Interaction of PD-L1 on tumor cells with PD-1 on tumor-specific T cells as a mechanism of immune evasion: implications for tumor immunotherapy

Programmed death receptor ligand 1 (PD-L1, also called B7-H1) is a recently described B7 family member. In contrast to B7-1 and B7-2, PD-L1 does not interact with either CD28 or CTLA-4. To date, one specific receptor has been identified that can be ligated by PD-L1. This receptor, programmed death receptor 1 (PD-1), has been shown to negatively regulate T-cell receptor (TCR) signaling. Upon ligating its receptor, PD-L1 has been reported to decrease TCR-mediated proliferation and cytokine production. PD-1 gene–deficient mice developed autoimmune diseases, which early led to the hypothesis of PD-L1 regulating peripheral tolerance. In contrast to normal tissues, which show minimal surface expression of PD-L1 protein, PD-L1 expression was found to be abundant on many murine and human cancers and could be further up-regulated upon IFN- stimulation. Thus, PD-L1 might play an important role in tumor immune evasion. This review discusses the currently available data concerning negative T-cell regulation via PD-1, the blockade of PD-L1/PD-1 interactions, and the implications for adoptive T-cell therapies.     

Dynamic cross-talk between tumor and immune cells in orchestrating the immunosuppressive network at the tumor microenvironment

Accumulating evidence indicates that a dynamic cross-talk between tumors and the immune system can regulate tumor growth and metastasis. Increased understanding of the biochemical nature of tumor antigens and the molecular mechanisms responsible for innate and adaptive immune cell activation has revolutionized the fields of tumor immunology and immunotherapy. Both the protective effects of the immune system against tumor cells (immunosurveillance) and the evasion of tumor cells from immune attack (tumor-immune escape) have led to the concept of cancer immunoediting, a proposal which infers that a bidirectional interaction between tumor and inflammatory/regulatory cells is ultimately responsible for orchestrating the immunosuppressive network at the tumor site. In this context, a major challenge is the potentiation or redirection of tumor antigen-specific immune responses. The success in reaching this goal is highly dependent on an improved understanding of the interactions and mechanisms operating during the different phases of the cancer immunoediting process. In this review, we discuss the multiple defense and counterattack strategies that tumors have devised in order to evade immune attack and to thwart the effectiveness of several immunotherapeutic approaches. Diego O. Croci, Mariano F. Zacarías Fluck contributed equally to this work. Gabriel A. Rabinovich, O. Graciela Scharovsky contributed equally to this work and should be considered as senior authors.     

Immune responses to p53 in patients with cancer:enrichment in tetramer+ p53 peptide-specific T cells and regulatory T cells at tumor sites

Objective: A majority of human cancers, including head and neck cancer (HNC), overexpress p53. Although T cells specific for wild-type (wt) sequence p53 peptides are detectable in the peripheral blood of patients with HNC, it is unknown whether such T cells accumulate in tumor-involved tissues. Also, the localization of regulatory T cells (Treg) to tumor sites in HNC has not been investigated to date. Methods: Tumor infiltrating lymphocytes (TIL), tumor-involved or non-involved lymph node lymphocytes (LNL) and peripheral blood mononuclear cells (PBMC) were obtained from 24 HLA-A2.1+ patients with HNC. Using tetramers and four-color flow cytometry, the frequency of Treg and CD3+CD8+ T cells specific for wt p53 epitopes as well as their functional attributes were determined. Results: The CD3+CD8+ tetramer+ cell frequency was significantly higher (P<0.001) in TIL than autologous PBMC as was the percentage of CD4+CD25+ T cells (P<0.003). TIL were enriched in FOXp3+, GITR+ and CTLA-4+ Treg. CD8+ TIL had low expression and produced little IFN- after ex vivo stimulation relative to autologous PBMC or PBMC from NC. Conclusions: Anti-wt p53 epitope-specific T cells and Treg preferentially localize to tumor sites in patients with HNC. However, despite enrichment in tumor peptide-specific T cells, the effector cell population (CD3+CD8+) in TIL or PBMC was unresponsive to activation in the tumor microenvironment enriched in Treg.     

1型调节性T细胞的表型和功能

1型调节性T细胞(Tr1)在免疫耐受的诱导和维持过程中发挥重要作用。Tr1细胞通常在免疫耐受的环境中由外来抗原诱导产生,通过产生高水平的IL-10而发挥免疫抑制作用。而CD4 CD25 调节性T细胞(Tregs)可在胸腺中天然产生也可在外周被抗原诱导产生,通过细胞接触发挥抑制作用。现对Tr1细胞的表型、功能及其抑制作用机制等方面的研究进展进行综述。     

Comprehensive analysis of tumor mutation burden and immune microenvironment in gastric cancer

Tumor mutation burden (TMB) was a promising marker for immunotherapy. We aimed to investigate the prognostic role of TMB and its relationship with immune cells infiltration in gastric cancer (GC). We analyzed the mutation landscape of all GC cases and TMB of each GC patient was calculated and patients were divided into TMB-high and TMB-low group. Differentially expressed genes (DEGs) between the two groups were identified and pathway analysis was performed. The immune cells infiltration in each GC patient was evaluated and Kaplan–Meier analysis was performed to investigate the prognostic role of immune cells infiltration. At last, hub immune genes were identified and a TMB prognostic risk score (TMBPRS) was constructed to predict the survival outcome of GC patients. The relationships between mutants of hub immune genes and immune infiltration level in GC was investigated. We found higher TMB was correlated with better survival outcome and female patients, patients with T1-2 and N0 had higher TMB score. Altogether 816 DEGs were harvested and pathway analysis demonstrated that patients in TMB-high group were associated with neuroactive ligand–receptor interaction, cAMP signaling pathway, calcium signaling pathway. The infiltration of activated CD4⁺ memory T cells, follicular helper T cells, resting NK cells, M0 and M1 macrophages and neutrophils in TMB-high group were higher compared than that in TMB-low group and high macrophage infiltration was correlated with inferior survival outcome of GC patients. Lastly, the TMBPRS was constructed and GC patients with high TMBPRS had poor prognosis.     

Relationship between regulatory and type 1 T cells in dogs with oral malignant melanoma

Recent data suggest a decreased prevalence of IFN‐γ‐producing T lymphocytes (Type 1 T cells) in tumor‐bearing hosts. Moreover, it has been reported that Treg have a strong impact on the activation and proliferation of CD4 (+) and CD8 (+) lymphocytes; however, no previous reports have described the relationship between Treg and the progression of tumor, or Type 1 T cell populations in dogs with malignant tumor. In this study, the percentage of Treg, Th1, and Tc1 in the peripheral blood of dogs with oral malignant melanoma and healthy dogs was measured and compared. Although the percentages of Th1 and Tc1 in dogs with oral malignant melanoma were less than those in healthy dogs (Th1: P < 0.01, Tc1: P < 0.05), the percentage of Treg was increased (P < 0.01). A significant inverse correlation between the percentage of Tc1 and the clinical tumor stage (P < 0.01), and a significant correlation between that of Treg and the clinical tumor stage (P < 0.001) was found. Moreover, there was a significant inverse correlation between the percentages of Treg and Th1 (P < 0.05) or Tc1 (P < 0.001). In conclusion, the percentage of Treg increases with the tumor stage in the peripheral blood of dogs with oral malignant melanoma. In dogs, Treg appears to suppress Type 1 immunity, which may be responsible for anti‐tumor responses.     

Studies of the T1 and T2 of intracellular water as a function of frequency in normal and transformed fetal cells

Frequency-dependent values of the spin-lattice relaxation time (T₁) and the spin-spin relaxation time (T₂) have been obtained for intracellular water in normal and transformed Syrian hamster fetal fibroblasts. Values of T₁ and T₂ were obtained for normal and transformed cells at 24.3 (0.57 T), 100 (2.4 T), 300 (7.0 T), and 400 MHz (9.4 T). At each frequency, values of T₁ were the same for both normal and transformed cells, whereas values of T₂ were lower for one passage of transformed cells. As expected, T₁ increased with frequency. However, T₂ decreased with frequency for both normal and transformed cells. The frequency dependence of T₂, was similar for all cells; thus, the ability of T₂ to make a distinction between normal and transformed cells did not change with field.     

Decreased number and impaired function of type 1 regulatory T cells in autoimmune diseases

Type 1 regulatory T (Tr1) cell is a special type of T regulatory cells with surface molecular markers such as lymphocyte-activation gene 3 and CD49b. A key property of Tr1 cells is the capability to produce high-level interleukin 10 (IL-10) upon activation, in a FOXP3-independent manner. The immunosuppressive function of IL-10 producing Tr1 cells has been extensively studied for many years. Autoimmune diseases (AIDs) are conditions in which the immune system breaks down and starts to attack the body. AIDs include inflammatory bowel disease, rheumatoid arthritis, multiple sclerosis (MS), type 1 diabetes mellitus, Greaves' disease, and so forth. In recent years, more and more studies have documented that the number of Tr1 cells is decreased and the function is inhibited in a variety of AIDs, among which MS is the most widely studied. The protocol for engineering Tr1 cell therapy has been established and is gradually being used in clinical practice in recent years. Tr1 cell therapy has been proven to be safe and effective, but it is mainly involved in myeloid leukemia, graft versus host disease currently. Its therapeutic role in AIDs still needs to be further explored. In this study, we will summarize the research advances of Tr1 cells in AIDs, which will provide useful information for treating AIDs through Tr1 cell therapy in the future.     

Contrasting effects of IGF binding protein-3 expression in mammary tumor cells and the tumor microenvironment

IGFBP-3 has both stimulatory and inhibitory effects on cancer progression. The growth of EO771 mammary carcinoma cells as syngeneic tumors in C57BL/6 mice is reduced in Igfbp3-null (BP3KO) mice, suggesting that systemic IGFBP-3 enhances tumor progression. In this study we assessed the growth of EO771 cells expressing human IGFBP-3 in BP3KO mice. Cells expressing hIGFBP-3 showed decreased proliferation in vitro and increased levels of IGF-1 receptor (IGF1R) protein but not mRNA, consistent with sequestration of endogenous IGF by IGFBP-3. The growth rate of these cells was restored by exposure to IGF-1 or analogues with reduced affinity for IGFBP-3 (long Arg³-IGF-1) or IGF1R (Leu²⁴-IGF-1). In EO771 cells implanted orthotopically into mice, hIGFBP-3 expression by the cells inhibited tumor establishment in BP3KO but not wild-type mice. For tumors that successfully established, final weight was not affected significantly by hIGFBP-3 expression. However, final tumor weight was inversely related to intratumoral T cell counts, and sera from BP3KO mice with tumors showed low-titer immunoreactivity against IGFBP-3. The contrasting effects on tumor establishment and progression of IGFBP-3 expressed by mammary carcinoma cells, compared to systemic stromal and circulating IGFBP-3, highlights the complexity of growth regulation by IGFBP-3 in mammary tumors.     

Targeting the immune system: novel therapeutic approaches in squamous cell carcinoma of the head and neck

Despite advances in surgery, radiotherapy, and chemotherapy, the overall survival rates for patients with squamous cell carcinoma of the head and neck (SCCHN) have not changed over the last decades. Clearly, novel therapeutic strategies are needed for this cancer, which is highly immunosuppressive. Therefore, biologic therapies able to induce and/or up-regulate antitumor immune responses could represent a complementary approach to conventional treatments. Because patients with SCCHN are frequently immunocompromised due to the elimination or dysfunction of critical effector cells of the immune system, it might be necessary to restore these immune functions to allow for the generation of more effective antitumor host responses. Simultaneously, to prevent tumor escape, it might be necessary to alter attributes of the malignant cells. The present review summarizes recent advances in the field of immunotherapy of SCCHN, including techniques of nonspecific immune stimulation, the use of monoclonal antibodies, advances in adoptive immunotherapy and genetic engineering, as well as anticancer vaccines. These biologic therapies, alone or in combination with conventional treatment, are likely to develop into useful future treatment options for patients with SCCHN.     

PD-1/PD-L1与Treg细胞相关性的研究新进展

PD-1和PD-L属于B7家族的共刺激分子,介导免疫反应的负性调节信号。Treg细胞是一个具有免疫调节作用的T细胞亚群,在机体的免疫耐受和免疫稳定中具有重要作用。本文就PD-1/PD-L1与Treg细胞的免疫调节作用及相关性研究进展作简要综述。     

Adenosine and Prostaglandin E2 Cooperate in the Suppression of Immune Responses Mediated by Adaptive Regulatory T Cells

Adaptive regulatory T cells (Tr1) are induced in the periphery upon encountering cognate antigens. In cancer, their frequency is increased; however, Tr1-mediated suppression mechanisms are not yet defined. Here, we evaluate the simultaneous involvement of ectonucleotidases (CD39/CD73) and cyclooxygenase 2 (COX-2) in Tr1-mediated suppression. Human Tr1 cells were generated from peripheral blood mononuclear cell-derived, sorted CD4⁺CD25⁻ T cells and incubated with autologous immature dendritic cells, irradiated COX-2⁺ or COX-2⁻ tumor cells, and IL-2, IL-10, and IL-15 (each at 10–15 IU/ml) for 10 days as described (Bergmann, C., Strauss, L., Zeidler, R., Lang, S., and Whiteside, T. L. (2007) Cancer Immunol. Immunother. 56, 1429–1442). Tr1 were phenotyped by multicolor flow cytometry, and suppression of proliferating responder cells was assessed in carboxyfluorescein diacetate succinimidyl ester-based assays. ATP hydrolysis was measured using a luciferase detection assay, and levels of adenosine or prostaglandin E₂ (PGE₂) in cell supernatants were analyzed by mass spectrometry or ELISA, respectively. Intracellular cAMP levels were measured by enzyme immunoassay. The COX-2⁺ tumor induced a greater number of Tr1 than COX-2⁻ tumor (p < 0.05). Tr1 induced by COX-2⁺ tumor were more suppressive, hydrolyzed more exogenous ATP (p < 0.05), and produced higher levels of adenosine and PGE₂ (p < 0.05) than Tr1 induced by COX-2⁻ tumor. Inhibitors of ectonucleotidase activity, A_2A and EP₂ receptor antagonists, or an inhibitor of the PKA type I decreased Tr1-mediated suppression (p < 0.05), whereas rolipram, a PDE₄ inhibitor, increased the intracellular cAMP level in responder cells and their susceptibility to Tr1-mediated suppression. Tr1 present in tumors or the peripheral blood of head and neck squamous cell carcinoma patients co-expressed COX-2, CD39, and CD73. A concomitant inhibition of PGE₂ and adenosine via the common intracellular cAMP pathway might be a novel approach for improving results of immune therapies for cancer.     

A listing of human tumor antigens recognized by T cells: March 2004 update

The technological advances occurred in the last few years have led to a great increase in the number of tumor associated antigens (TAA) that are currently available for clinical applications. In this review we provide a comprehensive list of human tumor antigens as reported in the literature updated at Feburary 2004. The list includes all T cell-defined epitopes, while excluding analogs or artificially modified epitopes, as well as virus-encoded and antibodies-recognized antigens. TAAs are listed in alphabetical order along with the epitope sequence and the HLA allele which restricts recognition by T cells. Data on the tissue distribution of each antigen are also provided together with an extensive bibliography that allows a rapid search for any additional information may be needed on each single antigen or epitope. Overall, the updated list is a database tool for clinicians, scientists and students who have an interest in the field of tumor immunology and immunotherapy.     

Cross-correlation suppressed T1 and NOE experiments for protein side-chain 13CH2 groups

Relaxation measurements of side-chain ¹³CH₂-groups of uniformly ¹³C labeled human ubiquitin were performed at 600 MHz and 800 MHz magnetic field strength at 30°C. Dipole-dipole cross-correlated relaxation effects in T₁ experiments were suppressed by the combination of radio-frequency pulses and pulsed field gradients during the relaxation delay leading to monoexponential relaxation decays that allow a more accurate extraction of the ¹³C T₁ relaxation times. Heteronuclear ¹H-¹³C NOEs obtained by using different proton saturation schemes indicate that the influence of cross-correlation is small. The experimental T₁ and NOE data were interpreted in a model-free way in terms of a generalized order parameter and an internal correlation time.     

A MAGE-1 antigenic peptide recognized by human cytolytic T lymphocytes on HLA-A2 tumor cells

Cancer-germline genes such as those of the MAGE family are expressed in many tumors and in male germline cells, but are silent in normal tissues. They encode shared tumor-specific antigens that have been used in therapeutic vaccination trials of cancer patients. It was previously demonstrated that MAGE-1 peptide KVLEYVIKV was presented by HLA-A 0201 molecules on the surface of a human breast carcinoma cell line, but no human specific CTL had been isolated so far. Here, we have used HLA-A2/MAGE-1 fluorescent multimers to isolate from blood cells three human CTL clones that recognized the MAGE-1 peptide. These clones killed efficiently HLA-A2 tumor cells expressing MAGE-1, whether or not they were treated with IFN-, suggesting that the MAGE-1 antigen is processed efficiently by both the standard proteasome and the immunoproteasome. These results indicate that the MAGE-1.A2 peptide can be used for antitumoral vaccination.     

Lysophosphatidic acid regulates inflammation-related genes in human endothelial cells through LPA1 and LPA3

Lysophosphatidic acid (LPA) is a low-molecular-weight lysophospholipid (LPL), which regulates endothelial cells participating in inflammation processes via interactions with endothelial differentiation gene (Edg) family G protein-coupled receptors. In this study, we attempted to determine which LPA receptors mediate the inflammatory response in human endothelial cells. Introduction of siRNA against LPA₁ significantly suppressed LPA-induced ICAM-1 mRNA, total protein, and cell surface expressions, and subsequent U937 monocyte adhesion to LPA-treated human umbilical endothelial cells (HUVECs). By knock down of LPA₁ and LPA₃ in HUVECs, LPA-enhanced IL-1β mRNA expression was significantly attenuated. Moreover, LPA₁ and LPA₃ siRNA also inhibited LPA-enhanced IL-1-dependent long-term IL-8 and MCP-1 mRNA expression, and subsequent THP-1 cell chemotaxis toward LPA-treated HUVEC-conditioned media. These results suggest that the expression of LPA-induced inflammatory response genes is mediated by LPA₁ and LPA₃. Our findings suggest the possible utilization of LPA₁ or LPA₃ as drug targets to treat severe inflammation.     

microRNA-342, microRNA-191 and microRNA-510 are differentially expressed in T regulatory cells of type 1 diabetic patients

Regulatory T cells (Tregs) are critical regulators of autoimmune diseases, including type 1 diabetes mellitus. It is hypothesised that Tregs’ function can be influenced by changes in the expression of specific microRNAs (miRNAs). Thus, we performed miRNAs profiling in a population of Tregs separated from peripheral blood of five type 1 diabetic patients and six healthy donors. For more detailed molecular characterisation of Tregs, we additionally compared miRNAs expression profiles of Tregs and conventional T cells. Tregs were isolated according to CD3+, CD4+, CD25^hi+ and CD127− by flow cytometry, and miRNA expression profiling was performed using TaqMan Array Human MicroRNA Panel-1 (384-well low density array). In Tregs of diabetic patients we found significantly increased expression of miRNA-510 (p = 0.05) and decreased expression of both miRNA-342 (p < 0.0001) and miRNA-191 (p = 0.0079). When comparing Tregs and T cells, we revealed that Tregs had significant higher expression of miRNA-146a and lower expression of eight specific miRNAs (20b, 31, 99a, 100, 125b, 151, 335, and 365). To our knowledge, this is the first study demonstrating changes in miRNA expression profiles occurring in Tregs of T1D patients and a miRNAs signature of adult Tregs.