TGF‐β blockade, we tested the effect of anti‐TGF‐β combined with an irradiated tumor vaccine in a subcutaneous CT26 colon carcinoma tumor model. The irradiated tumor cell vaccine alone in prophylactic setting significantly delayed tumor growth, whereas anti‐TGF‐β antibodies alone did not show any antitumor effect. However, tumor growth was inhibited significantly more in vaccinated mice treated with anti‐TGF‐β antibodies compared to vaccinated mice without anti‐TGF‐β, suggesting that anti‐TGF‐β synergistically enhanced irradiated tumor vaccine efficacy. CD8⁺ T‐cell depletion completely abrogated the vaccine efficacy, and so protection required CD8⁺ T cells. Depletion of CD25⁺ T regulatory cells led to the almost complete rejection of tumors without the vaccine, whereas anti‐TGF‐β did not change the number of CD25⁺ T regulatory cells in unvaccinated and vaccinated mice. Though the abrogation of CD1d‐restricted NKT cells, which have been reported to induce TGF‐β production by MDSC through an IL‐13‐IL‐4R‐STAT6 pathway, partially enhanced antitumor immunity regardless of vaccination, abrogation of the NKT cell‐IL‐13‐IL‐4R‐STAT‐6 immunoregulatory pathway did not enhance vaccine efficacy. Taken together, these data indicated that anti‐TGF‐β enhances efficacy of a prophylactic vaccine in normal individuals despite their not having the elevated TGF‐β levels found in patients with cancer and that the effect is not dependent on TGF‐β solely from CD4⁺CD25⁺ T regulatory cells or the NKT cell‐IL‐13‐IL‐4R‐STAT‐6 immunoregulatory pathway.     

Human effector T cells derived from central memory cells rather than CD8T cells modified by tumor-specific TCR gene transfer possess superior traits for adoptive immunotherapy

Adoptive cell therapy provides an attractive treatment of cancer, and our expanding capacity to target tumor antigens is driven by genetically engineered human T lymphocytes that express genes encoding tumor-specific T cell receptors (TCRs). The intrinsic properties of cultured T cells used for therapy were reported to have tremendous influences on their persistence and antitumor efficacy in vivo. In this study, we isolated CD8⁺ central memory T cells from peripheral blood lymphocytes of healthy donors, and then transferred with the gene encoding TCR specific for tumor antigen using recombinant adenovirus vector Ad5F35-TRAV-TRBV. We found effector T cells derived from central memory T cells improved cell viability, maintained certain level of CD62L expression, and reacquired the CD62L⁺CD44^high phenotype of central memory T cells after effector T cells differentiation. We then compared the antitumor reactivity of central memory T cells and CD8⁺T cells after TCR gene transferred. The results indicated that tumor-specific TCR gene being transferred to central memory T cells effectively increased the specific killing of antigen positive tumor cells and the expression of cytolytic granule protein. Furthermore, TCR gene transferred central memory T cells were more effective than TCR gene transferred CD8⁺T cells in CTL activity and effector cytokine secretion. These results implicated that isolating central memory T cells rather than CD8⁺T cells for insertion of gene encoding tumor-specific TCR may provide a superior tumor-reactive T cell population for adoptive transfer.     

Targeting of SIRPα as a potential therapy for Langerhans cell histiocytosis

Langerhans cell histiocytosis (LCH) is a rare neoplastic disorder characterized by inflammatory lesions arising from the anomalous accumulation of pathogenic CD1a⁺CD207⁺ dendritic cells (DCs). SIRPα is a transmembrane protein highly expressed in myeloid cells such as DCs and macrophages. Here we show that SIRPα is a potential therapeutic target for LCH. We found that SIRPα is expressed in CD1a⁺ cells of human LCH lesions as well as in CD11c⁺ DCs in the spleen, liver, and lung of a mouse model of LCH (BRAFV600E^CD11c mouse), in which an LCH-associated active form of human BRAF is expressed in a manner dependent on the mouse Cd11c promoter. BRAFV600E^CD11c mice manifested markedly increased numbers of CD4⁺ T cells, regulatory T cells, and macrophages as well as of CD11c⁺MHCII⁺ DCs in the spleen. Monotherapy with a mAb to SIRPα greatly reduced the percentage of CD11c⁺MHCII⁺ DCs in peripheral blood, LCH-like lesion size in the liver, and the number of CD11c⁺MHCII⁺ DCs in the spleen of the mutant mice. Moreover, this mAb promoted macrophage-mediated phagocytosis of CD11c⁺ DCs from BRAFV600E^CD11c mice, whereas it had no effects on the viability or CCL19-dependent migration of such CD11c⁺ DCs or on their expression of the chemokine genes Ccl5, Ccl20, Cxcl11, and Cxcl12. Our results thus suggest that anti-SIRPα monotherapy is a promising approach to the treatment of LCH that is dependent in part on the promotion of the macrophage-mediated killing of LCH cells.     

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.     

CD4+ and CD8+ T-Cell Skewness in Classic Kaposi Sarcoma

It is widely accepted that a deranged immune system plays a key role in the onset and evolution of classic Kaposi sarcoma (CKS). Nevertheless, the usage of the T-cell receptor (TCR) β-variable (BV) chain repertoire expressed by peripheral blood lymphocytes in patients with CKS is still unknown. With the aim of providing some further insights into the complex role of the immune system in CKS pathogenesis, we performed an extensive analysis of the TCR BV repertoire in both CD4⁺ and CD8⁺ T cells in 30 human herpesvirus 8-positive Sardinian patients with CKS and an equal number of age-matched healthy controls. We used a panel of monoclonal antibodies covering approximately 70% of human BV subfamilies and third complementarity determining region (CDR3) spectratyping. Patients with CKS showed an increased frequency of BV expansions in both CD4⁺ and CD8⁺ lymphocytes, with no prevalent clones. On spectratyping analysis, most of the 720 BV CDR3 profiles obtained from both CD4⁺ and CD8⁺ T cells in patients with CKS were skewed. In particular, the surprising increase of BV skewing observed in CD4⁺ lymphocytes mimics the pattern of progressive TCR BV narrowing described in responses to persistent viral antigen stimulations. Our findings support the hypothesis that CKS evolution is associated with inadequate activation rather than impairment of the immune system.     

CD8+Foxp3+ tumor infiltrating lymphocytes accumulate in the context of an effective anti‐tumor response

The composition of tumor infiltrating lymphocytes (TIL) is heterogeneous. In addition, the ratio of various subpopulations in the tumor microenvironment is highly dependent on the nature of the host's immune response. Here, we characterize Foxp3‐expressing CD8⁺ T cells in the tumor that demonstrate effector function and accumulate in the context of an effective anti‐tumor response. CD8⁺Foxp3⁺ T cells are induced in TIL in regressing tumors of FVB/N mice treated with a GM‐CSF secreting HER‐2/neu targeted whole cell vaccine. Foxp3 expression in tumor antigen‐specific CD8 T cells is restricted to the tumor microenvironment and influenced by cues in the tumor. Interestingly, Foxp3⁺ and Foxp3^? CD8⁺ T cells have similar IFN‐γ production and antigen‐specific degranulation after stimulation with RNEU_420–429, the immunodominant HER‐2/neu (neu) epitope in this model. Adoptive transfer studies, using RNEU_(420–429)‐specific effector T cells into neu‐N mice (a model that results in immune tolerance to neu), confirm that CD8⁺Foxp3⁺ T cells are present in tumors only if there is an existing pool of tumor‐rejecting effector T cells. CD8⁺Foxp3⁺ TILs mark the presence of tumor‐rejecting antigen‐specific T cells and their accumulation serves as a marker for an effective T cell response.     

Recombinant E. coli LLO/OVA vaccination effectively inhibits murine melanoma metastasis to lung by CD8+T cells immunity

Objective  To construct recombinant E.coli LLO/OVA and investigate its tumor metastatic inhibition effect in B16 OVA melanoma challenged mice. Methods  Recombinant E.coli LLO/OVA was constructed and the expression of listeriolysin O (LLO) and ovalbumin (OVA) of the vaccine was determined by coomassie brilliant blue staining and western blotting. After 3 subcutaneous injections of E.coli LLO/OVA, the percentages of CD3⁺CD4⁺T, CD4⁺CD25⁺T, CD3⁺CD8⁺T and OVA_257–264 SIINFEKL specific CD8⁺T cells were determined by flow cytomytry, and the tumor metastatic inhibition effect in B16 OVA melanoma challenged mice was observed. Results  Recombinant E.coli LLO/OVA was successfully constructed, and the expression of LLO and OVA of the vaccine was confirmed. After 3 subcutaneous injections of E.coli LLO/OVA and E.coli OVA in mice, the percentages of CD3⁺CD4⁺T, CD4⁺CD25⁺T and CD3⁺CD8⁺T cells were equivalent in the two groups of mice. However, there were significantly more OVA_257–264 SIINFEKL specific CD8⁺T cells in E.coli LLO/OVA vaccinated mice than that in E.coli OVA vaccinated mice. The prophylactic E.coli LLO/OVA vaccination effectively prevented the tumor metastasis to lungs in B16 OVA melanoma challenged mice. Depletion of CD8⁺T cells significantly impaired the tumor inhibition effect of the vaccine in B16 OVA challenged mice. The therapeutic vaccination of E.coli LLO/OVA significantly prevented melanoma metastasis to lungs in B16 OVA challenged mice too. Conclusion  Recombinant E.coli LLO/OVA vaccination is highly effective in inhibiting murine malignant melanoma metastasis by promoting CD8⁺T cell immunity. This work was supported by the State Scholarship Fund from China Scholarship Council (No.2003850064) and Chongqing Educational Committee Foundation (cq20070319).     

Glutamine deprivation impairs function of infiltrating CD8+ T cells in hepatocellular carcinoma by inducing mitochondrial damage and apoptosis

BACKGROUNDThe functions of infiltrating CD8⁺ T cells are often impaired due to tumor cells causing nutrient deprivation in the tumor microenvironment. Thus, the mechanisms of CD8⁺ T cell dysfunction have become a hot research topic, and there is increased interest on how changes in metabolomics correlate with CD8⁺ T cell dysfunction.AIMTo investigate whether and how glutamine metabolism affects the function of infiltrating CD8⁺ T cells in hepatocellular carcinoma.METHODSImmunohistochemical staining and immunofluorescence were performed on surgically resected liver tissues from patients. Differentially expressed genes in infiltrating CD8⁺ T cells in hepatocellular carcinoma were detected using RNA sequencing. Activated CD8⁺ T cells were co-cultured with Huh-7 cells for 3 d. The function and mitochondrial status of CD8⁺ T cells were analyzed by flow cytometry, quantitative real-time polymerase chain reaction, and transmission electron microscopy. Next, CD8⁺ T cells were treated with the mitochondrial protective and damaging agents. Functional alterations in CD8⁺ T cells were detected by flow cytometry. Then, complete medium without glutamine was used to culture cells and their functional changes and mitochondrial status were detected.RESULTSThere were a large number of infiltrating PD-1⁺CD8⁺ T cells in liver cancer tissues. Next, we co-cultured CD8⁺ T cells and Huh-7 cells to explore the regulatory effect of hepatoma cells on CD8⁺ T cells. Flow cytometry results revealed increased PD-1 expression and decreased secretion of perforin (PRF1) and granzyme B (GZMB) by CD8⁺ T cells in the co-culture group. Meanwhile, JC-1 staining was decreased and the levels of reactive oxygen species and apoptosis were increased in CD8⁺ T cells of the co-culture group; additionally, the mitochondria of these cells were swollen. When CD8⁺ T cells were treated with the mitochondrial protective and damaging agents, their function was restored and inhibited, respectively, through the mitochondrial damage and apoptotic pathways. Subsequently, complete medium without glutamine was used to culture cells. As expected, CD8⁺ T cells showed functional downregulation, mitochondrial damage, and apoptosis.CONCLUSIONGlutamine deprivation impairs the function of infiltrating CD8⁺ T cells in hepatocellular carcinoma through the mitochondrial damage and apoptotic pathways.