Proliferation Of Effector Cells Research Articles

T-regulatory cells in transplantology: from preparation to clinical applications

The intensive study of the cellular approaches for the correction of different pathologies, including immunopathological processes and oncology, as well as the investigation of the immunosuppressive role of the regulatory T-cells (Tregs) made conditions for the development of techniques of the cell-based correction of immune-mediated states, such as autoimmune pathology or transplantation. Since there are few Tregs in periphery and no specific Treg marker is known, the sorting of the whole blood yields insufficient number of cells. That is why it is emerging to search for optimal conditions of Treg generation and expansion using proliferation stimulators and targeted differentiation of the “pure” Treg population that does not involve the proliferation of effector cells. To date, promising results of the Treg immunotherapy to induce allospecific tolerance in recipients having transplanted organs or tissues were obtained in laboratory experiments and clinical trials. The key problems of this therapy are the lack of knowledge about the mechanism of action and the specific phenotype of the most efficient tolerance-inducing Tregs, and the difficulties to obtain stable populations of functional Tregs. Moreover, it is still unknown how to obtain antigen-specific Treg populations and what the mechanism of their action is. In this review the protocols of Treg generation are summarized and the clinical data on the Treg use for the induction of allo-specific tolerance in transplantation are analyzed.

Heterodimeric Fc-fused IL12 shows potent antitumor activity by generating memory CD8+ T cells

ABSTRACTInterleukin-12 (IL12) (p35/p40 complex) is a heterodimeric cytokine with potent anti-tumor activity. However, its short serum half-life and high dose-related toxicities limit its clinical efficacy. Here, we constructed heterodimeric immunoglobulin Fc-fused mouse IL12 (mIL12) in a monovalent binding format (mono-mIL12-Fc) to generate long-acting mIL12 in the naturally occurring heterodimeric form. Mono-mIL12-Fc exhibited a much longer plasma half-life than recombinant mIL12, enabling twice-weekly systemic injections to remove established tumors in syngeneic mouse models. Mono-mIL12-Fc was more potent than wild-type Fc-based bivalent-binding IL12-Fc (bi-mIL12-Fc) for eradicating large established immunogenic tumors without noticeable toxicities by enhancing interferon-γ production and the proliferation of immune effector cells in tumors. More importantly, mono-mIL12-Fc triggered weaker IL12 signaling than bi-mIL12-Fc, favoring the generation of functional and protective memory CD8+ T cells. Our results demonstrate that heterodimeric-Fc-fused IL12 is a suitable format for augmenting adaptive CD8+ T cell immune responses, providing a practical alternative to the systemic administration of IL12 for antitumor therapy.

Immune Responses to Neoadjuvant Chemotherapy in Muscle Invasive Bladder Cancer.

The secondary effects of chemotherapy, with bone marrow depression and risk of leukopenia, has traditionally been considered being detrimental for the immune system. However, growing evidence suggests a main role for chemotherapy in antitumor immunomodulation. With reference to cisplatin, which is the basis of neoadjuvant chemotherapy in muscle invasive bladder cancer, four different aspects of immunomodulation has thus far been described; increased MHC class I expression, recruitment and proliferation of effector cells, enhancement of tumor-lytic activity of cytotoxic effectors and downregulation of immunosuppressive actors in the microenvironment. Consequently, the role of chemotherapy in cancer is changing from a therapy solely aimed at inducing tumor cell death, to a potent inducer of immune responses and a potential future major partaker in cancer immunotherapy. This is a great opportunity for the urological community to broaden research in this field in order to increase knowledge, optimize and improve the neoadjuvant regimens of muscle invasive bladder cancer to ultimately improve patient outcome.

Characterization of alpha-fetoprotein effects on dendritic cell and its function as effector immune response activator.

Tumor antigen alpha-fetoprotein (AFP) can promote immune tolerance toward tumor cells by inducing regulatory functions of the immune system. The purpose of this study was to characterize the effects of AFP on dendritic cells (DC) in their antitumor immune response stimulation and subsequent immune tolerance toward tumor cells. Monocytes were cultured in medium with GM-CSF and IL-4 and incubated for 6 days to generate immature DC (imDC). AFP was added into the treatment group at the beginning of the monocyte-derived DC culture. Mature DC (mDC) were generated by an addition of lipopolysaccharide (LPS) into the culture and incubation for another 48 hours. We observed that the addition of AFP in early DC culture was able to decrease the binding of LPS onto imDC surface, which lowered the strength of stimulation and consequently the maturity of DC. As expected, the expression of mDC surface markers, which are known to be crucial in effector cell proliferation and activation such as HLA-DR, CD40, CD80, CD83, and CD86, were confirmed to be reduced on AFP-exposed DC. DC potential in stimulating proliferation of CD4+ T cells was decreased, in line with the reduction of surface markers’ expression. Additionally, an increased secretion of cytokine TGF-β by DC was observed. In summary, AFP inhibited the effector immune responses while increasing the regulatory immune responses in DC. This might lead to tolerance toward antigens and tumor cell survival, such as in cases of hepatocellular carcinoma patients with high levels of AFP.

Single and combined effect of retinoic acid and rapamycin modulate the generation, activity and homing potential of induced human regulatory T cells.

Adoptive transfer of CD4+CD25+FOXP3+ regulatory T cells (Treg cells) has been successfully utilized to treat graft versus host disease and represents a promising strategy for the treatment of autoimmune diseases and transplant rejection. The aim of this study was to evaluate the effects of all-trans retinoic acid (atRA) and rapamycin (RAPA) on the number, phenotype, homing markers expression, DNA methylation, and function of induced human Treg cells in short-term cultures. Naive T cells were polyclonally stimulated and cultured for five days in the presence of different combinations of IL-2, TGF-β1, atRA and RAPA. The resulting cells were characterized by the expression of FOXP3, activation, surface and homing markers. Methylation of the Conserved Non-coding Sequence 2 was also evaluated. Functional comparison of the different culture conditions was performed by suppression assays in vitro. Culturing naive human T cells with IL-2/TGFβ1 resulted in the generation of 54.2% of Treg cells (CD4+CD25+FOXP3+) whereas the addition of 100 nM atRA increased the yield of Treg cells to 66% (p = 0.0088). The addition of RAPA did not increase the number of Treg cells in any of these settings. Treg cells generated in the presence of atRA had an increased expression of the β7 integrin to nearly 100% of the generated Treg cells, while RAPA treated cells showed enhanced expression of CXCR4. The differential expression of homing molecules highlights the possibility of inducing Treg cells with differential organ-specific homing properties. Neither atRA nor RAPA had an effect on the highly methylated CNS2 sites, supporting reports that their contribution to the lineage stability of Treg cells is not mediated by methylation changes in this locus. Treg cells generated in the presence of RAPA show the most potent suppression effect on the proliferation of effector cells.

In Vitro Induction of Human Regulatory T Cells Using Conditions of Low Tryptophan Plus Kynurenines.

Thymic regulatory T cells (tTregs) and induced regulatory T cells (iTregs) suppress murine acute graft-versus-host disease (GVHD). Previously, we demonstrated that the plasmacytoid dendritic cell indoleamine 2,3-dioxygenase (IDO) fosters the in vitro development of human iTregs via tryptophan depletion and kynurenine (Kyn) metabolites. We now show that stimulation of naïve CD4+ T cells in low tryptophan (low Trp) plus Kyn supports human iTreg generation. In vitro, low Trp + Kyn iTregs and tTregs potently suppress T effector cell proliferation equivalently but are phenotypically distinct. Compared with tTregs or T effector cells, bioenergetics profiling reveals that low Trp + Kyn iTregs have increased basal glycolysis and oxidative phosphorylation and use glutaminolysis as an energy source. Low Trp + Kyn iTreg viability was reliant on interleukin (IL)-2 in vitro. Although in vivo IL-2 administration increased low Trp + Kyn iTreg persistence on adoptive transfer into immunodeficient mice given peripheral blood mononuclear cells to induce GVHD, IL-2-supported iTregs did not improve recipient survival. We conclude that low Trp + Kyn create suppressive iTregs that have high metabolic needs that will need to be addressed before clinical translation.

Utilizing IL-7 to generate melanoma antigen T cell receptor-modified cells for adoptive transfer in the absence of activation.

e14552 Background: Metastatic melanoma remains a fatal disease as many patients fail to respond to novel therapies. Presently, we have an ongoing trial utilizing T cell receptor (TCR)-modified cells reactive to the melanoma antigen, tyrosinase, for treatment of metastatic melanoma (NCT01586403). Current protocols using viral vectors to generate cells for adoptive transfer require activation in the presence of IL-2. While IL-2 stimulates proliferation and production of mature effector cells, it also induces T cell exhaustion. We hypothesize that tyrosinase-reactive TCR-modified cells generated in the absence of activation have an increased potential for expansion, persistence, and anti-melanoma activity. In these studies, we investigate whether IL-7, a homeostatic regulator, facilitates the transfer of a tyrosinase-reactive TCR to lymphocytes without activation. Methods: Gene-modified cells were generated using a lentiviral vector including alpha and beta chains of a TCR reactive to tyrosinase and a truncated CD34 molecule as a transduction marker. Peripheral lymphocytes from normal human donors were transduced with lentivirus following no treatment, treatment with IL-7, or activation in the presence of IL-2. Transduced CD34 positive cells were isolated using magnetic activated cell sorting (MACS) and injected intravenously into NOD scid gamma (NSG) mice. Results: We found that IL-7 treatment improved transduction in non-activated cells and resulted in expression levels of CD34 and TCR variable beta chain 12 (vbeta12) comparable to cells activated in the presence of IL-2. When non-activated tyrosinase-reactive TCR-modified human T cells were adoptively transferred into NOD NSG mice, the cells were detectable in the peripheral blood of mice up to 7 days after transfer. Conclusions: These findings suggest that generating melanoma antigen TCR-modified cells in the absence of activation for adoptive transfer is feasible with IL-7 treatment. Ongoing studies in NSG mice transplanted with melanoma will help determine whether non-activated gene-modified cells will improve the efficacy and longevity of cell therapy for melanoma and other malignancies.

Induction Therapy With Basiliximab in Heart-Transplanted Children Impairs Regulatory T-Cell Population (Treg)

Introduction: Following transplantation, many transplant programs employ induction immunosuppression, a relatively intense prophylactic therapy used at the time of transplant to prevent early acute rejection. Of the induction drugs, the interleukin-2 receptor (CD25) antagonist basiliximab (BXM) is frequently used. The targets of BXM are activated effector T cells, that upregulate CD25 upon activation and undergo IL-2 mediated T-cell proliferation, compromising allograft survival [1]. However, regulatory T cells (Treg) also express high levels of CD25 constitutively, and they rely on CD25 not only for their own proliferation and survival but also for detection of excessive proliferation of effector cells [2][3]. Therefore, BXM may be able to prevent acute rejection by hindering IL-2 mediated T effector expansion, but it could also compromise the likelihood of tolerance by impairing Treg proliferation and function. Methods: We enrolled 6 children who received a heart transplant and were treated with an established immunosuppression regimen. Two of the children were treated with BXM (20 mg on Day 0 and +4 after transplant), and 4 children did not receive BXM as induction therapy. We performed an exhaustive immunologic analysis at different time points including Tregs to analyze the effect of BXM on immune cells. Results: We show that the use of BXM during the first 4 days post-transplant completely blocks CD25 on T cells including Tregs for at least 45 days. Moreover, Treg numbers and Foxp3 expression were decreased in BXM-treated patients compared to patients not treated with BXM. We confirm the direct effect of BXM on Tregs by treating in vitro PBMC from healthy donors with BXM (n = 6). Basiliximab produced a marked Treg deregulation inducing a decrease in Foxp3 expression, a reduction in IL-10 production and a diminution in Treg proliferation under α-CD3/α-CD28 activation. Treg cells were more sensitive to the inhibitory effect of BXM than total CD4+ or CD8+ T cells. Conclusion: The use of basiliximab induction in transplanted children could compromise Treg-mediated tolerance during the first months post-transplant, which is the period with highest incidence of acute rejection, and could contribute to deregulation of immune homeostasis. This impact of BXM must be considered when making clinical decisions regarding induction immunosuppression.FIGURE 1: Frequency of Foxp3+CD25+CD4+ Treg cells in heart transplated children receiving induction therapy with Basiliximab (+BXM, n=2), or patients not treated with basilimab (-BXM, n=4). Mean values ±SEM are showed.This work was supported by grants from Instituto de Salud Carlos III (ISCIII) co-financed by FEDER funds (PI15/00011; ICI14/00282).FIGURE 2: Changes (%) from pre-transplant values (BL=0) in absolute counts (cells per uL of blood) of Foxp3+CD4+ cells in heart transplanted children receiving induction therapy with basilimab (+BXM, n=2), or patients not treated with basiliximab (-BXM, n=4). Mean values ±SEM are showed.FIGURE 3: CFrequencies of CDA+Foxp3+ (Treg) cells, CD4+ and CD8+ T cells within untreated (clear box) or BXM treated (solid box) PBMC stimulated with anti-CD3/CD28 dynabeads for 72h. Average and error bars indicating the 90th and 10th percentiles of 6 experiments are represented. *=<0.05 when comparing BXM-treated and non treated conditions.

Human intrahepatic regulatory T cells are functional, require IL-2 from effector cells for survival, and are susceptible to Fas ligand-mediated apoptosis.

Regulatory T cells (Treg) suppress T effector cell proliferation and maintain immune homeostasis. Autoimmune liver diseases persist despite high frequencies of Treg in the liver, suggesting that the local hepatic microenvironment might affect Treg stability, survival, and function. We hypothesized that interactions between Treg and endothelial cells during recruitment and then with epithelial cells within the liver affect Treg stability, survival, and function. To model this, we explored the function of Treg after migration through human hepatic sinusoidal‐endothelium (postendothelial migrated Treg [PEM Treg]) and the effect of subsequent interactions with cholangiocytes and local proinflammatory cytokines on survival and stability of Treg. Our findings suggest that the intrahepatic microenvironment is highly enriched with proinflammatory cytokines but deficient in the Treg survival cytokine interleukin (IL)‐2. Migration through endothelium into a model mimicking the inflamed liver microenvironment did not affect Treg stability; however, functional capacity was reduced. Furthermore, the addition of exogenous IL‐2 enhanced PEM Treg phosphorylated STAT5 signaling compared with PEMCD8. CD4 and CD8 T cells are the main source of IL‐2 in the inflamed liver. Liver‐infiltrating Treg reside close to bile ducts and coculture with cholangiocytes or their supernatants induced preferential apoptosis of Treg compared with CD8 effector cells. Treg from diseased livers expressed high levels of CD95, and their apoptosis was inhibited by IL‐2 or blockade of CD95. Conclusion: Recruitment through endothelium does not impair Treg stability, but a proinflammatory microenvironment deficient in IL‐2 leads to impaired function and increased susceptibility of Treg to epithelial cell‐induced Fas‐mediated apoptosis. These results provide a mechanism to explain Treg dysfunction in inflamed tissues and suggest that IL‐2 supplementation, particularly if used in conjunction with Treg therapy, could restore immune homeostasis in inflammatory and autoimmune liver disease. (Hepatology 2016;64:138–150)

The factors of dysregualtion of the immune response (at different stage of its implementation) in patients with pulmonary tuberculosis

The article provides an overview of scientific research to explain the mechanisms of inefficient implementation of antigen-specific immune response in patients with tuberculosis infection depending on the clinical form (infiltrative and disseminated pulmonary tuberculosis) and the course (drug-sensitive and drug-resistant pulmonary tuberculosis) of the disease. The mechanisms of immune imbalance in patients with pulmonary tuberculosis are associated with abnormal co-stimulation of a signal required for the activation of T-lymphocytes and immunosuppressive influence of regulatory T-cells both when they interact with dendritic cells at the inductive stage of the immune response, and in the process of differentiation and proliferation of effector cells with formation of suppressive mode of immune-regulation.

Effects of Human Mesenchymal Stem Cells Transduced with Superoxide Dismutase on Imiquimod-Induced Psoriasis-Like Skin Inflammation in Mice

The immunomodulatory and anti-inflammatory properties of mesenchymal stem cells (MSCs) have been proposed in several autoimmune diseases and successfully tested in animal models, but their contribution to psoriasis and underlying pathways remains elusive. Likewise, an increased or prolonged presence of reactive oxygen species and aberrant antioxidant systems in skin are known to contribute to the development of psoriasis and therefore effective antioxidant therapy is highly required. We explored the feasibility of using extracellular superoxide dismutase (SOD3)-transduced allogeneic MSCs as a novel therapeutic approach in a mouse model of imiquimod (IMQ)-induced psoriasis-like inflammation and investigated the poorly understood underlying mechanism. In addition, the chronicity and late-phase response of inflammation were evaluated during continued activation of antigen receptors by applying a booster dose of IMQ. Subcutaneous injection of allogeneic SOD3-transduced MSCs significantly prevented psoriasis development in our IMQ-induced mouse model, likely through a suppression of proliferation and infiltration of various effector cells into skin with a concomitant modulated cytokine and chemokine expression and inhibition of signaling pathways such as toll-like receptor-7, nuclear factor-kappa B, p38 mitogen-activated kinase, and Janus kinase-signal transducer and activator of transcription, as well as adenosine receptor activation. Our data offer a novel therapeutic approach to chronic inflammatory skin diseases such as psoriasis by leveraging immunomodulatory effects of MSCs as well as SOD3 expression.

Authentic GITR Signaling Fails To Induce Tumor Regression unless Foxp3+ Regulatory T Cells Are Depleted.

The glucocorticoid-induced TNFR family-related protein (GITR, TNFRSF18, CD357) is expressed on effector and regulatory T (Treg) cells. Previous studies demonstrated that GITR triggering by anti-GITR mAb enhanced T and B cell-mediated immune responses. GITR-deficient T cells, however, also proliferate more than normal T cells, and this effect is unexplained. Because the activities of mAbs are controlled by their Fc regions, the true effect of GITR signaling needs to be determined by examining its interaction with authentic ligand. Therefore, we generated a pentamerized form of the GITRL extracellular domain (pGITRL) for ligation to GITR and compared its effect on T cells with that of anti-GITR mAb. The pGITRL was more effective than anti-GITR mAb in enhancing the proliferation of effector and regulatory cells in vitro and in vivo. Nonetheless, the growth of MC38 adenocarcinoma cells in vivo was only suppressed for initial 15 d by pGITRL, whereas it was suppressed indefinitely by anti-GITR mAb. Detailed analysis revealed that pGITRL induced extensive proliferation of Foxp3(+)CD4(+) Treg cells and led to the accumulation of activated Treg cells in tumor tissue and draining lymph nodes. Because GITR signaling could not neutralize the suppressive activity of activated Treg cells, pGITRL seems to lose its adjuvant effect when sufficient activated Treg cells have accumulated in the lymph nodes and tumor tissue. Indeed, the antitumor effects of pGITRL were markedly enhanced by depleting CD4(+) cells. These results suggest that GITR signaling has stimulatory effects on effector T cells and inhibitory effects through Treg cells.

Suppression of experimental autoimmune myasthenia gravis by autologous T regulatory cells

Adoptive transfer of regulatory T (Treg) cells have been employed effectively for suppression of several animal models for autoimmune diseases. In order to employ Treg cell therapy in patients, it is necessary to generate Treg cells from the patient's own cells (autologous) that would be able to suppress effectively the disease in vivo, upon their reintroduction to the patient. Towards this objective, we report in the present study on a protocol for a successful immune-regulation of experimental autoimmune myasthenia gravis (EAMG) by ex vivo – generated autologous Treg cells. For this protocol bone marrow (BM) cells, are first cultured in the presence of GM-CSF, giving rise to a population of CD11c+MHCII+CD45RA+CD8– DCs (BMDCs). Splenic CD4+ T cells are then co-cultured with the differentiated BM cells and expand to 90% of Foxp3+ Treg cells. In vitro assay exhibits a similar dose dependent manner in the suppression of T effector cells proliferation between Treg cells obtained from either healthy or sick donors. In addition, both Treg cells inhibit similarly the secretion of IFN-γ from activated splenocytes. Administration of 1 × 106 ex-vivo generated Treg cells, I.V, to EAMG rats, modulates the disease following a single treatment, given 3 days or 3 weeks after disease induction. Similar disease inhibition was achieved when CD4 cells were taken from either healthy or sick donors. The disease suppression was accompanied by reduced levels of total AChR specific antibodies in the serum. Moreover, due to the polyclonality of the described Treg cell, we have examined whether this treatment approach could be also employed for the treatment of other autoimmune diseases involving Treg cells. Indeed, we demonstrated that the ex-vivo generated autologous Treg cells suppress Adjuvant Arthritis (AA) in rats.This study opens the way for the application of induced autologous Treg cell therapy for myasthenia gravis, as well as for other human autoimmune diseases involving Treg cells.

In vitro evaluation of γδ T cells regulatory function in Behçet’s disease patients and healthy controls

CD8-positive γδ T lymphocytes (GDCD8+) are specifically increased in peripheral blood of Behçet’s disease (BD) patients. GDCD8+ have shown a T regulatory (Treg) function in autoimmune experimental models, human tumor infiltrates and intestinal intraepithelial lymphocytes from celiac patients. The aim of this study was to evaluate the Treg function of GDCD8+ and GDCD8−, freshly isolated from peripheral blood, in comparison to CD4+CD25high naturally occurring Treg cells (nTreg) in BD and healthy controls (HC).We tested their suppressive activity on CD4+CD25− T effector cells (Teff) proliferation by a CFSE dilution protocol, after suboptimal activation with anti-CD3, in the absence or presence of IL-2. Furthermore, secreted cytokines and suppressive latency associated peptide (LAP)-TGFβ surface upregulation were determined after GD activation.We found that Vδ1 chains contribution to GDCD8+ was higher in BD than in HC, but neither GDCD8+ nor GDCD8−; (i) suppressed Teff proliferation, (ii) expressed LAP-TGFβ (iii) nor secreted IL-10, in either group. Moreover, GD presented a proinflammatory cytokine profile, mainly producing IFNγ and TNFα, in contrast to nTregs.In conclusion, peripheral GD could contribute more to the dysregulation of TH1 type of cytokines than to exerting a Treg function in BD.

Abstract IA07: Enhancing immune effector function via antibody engineering

Abstract Therapeutic antibodies of the IgG1 isotype have been established as important modality for cancer therapy in the clinic. They mediate their anti-tumoral efficacy largely by direct effects such as apoptosis/cell death induction or signaling inhibition, and by recruiting innate immune effector cells such as NK cells, macrophages and monocytes for antibody dependent cellular cytotoxicity and phagocytosis (ADCC, ADCP). Recently, methods to improve the immune effector function of therapeutic antibodies have been developed by enhancing FcgRIII binding including Fc-engineering and engineering the carbohydrate portion of the antibody. We have developed and applied a technology known as “glycoengineering” to improve the immune effector function of therapeutic antibodies. Obinutuzumab (GA101) is a glycoengineered Type II anti CD20 monoclonal antibody of the IgG1 isotype derived by humanization and elbow hinge optimization of the parental B-Ly1 mouse antibody that has recently been approved by the FDA and EMA for first line therapy of chronic lymphocytic leukemia (CLL) patients in combination with chlorambucil. In nonclinical studies obinutuzumab mediates enhanced direct (caspase independent) cell death induction with a concomitant reduction of complement dependent cytotoxicity compared to rituximab or ofatumumab by virtue of being a Type II CD20 antibody. As a consequence of glycoengineering the Fc region the core fucosylation of obinutuzumab is reduced and it has enhanced affinity for low and high affinity FcγRIIIa receptors resulting in enhanced NK cell mediated antibody dependent cellular cytotoxicity (ADCC). Recently, we demonstrated that glycoengineering not only enhances ADCC, but also results in enhanced antibody dependent cellular phagocytosis (ADCP) as compared to non-glycoengineered antibodies when physiological amounts of immunoglobulins are present. Furthermore, through enhanced affinity for FcγRIIIb that is expressed on neutrophils, obinutuzumab also mediates enhanced activation of neutrophils and neutrophil mediated phagocytosis. Finally, we have shown that in contrast to rituximab or non-glycoengineered obinutuzumab, ADCC mediated by obinutuzumab is largely unaffected by inhibitory KIR/HLA interactions. Thus, the modification of the Fc fragment of obinutuzumab appears as an effective strategy to enhance ADCC and ADCP by NK cells, macrophages/monocytes and neutrophils independent of inhibitory KIR expression. In whole blood assays derived from healthy donors and chronic lymphocytic leukemia (CLL) patients these properties result in superior depletion of normal and malignant B cells in terms of potency and absolute B cell depletion as compared to rituximab. In order to further enhance the efficacy of ADCC competent therapeutic antibodies we have developed a novel class of tumor-targeted IL2v immunocytokines with the aim to activate and induce the proliferation of innate and adaptive immune effector cells in the tumor. In the FAP-IL2v immunocytokine an IL-2 variant (IL2v) with abolished CD25 binding is fused to the C-terminus of one of the Fc chains of a tumor stroma-targeted FAP antibody. FAP-IL2v lacks binding to CD25, but retains IL-2Rßγ binding so that is no longer preferentially activates Treg cells and reduces sensitivity for Fas-mediated apoptosis as compared to wildtype IL-2-based FAP-IL2wt immunocytokine. In C56BL/6 mice, FAP-IL2v strongly expands and activates NK cells and CD8+ T cells, and skews the CD4:CD8 ratio towards CD8+ T cells. In vitro, FAP-IL2v induces the proliferation and activation of NK cells and enhances the cytotoxic activity of NK cells in combination with ADCC competent antibodies. Similarily, in xenograft (hCD16 transgenic SCID mice) and BALB-neuT (immunocompetent ratHER2 transgenic mice) models, FAP-IL2v strongly enhances the antitumoral efficacy and survival mediated by HER2 antibodies making it a promising combination partner for ADCC-competent antibodies in future clinical investigation. Citation Format: Christian Klein. Enhancing immune effector function via antibody engineering. [abstract]. In: Proceedings of the AACR Special Conference: Tumor Immunology and Immunotherapy: A New Chapter; December 1-4, 2014; Orlando, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2015;3(10 Suppl):Abstract nr IA07.

TGF-β1 gene − 509C > T promoter polymorphism modulates TGF-β1 levels in hepatitis E patients

Elevated levels of transforming growth factor-β1 (TGF-β1) and its positive correlation with Foxp3 expression in hepatitis E patients have indicated involvement of TGF-β1 in hepatitis E pathogenesis. The current study determined polymorphisms in TGF-β1 gene, plasma TGF-β1 levels and T effector (Teff) cell proliferation and explored their association in a case control study. Polymorphisms in three selected sites (−509C>T, +869T>C and +915G>C) of TGF-β1 gene by PCR & restriction fragment length polymorphism methods, plasma TGF-β1 quantitation by ELISA and Teff (CD4+CD25−) cell proliferation by CFSE method were carried out in 277 hepatitis E patients (HE) with self-limiting infection and 233 ethnically matched healthy controls (HCs) from western India. Frequency of CT genotype of −509C>T site was significantly higher in hepatitis E patients compared to healthy controls (p=0.017; OR 1.53, 95% CI 1.07–2.17). Plasma TGF-β1 levels were significantly higher in HE compared to HCs. TGF-β1 level of patient group having CT genotype of −509C>T site was significantly higher compared to those having CC or TT genotypes. Teff cell proliferation was negatively correlated with plasma TGF-β1 levels in HE patients (r=−0.568; p=0.014). Influence of TGF-β1 promoter (−509C>T) polymorphism on plasma TGF-β1 levels and inverse correlation of Teff cell proliferation with plasma TGF-β1 levels in self-limiting hepatitis E patients suggest key role of TGF-β1 in augmentation of reported T regulatory cell mediated pathogenesis in hepatitis E.

Macrophages play an essential role in antigen-specific immune suppression mediated by T CD8⁺ cell-derived exosomes.

Murine contact sensitivity (CS) reaction could be antigen-specifically regulated by T CD8(+) suppressor (Ts) lymphocytes releasing microRNA-150 in antibody light-chain-coated exosomes that were formerly suggested to suppress CS through action on macrophages (Mφ). The present studies investigated the role of Mφ in Ts cell-exosome-mediated antigen-specific suppression as well as modulation of Mφ antigen-presenting function in humoral and cellular immunity by suppressive exosomes. Mice depleted of Mφ by clodronate liposomes could not be tolerized and did not produce suppressive exosomes. Moreover, isolated T effector lymphocytes transferring CS were suppressed by exosomes only in the presence of Mφ, demonstrating the substantial role of Mφ in the generation and action of Ts cell regulatory exosomes. Further, significant decrease of number of splenic B cells producing trinitrophenyl (TNP) -specific antibodies with the alteration of the ratio of serum titres of IgM to IgG was observed in recipients of exosome-treated, antigen-pulsed Mφ and the significant suppression of CS was demonstrated in recipients of exosome-treated, TNP-conjugated Mφ. Additionally, exosome-pulsed, TNP-conjugated Mφ mediated suppression of CS in mice pre-treated with a low-dose of cyclophosphamide, suggesting de novo induction of T regulatory (Treg) lymphocytes. Treg cell involvement in the effector phase of the studied suppression mechanism was proved by unsuccessful tolerization of DEREG mice depleted of Treg lymphocytes. Furthermore, the inhibition of proliferation of CS effector cells cultured with exosome-treated Mφ in a transmembrane manner was observed. Our results demonstrated the essential role of Mφ in antigen-specific immune suppression mediated by Ts cell-derived exosomes and realized by induction of Treg lymphocytes and inhibition of T effector cell proliferation.

The immunosuppressive enzyme IL4I1 promotes FoxP3(+) regulatory T lymphocyte differentiation.

IL4I1 (interleukin-4-induced gene 1) is a phenylalanine oxidase produced mainly by APCs of myeloid origin, and converts phenylalanine (Phe) to phenylpyruvate, hydrogen peroxide, and ammonia. We have previously shown that IL4I1 is highly expressed by tumor-associated macrophages from various human cancers and facilitates immune evasion from the cytotoxic response in a murine tumor model. Indeed, IL4I1 inhibits T-cell proliferation via hydrogen peroxide toxicity on effector/memory Tcells. Here, we explored the effect of IL4I1 on naïve CD4(+) T-cell differentiation. We show that IL4I1 stimulates the generation of Foxp3(+) regulatory T (Treg) cells in vitro from human and mouse Tcells. This effect was observed with IL4I1 from different sources, including the naturally produced enzyme. Conversely, IL4I1 limits Th1 and Th2 polarization while modifying the Th17 phenotype, in particular, by inducing its own production. Analysis of Treg-cell induction under conditions of Phe deprivation and hydrogen peroxide addition suggests that Phe consumption by the enzyme participates in Treg-cell enrichment. In line with this hypothesis, IL4I1 inhibits mTORC1 signaling shortly after T-cell activation. Thus, the IL4I1 enzyme may act on Tcells both by direct inhibition of effector cell proliferation and by indirect immunoregulation mediated by Treg-cell induction.

Mesenchymal Stromal Cells and Viral Infection.

Mesenchymal Stromal Cells (MSCs) are a subset of nonhematopoietic adult stem cells, readily isolated from various tissues and easily culture-expanded ex vivo. Intensive studies of the immune modulation and tissue regeneration over the past few years have demonstrated the great potential of MSCs for the prevention and treatment of steroid-resistant acute graft-versus-host disease (GvHD), immune-related disorders, and viral diseases. In immunocompromised individuals, the immunomodulatory activities of MSCs have raised safety concerns regarding the greater risk of primary viral infection and viral reactivation, which is a major cause of mortality after allogeneic transplantation. Moreover, high susceptibilities of MSCs to viral infections in vitro could reflect the destructive outcomes that might impair the clinical efficacy of MSCs infusion. However, the interplay between MSCs and virus is like a double-edge sword, and it also provides beneficial effects such as allowing the proliferation and function of antiviral specific effector cells instead of suppressing them, serving as an ideal tool for study of viral pathogenesis, and protecting hosts against viral challenge by using the antimicrobial activity. Here, we therefore review favorable and unfavorable consequences of MSCs and virus interaction with the highlight of safety and efficacy for applying MSCs as cell therapy.

Targeting Immune Suppressive Microenvironment By Immune Checkpoint Blockade in Multiple Myeloma

Bidirectional interaction between MM cells and accessory cells regulates tumor development on the one hand, while transforming the BM microenvironment into a tumor promoting and immune suppressive milieu on the other. Recent developments in targeted therapies have indicated that generation of the most effective therapeutic strategies requires not only targeting tumor or stroma, but also methods to overcome blockade of anti-tumor immune responses. Tumor associated immune suppressor cells such as Treg and myeloid derived suppressor cells (MDSC) can effectively block anti-tumor immune responses, thereby representing an important obstacle for immunotherapy. Co-inhibitory molecules programmed cell death-1 (PD-1) and its ligand (PD-L1) play a fundamental role in tumor immune escape by inhibiting immune effector functions. Since PD-1/PD-L1 signaling promotes tumor growth while inhibiting effector cell mediated anti-tumor immune responses, we here assessed the impact of PD-1/PD-L1 blockade, alone or in combination with lenalidomide (Len), on accessory and immune cells function, as well as tumor cell growth of MM in the BM milieu.Methods: PD-L1 gene expression was determined in IFM MM data set (n=170). Cell surface expression of PD-L1 in CD138+ MM cells, stroma, and MDSCs; as well as PD-1 expression on effector cells (CD4T, CD8T, NK, NKT and monocytes/macrophages) were determined in the bone marrow (BM) and peripheral blood (PB) of patients with MGUS (n=3), newly diagnosed (ND-MM, n=5) and relapsed/refractory (RR-MM, n=11) compared to healthy donors (HD, n=10). PD-1/PD-L1 signaling was assessed in the autologous cocultures of patient MM cells or MM cell lines with stroma (BMSC) or MDSCs and effector cells, in the presence or absence of PD-1/PD-L1 blockade and Len. Len effect on PD-1 expression on effector cells and PD-L1 expression on tumor, BMSC and MDSCs was determined by flow cytometry analysis. BMSC and MDSC induced MM growth/viability was measured by MTT, 3H-Thy and CFSE flow cytometry analysis. Effector cell-mediated MM cytotoxicity was measured by CFSE/PI flow cytometry. The effect of PD-1/PD-L1 blockade with or without Len on cytokine pattern was determined by intracellular cytokine flow cytometry analysis.Results: Statistical analysis of IFM MM data demonstrated that the majority of patient MM cells have increased PD-L1 mRNA compared to HD (p=0.0064). Cell surface expression of PD-L1 was also significantly increased in ND-MM cells (median 65%) and even higher in RR-MM cells. Correspondingly, there was a significant increase in PD-1 expression on CD8T and NK cells in ND-MM and RR-MM. Moreover, PD-L1 expression was significantly higher on both mMDSC and nMDSC than APCs in ND-MM and RR-MM. Coculture with BMSC significantly increased expression of PD-L1 on MM cells. PD-1/ PD-L1 blockade overcame BMSC-induced tumor cell growth in both patient MM cells and MM cell lines. Importantly, Len significantly reduced PD-L1 expression on MM cells; and combined blockade of PD-1/PD-L1 with Len further decreased BMSC-induced MM growth. Immunomodulatory effects of PD-1/PD-L1 blockade were also evaluated in autologous cocultures of immune effector cells with MM cells. Even though there was no change in effector cell proliferation, PD-1/PD-L1 blockade significantly induced cytotoxic activity of autologous T cells, NK cells, and macrophages cultured with MM cells; and Len further enhanced effector cell-mediated cytotoxicity. PD-1/PD-L1 blockade induced intracellular expression of cytotoxic cytokines IFNg and Granzyme B (Gzm B) in CD4T cells, CD8T cells, NK cells and Macrophages. Furthermore, MDSC-mediated MM growth was significantly decreased by PD-1/ PD-L1 blockade. Finally, PD-1/PD-L1 blockade induced intracellular expression of IFNg and Gzm B in T cells, NK cells and NKT cells cultured with autologous MDSC; and Len further enhanced this effector cell activation.Conclusion: Our data demonstrated that immune checkpoint signaling plays an important role in providing the tumor promoting, immune suppressive microenvironment in MM. Blockade of PD-1/PD-L1 signaling induces anti-MM immune responses that can be enhanced by Len. Targeting checkpoint signaling using PD-1 and PD-L1 blocking antibodies, particularly in combination with Len, therefore represents a promising novel immune-based therapeutic strategy to both inhibit tumor cell growth and restore host immune function in MM. DisclosuresKikuchi:The ITO Foundation for the Promotion of Medical Science: Research Funding. Hideshima:Acetylon Pharmaceuticals: Consultancy. Raje:novartis, Amgen, Celgene, Millenium, Onyx: Consultancy; Eli Lilly, Acetylon: Research Funding. Anderson:Celgene: Consultancy; Onyx: Consultancy; Gilead Sciences: Consultancy; Sanofi-Aventis US: Consultancy; Acetylon: Scientific Founder Other; Oncoprep: Scientific Founder Other.