Targeting of epidermal Langerhans cells with antigenic proteins: attempts to harness their properties for immunotherapy

Langerhans cells, a subset of skin dendritic cells in the epidermis, survey peripheral tissue for invading pathogens. In recent functional studies it was proven that Langerhans cells can present exogenous antigen not merely on major histocompatibility complexes (MHC)-class II molecules to CD4⁺ T cells, but also on MHC-class I molecules to CD8⁺ T cells. Immune responses against topically applied antigen could be measured in skin-draining lymph nodes. Skin barrier disruption or co-application of adjuvants was required for maximal induction of T cell responses. Cytotoxic T cells induced by topically applied antigen inhibited tumor growth in vivo, thus underlining the potential of Langerhans cells for immunotherapy. Here we review recent work and report novel observations relating to the potential use of Langerhans cells for immunotherapy. We investigated the potential of epicutaneous immunization strategies in which resident skin dendritic cells are loaded with tumor antigen in situ. This contrasts with current clinical approaches, where dendritic cells generated from progenitors in blood are loaded with tumor antigen ex vivo before injection into cancer patients. In the current study, we applied either fluorescently labeled protein antigen or targeting antibodies against DEC-205/CD205 and langerin/CD207 topically onto barrier-disrupted skin and examined antigen capture and transport by Langerhans cells. Protein antigen could be detected in Langerhans cells in situ, and they were the main skin dendritic cell subset transporting antigen during emigration from skin explants. Potent in vivo proliferative responses of CD4⁺ and CD8⁺ T cells were measured after epicutaneous immunization with low amounts of protein antigen. Targeting antibodies were mainly transported by langerin⁺ migratory dendritic cells of which the majority represented migratory Langerhans cells and a smaller subset the new langerin⁺ dermal dendritic cell population located in the upper dermis. The preferential capture of topically applied antigen by Langerhans cells and their ability to induce potent CD4⁺ and CD8⁺ T cell responses emphasizes their potential for epicutaneous immunization strategies. This article is a symposium paper from the conference “Immunotherapy—From Basic Research to Clinical Applications,” Symposium of the Collaborative Research Center (SFB) 685, held in Tübingen, Germany, 6–7 March 2008.     

Dendritic Cell-Directed Vaccination with a Lentivector Encoding PSCA for Prostate Cancer in Mice

Many studies have demonstrated that prostate stem cell antigen (PSCA) is an attractive target for immunotherapy based on its overexpression in prostate tumor tissue, especially in some metastatic tissues. In this study, we evaluated dendritic cell (DC)-directed lentiviral vector (DCLV) encoding murine PSCA (DCLV-PSCA) as a novel tumor vaccine for prostate cancer in mouse models. We showed that DCLV-PSCA could preferentially deliver the PSCA antigen gene to DC-SIGN-expressing 293T cells and bone marrow-derived DCs (BMDCs). Direct immunization with the DCLV-PSCA in male C57BL/6 mice elicited robust PSCA-responsive CD8⁺ and CD4⁺ T cells in vivo. In a transgenic adenocarcinoma mouse prostate cell line (TRAMP-C1) synergetic tumor model, we further demonstrated that DCLV-PSCA-vaccinated mice could be protected from lethal tumor challenge in a prophylactic model, whereas slower tumor growth was observed in a therapeutic model. This DCLV-PSCA vaccine also showed efficacy in inhibiting tumor metastases using a PSCA-expressing B16-F10 model. Taken together, these data suggest that DCLV is a potent vaccine carrier for PSCA in delivering anti-prostate cancer immunity.     

Lung carcinomas do not induce T-cell apoptosis via the Fas/Fas ligand pathway but down-regulate CD3 epsilon expression

Background Non-small cell lung carcinoma (NSCLC) patients have impaired cellular immune responses. It has been hypothesized that tumor cells expressing Fas Ligand (FasL) induce in T lymphocytes: (a) apoptosis (tumor counterattack) and (b) down-regulation of CD3ζ expression. However, the hypothesis of tumor counterattack is still controversial. Methods We analyzed FasL expression on NSCLC cell lines and on tumor cells from lung adenocarcinoma patients by flow cytometry and immunocytochemistry. FasL mRNA expression was detected in NSCLC cell lines using RT-PCR, and functional FasL was evaluated on Fas-expressing Jurkat T-cells by annexin-V-FITC staining and by SubG₁ peak detection. Also, the proapoptotic effect of microvesicles released from NSCLC cell lines in Jurkat T-cells was studied. Alterations in the expression levels of CD3ζ, CD3ε, and CD28 [measured as mean fluorescence intensity (MFI)] were determined in Jurkat T-cells after co-culture with NSCLC cell lines or tumor-derived microvesicles. Furthermore, the expression levels of CD3ζ and CD3ε in CD4+T and CD8+T lymphocytes from lung adenocarcinoma patients was studied. Results Our results indicate that NSCLC cells neither FasL expressed nor induced apoptosis in Jurkat T-cells. Tumor-derived microvesicles did not induce apoptosis in Jurkat T-cells. In contrast, NSCLC cell lines down-regulated CD3ε but not CD3ζ chain expression in Jurkat T-cells; this effect was induced by soluble factors but not by microvesicles. In lung adenocarcinoma patients, significant decreases of MFI values for CD3ε, but not CD3ζ, were found in CD4+T and CD8+T cells from pleural effusion compared to peripheral blood and in peripheral blood of patients compared to healthy donors. Conclusions Our data do not support the tumor counterattack hypothesis for NSCLC. Nonetheless, down-regulation of CD3ε in T-cells induced by NSCLC cells might lead to T-cell dysfunction.     

Tumor Mouse Model Confirms MAGE-A3 Cancer Immunotherapeutic As an Efficient Inducer of Long-Lasting Anti-Tumoral Responses

Purpose

MAGE-A3 is a potential target for immunotherapy due to its tumor-specific nature and expression in several tumor types. Clinical data on MAGE-A3 immunotherapy have raised many questions that can only be addressed by using animal models. In the present study, different aspects of the murine anti-tumor immune responses induced by a recombinant MAGE-A3 protein (recMAGE-A3) in combination with different immunostimulants (AS01, AS02, CpG7909 or AS15) were investigated.

Experimental Design and Results

Based on cytokine profile analyses and protection against challenge with MAGE-A3-expressing tumor, the combination recMAGE-A3+AS15 was selected for further experimental work, in particular to study the mechanisms of anti-tumor responses. By using MHC class I-, MHC class II-, perforin-, B-cell- and IFN-γ- knock-out mice and CD4⁺ T cell-, CD8⁺ T cell- and NK cell- depleted mice, we demonstrated that CD4⁺ T cells and NK cells are the main anti-tumor effectors, and that IFN-γ is a major effector molecule. This mouse tumor model also established the need to repeat recMAGE-A3+AS15 injections to sustain efficient anti-tumor responses. Furthermore, our results indicated that the efficacy of tumor rejection by the elicited anti-MAGE-A3 responses depends on the proportion of tumor cells expressing MAGE-A3.

Conclusions

The recMAGE-A3+AS15 cancer immunotherapy efficiently induced an antigen-specific, functional and long-lasting immune response able to recognize and eliminate MAGE-A3-expressing tumor cells up to several months after the last immunization in mice. The data highlighted the importance of the immunostimulant to induce a Th1-type immune response, as well as the key role played by IFN-γ, CD4⁺ T cells and NK cells in the anti-tumoral effect.     

Ectonucleotidase CD39 and Checkpoint Signalling Receptor Programmed Death 1 are Highly Elevated in Intratumoral Immune Cells in Non–small-cell Lung Cancer

Lung cancer is the leading cause of cancer death in both sexes worldwide and has a predicted 5-year survival rate of <20%. Immunotherapy targeting immune checkpoints such as the programmed death 1 (PD-1) signaling pathway has led to a shift of paradigm in the treatment of advanced non–small-cell lung cancer (NSCLC) but remains without effect in ∼80% of patients. Accumulating evidence suggests that several immunosuppressive mechanisms may work together in NSCLC. The contribution and cooperation between different immunosuppressive mechanisms in NSCLC remain unknown. Recently, the CD39-adenosine pathway has gained increasing attention as a crucial immunosuppressive mechanism and possible target for immunotherapy. Immune cells were extracted from lung and tumor tissue after lung resection in 12 patients by combined enzymatic and mechanical tissue disaggregation. A multiparameter flow cytometry panel was established to investigate the expression and coexpression of CD39 and PD-1 on key lymphocyte subtypes. Frequencies of CD39⁺, PD-1⁺, and CD39⁺/PD-1⁺cells were higher among both CD4⁺ and CD8⁺ T cells isolated from NSCLC tumor tissue than in T cells from normal lung tissue. Similarly, the frequency of FoxP3⁺ CD4⁺ T cells (Tregs) was highly significantly elevated in tumor tissue compared to adjacent lung tissue. The consistent upregulation of CD39 on immune cells in tumor microenvironment indicates that the CD39 signaling pathway may, in addition to the PD-1 pathway, represent another important mechanism for tumor-induced immunosuppression in NSCLC. In addition, the present study indicates that a comprehensive immune response profiling with flow cytometry may be both feasible and clinically relevant.     

Surgical Stress Abrogates Pre-Existing Protective T Cell Mediated Anti-Tumor Immunity Leading to Postoperative Cancer Recurrence

Anti-tumor CD8⁺ T cells are a key determinant for overall survival in patients following surgical resection for solid malignancies. Using a mouse model of cancer vaccination (adenovirus expressing melanoma tumor-associated antigen (TAA)—dopachrome tautomerase (AdDCT) and resection resulting in major surgical stress (abdominal nephrectomy), we demonstrate that surgical stress results in a reduction in the number of CD8⁺ T cell that produce cytokines (IFNγ, TNFα, Granzyme B) in response to TAA. This effect is secondary to both reduced proliferation and impaired T cell function following antigen binding. In a prophylactic model, surgical stress completely abrogates tumor protection conferred by vaccination in the immediate postoperative period. In a clinically relevant surgical resection model, vaccinated mice undergoing a positive margin resection with surgical stress had decreased survival compared to mice with positive margin resection alone. Preoperative immunotherapy with IFNα significantly extends survival in surgically stressed mice. Importantly, myeloid derived suppressor cell (MDSC) population numbers and functional impairment of TAA-specific CD8⁺ T cell were altered in surgically stressed mice. Our observations suggest that cancer progression may result from surgery-induced suppression of tumor-specific CD8⁺ T cells. Preoperative immunotherapies aimed at targeting the prometastatic effects of cancer surgery will reduce recurrence and improve survival in cancer surgery patients.     

B7-H1 signaling is integrated during CD8+ T cell priming and restrains effector differentiation

A promising strategy in tumor immunotherapy is the use of activated dendritic cells as vehicles for tumor vaccines with the goal of activating anti-tumor T cell responses. Current formulations for dendritic cell-based immunotherapies have limited effects on patient survival, providing motivation for further investigation of ways to enhance dendritic cell priming of anti-tumor T cell responses. Using a brief in vitro priming model, we have found that B7-H1 expressed by activated dendritic cells is integrated during priming of naïve CD8⁺ T cells and functions to limit the differentiation of effector T cell responses. CD8⁺ T cells primed by B7-H1-deficient dendritic cells exhibit increased production of IFN-γ, enhanced target cell killing, and improved anti-tumor activity. Additionally, enhanced memory populations arise from CD8⁺ T cells primed by B7-H1-deficient dendritic cells. Based on these findings, we suggest that early blockade of B7-H1 signaling should be investigated as a strategy to improve dendritic cell-based anti-tumor immunotherapy.     

Identification of new cytotoxic T-lymphocyte epitopes from cancer testis antigen HCA587

The cancer testis (CT) antigen HCA587 is highly expressed in human hepatocellular carcinoma (HCC) and induces specific T-cell responses in a significant proportion of HCC patients. To explore its potential in cancer immunotherapy, a reverse immunology approach was adopted to identify HCA587-derived HLA-A^∗0201-restricted epitopes. Multiple peptides with a top ranking in various prediction programs were thus synthesized and three of them—p248-256, p140-149 and p144-152—were found to bind to HLA-A^*0201 molecules with a high affinity and effectively induced a recall response of CD8⁺ T cells, which were either primed in vitro with the HCA587 antigen or directly isolated from HCC patients bearing HCA587⁺ tumors. Notably, these peptide-specific CD8⁺ T cells exhibited potent cytotoxic activity over HCA587⁺ tumor cells. Taken together, the present study has identified three new HLA-A^*0201-restricted cytotoxic T cell epitopes in the CT antigen HCA587, which may serve as targets for peptide-based immunotherapy for HCC patients.     

STEAP, a prostate tumor antigen, is a target of human CD8+ T cells

STEAP is a recently identified protein shown to be particularly overexpressed in prostate cancer and also present in numerous human cancer cell lines from prostate, pancreas, colon, breast, testicular, cervical, bladder and ovarian carcinoma, acute lymphocytic leukemia and Ewing sarcoma. This expression profile renders STEAP an appealing candidate for broad cancer immunotherapy. In order to investigate if STEAP is a tumor antigen that can be targeted by specific CD8⁺ T cells, we identified two high affinity HLA-A*0201 restricted peptides (STEAP_86–94 and STEAP_262–270). These peptides were immunogenic in vivo in HLA-A*0201 transgenic HHD mice. Peptide specific murine CD8 T cells recognized COS-7 cells co-transfected with HHD (HLA-A*0201) and STEAP cDNA constructs and also HLA-A*0201⁺ STEAP⁺ human tumor cells. Furthermore, STEAP_86–94 and STEAP_262–270 stimulated specific CD8⁺ T cells from HLA-A*0201⁺ healthy donors, and these peptide specific CD8⁺ T cells recognized STEAP positive human tumor cells in an HLA-A*0201-restricted manner. Importantly, STEAP_86–94-specific T cells were detected and reactive in the peripheral blood mononuclear cells in NSCLC and prostate cancer patients ex vivo. These results show that STEAP can be a target of anti-tumor CD8⁺ T cells and that STEAP peptides can be used for a broad-spectrum-tumor immunotherapy.     

The generation of anti-tumoral cells using dentritic cells from the peripheral bloood of patients with malignant brain tumors

 Dendritic cells (DCs) can be the principal initiators of antigen-specific immune responses. We analyzed the in vitro-responses against brain tumor cells using DCs from the peripheral blood of patients with brain tumors. Peripheral blood mononuclear cells (PBMC) were obtained from 19 patients with malignant brain tumors: 12 metastatic brain tumors of lung adenocarcinoma, 7 high-grade astrocytomas. PBMC were cultured with 100 ng/ml of GM-CSF and 10 ng/ml of IL-4 for 5–7 days in order to produce mature DCs. The autologous tumor lysate (5 mg/ml, containing 1 × 10⁶ cells) was then added to the cultured DCs. Using the DCs generated by these treatments, we assessed the changes that occurred in their immune responses against brain tumor via ⁵¹Cr-release and lymphocyte proliferation assays. We found that the matured DCs displayed the typical surface phenotype of CD3⁺, CD45⁺, CD80⁺ and CD86⁺. After the pulsation treatment with tumor lysate, DCs were found to have strong cytotoxic T lymphocyte activity, showing 42.5 ± 12.7% killing of autologous tumor cells. We also found an enhancement of allogeneic T cell proliferation after pulsing the DC with tumor lysate. These data support the efficacy of DC-based immunotherapy for patients with malignant brain tumors. Received: 2 October 2000 / Accepted: 26 April 2001     

Phenotypic T Cell Exhaustion in a Murine Model of Bacterial Infection in the Setting of Pre-Existing Malignancy

While much of cancer immunology research has focused on anti-tumor immunity both systemically and within the tumor microenvironment, little is known about the impact of pre-existing malignancy on pathogen-specific immune responses. Here, we sought to characterize the antigen-specific CD8⁺ T cell response following a bacterial infection in the setting of pre-existing pancreatic adenocarcinoma. Mice with established subcutaneous pancreatic adenocarcinomas were infected with Listeria monocytogenes, and antigen-specific CD8⁺ T cell responses were compared to those in control mice without cancer. While the kinetics and magnitude of antigen-specific CD8⁺ T cell expansion and accumulation was comparable between the cancer and non-cancer groups, bacterial antigen-specific CD8⁺ T cells and total CD4⁺ and CD8⁺ T cells in cancer mice exhibited increased expression of the coinhibitory receptors BTLA, PD-1, and 2B4. Furthermore, increased inhibitory receptor expression was associated with reduced IFN-γ and increased IL-2 production by bacterial antigen-specific CD8⁺ T cells in the cancer group. Taken together, these data suggest that cancer''s immune suppressive effects are not limited to the tumor microenvironment, but that pre-existing malignancy induces phenotypic exhaustion in T cells by increasing expression of coinhibitory receptors and may impair pathogen-specific CD8⁺ T cell functionality and differentiation.     

PD-1抑制剂Pembrolizumab联合培美曲塞和卡铂对NSCLC患者T淋巴细胞亚群及血清肿瘤标志物的影响

摘要 目的：探讨PD-1抑制剂Pembrolizumab联合培美曲塞和卡铂对非小细胞肺癌（NSCLC）患者T淋巴细胞亚群及血清肿瘤标志物的影响。方法：将苏州大学附属第一医院2018年1月到2020年1月期间收治的80例NSCLC患者根据随机数字表法分为对照组（40例,培美曲塞和卡铂治疗）和观察组（40例,对照组基础上联合PD-1抑制剂Pembrolizumab治疗）。对比两组疗效、T淋巴细胞亚群、血清肿瘤标志物、生存质量及不良反应。结果：观察组的客观有效率、疾病控制率均高于对照组（P<0.05）。与对照组治疗后相比,观察组Karnofsky功能状态（KPS）评分更高（P<0.05）。与对照组治疗后相比,观察组CD3⁺、CD4⁺、CD4⁺/CD8⁺更高,CD8⁺更低（P<0.05）。与对照组治疗后相比,观察组癌胚抗原（CEA）、糖类抗原125（CA125）及细胞角质素片段抗原21-1（CYFRA 21-1）更低（P<0.05）。两组不良反应发生率比较未见差异（P>0.05）。结论：NSCLC患者在化疗基础上联合PD-1抑制剂Pembrolizumab治疗,可提高其治疗效果及生存质量,减轻免疫抑制,阻止肿瘤进展,且安全性较好。     

Identification of tumor antigens as potential target antigens for immunotherapy by serological expression cloning

The presence of tumor infiltrating T cells has been shown to be associated with a favorable prognosis in different tumor types. Several strategies have been developed to identify relevant tumor antigens which can be used for active immunotherapy strategies. The SEREX technique (serological analysis of cDNA expression libraries) identifies tumor antigens based on a spontaneous humoral immune response in cancer patients. This technique is not limited to tumor types that can be grown in cell culture or depends on established T cell clones recognizing the autologous tumor. Several steps of analysis are mandatory to evaluate SEREX-defined antigens before they become new target antigens for active immunotherapy: expression analysis; serological analysis with sera from tumor patients and normal individuals; identification of potential peptide epitopes for CD8 T cells and evaluation in T cell assays. This article summarizes our approach of antigen identification and evaluation giving the example of the recently cloned breast cancer antigen NY-BR-1.This work was presented at the first Cancer Immunology and Immunotherapy Summer School, 8–13 September 2003, Ionian Village, Bartholomeio, Peloponnese, Greece.     

Tumor-infiltrating T cells correlate with NY-ESO-1-specific autoantibodies in ovarian cancer

Background

Tumor-infiltrating CD8+ T cells are correlated with prolonged progression-free and overall survival in epithelial ovarian cancer (EOC). A significant fraction of EOC patients mount autoantibody responses to various tumor antigens, however the relationship between autoantibodies and tumor-infiltrating T cells has not been investigated in EOC or any other human cancer. We hypothesized that autoantibody and T cell responses may be correlated in EOC and directed toward the same antigens.

Methodology and Principal Findings

We obtained matched serum and tumor tissue from 35 patients with high-grade serous ovarian cancer. Serum samples were assessed by ELISA for autoantibodies to the common tumor antigen NY-ESO-1. Tumor tissue was examined by immunohistochemistry for expression of NY-ESO-1, various T cell markers (CD3, CD4, CD8, CD25, FoxP3, TIA-1 and Granzyme B) and other immunological markers (CD20, MHC class I and MHC class II). Lymphocytic infiltrates varied widely among tumors and included cells positive for CD3, CD8, TIA-1, CD25, FoxP3 and CD4. Twenty-six percent (9/35) of patients demonstrated serum IgG autoantibodies to NY-ESO-1, which were positively correlated with expression of NY-ESO-1 antigen by tumor cells (r = 0.57, p = 0.0004). Autoantibodies to NY-ESO-1 were associated with increased tumor-infiltrating CD8+, CD4+ and FoxP3+ cells. In an individual HLA-A2+ patient with autoantibodies to NY-ESO-1, CD8+ T cells isolated from solid tumor and ascites were reactive to NY-ESO-1 by IFN-γ ELISPOT and MHC class I pentamer staining.

Conclusion and Significance

We demonstrate that tumor-specific autoantibodies and tumor-infiltrating T cells are correlated in human cancer and can be directed against the same target antigen. This implies that autoantibodies may collaborate with tumor-infiltrating T cells to influence clinical outcomes in EOC. Furthermore, serological screening methods may prove useful for identifying clinically relevant T cell antigens for immunotherapy.     

Galectins and their ligands: negative regulators of anti-tumor immunity

Cytotoxic CD8⁺ T cells are major players of anti-tumor immune responses, as their functional activity can limit tumor growth and progression. Data show that cytotoxic T cells efficiently control the proliferation of tumor cells through major histocompatibility complex class I-mediated mechanisms; nevertheless, the presence of tumor-infiltrating CD8⁺ T cells in lesional tissue does not always correlate with better prognosis and increased survival of cancer patients. Similarly, adoptive transfer of tumor-specific cytotoxic T cells has only shown marginal improvement in life spans of patients with metastatic disease. In this report, we discuss experimental evidence showing that expression of tumor-derived galectins, galectin (Gal)-1, Gal-3 and Gal-9, and concomitant presence of their ligands on the surface of anti-tumor immunocytes directly compromise anti-tumor CD8⁺ T cell immune responses and, perhaps, undermine the promise of adoptive CD8⁺ T cell immunotherapy. Furthermore, we describe novel strategies designed to counteract Gal-1-, Gal-3- and Gal-9-mediated effects and highlight their targeting potential for creating more effective anti-tumor immune responses. We believe that Gal and their ligands represent an efficacious targeted molecular paradigm that warrants clinical evaluation.     

Induction of tumor-reactive T helper responses by a posttranslational modified epitope from tumor protein p53

Posttranslational modifications regulate the function and stability of proteins, and the immune system is able to recognize some of these modifications. Therefore, the presence of posttranslational modifications increases the diversity of potential immune responses to a determinant antigen. The stimulation of tumor-specific CD4⁺ helper T lymphocytes (HTLs) is considered important for the production of anti-tumor antibodies by B cells and for the generation and persistence of CD8⁺ cytotoxic T lymphocytes, and in some instances, HTLs can directly reduce tumor cell growth. Identification of MHC class II-restricted peptide epitopes from tumor-associated antigens including those generated from posttranslational protein modifications should enable the improvement of peptide-based cancer immunotherapy. We describe here an MHC class II binding peptide from the tumor protein p53, which possesses an acetylated lysine at position 120 (p53_{110-124/AcK120}) that is effective in eliciting CD4⁺ T cell responses specific for the acetylated peptide. Most importantly, the acetylated peptide-reactive CD4 HTLs recognized the corresponding naturally processed posttranslational modified epitope presented by either dendritic cells loaded with tumor cell lysates or directly on tumors expressing p53 and the restricting MHC class II molecules. Treatment of tumor cells with a histone deacetylase inhibitor augmented their recognition by the p53_{110-124/AcK120}-reactive CD4⁺ T cells. These findings prove that the epitope p53_{110-124/AcK120} is immunogenic for anti-tumor responses and is likely to be useful for cancer immunotherapy.     

Identification and HLA-Tetramer-Validation of Human CD4+ and CD8+ T Cell Responses against HCMV Proteins IE1 and IE2

Human cytomegalovirus (HCMV) is an important human pathogen. It is a leading cause of congenital infection and a leading infectious threat to recipients of solid organ transplants as well as of allogeneic hematopoietic cell transplants. Moreover, it has recently been suggested that HCMV may promote tumor development. Both CD4⁺ and CD8⁺ T cell responses are important for long-term control of the virus, and adoptive transfer of HCMV-specific T cells has led to protection from reactivation and HCMV disease. Identification of HCMV-specific T cell epitopes has primarily focused on CD8⁺ T cell responses against the pp65 phosphoprotein. In this study, we have focused on CD4⁺ and CD8⁺ T cell responses against the immediate early 1 and 2 proteins (IE1 and IE2). Using overlapping peptides spanning the entire IE1 and IE2 sequences, peripheral blood mononuclear cells from 16 healthy, HLA-typed, donors were screened by ex vivo IFN-γ ELISpot and in vitro intracellular cytokine secretion assays. The specificities of CD4⁺ and CD8⁺ T cell responses were identified and validated by HLA class II and I tetramers, respectively. Eighty-one CD4⁺ and 44 CD8⁺ T cell responses were identified representing at least seven different CD4 epitopes and 14 CD8 epitopes restricted by seven and 11 different HLA class II and I molecules, respectively, in total covering 91 and 98% of the Caucasian population, respectively. Presented in the context of several different HLA class II molecules, two epitope areas in IE1 and IE2 were recognized in about half of the analyzed donors. These data may be used to design a versatile anti-HCMV vaccine and/or immunotherapy strategy.     

Manganese is critical for antitumor immune responses via cGAS-STING and improves the efficacy of clinical immunotherapy

CD8⁺ T cell-mediated cancer clearance is often suppressed by the interaction between inhibitory molecules like PD-1 and PD-L1, an interaction acts like brakes to prevent T cell overreaction under normal conditions but is exploited by tumor cells to escape the immune surveillance. Immune checkpoint inhibitors have revolutionized cancer therapeutics by removing such brakes. Unfortunately, only a minority of cancer patients respond to immunotherapies presumably due to inadequate immunity. Antitumor immunity depends on the activation of the cGAS-STING pathway, as STING-deficient mice fail to stimulate tumor-infiltrating dendritic cells (DCs) to activate CD8⁺ T cells. STING agonists also enhance natural killer (NK) cells to mediate the clearance of CD8⁺ T cell-resistant tumors. Therefore STING agonists have been intensively sought after. We previously discovered that manganese (Mn) is indispensable for the host defense against cytosolic dsDNA by activating cGAS-STING. Here we report that Mn is also essential in innate immune sensing of tumors and enhances adaptive immune responses against tumors. Mn-insufficient mice had significantly enhanced tumor growth and metastasis, with greatly reduced tumor-infiltrating CD8⁺ T cells. Mechanically, Mn²⁺ promoted DC and macrophage maturation and tumor-specific antigen presentation, augmented CD8⁺ T cell differentiation, activation and NK cell activation, and increased memory CD8⁺ T cells. Combining Mn²⁺ with immune checkpoint inhibition synergistically boosted antitumor efficacies and reduced the anti-PD-1 antibody dosage required in mice. Importantly, a completed phase 1 clinical trial with the combined regimen of Mn²⁺ and anti-PD-1 antibody showed promising efficacy, exhibiting type I IFN induction, manageable safety and revived responses to immunotherapy in most patients with advanced metastatic solid tumors. We propose that this combination strategy warrants further clinical translation.Subject terms: Pattern recognition receptors, Immunosurveillance