Helper Activity Research Articles

Long Non-Coding RNA Function in CD4+ T Cells: What We Know and What Next?

The non-coding genome has previously been regarded as “junk” DNA; however, emerging evidence suggests that the non-coding genome accounts for some of the greater biological complexity observed in mammals. Research into long non-coding RNAs (lncRNAs) has gathered speed in recent years, and a growing body of evidence has implicated lncRNAs in a vast range of cellular functions including gene regulation, chromosome organisation and splicing. T helper cells offer an ideal platform for the study of lncRNAs given they function as part of a complex cellular network and undergo remarkable and finely regulated gene expression changes upon antigenic stimulation. Using various knock down and RNA interaction studies several lncRNAs have been shown to be crucial for T helper cell differentiation, activation and function. Given that RNA targeting therapeutics are rapidly gaining attention, further understanding the mechanistic role of lncRNAs in a T helper context is an exciting area of research, as it may unearth a wide range of new candidate targets for treatment of CD4+ mediated pathologies.

Abstract 3213: Combination of anti-IL-6 and anti-PD-L1 antibodies synergistically reinvigorates cancer immunity by activating both CTL and Th1 cells in mice

Abstract We previously demonstrated that Th1 differentiation of tumor-specific CD4+ T cells was attenuated in tumor-bearing or aged mice in an IL-6-dependent manner (Nat. Comm. 6: e6702, 2015, Cancer Immunol. Res. 1: 64, 2013). Furthermore we found that accompanied by the systemic increase of soluble IL-6 receptor (sIL-6R) in cancer patients, inhibition of IL-6 trans-signaling mediated through the sIL-6R restored the Th1 responses, their helper activity toward CD8+ T cells, and anti-tumor activity in tumor-bearing mice (Cancer Res. 77: 2279, 2017). The systemically increased sIL-6Rs were mainly produced by myeloid cells. Moreover, cMaf-deficient CD4+ T cells were resistant to Th1 suppression and impairment of T cell-mediated anti-tumor immunity induced by IL-6/sIL-6R indicating that c-Maf activity was responsible for Th1 suppression. Myeloid cell-derived sIL-6R was also possibly associated with Th1 suppression and c-Maf expression in head and neck cancer patients. These results suggest that targeting a pro-inflammatory cytokine, IL-6 enhanced the tumor-specific Th1 responses and subsequent anti-tumor effects. However, IL-6 blockade in turn up-regulated the expression of immune-checkpoint molecule, PD-L1 on melanoma cells. This PD-L1 induction was canceled in IFN-γ-deficient mice or mice depleted of CD4+ T cells, suggesting an important role of CD4+ T cell-derived IFN-γ in the PD-L1 induction in tumor-bearing hosts. On the other hand, in some patients with melanoma, anti-PD-1 antibody, Nivolumab treatment increased the systemic level of IL-6, which were associated with their poor clinical responses. This PD-L1 blockade-evoked IL-6 induction was also observed in melanoma-bearing mice (Cancer Res. 78: 5011, 2018). Considering the mechanistic linkage between IL-6 and PD-1/PD-L1 signals, we found that PD-1/PD-L1 blockade prompted PD-1+macrophages to produce IL-6 in tumor microenvironment. Depletion of macrophages in melanoma-bearing mice revealed that macrophages functioned as a source of IL-6 during PD-L1 blockade, which was responsible for the defective Th1 response. Furthermore, combined blockade of the mutually regulated-immunosuppressive activities mediated by IL-6 and PD-1/PD-L1 signals enhanced the infiltration of IFN-γ-producing CD4+ T cells in tumor tissues, and exerted a synergistic anti-tumor effect, whereas PD-L1 blockade alone did not promote Th1 response. Collectively, these findings suggest that IL-6 is considered to be a rational immunosuppressive target to overcome a narrow therapeutic window of anti-PD-1/PD-L1 therapy. [ This research was financially supported by the JSPS KAKENHI grant nos. 26430165 and 18K07325 to HT, nos. 15H04311 and 16H06498 to YN, and the P-CREATE from the AMED, Japan to YN and HT. ] Citation Format: Yasuharu Nishimura, Koji Fujieda, Azusa Miyashita, Satoshi Fukushima, Tokunori Ikeda, Yosuke Kubo, Satoru Senju, Hironobu Ihn, Hiroyuki Oshiumi, Hirotake Tsukamoto. Combination of anti-IL-6 and anti-PD-L1 antibodies synergistically reinvigorates cancer immunity by activating both CTL and Th1 cells in mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3213.

Therapeutic effects of artesunate on lupus-prone MRL/lpr mice are dependent on T follicular helper cell differentiation and activation of JAK2–STAT3 signaling pathway

BackgroundAnti-malarial drug artesunate (ART), a semi-synthetic derivative of artemisnin, has immunosuppressive effects on several autoimmune diseases, including Systemic lupus erythematosus (SLE), Rheumatoid arthritis (RA), and Colitis. However, molecular mechanisms of ART, especially on follicular helper T cells (Tfh), central players in SLE pathology, are far from clear. PurposeThe object for this work is to investigate the therapeutic effect of ART on lupus-prone MRL/lpr mice and its regulatory function on Tfh cells. Study design and methodsMRL/lpr mice were used to explore therapeutic effects of ART on lupus-prone MRL/lpr mice and its regulatory functions on Tfh cells. Then, experiments of renal function were accomplished using the biochemical kits. Effects of ART on histopathology of kidneys, inflammatory factors and autoantibodies were examined using H&E staining, ELISA and real-time PCR. Flow cytometry and western blot analysis were used to examine effects of ART on Tfh differentiation and Jak2–Stat3 signaling pathway. ResultsUpon oral administration, ART significantly prolonged the survival of MRL/lpr mice, ameliorated the lupus nephritis symptoms, decreased the levels of anti-dsDNA antibodies deposited in the kidney, and the levels of pathogenic cytokines (IL-6, IFN-γ and IL-21). After ART treatment, T-cell compartment in the spleen of MRL/lpr mice was restored in terms of reduction in the number of Tfh cells and in the maintenance of the ratio of Tfr to follicular regulatory T cells (Tfh). In addition, ART has significantly inhibited the phosphorylation levels of Jak2 and Stat3 in the MRL/lpr mice. ConclusionART showed therapeutic effects on lupus-prone MRL/lpr mice by inhibiting the differentiation of Tfh cells as well as altering the activation status of Jak2–Stat3 signaling cascade.

Dendritic cells dysfunction in tumor-draining lymph nodes

Abstract An effective anti-tumor response requires activation of both CD4 helper T cells (through MHC class II) and CD8 cytotoxic T cells (through MHC class I). Activation of tumor-specific CD4 and CD8 T cells depends on tumor antigen presentation by DCs. However, it is known that the tumor microenvironment largely suppresses immune responses. The focus of our study is to characterize the functional status of DCs in tumor-draining lymph nodes and to identify the mechanisms that lead to defective MHC class II presentation of tumor antigens. We injected either B16-F10 melanoma or MB49 bladder cancer adenocarcinoma cells into the flank of mice and isolated DCs from both tumor-draining lymph node (TDLN) and non-draining lymph nodes (NDLN) as our model system. Cross-presentation of OVA to OVA-specific CD8 T cells was not suppressed in TDLN in comparison to the NDLN either in vivo and ex vivo. However, both in vivo and ex vivo CD4 T cell activation by DCs from TDLNs was defective. Subset analysis revealed that both migratory and resident CD8-positive and CD8-negative DCs in TDLN show a less mature phenotype as compared to their counterparts isolated from NDLN. Examination of antigen uptake and processing also showed defects in DCs in TDLN. RNA-seq revealed many differences in gene expression between both resident and migratory DCs isolated from TDLN and NDLN. Characterized phenotypical and functional differences, confirmed by gene expression studies, may explain the dysfunction of DCs in tumor-bearing mice. Further analysis should enable moving towards “correcting” DC defects that would allow proper CD4 T cell priming to provide help to anti-tumor CD8 T cells.

IL-12 signaling drives the differentiation and function of a TH1-derived TFH1-like cell population

Abstract CD4+ T follicular helper (TFH) cells play key roles in the generation of humoral immunity, as they provide T cell help to B cells that ultimately results in the production of pathogen-specific antibodies. A subset of TFH cells that shares features with TH1 cells, referred to as (TH1)-biased TFH (TFH1) cells, has recently been shown to play key roles in responses to chronic infections including HIV and malaria, as well as the onset of some autoimmune diseases. Despite the apparent importance of these cells to comprehensive immune responses and human health, many questions remain regarding their origin, including the molecular mechanisms that regulate their differentiation. Here, we describe a population of TH1-derived TFH1-like cells that produce both the TH1 cytokine interferon-γ and the TFH-associated cytokine interleukin-21 (IL-21). Interestingly, this TFH1-like population exhibits increased B cell helper activity compared to IL-6 derived TFH-like cells. We further demonstrate that IL-12 signaling is required for both the differentiation and functional properties of the TFH1-like cell population. Specifically, IL-12-dependent activation of STAT4, and unexpectedly STAT3, result in both the upregulation of the TFH lineage-defining transcription factor Bcl-6, as well as the increased production of IL-21. Finally, we show that IL-12 signaling is required for TFH1-like cell expression of Icos, a receptor that is critical for the ability of T cells to provide help to B cells. Taken together, our findings provide potential insight into the genesis and regulatory requirements of recently described TFH1 cells with emerging roles in human health and disease.

Cutaneous Forkhead Box P3 (FOXP3) Immune-Expression in Vitiligo Patients

The exact etiology of vitiligo remains obscure. Studies have indicated a role for cellular immunity in the pathogenesis of vitiligo. The aim of this study is to assess tissue FOXP3+ natural regulatory T-cells (Tregs) in active vs. stable nonsegmental vitiligo. Immunohistochemical staining for expression of FoxP3 in lesional, marginal, and nonlesional skin of nonsegmental vitiligo was used to evaluate the abundance of Tregs in active and stable cases of vitiligo. Reduction in the number of FOXP3-expressing Tregs was detected in marginal skin in both stable and active vitiligo cases. The results revealed that FOXP3+ T-cells were lowest in marginal skin of active cases in comparison with lesional and nonlesional skin of the same group. Tregs were lower in marginal and nonlesional skin of stable vitiligo compared with lesional skin of the same group. Tregs were mostly present at the DEJ and upper dermis. The reduction in the number of FOXP3+ cells in the marginal skin suggests that this is the site where regulatory activity is needed to suppress the activity of helper and cytotoxic T-cells that are actively contributing to depigmentation.

Pathways- and epigenetic-based assessment of relative immune infiltration in various types of solid tumors.

Recent clinical studies document the power of immunotherapy in treating subsets of patients with advanced cancers. In this context and with multiple cancer immunotherapeutics already evaluated in the clinic and a large number in various stages of clinical trials, it is imperative to comprehensively examine genomics data to better comprehend the role of immunity in different cancers in predicting response to therapy and in directing appropriate therapies. The approach we chose is to scrutinize the pathways and epigenetic factors predicted to drive immune infiltration in different cancer types using publicly available TCGA transcriptional and methylation datasets, along with accompanying clinico-pathological data. We observed that the relative activation of T cells and other immune signaling pathways differs across cancer types. For example, pathways related to activation and proliferation of helper and cytotoxic T cells appear to be more highly enriched in kidney, skin, head and neck, and esophageal cancers compared to those of lung, colorectal, and liver or bile duct cancers. The activation of these immune-related pathways positively associated with prognosis in certain cancer types, most notably melanoma, head and neck, and cervical cancers. Integrated methylation and expression data (along with publicly available, ENCODE-generated histone ChIP Seq and DNAse hypersensitivity data) predict that epigenetic regulation is a primary factor driving transcriptional activation of a number of genes crucial to immunity in cancer, including T cell receptor genes (e.g., CD3D, CD3E), CTLA4, and GZMA. However, the extent to which epigenetic factors (primarily methylation at promoter regions) affect transcription of immune-related genes may vary across cancer types. For example, there is a high negative correlation between promoter CpG methylation and CD3D expression in renal and thyroid cancers, but not in brain tumors. The types of analyses we have undertaken provide insights into the relationships between immune modulation and cancer etiology and progression, offering clues into ways of therapeutically manipulating the immune system to promote immune recognition and immunotherapy.

Ezh2 programs TFH differentiation by integrating phosphorylation-dependent activation of Bcl6 and polycomb-dependent repression of p19Arf

Ezh2 is an histone methyltransferase (HMT) that catalyzes H3K27me3 and functions in TH1, TH2, and Treg cells primarily via HMT activity. Here we show that Ezh2 ablation impairs T follicular helper (TFH) cell differentiation and activation of the TFH transcription program. In TFH cells, most Ezh2-occupied genomic sites, including the Bcl6 promoter, are associated with H3K27ac rather than H3K27me3. Mechanistically, Ezh2 is recruited by Tcf1 to directly activate Bcl6 transcription, with this function requiring Ezh2 phosphorylation at Ser21. Meanwhile, Ezh2 deploys H3K27me3 to repress Cdkn2a expression in TFH cells, where aberrantly upregulated p19Arf, a Cdkn2a protein product, triggers TFH cell apoptosis and antagonizes Bcl6 function via protein-protein interaction. Either forced expression of Bcl6 or genetic ablation of p19Arf in Ezh2-deficient cells improves TFH cell differentiation and helper function. Thus, Ezh2 orchestrates TFH-lineage specification and function maturation by integrating phosphorylation-dependent transcriptional activation and HMT-dependent gene repression.

Mechanisms of CD4 T Cell Tumor Immunity in a Preclinical Model of Multiple Myeloma

The majority of immune therapies for cancer have focused on generating tumor-specific CD8+ T cells that directly engage and kill tumor cells. However, recent data demonstrate a critical role of CD4 T cells in tumor immunity. Animal models showed that MHC class II epitopes derived from mutated tumor-associated proteins (neo-antigens, neoAg) conferred protection against different mouse tumor types. Clinical studies of neoAg vaccines demonstrated anti-tumor activity, with the majority of neoAg-specific immune responses being CD4 T cells. These tumor Ag-specific CD4 T cells could confer protective anti-tumor immunity even in the absence of CD8 T cells directed against the same antigen. These findings are even more striking because most tumor cells do not express MHC class II molecules and CD4 T cells cannot directly kill tumor cells.In order to understand how CD4 T cells eliminate tumors in the absence of direct cytolytic activity and vaccine specific CD8 T cells, we used an animal model of multiple myeloma, MOPC315.BM (MOPC) cell line derived from BalB/C background, that mimics many of the features of the human disease. MOPC cells do not express MHCII and produce an IgA with a unique mutated neoAg in the l2 light chain called Idiotype l2.315 (Id). This Id antigen is well characterized and known to activate CD4 T cells, however, when used as a peptide vaccine alone, it is weakly immunogenic and fails to elicit protective immune responses. We used an Id peptide sequence fused with a high affinity HSP70 binding site that elicited strong Id-specific CD4 T cell IFNg responses that protect against MOPC tumor growth. No Id-specific CD8 T cell or B cell responses were detected, and Id-specific CD4 cells did not directly lyse MOPC cells in vitro. These CD4 responses were specific to the neoAg, as they did not cross react with the wild type peptide fused to the same HSP70 binding sequence. These findings lead to the hypothesis that Id-specific CD4 T cells confer protective immunity by promoting cross-priming of CD8 T cells against non-Id, MOPC-associated antigens.To investigate this hypothesis, splenocytes and bone marrow cells were harvested from mice either challenged with MOPC cells only (No vax + MOPC), vaccinated with the Id peptide only (Id vax+No MOPC) or Id vaccinated and challenged with MOPC cells (Id vax + MOPC). CD4 and CD8 T cell activation was assessed by measuring IFNg and TNFa production by intracellular cytokine staining (ICS) after eight days of in vitro restimulation with Id peptide and irradiated MOPC cells. As shown in fig.1, in the absence of Id vaccination, no or very little immune activation was observed in the spleen and bone marrow. Id vaccination alone induced CD4 T cells in both compartments. In mice that received both Id vax and MOPC challenge, there was induction of CD8 T cell, with a notable trend towards high levels of CD8 activity in the tumor microenvironment (bone marrow). These CD8 T cells were not detected when cells from the same animals were restimulated with the Id peptide alone, indicating that these CD8 responses were against non-Id, MOPC-associated Ags (data not shown). All together these data support a model in which Id-specific CD4 T cell provide helper activity to promote cross-priming of CD8 T cell against MOPC-associated Ags only in the presence of tumor challenge. These responses are present both systemically and in the tumor site. We are currently studying the specificity of these CD8 T cells and the potential role of innate immune cells such as NK cells and macrophages in the anti-tumor immune response initiated by Id-specific CD4 T cells. These findings will help better understand the mechanisms of CD4 mediated anti-tumor immunity and promote the development of combination immunotherapy strategies to improve the efficacy of vaccines targeting CD4 antigens. [Display omitted] DisclosuresBekri:Agenus Inc: Research Funding. Levey:Agenus Inc: Employment, Equity Ownership. Cho:BMS: Membership on an entity's Board of Directors or advisory committees; Janssen: Consultancy; Agenus Inc.: Research Funding; F. Hoffmann-La Roche Ltd: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; J & J: Consultancy; Genentech Inc: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.

Abstract B055: Optimized human monocyte-derived macrophage cultures accurately reflect macrophage immunomodulatory function

Abstract Prostate cancer tumor growth and disease progression is highly influenced by noncancerous host cells within the tumor microenvironment. Tumor-associated macrophages (TAMs) are a critical component of this microenvironment, and high TAM infiltrate correlates with poor prognosis in prostate cancer. Macrophages can inhibit or support tumor growth depending on their polarization to an M1 or M2 state. M1 macrophages, as part of the Th1 immune response that fights bacterial or viral infection, promote helper and killer T-cell recruitment and activation and induce cell death of infected cells. In the tumor, M1s and their recruited T cells target the cancer cells for cell death. Conversely, as part of the Th2 immune response that repairs tissue injury, M2 macrophages promote cell growth and proliferation, inhibit helper and killer T-cell function, and induce regulatory T-cell differentiation. Protumor M2s TAMS make up the majority of macrophage infiltrate in prostate cancer tumors and protect the cancer cells from attack by the immune system. Techniques for studying TAMs in vitro are limited, which is confounded by the lack of procedure consensus for in vitro macrophage differentiation and polarization. The Pienta Lab established an optimized protocol for generating homogenous populations of M1 and M2 macrophages from human monocytes isolated from whole blood (doi: 10.2144/000114435). While these cell populations have been well characterized by their marker expression, their functional ability to modulate T-cell activation is critical to determining their validity as an in vitro TAM model. Following our optimized protocol, monocytes isolated from human blood were differentiated and cultured as resting macrophages or polarized into M1 or M2 macrophages. Conditioned media were collected at 24, 48, and 72 hours. The immunomodulatory capacity of the macrophages was evaluated by measuring the secreted levels of Th1 cytokines IL-1B and TNF known to recruit and activate helper and killer T cells and Th2 cytokine IL-10 known to prevent helper and killer T-cell function and promote regulatory T-cell induction. The in vitro-polarized human M1s secreted high levels of Th1 cytokines IL-1B and TNF, while levels from both the resting and M2-polarized macrophages were below the limit of detection. Likewise, conditioned media from M2-polarized macrophages showed high levels of Th2 cytokine IL-10, but none was detected in resting or M1 cultures. Taken together, this indicates that M1 and M2 macrophages isolated and polarized using our optimized protocol accurately reflect anticipated macrophage immunomodulatory function. Further work will confirm these data by RT-qPCR of RNA taken from M1, M2, and resting macrophage cultures, and conditioned media from serum-free cultures will be assessed to determine the secretion of additional immunomodulatory cytokines present at abundant levels in serum (e.g., TGFB1). Citation Format: Amber E. de Groot, Kenneth J. Pienta. Optimized human monocyte-derived macrophage cultures accurately reflect macrophage immunomodulatory function [abstract]. In: Proceedings of the AACR Special Conference: Prostate Cancer: Advances in Basic, Translational, and Clinical Research; 2017 Dec 2-5; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(16 Suppl):Abstract nr B055.

CD4hiCD8low Double-Positive T Cells Are Associated with Graft Rejection in a Nonhuman Primate Model of Islet Transplantation.

Peripheral CD4/CD8 double-positive (DP) T cells are associated with autoimmune disorders, cancer, and viral infection. However, the relationship between organ transplantation and DP T cells is unclear. Here, we examined the functional characteristics of peripheral DP T cells and analyzed their significance with respect to islet graft rejection in a nonhuman primate model of islet transplantation. DP T cells were functionally equivalent to conventional CD4 and CD8 T cells in terms of helper and cytotoxic activity, respectively. DP T cells expressed high levels of CXCR5 and PD-1 and secreted IFN-γ, IL-4, and IL-21 in amounts equivalent to those secreted by CD4 or CD8 T cells; also, they produced large amounts of granzyme B and perforin. In addition, under steady-state conditions, DP T cells expressed eomesodermin (Eomes) and promyelocytic leukemia zinc finger (PLZF) proteins, both of which act as transcription factors in innate/memory-like T cells. The number of peripheral DP T cells in the islet transplantation model was high in the group that experienced graft rejection; this was not the case in the long-term survival group. Interestingly, numbers of effector memory T cells (TEM) within the DP T cell population increased significantly during islet graft rejection, as did those of TEM within the cytotoxic CD8 T cells. Furthermore, the most conspicuous of which was the increase of CD4hiCD8low T cell subpopulation at that point. Taken together, the data suggest that peripheral DP T cells showing an innate/memory-like phenotype have both helper and cytotoxic activity in vitro and that they may act as a novel biomarker for graft rejection after islet transplantation.

Abstract 2745: EpCAM targeted immunotoxin prolongs survival of metastatic colorectal cancer patients by a previously unknown immunostimulatory mechanism

Abstract A first-in-man study of the anti-EpCAM immunotoxin MOC31PE with and without parallel immunosuppression with Cyclosporin A (CsA) was performed in metastatic colorectal cancer (CRC) patients. The safety and maximum tolerable doses in patients with EpCAM-positive tumors were determined, and only mild and reversible dose-limiting liver toxicity was seen, with no subjective side effects to the patients. Although no complete or partial responses were observed on CT scans at eight weeks, upon evaluation of the overall survival (OS) of the patients we surprisingly found that CRC patients treated with immunotoxin alone (n=15) had a median OS of 16.3 months (95% CI=5.6-27.0) compared to 6.0 months (CI=5.8-6.2) (p<0.001) for the combination group (n=18) (hazard ratio (HR): 0.248, 95% credible interval (CrI): 0.109-0.564). The 6.0 months OS for the patients treated with CsA in addition to the immunotoxin is comparable to OS of patients with progressive disease on last line of standard chemotherapy. The Th1 cytokine profile in sera from patients treated with MOC31PE alone indicated that the immuntoxin induces an immunogenic cell death resulting in T-cell activation, and that this effect disappeared in the patients who had received simultaneous treatment with CsA. Further, we demonstrate in in vitro experiments that the immunogenic cell death factors ATP and HMGB1 were secreted from CRC cell lines treated with MOC31PE alone. Finally, conditioned media from MOC31PE treated CRC cells induced maturation of dendritic cells and promoted activation of killer (CD8+) and helper (CD4+) T cells, showing that MOC31PE treatment induces release of immune stimulating factors. The present results demonstrate the novel and exciting finding that MOC31PE induces an immunogenic effect resulting in prolonged overall survival of patients with metastatic CRC, an effect obliterated by the parallel immunosuppression of CsA. Citation Format: Øystein Fodstad, Svein Dueland, Kjersti Flatmark, Else Marit Inderberg, Gunnar Kvalheim, Yvonne Andersson. EpCAM targeted immunotoxin prolongs survival of metastatic colorectal cancer patients by a previously unknown immunostimulatory mechanism [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2745.

Immune Responses in Renal Transplant Recipients at Risk of BK-Virus Associated Nephropathy – Impact of Three Immunosuppressive Regimens

Introduction Early detection of patients at risk of BK virus-associated graft nephropathy (BKAN) would allow prevention by early adjusting immunosuppressive treatment. Materials and Methods We analyzed intracellular cytokine responses, CD4+ T helper function and T-cell dependent and independent B cell responses up to 24 months post-transplant in a prospective study of 105 renal transplant recipients, who were randomized to a CsA/MMF (n=35), Tacr/MMF (n=37) and Tacr/ERL (n=33) regimen, respectively. 18 drop-outs were excluded from analysis. BK virus (BKV) screening was performed by real time PCR with hybridization probes in serum and urine specimens. Results Risk of BK viremia was lowest in Tacr/ERL patients (4/30 (13%), CsA/MMF; 14/36 (39%), Tacr/MMF; 1/21 (5%), Tacr/ERL; p=0.005). BKAN was histologically proven in 6 patients (n=1, CsA/MMF; n=4, Tacr/MMF; n=1, Tacr/ERL). Tacr/ERL patients showed increased T-dependent B cell responses (p=0.004, 4 months; p=0.019, 1 year), increased CD4 cell IL4 (PMA; p=0.032, 1 year), CD14 IL6 (LPS; p=0.044, 4 months) and lowest CD4, CD19 and CD14 cell IL10 responses (CD4: PMA; p=0.009, 1year; CD19: PMA; p=0.002, 4 months; CD14: LPS; p=0.048, 2 years) "compared to CsA/MMF and Tacr/MMF patients". Intracellular CD4 and CD8 cell IL-2, IL-4 and IFN-γ production as well as B cell responses did not differ between patients with and without BK viremia. Pretransplant, increased IL-10R expression was detected in patients developing BK viremia (CD4+ T cells, p=0.005; CD8+ T cells, p=0.004; CD14+ monocytes, p=0.004). Post-transplant, we found an increased CD4 helper activity (p=0.008, 4 months), a decreased IL1β response of CD14+ monocytes (LPS; p=0.008, 4 months) and increased IL10 responses of CD4+ and CD8+ T cells (PMA; CD4: p=0.003, 1 year; p=0.038, 2 years; CD8: p=0.013, 1 year) in these patients. Conclusion Our data demonstrate a significantly lower risk of BK viremia on Tacr/ERL compared to CsA/MMF and Tacr/MMF treatment. The previously reported increased risk of donor-specific antibody (DSA) formation on everolimus treatment is supported by our finding of significantly increased T-dependent B cell responses but not the low CD4, CD8 and CD14 cell IL-10 production. Whereas pretransplant increased IL-10R expression may predict an increased risk of developing BK viremia, the increased post-transplant Th2 responses (CD4 helper activity and IL10 responses) found in these patients may increase the risk of DSA formation.

Activation and Regulation of B Cell Responses by Invariant Natural Killer T Cells.

CD1d-restricted invariant natural killer T (iNKT) cells play central roles in the activation and regulation of innate and adaptive immunity. Cytokine-mediated and CD1d-dependent interactions between iNKT cells and myeloid and lymphoid cells enable iNKT cells to contribute to the activation of multiple cell types, with important impacts on host immunity to infection and tumors and on the prevention of autoimmunity. Here, we review the mechanisms by which iNKT cells contribute to B cell maturation, antibody and cytokine production, and antigen presentation. Cognate interactions with B cells contribute to the rapid production of antibodies directed against conserved non-protein antigens resulting in rapid but short-lived innate humoral immunity. iNKT cells can also provide non-cognate help for the generation of antibodies directed against protein antigens, by promoting the activation of follicular helper T cells, resulting in long-lasting adaptive humoral immunity and B cell memory. iNKT cells can also regulate humoral immunity by promoting the development of autoreactive B cells into regulatory B cells. Depletions and functional impairments of iNKT cells are found in patients with infectious, autoimmune and malignant diseases associated with altered B cell function and in murine models of these conditions. The adjuvant and regulatory activities that iNKT cells have for B cells makes them attractive therapeutic targets for these diseases.

Hepatotoxicity of immune checkpoint inhibitors: a histology study of seven cases in comparison with autoimmune hepatitis and idiosyncratic drug-induced liver injury

AbstractThe adverse effects of immune checkpoint inhibitors in various organs may be attributed to immune-mediated processes triggered by disrupted self-tolerance; however, it remains unclear whether they are similar or dissimilar to classic organ-specific autoimmune diseases. The present study aimed to compare clinicopathologic features between checkpoint inhibitor-induced liver injury and acutely presenting autoimmune hepatitis or idiosyncratic drug-induced liver injury. Seven patients treated with nivolumab (n = 5) or ipilimumab (n = 2) presented with liver dysfunction a median of 41 days (range 21–120) after the initiation of immunotherapy. All patients had elevated liver enzymes, whereas hyper-bilirubinemia was less common. None of the patients had antinuclear antibodies or IgG elevations. Stopping the immunotherapy and additional immunosuppression with corticosteroids normalized or decreased liver enzymes in all patients treated. Histologically, all biopsies showed predominantly lobular hepatitis with milder portal inflammation. Centrilobular confluent necrosis and plasmacytosis were observed in a single case, and were markedly less common and milder than those in autoimmune hepatitis (p = 0.017 and p < 0.001, respectively). Bile duct injury, micro-abscesses, and extramedullary hematopoiesis were also found in one case each. Immunostaining revealed the presence of large numbers of CD3+ and CD8+ lymphocytes, whereas CD20+ B cells and CD4+ T cells were fewer in checkpoint inhibitor-induced liver injury than in autoimmune hepatitis or drug-induced liver injury. In conclusion, liver injury caused by cancer immunotherapy shares some features with injury of autoimmune hepatitis; however, there are obvious differences between the two conditions. Checkpoint inhibitor-induced liver injury may represent an immune-mediated, less zone-selective hepatocyte necrosis not requiring the strong activation of helper T cells and immunoglobulin production.

Therapeutic Vaccines for Hypertension: a New Option for Clinical Practice.

Vaccines are commonly used as preventive methods, primarily against infectious diseases. The goal of our study is to develop the therapeutic vaccine for hypertension. We and others recently reported that an angiotensin II (AngII) vaccine for hypertension successfully attenuated elevated blood pressures in an animal model without any immunogenic side effects. In this system, an immunogenic molecule (i.e., KLH) with adjuvants provides an antigen that supports the activation of helper T cells. In addition, pretreatment with the AngII vaccine exerts neuroprotective effects in a cerebral ischemia model and cardioprotective effects in a myocardial infarction model. In the early phase of clinical trial, the administration of an AngII vaccine (AngQb-Cyt006) successfully decreased blood pressure in hypertensive patients with the increase of anti-AngII antibody titer. Increasing the effectiveness of drug adherence interventions in the clinical setting may have a large impact on the health of the population, which can be improved by using successful therapeutic vaccines. In this review, we describe the concept of therapeutic vaccines for hypertension and future directions for therapeutic vaccines.

Molecular Mechanism of Atopic Dermatitis Induction Following Sensitization and Challenge with 2,4-Dinitrochlorobenzene in Mouse Skin Tissue

Laboratory animal models have been developed to investigate preventive or therapeutic effect of medicinal products, or occurrence or progression mechanism of atopic dermatitis (AD), a pruritic and persistent inflammatory skin disease. The murine model with immunologic phenomena resembling human AD was introduced, which demonstrated skewedness toward predominance of type-2 helper T cell reactivity and pathophysiological changes similar as human AD following 2,4-dinitrochlorobenzene (DNCB) sensitization and challenge. Molecular mechanism on the DNCB-mediated AD was further evaluated. Skin tissues were collected from mice treated with DNCB, and each tissue was equally divided into two sections; one for protein and the other for mRNA analysis. Expression of filaggrin, an important protein for keratinocyte integrity, was evaluated through SDS-PAGE. Level of mRNA expression for cytokines was determined through semi-quantitative reverse transcriptase polymerase chain reaction. Expression of filaggrin protein was significantly enhanced in the mice treated with DNCB compared with the vehicle (acetone : olive oil = 4 : 1 mixture) treatment group or the normal group without any treatment. Level of tumor necrosis factor-alpha and interleukin-18 mRNA expression, cytokines involved in activity of type-1 helper T (TH1) cell, was significantly downregulated in the AD group compared with other control groups. These results suggest that suppression of TH1 cell-mediated immune response could be reflected into the skin tissue of mice treated with DNCB for AD induction, and disturbance of keratinocyte integrity might evoke a compensatory mechanism.

MicroRNA-181 Variants Regulate T Cell Phenotype in the Context of Autoimmune Neuroinflammation.

Recent studies have revealed that multiple sclerosis (MS) lesions have distinct microRNA (miRNA) expression profiles. miR-181 family members show altered expression in MS tissues although their participation in MS pathogenesis remains uncertain. Herein, we investigated the involvement of miR-181a and miR-181b in the pathogenesis of MS and its animal model, experimental autoimmune encephalomyelitis (EAE). miR-181a and -b levels were measured in the central nervous system (CNS) of patients with MS and mice with EAE as well as relevant leukocyte cultures by real-time RT-PCR. To examine the role of the miRNAs in leukocyte differentiation and function, miR-181a and -b mimic sequences were transfected into cultured primary macrophages and purified CD4+ T cells which were then analyzed by RT-PCR and flow cytometry. Luciferase reporter assays were performed to investigate the interaction of miR-181a and -b with the 3'-UTR of potential target transcripts, and the expression of target genes was measured in the CNS of EAE mice, activated lymphocytes, and macrophages. Expression analyses revealed a significant decrease in miR-181a and -b levels in brain white matter from MS patients as well as in spinal cords of EAE mice during the acute and chronic phases of disease. Suppression of miR-181a was observed following antigen-specific or polyclonal activation of lymphocytes as well as in macrophages following LPS treatment. Overexpression of miR-181a and -b mimic sequences reduced proinflammatory gene expression in macrophages and polarization toward M1 phenotype. miR-181a and -b mimic sequences inhibited Th1 generation in CD4+ T cells and miR-181a mimic sequences also promoted Treg differentiation. Luciferase assays revealed Suppressor of mothers against decapentaplegic 7 (Smad7), as a direct target of miR-181a and -b. Our data highlight the anti-inflammatory actions of miR-181a and -b in the context of autoimmune neuroinflammation. miR-181a and -b influence differentiation of T helper cell and activation of macrophages, providing potential therapeutic options for controlling inflammation in MS.

Achaete-scute complex homologue 2 accelerates the development of Sjögren’s syndrome-like disease in the NOD/ShiLtJ mouse

Achaete-scute complex homologue 2 (Ascl2) has been reported to induce the differentiation and activation of follicular helper T (TFH) cells, which are essential for development of Sjögren’s syndrome (SS). This study examined whether Ascl2 plays a role in the development of SS. NOD/ShiLtJ mice were injected with an Ascl2-overexpression vector, and the infiltration of lymphocytes into salivary and lacrimal glands was assessed. The expression of inflammatory cytokines and chemoattractants for T or B cells was measured. The activation of TFH cells was assessed using a specific marker of TFH cells. Ascl2 level was also measured in SS patients. Overexpression of Ascl2 increased the expression of C-X-C chemokine receptor type 5 (CXCR5) in both salivary and lacrimal glands (p<0.0001). Overexpression of Ascl2 also increased the expression of proinflammatory cytokines and chemoattractants including interleukin 6 (IL-6), tumor necrosis factor-α, IL-8, programmed cell death 1 (PD-1), IL-21, and B-cell lymphoma 6 (Bcl-6). Overexpression of Ascl2 increased the populations of CD4+CXCR5+, CD4+ICOS+, and CD4+PD-1+ cells. The Ascl2 level was higher in peripheral blood mononuclear cells from SS patients compared with those from healthy controls. Our findings suggest that Ascl2 may play a role in the development and progression of SS and may be a therapeutic target in the treatment of SS.

Biologic Therapy and Novel Molecular Targets of Severe Asthma

Treatment options for severe or uncontrolled asthma are increasing, especially pertaining to novel biologic therapies. The 2 primary asthma endotypes, T2 high and T2 low, are defined by the level of type2 T helper and innate lymphoid cell activity and mediators. Most therapies for severe asthma target T2 high asthma, including the 3 biologics approved for use in the United States and Europe: omalizumb, mepolizumb, and reslizumab. Other biologics, with various molecular targets, are under investigation. Unfortunately, treatment options for T2 low asthma are limited. Although these therapies may improve asthma symptoms, exacerbation rates, and lung function parameters, they have not been shown to modify the disease process or provide lasting benefits after discontinuation. Biomarkers identified thus far to help guide individualized therapy in severe asthma are helpful, but imperfect discriminators for picking the best option for individual patients. This review will discuss the mechanisms of action, indications, and therapeutic effects of currently available and emerging biologics for the treatment of severe or uncontrolled asthma.