Epigenetic Reprogramming of CD4+ Helper T Cells as a Strategy to Improve Anticancer Immunotherapy.

Evidences highlight the role of various CD4+ helper T cells (CD4+ Th) subpopulations in orchestrating the immune responses against cancers. Epigenetics takes an important part in the regulation of CD4+ Th polarization and plasticity. In this review, we described the epigenetic factors that govern CD4+ T cells differentiation and recruitment in the tumor microenvironment and their subsequent involvement in the antitumor immunity. Finally, we discussed how to manipulate tumor reactive CD4+ Th responses by epigenetic drugs to improve anticancer immunotherapy.

EGFR-HIF1α signaling positively regulates the differentiation of IL-9 producing T helper cells.

Interleukin 9 (IL-9)-producing helper T (Th9) cells are essential for inducing anti-tumor immunity and inflammation in allergic and autoimmune diseases. Although transcription factors that are essential for Th9 cell differentiation have been identified, other signaling pathways that are required for their generation and functions are yet to be explored. Here, we identify that Epidermal Growth Factor Receptor (EGFR) is essential for IL-9 induction in helper T (Th) cells. Moreover, amphiregulin (Areg), an EGFR ligand, is critical for the amplification of Th9 cells induced by TGF-ß1 and IL-4. Furthermore, our data show that Areg-EGFR signaling induces HIF1α, which binds and transactivates IL-9 and NOS2 promoters in Th9 cells. Loss of EGFR or HIF1α abrogates Th9 cell differentiation and suppresses their anti-tumor functions. Moreover, in line with its reliance on HIF1α expression, metabolomics profiling of Th9 cells revealed that Succinate, a TCA cycle metabolite, promotes Th9 cell differentiation and Th9 cell-mediated tumor regression.

Skin and gut imprinted helper T cell subsets exhibit distinct functional phenotypes in central nervous system autoimmunity.

Multidimensional single-cell analyses of T cells have fueled the debate about whether there is extensive plasticity or 'mixed' priming of helper T cell subsets in vivo. Here, we developed an experimental framework to probe the idea that the site of priming in the systemic immune compartment is a determinant of helper T cell-induced immunopathology in remote organs. By site-specific in vivo labeling of antigen-specific T cells in inguinal (i) or gut draining mesenteric (m) lymph nodes, we show that i-T cells and m-T cells isolated from the inflamed central nervous system (CNS) in a model of multiple sclerosis (MS) are distinct. i-T cells were Cxcr6+, and m-T cells expressed P2rx7. Notably, m-T cells infiltrated white matter, while i-T cells were also recruited to gray matter. Therefore, we propose that the definition of helper T cell subsets by their site of priming may guide an advanced understanding of helper T cell biology in health and disease.

Murine T-Cell Transfer Colitis as a Model for Inflammatory Bowel Disease.

Inflammatory bowel disease (IBD) is a group of severe chronic inflammatory conditions of the human gastrointestinal tract. Murine models of colitis have been invaluable tools to improve the understanding of IBD development and pathogenesis. While the disease etiology of IBD is complex and multifactorial, CD4+ T helper cells have been shown to strongly contribute to the disease pathogenesis of IBD. Here, we present a detailed protocol of the preclinical model of T-cell transfer colitis, which can easily be utilized in the laboratory to study T helper cell functions in intestinal inflammation.

The Anticancer Potential of T Cell Receptor-Engineered T Cells.

Adoptively transferred T cell receptor (TCR)-transgenic T cells (TCR-T cells) are not restricted by cell surface expression of their targets and are therefore poised to become a main pillar of cellular cancer immunotherapies. Addressing clinical and laboratory data, we discuss emerging features for the efficient deployment of novel TCR-T therapies, such as selection of ideal TCRs targeting validated epitopes with well-characterized cancer cell expression and processing, enhancing TCR-T effector function, trafficking, expansion, persistence, and memory formation by strategic selection of substrate cells, and gene-engineering with synthetic co-stimulatory circuits. Overall, a better understanding of the relevant mechanisms of action and resistance will help prioritize the vast array of potential TCR-T optimizations for future clinical products.

Proteome analysis revealed the essential functions of protein phosphatase PP2A in the induction of Th9 cells.

Proteomic analysis identifies post-translational functions of proteins, which remains obscure in transcriptomics. Given the important functions of Th9 cells in anti-tumor immunity, we performed proteome analysis of Th9 cells to understand the involvement of proteins that might be crucial for the anti-tumor functions of Th9 cells. Here we performed a comprehensive proteomic analysis of murine Th0 and Th9 cells, and identified proteins that are enriched in Th9 cells. Pathway analysis identified an abundance of phosphoproteins in the proteome of Th9 cells as compared to Th0 cells. Among upregulated phosphoproteins, Ppp2ca (catalytic subunit of protein phosphatase, PP2A) was found to be highly enriched in Th9 cells. Although the role of PP2A has been shown to regulate the differentiation and functions of Th1, Th2, Th17 and Tregs, its role in the differentiation and functions of Th9 cells is not identified yet. Here we found that PP2A is required for the induction of Th9 cells, as PP2A inhibition leads to the suppression of IL-9 and expression of key transcription factors of Th9 cells. PP2A inhibition abrogates Th9 cell-mediated anti-tumor immune response in B16-OVA melanoma tumor model. Thus, we report that PP2A is essential for the differentiation and anti-tumor functions of Th9 cells.

Class-switch recombination to IgA in the Peyer's patches requires natural thymus-derived Tregs and appears to be antigen independent.

Our understanding of how class-switch recombination (CSR) to IgA occurs in the gut is still incomplete. Earlier studies have indicated that Tregs are important for IgA CSR and these cells were thought to transform into follicular helper T cells (Tfh), responsible for germinal center formation in the Peyer's patches (PP). Following adoptive transfer of T-cell receptor-transgenic (TCR-Tg) CD4 T cells into nude mice, we unexpectedly found that oral immunization did not require an adjuvant to induce strong gut IgA and systemic IgG responses, suggesting an altered regulatory environment in the PP. After sorting of splenic TCR-Tg CD4 T cells into CD25+ or CD25- cells we observed that none of these fractions supported a gut IgA response, while IgG responses were unperturbed in mice receiving the CD25- cell fraction. Hence, while Tfh functions resided in the CD25- fraction the IgA CSR function in the PP was dependent on CD25+ Foxp3+ Tregs, which were found to be Helios+ neuropilin-1+ thymus-derived Tregs. This is the first study to demonstrate that Tfh and IgA CSR functions are indeed, unique, and separate functions in the PP with the former being TCR-dependent while the latter appeared to be antigen independent.

T follicular helper cells restricted by IRF8 contribute to T cell-mediated inflammation.

The follicular helper T cell (TFH) are established regulators of germinal center (GC) B cells, whether TFH have pathogenic potential independent of B cells is unknown. Based on in vitro TFH cell differentiation, in vivo T cell transfer animal colitis model, and intestinal tissues of inflammatory bowel disease (IBD) patients, TFH and its functions in colitis development were analyzed by FACS, ChIP, ChIP-sequencing, WB, ELISA and PCR. Herein we demonstrate that intestinal tissues of patients and colon tissues obtained from Rag1-/- recipients of naïve CD4+ T cells with colitis, each over-express TFH-associated gene products. Adoptive transfer of naïve Bcl6-/- CD4+ T cells into Rag1-/- recipient mice abrogated development of colitis and limited TFH differentiation in vivo, demonstrating a mechanistic link. In contrast, T cell deficiency of interferon regulatory factor 8 (IRF8) resulted in augmentation of TFH induction in vitro and in vivo. Functional studies showed that adoptive transfer of IRF8 deficient CD4+ T cells into Rag1-/- recipients exacerbated colitis development associated with increased gut TFH-related gene expression, while Irf8-/-/Bcl6-/- CD4+ T cells abrogated colitis, together indicating that IRF8-regulated TFH can directly cause colon inflammation. Molecular analyses revealed that IRF8 suppresses TFH differentiation by inhibiting transcription and transactivation of the TF IRF4, which is also known to be essential for TFH induction. Our documentation showed that IRF8-regulated TFH can function as B-cell-independent, pathogenic, mediators of colitis suggests that targeting TFH could be effective for treatment of IBD.

PD1-CD28 Fusion Protein Enables CD4+ T Cell Help for Adoptive T Cell Therapy in Models of Pancreatic Cancer and Non-hodgkin Lymphoma.

Background: Interaction of the programmed death receptor 1 (PD-1) and its ligand, PD-L1, suppresses T cell activity and permits tumors to evade T cell-mediated immune surveillance. We have recently demonstrated that antigen-specific CD8+ T cells transduced with a PD1-CD28 fusion protein are protected from PD-1-mediated inhibition. We have now investigated the potential of PD1-CD28 fusion protein-transduced CD4+ T cells alone or in combination with CD8+ T cells for immunotherapy of pancreatic cancer and non-Hodgkin lymphoma. Methods: OVA-specific CD4+ and CD8+ were retrovirally transduced with the PD1-CD28 fusion protein. Cytokine release, proliferation, cytotoxic activity, and phenotype of transduced T cells were assessed in the context of Panc02-OVA (murine pancreatic cancer model) and E.G7-PD-L1 (murine T cell lymphoma model) cells. Results: Stimulation of PD1-CD28 fusion protein-transduced CD4+ T cells with anti-CD3 and recombinant PD-L1 induced specific T cell activation, as measured by IFN-y release and T cell proliferation. Coculture with Panc02-OVA or E.G7-PD-L1 tumor cells also led to specific activation of CD4+ T cells. Cytokine release and T cell proliferation was most effective when tumor cells simultaneously encountered genetically engineered CD4+ and CD8+ T cells. Synergy between both cell populations was also observed for specific tumor cell lysis. T cell cytotoxicity was mediated via granzyme B release and mediated enhanced tumor control in vivo. Transduced CD4+ and CD8+ T cells in co-culture with tumor cells developed a predominant central memory phenotype over time. Different ratios of CD4+ and CD8+ transduced T cells led to a significant increase of IFN-y and IL-2 secretion positively correlating with CD4+ T cell numbers used. Mechanistically, IL-2 and MHC-I were central to the synergistic activity of CD4+ and CD8+ T cells, since neutralization of IL-2 prevented the crosstalk between these cell populations. Conclusion: PD1-CD28 fusion protein-transduced CD4+ T cells significantly improved anti-tumoral effect of fusion protein-transduced CD8+ T cells. Thus, our results indicate that PD1-CD28 fusion protein-transduced CD4+ T cells have the potential to overcome the PD-1-PD-L1 immunosuppressive axis in pancreatic cancer and non-Hodgkin lymphoma.

Iron Drives T Helper Cell Pathogenicity by Promoting RNA-Binding Protein PCBP1-Mediated Proinflammatory Cytokine Production.

Iron deposition is frequently observed in human autoinflammatory diseases, but its functional significance is largely unknown. Here we showed that iron promoted proinflammatory cytokine expression in T cells, including GM-CSF and IL-2, via regulating the stability of an RNA-binding protein PCBP1. Iron depletion or Pcbp1 deficiency in T cells inhibited GM-CSF production by attenuating Csf2 3' untranslated region (UTR) activity and messenger RNA stability. Pcbp1 deficiency or iron uptake blockade in autoreactive T cells abolished their capacity to induce experimental autoimmune encephalomyelitis, an animal model for multiple sclerosis. Mechanistically, intracellular iron protected PCBP1 protein from caspase-mediated proteolysis, and PCBP1 promoted messenger RNA stability of Csf2 and Il2 by recognizing UC-rich elements in the 3' UTRs. Our study suggests that iron accumulation can precipitate autoimmune diseases by promoting proinflammatory cytokine production. RNA-binding protein-mediated iron sensing may represent a simple yet effective means to adjust the inflammatory response to tissue homeostatic alterations.

Adoptive cancer immunotherapy using DNA-demethylated T helper cells as antigen-presenting cells.

In cancer cells, cancer/testis (CT) antigens become epigenetically derepressed through DNA demethylation and constitute attractive targets for cancer immunotherapy. Here we report that activated CD4+ T helper cells treated with a DNA-demethylating agent express a broad repertoire of endogenous CT antigens and can be used as antigen-presenting cells to generate autologous cytotoxic T lymphocytes (CTLs) and natural killer cells. In vitro, activated CTLs induce HLA-restricted lysis of tumor cells of different histological types, as well as cells expressing single CT antigens. In a phase 1 trial of 25 patients with recurrent glioblastoma multiforme, cytotoxic lymphocytes homed to the tumor, with tumor regression ongoing in three patients for 14, 22, and 27 months, respectively. No treatment-related adverse effects were observed. This proof-of-principle study shows that tumor-reactive effector cells can be generated ex vivo by exposure to antigens induced by DNA demethylation, providing a novel, minimally invasive therapeutic strategy for treating cancer.

The intestinal microbiota determines the colitis-inducing potential of T-bet-deficient Th cells in mice.

Conflicting evidence has been provided as to whether induction of intestinal inflammation by adoptive transfer of naïve T cells into Rag-/- mice requires expression of the transcription factor T-bet by the T cells. Here, we formally show that the intestinal microbiota composition of the Rag-/- recipient determines whether or not T-bet-deficient Th cells can induce colitis and we have resolved the differences of the two microbiomes, permissive or non-permissive to T-bet-independent colitis. Our data highlight the dominance of the microbiota over particular T cell differentiation programs in the pathogenesis of chronic intestinal inflammation.

Vaccines targeting helper T cells for cancer immunotherapy.

There are compelling arguments for designing cancer vaccines specifically to induce CD4+ helper T cell responses. Recent studies highlight the crucial role of proliferating, activated effector memory Th1 CD4+ T cells in effective antitumor immunity and reveal that CD4+ T cells induce more durable immune-mediated tumor control than CD8+ T cells. CD4+ T cells promote antitumor immunity by numerous mechanisms including enhancing antigen presentation, co-stimulation, T cell homing, T cell activation, and effector function. These effects are mediated at sites of T cell priming and at the tumor microenvironment. Several cancer vaccine approaches induce durable CD4+ T cell responses and have promising clinical activity. Future work should further optimize vaccine adjuvants and combination therapies incorporating helper peptide vaccines.

CD40-mediated signalling influences trafficking, T-cell receptor expression, and T-cell pathogenesis, in the NOD model of type 1 diabetes.

CD40 plays a critical role in the pathogenesis of type 1 diabetes (T1D). The mechanism of action, however, is undetermined, probably because CD40 expression has been grossly underestimated. CD40 is expressed on numerous cell types that now include T cells and pancreatic ß cells. CD40+ CD4+ cells [T helper type 40 (TH40)] prove highly pathogenic in NOD mice and in translational human T1D studies. We generated BDC2.5.CD40-/- and re-derived NOD.CD154-/- mice to better understand the CD40 mechanism of action. Fully functional CD40 expression is required not only for T1D development but also for insulitis. In NOD mice, TH40 cell expansion in pancreatic lymph nodes occurs before insulitis and demonstrates an activated phenotype compared with conventional CD4+ cells, apparently regardless of antigen specificity. TH40 T-cell receptor (TCR) usage demonstrates increases in several Vα and Vß species, particularly Vα3.2+ that arise early and are sustained throughout disease development. TH40 cells isolated from diabetic pancreas demonstrate a relatively broad TCR repertoire rather than restricted clonal expansions. The expansion of the Vα/Vß species associated with diabetes depends upon CD40 signalling; NOD.CD154-/- mice do not expand the same TCR species. Finally, CD40-mediated signals significantly increase pro-inflammatory Th1- and Th17-associated cytokines whereas CD28 co-stimulus alternatively promotes regulatory cytokines.

Autologous bone marrow Th cells can support multiple myeloma cell proliferation in vitro and in xenografted mice.

Multiple myeloma (MM) is a plasma cell malignancy where MM cell growth is supported by the bone marrow (BM) microenvironment with poorly defined cellular and molecular mechanisms. MM cells express CD40, a receptor known to activate autocrine secretion of cytokines and elicit proliferation. Activated T helper (Th) cells express CD40 ligand (CD40L) and BM Th cells are significantly increased in MM patients. We hypothesized that activated BM Th cells could support MM cell growth. We here found that activated autologous BM Th cells supported MM cell growth in a contact- and CD40L-dependent manner in vitro. MM cells had retained the ability to activate Th cells that reciprocated and stimulated MM cell proliferation. Autologous BM Th cells supported MM cell growth in xenografted mice and were found in close contact with MM cells. MM cells secreted chemokines that attracted Th cells, secretion was augmented by CD40-stimulation. Within 14 days of culture of whole BM aspirates in autologous serum, MM cells and Th cells mutually stimulated each other, and MM cells required Th cells for further expansion in vitro and in mice. The results suggest that Th cells may support the expansion of MM cells in patients.

Adequate immune response ensured by binary IL-2 and graded CD25 expression in a murine transfer model.

The IL-2/IL-2Ralpha (CD25) axis is of central importance for the interplay of effector and regulatory T cells. Nevertheless, the question how different antigen loads are translated into appropriate IL-2 production to ensure adequate responses against pathogens remains largely unexplored. Here we find that at single cell level, IL-2 is binary (digital) and CD25 is graded expressed whereas at population level both parameters show graded expression correlating with the antigen amount. Combining in vivo data with a mathematical model we demonstrate that only this binary IL-2 expression ensures a wide linear antigen response range for Teff and Treg cells under real spatiotemporal conditions. Furthermore, at low antigen concentrations binary IL-2 expression safeguards by its spatial distribution selective STAT5 activation only of closely adjacent Treg cells regardless of their antigen specificity. These data show that the mode of IL-2 secretion is critical to tailor the adaptive immune response to the antigen amount.

Combination of TLR1/2 and TLR3 ligands enhances CD4(+) T cell longevity and antibody responses by modulating type I IFN production.

Despite the possibility of combining Toll-like receptor (TLR) ligands as adjuvants to improve vaccine efficacy, it remains unclear which combinations of TLR ligands are effective or what their underlying mechanisms may be. Here, we investigated the mechanism of action of L-pampo, a proprietary adjuvant composed of TLR1/2 and TLR3 ligands. L-pampo dramatically increased humoral immune responses against the tested target antigens, which was correlated with an increase in follicular helper T cells and the maintenance of antigen-specific CD4(+) T cells. During the initial priming phase, in contrast to the induction of type I interferon (IFN) and pro-inflammatory cytokines stimulated by polyI:C, L-pampo showed a greatly diminished induction of type I IFN, but not of other cytokines, and remarkably attenuated IRF3 signaling, which appeared to be critical to L-pampo-mediated adjuvanticity. Collectively, our results demonstrate that the adjuvant L-pampo contributes to the promotion of antigen-specific antibodies and CD4(+) T cell responses via a fine regulation of the TLR1/2 and TLR3 signaling pathways, which may be helpful in the design of improved vaccines.

Isolation and Characterization of an HLA-DPB1*04: 01-restricted MAGE-A3 T-Cell Receptor for Cancer Immunotherapy.

Long-term tumor regressions have been observed in patients following the adoptive transfer of autologous tumor-infiltrating lymphocytes or genetically modified T cells expressing MHC class I-restricted T-cell receptors (TCRs), but clinical trials have not evaluated responses to genetically modified T cells expressing antitumor MHC class II-restricted TCRs. As studies carried out in a murine tumor model system have demonstrated that the adoptive transfer of CD4 T cells could lead to the regression of established tumors, we plan to test the hypothesis that CD4 T cells can also induce tumor regressions in cancer patients. In this study, 2 MAGE-A3-specific TCRs were isolated from a regulatory T-cell clone (6F9) and an effector clone (R12C9), generated from the peripheral blood of 2 melanoma patients after MAGE-A3 vaccination. The results indicated that T cells transduced with 6F9 TCR mediated stronger effector functions than R12C9 TCR. The 6F9 TCR specifically recognized MAGE-A3 and the closely related MAGE-A6 gene product, but not other members of the MAGE-A family in the context of HLA-DPB1*04:01. To test the feasibility of a potential clinical trial using this TCR, a clinical-scale procedure was developed to obtain a large number of purified CD4 T cells transduced with 6F9 TCR. Because HLA-DPB1*04:01 is present in ∼60% of the Caucasian population and MAGE-A3 is frequently expressed in a variety of cancer types, this TCR immunotherapy could potentially be applicable for a significant portion of cancer patients.

Foxp3-expressing sensitized Teff cells prolong survival of corneal allograft in corneal allograft transplantation mouse model.

OBJECTIVE: The study aimed to investigate whether Foxp3-expressing sensitized Teff cells could inhibit allograft rejection in corneal allograft transplantation mouse model. METHODS: Foxp3-expressing sensitized Teff cells were constructed by transfection of retroviral expression plasmid expressing Foxp3 into the sensi-Teff cells from a Balb/c mouse immunized by C57BL/6(H2b) mouse splenocytes. Balb/c mice were randomly divided into 5 groups: Four groups received tail vein injection of Foxp3-expressing sensitized Teff cells, or Foxp3-expressing Teff cells, or Treg cells or no intervention 1 day prior to corneal allograft transplantation. C57BL/6(H2b) was the donor mouse. The last group received corneal autograft transplantation. Corneal allograft survival time and percentage of CD4(+) T cells were detected. ELISPOT and Footpad swelling test were used to measure IL-2 and IFN-γ, and delayed-type hypersensitivity (DTH) response, respectively. RESULTS: Mice that had received an injection of Foxp3-expressing sensitized T cells prior to an allograft corneal transplantation, showed significantly longer survival time of corneal allograft, decreased percentage of CD4(+) T cells, IL-2 and IFN-γ, and alleviated footpad swelling than the mice that had received either Foxp3-Teff or Treg cells. CONCLUSION: Foxp3-sensi-Teff cell treatment that prolongs corneal allograft survival in the mouse model, might partly through suppressing CD4(+) T cells, IL-2 and IFN-γ.