Activation Of Effector CD8 Research Articles

Abstract 1734: Modulation of immune responses to cancer by bi-specific IL-2/IL-13 superkines

Abstract Introduction: The IL-2 pathway plays a vital role in stimulating a pro-inflammatory (Th1) response against cancer through expansion and activation of effector CD8 T and NK cells. In contrast, the IL-4/IL-13 pathway stimulates myeloid derived suppressor cells (MDSCs) and M2 skewing of tumor associated macrophage (TAM) to foster an anti-inflammatory (Th2) response that is often exploited by cancers as a means to dampen the effects of the Th1 pathway. Therefore, suppression of MDSC and M2 TAM through inhibition of the IL-4/IL-13 pathway together with stimulation of effector immune cells through activation of the IL-2 pathway have the potential to invigorate a pro-inflammatory response in an otherwise immune suppressive tumor microenvironment (TME). To achieve this goal, we leveraged the versatility of our IL-2 and IL-13 superkine platforms to engineer long-acting bi-specific constructs to co-target surface receptors of these respective pathways. Experimental Procedure: Studies included binding analyses by BLI/Octet and Biacore/SPR, signaling analyses using IL-2 and IL-4/IL-13 reporter assays, signaling analysis in human PBMC, and in vitro M1/M2 macrophage polarization assay. Summary of Data: sIL2M-Fc-sIL13M is a bi-specific superkine composed of an IL-2 super-agonist (sIL2M) and IL-13 super-antagonist (sIL13M) linked together by human IgG1 Fc. sIL2M binds CD122 with superior affinity over IL-2 but does not engage CD25, which is expressed on immune-suppressive Tregs. sIL13M has higher affinity than IL-13 for IL13Rα1, which together with IL-4Rα forms a functional receptor complex. In pSTAT5 assay using primary human PBMCs, sIL2M-Fc-sIL13M showed enhanced potency over IL-2 in the activation of CD8 T-cells and NK cells while exhibiting limited activity on Treg. In an IL-4/IL-13 dependent pSTAT6 reporter assay, sIL2M-Fc-sIL13M demonstrated dose-dependent antagonism against stimulatory activity of both IL-4 and IL-13. This antagonistic effect was further validated with inhibition of IL-13 induced M2 polarization of macrophages in vitro. Conclusion: sIL2M-Fc-sIL13M is a bi-specific superkine capable of concomitantly stimulating a Th1 response through activation of the IL-2 pathway and suppressing a Th2 response through inhibition of the IL-4/IL-13 pathway. Additional bi-specific superkine constructs are currently under testing, including those designed to enable accumulation in TME by engaging the decoy IL-13Rα2 that is overexpressed on a number of different tumor types. Citation Format: Fahar Merchant, Minh To. Modulation of immune responses to cancer by bi-specific IL-2/IL-13 superkines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1734.

Multiple sclerosis patients have reduced resting and increased activated CD4+CD25+FOXP3+T regulatory cells

Resting and activated subpopulations of CD4+CD25+CD127loT regulatory cells (Treg) and CD4+CD25+CD127+ effector T cells in MS patients and in healthy individuals were compared. Peripheral blood mononuclear cells isolated using Ficoll Hypaque were stained with monoclonal antibodies and analysed by flow cytometer. CD45RA and Foxp3 expression within CD4+ cells and in CD4+CD25+CD127loT cells identified Population I; CD45RA+Foxp3+, Population II; CD45RA−Foxp3hi and Population III; CD45RA−Foxp3+ cells. Effector CD4+CD127+ T cells were subdivided into Population IV; memory /effector CD45RA− CD25−Foxp3− and Population V; effector naïve CD45RA+CD25−Foxp3−CCR7+ and terminally differentiated RA+ (TEMRA) effector memory cells. Chemokine receptor staining identified CXCR3+Th1-like Treg, CCR6+Th17-like Treg and CCR7+ resting Treg. Resting Treg (Population I) were reduced in MS patients, both in untreated and treated MS compared to healthy donors. Activated/memory Treg (Population II) were significantly increased in MS patients compared to healthy donors. Activated effector CD4+ (Population IV) were increased and the naïve/ TEMRA CD4+ (Population V) were decreased in MS compared to HD. Expression of CCR7 was mainly in Population I, whereas expression of CCR6 and CXCR3 was greatest in Populations II and intermediate in Population III. In MS, CCR6+Treg were lower in Population III. This study found MS is associated with significant shifts in CD4+T cells subpopulations. MS patients had lower resting CD4+CD25+CD45RA+CCR7+ Treg than healthy donors while activated CD4+CD25hiCD45RA−Foxp3hiTreg were increased in MS patients even before treatment. Some MS patients had reduced CCR6+Th17-like Treg, which may contribute to the activity of MS.

Targeted Deletion of CXCR2 in Myeloid Cells Alters the Tumor Immune Environment to Improve Antitumor Immunity.

Recruitment of myeloid-derived suppressor cells (MDSC) into the tumor microenvironment (TME) contributes to cancer immune evasion. MDSCs express the chemokine receptor CXCR2, and inhibiting CXCR2 suppresses the recruitment of MDSCs into the tumor and the premetastatic niche. Here, we compared the growth and metastasis of melanoma and breast cancer xenografts in mice exhibiting or not exhibiting targeted deletion of Cxcr2 in myeloid cells (CXCR2myeΔ/Δ vs. CXCR2myeWT). Detailed analysis of leukocyte populations in peripheral blood and in tumors from CXCR2myeΔ/Δ mice revealed that loss of CXCR2 signaling in myeloid cells resulted in reduced intratumoral MDSCs and increased intratumoral CXCL11. The increase in intratumoral CXCL11 was derived in part from tumor-infiltrating B1b cells. The reduction in intratumoral MDSCs coupled with an increase in intratumoral B1b cells expressing CXCL11 resulted in enhanced infiltration and activation of effector CD8+ T cells in the TME of CXCR2myeΔ/Δ mice, accompanied by inhibition of tumor growth in CXCR2myeΔ/Δ mice compared with CXCR2myeWT littermates. Treatment of tumor-bearing mice with a CXCR2 antagonist (SX-682) also inhibited tumor growth, reduced intratumoral MDSCs, and increased intratumoral B1b cells expressing CXCL11, leading to an increase in activated CD8+ T cells in the tumor. Depletion of B220+ cells or depletion of CD8+ T cells reversed the tumor-inhibitory properties in CXCR2myeΔ/Δ mice. These data revealed a mechanism by which loss of CXCR2 signaling in myeloid cells modulates antitumor immunity through decreasing MDSCs and enriching CXCL11-producing B1b cells in the TME, which in turn increases CD8+ T-cell recruitment and activation in tumors.

Continuous inertial cavitation evokes massive ROS for reinforcing sonodynamic therapy and immunogenic cell death against breast carcinoma

Intratumoral immunosuppressive microenvironment (ISM) remains the dominant limitation to disable oncological immunotherapy such as immunogenic cell death (ICD). To resolve the immune escape, a sonodynamic therapy (SDT)-based nanoplatform featuring continuous CO2 bubbling has been engineered to enforce continuous ultrasound-triggered inertial cavitation (UIC) for augmenting ROS production. Systematic in vitro and in vivo results demonstrate that the continuous UIC expedites massive production of reactive oxygen species (ROS), consequently enabling multiple enhancements of SDT under only one administration. More significantly, the highly-accumulative ROS arising from continuous UIC have been demonstrated to induce robust ICD that is typically represented by more antigen exposure and presentation, augmented DCs maturation and more activated effector CD8+ T cells infiltration in vitro &in vivo. Concurrently, the most ISM alleviation via releasing more pro-inflammatory cytokines and facilitating pro-tumorigenic M2-like macrophage polarization into anti-tumorigenic M1-like counterparts is accompanied, enabling immune escape blockade. Contributed by the significant ISM alleviation and massive ROS production for enhancing SDT and ICD, such SDT-based composite nanoplatforms harvest the most substantially enhanced inhibitory consequences against primary and metastatic tumors, which, thus, provide a profound attribute for T cell-based immunotherapy against tumor.

Cytokine driven effector functions of CD8 cells: application to protease-activated cytokine therapy for cancer

Abstract Cytokines are potent immune mediators and play a crucial role in the differentiation and activation of effector CD8 T cells which are critical in anti-tumor responses. Here we tested the ability of IL-2, IL-12, and IL-18 to drive expression of gamma interferon (IFNg) in the absence of intentional antigen stimulation. We show in vitro that spleen cells from both BALB/c and C57BL/6 mice produced large amounts of IFNg and downstream mediators in response to these cytokines individually or synergistically in combination. Moreover, CD8 cells purified from Colon38 tumors can be similarly activated, demonstrating that they are not permanently inactivated or exhausted. These data illustrate innate-like function of CD8 cells, including those in the tumor microenvironment (TME). To address concerns of severe toxicities that can accompany systemic delivery of cytokines, we designed protease-activated cytokine fusion proteins (FPs) which exploit the overexpression of proteases in tumors to release active cytokine in the TME. Using gene transfection techniques, we show that an IL-2FP with a matrix metalloproteinase 2, 9 cleavage sequence can be activated in the TME and reduce tumor growth. In contrast, a FP with a scrambled non-cleavable sequence does not reduce tumor growth, illustrating the specificity of cleavage of the FP in vivo. Additionally, we demonstrate that systemic expression of the IL-2FP elicits little systemic toxicity in striking contrast to the delivery of free IL-2. Collectively, these data support the development of cytokine fusions proteins as a new class of therapeutics to promote anti-tumor effectors with limited detrimental cytokine side effects.

Abstract B046: Addition of anti-VEGF overcomes the anti-PD-L1 refractoriness in a OV2944-HM-1 tumor model

Abstract Background: Efficacy of combination therapy with atezolizumab plus bevacizumab was shown in the IMpower150 trial. In this study, we use the anti-PD-L1 sensitive Colon 38 mouse tumor model and the anti-PD-L1 insensitive OV2944-HM-1 (HM-1) mouse tumor model to investigate the efficacy and the mechanisms of this combination. Methods: Colon 38 and HM-1 tumor cells were subcutaneously inoculated into the right flank of C57BL/6J and B6C3F1 mice, respectively. Mice with established tumors were randomized into treatment groups (day1). Anti-PD-L1 mAb (anti-PD-L1, clone 6E11) or mouse IgG was administrated intraperitoneally to the mice at a dose of 5 mg/kg twice a week from day 1. Anti-VEGF mAb (anti-VEGF, clone B20-4.1.1) or mouse IgG was administrated intraperitoneally at a dose of 10 mg/kg weekly from day 1. Anti-CD8 mAb (anti-CD8, clone 116-13.1) was intraperitoneally administered at a dose of 100 µg/head twice a week from day -10. Anti-CXCR3 mAb (anti-CXCR3, clone CXCR3-173) was intraperitoneally administered at a dose of 100 µg/head twice a week from day 1. Tumor volume was measured twice a week. The tumors were sampled on day 1 and were subjected to immunohistochemical staining using the anti-CD8 mAb (anti-CD8, clone KT15) and anti-B7-H1/PD-L1 polyclonal antibody. The tumors were sampled on day 4 or 8 and were subjected to flow cytometry analysis using the antibody for CD8 and Granzyme B, and ELISA analysis for CXCL9. Results: HM-1 tumors showed lower PD-L1 expression under conditions where Colon 38 tumors were partially PD-L1 positive. CD8+ T cells were hardly observed in HM-1 tumors, while CD8+ T cells were prominently observed in Colon 38 tumors. In the Colon 38 model, combination treatment with anti-PD-L1 plus anti-VEGF significantly inhibited the tumor growth compared with the single agent treatments which themselves significantly inhibited the tumor growth compared with control treatment. In the HM-1 model, combination treatment with anti-PD-L1 plus anti-VEGF significantly inhibited the tumor growth compared with the anti-VEGF treatment which itself significantly inhibited the tumor growth compared with control treatment. In the HM-l model, significantly larger number of CD8+ T cells and Granzyme B+CD8+ T cells and a significantly higher level of CXCL9 were observed in tumor tissue with anti-PD-L1 treatment compared with control treatment on day 4. However, these statuses were not maintained on day 8. On the other hand, these statuses were observed in tumor tissue with combination treatment both on day 4 and day 8. Anti-CD8 canceled the potentiation of the antitumor effect in combination treatment to the level of anti-VEGF treatment. Anti-CXCR3 canceled the increased number of intratumoral CD8+ T cells in combination treatment to the level of control treatment. Conclusions: This is the first study to show that the combination treatment of anti-PD-L1 plus anti-VEGF exhibits more potent anti-tumor activity compared with single agent treatments, not only in an anti-PD-L1 sensitive model but also in an anti-PD-L1 insensitive model with PD-L1 low and immune desert like properties. At least in the anti-PD-L1 insensitive model, this can be mainly explained by the maintained increase of intratumoral CXCL9 leading to the increased infiltration of activated effector CD8+ T cells into tumor tissue. Conflict of interest: The authors are employees of Chugai Pharmaceutical Co., Ltd. Citation Format: Nobuyuki Ishikura, Keigo Yorozu, Mitsue Kurasawa, Masamichi Sugimoto, Osamu Kondoh. Addition of anti-VEGF overcomes the anti-PD-L1 refractoriness in a OV2944-HM-1 tumor model [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr B046. doi:10.1158/1535-7163.TARG-19-B046

Conditional Upregulation of IFN-α Alone Is Sufficient to Induce Systemic Lupus Erythematosus.

The cause of systemic lupus erythematosus (SLE) is unknown. IFN-α has been suggested as a causative agent of SLE; however, it was not proven, and to what extent and how IFN-α contributes to the disease is unknown. We studied the contribution of IFN-α to SLE by generating inducible IFN-α transgenic mice and directly show that conditional upregulation of IFN-α alone induces a typical manifestation of SLE in the mice not prone to autoimmunity, such as serum immune complex, autoantibody against dsDNA (anti-dsDNA Ab), and the organ manifestations classical to SLE, such as immune complex-deposited glomerulonephritis, classical splenic onion-skin lesion, alopecia, epidermal liquefaction, and positive lupus band test of the skin. In the spleen of mice, activated effector CD4 T cells, IFN-γ-producing CD8 T cells, B220+CD86+ cells, and CD11c+CD86+ cells were increased, and the T cells produced increased amounts of IL-4, IL-6, IL-17, and IFN-γ and decreased IL-2. In particular, activated CD3+CD4-CD8- double-negative T cells positive for TCRαβ, B220, CD1d-teteramer, PD-1, and Helios (that produced increased amounts of IFN-γ, IL-4, IL-17, and TNF-α) were significantly expanded. They infiltrated into kidney and induced de novo glomerulonephritis and alopecia when transferred into naive recipients. Thus, sole upregulation of IFN-α is sufficient to induce SLE, and the double-negative T cells expanded by IFN-α are directly responsible for the organ manifestations, such as lupus skin disease or nephritis.

MALT1 Proteolytic Activity Suppresses Autoimmunity in a T Cell Intrinsic Manner.

MALT1 is a central signaling component in innate and adaptive immunity by regulating NF-κB and other key signaling pathways in different cell types. Activities of MALT1 are mediated by its scaffold and protease functions. Because of its role in lymphocyte activation and proliferation, inhibition of MALT1 proteolytic activity is of high interest for therapeutic targeting in autoimmunity and certain lymphomas. However, recent studies showing that Malt1 protease-dead knock-in (Malt1-PD) mice suffer from autoimmune disease have somewhat tempered the initial enthusiasm. Although it has been proposed that an imbalance between immune suppressive regulatory T cells (Tregs) and activated effector CD4+ T cells plays a key role in the autoimmune phenotype of Malt1-PD mice, the specific contribution of MALT1 proteolytic activity in T cells remains unclear. Using T cell-conditional Malt1 protease-dead knock-in (Malt1-PDT) mice, we here demonstrate that MALT1 has a T cell-intrinsic role in regulating the homeostasis and function of thymic and peripheral T cells. T cell-specific ablation of MALT1 proteolytic activity phenocopies mice in which MALT1 proteolytic activity has been genetically inactivated in all cell types. The Malt1-PDT mice have a reduced number of Tregs in the thymus and periphery, although the effect in the periphery is less pronounced compared to full-body Malt1-PD mice, indicating that also other cell types may promote Treg induction in a MALT1 protease-dependent manner. Despite the difference in peripheral Treg number, both T cell-specific and full-body Malt1-PD mice develop ataxia and multi-organ inflammation to a similar extent. Furthermore, reconstitution of the full-body Malt1-PD mice with T cell-specific expression of wild-type human MALT1 eliminated all signs of autoimmunity. Together, these findings establish an important T cell-intrinsic role of MALT1 proteolytic activity in the suppression of autoimmune responses.

Abstract A156: PDA tumor cell death as following combination anti-PD-1 blockade and CXCR4 blockade is a direct effect of CD8+ T-cells

Abstract Introduction: Pancreatic ductal adenocarcinoma (PDA) is the fourth most common cause of cancer death in the United States. Despite the fact that the human PDA microenvironment contains a mixture of infiltrating immune cells, including activated effector CD8+ T-cells, attempts to apply immune checkpoint inhibitor therapy have not yet been successful in achieving a clinical response (1). Combining blockade of the immune checkpoint receptor PD-1 with blockade of the chemokine receptor CXCR4 in a mouse model of PDA produced T-cell-mediated tumor cell killing (2). We therefore hypothesized that, in a human PDA slice culture model system that maintains the intact tumor microenvironment (3), combination of CXCR4 blockade with PD-1 blockade would lead to anti-tumor T-cell activation. Methods: Cores (6 mm) were taken from freshly resected, sterile human PDA specimens and cut into 250 µm thick slices using a vibratome. The slices were treated with either IgG antibody isotype control, AMD3100 (a CXCR4 blocking small molecule drug) plus antibody isotype control, anti-PD-1 antibody, or a combination of anti-PD-1 antibody and AMD3100 for two days. The slices were then stained with fluorescently labelled antibodies for CD8 and EpCAM, as well as with SR-FLICA, a reagent that produces a fluorescent signal when bound to activated Caspase 3 and 7 enzymes. Live slices were maintained in a humidified, temperature-controlled CO2 chamber for time-lapse confocal imaging for 1 hour. Similarly, untreated slices were imaged for 90 minutes prior to and following combined anti-PD-1 and AMD3100 drug treatment. Each slice was imaged at 3 different positions to observe CD8+ T-cells, EpCAM+ tumor cells, and SR-FLICA+ apoptotic cells throughout the slice. Each cell type was counted at each position over the entire time course imaged. Results: Compared to antibody isotype control or monotherapy, slices treated with combined PD-1 and CXCR4 blockade contained a greater fraction of EpCAM+ tumor cells that were undergoing apoptosis, indicated by SR-FLICA positivity. This treatment also increased the proportion of EpCAM+ tumor cells with a CD8+ T-cell within 20 µm, and EpCAM+ cells had increased evidence of apoptosis when a CD8+ cell was nearby. When individual slices were imaged before and after combined PD-1 and CXCR4 blockade, a greater proportion of EpCAM+ cells were found to have a CD8+ cell nearby after treatment compared to before treatment, and the tumor cells with a CD8+ cell nearby were more like to be undergoing apoptosis compared to before treatment. Conclusion: Direct visualization of live pancreatic cancer slice cultures demonstrates that combined PD-1 and CXCR4 blockade enhances both the migration of CD8+ T-cells towards epithelial tumor cells and their anti-tumor cytotoxic activity.

Injectable polypeptide hydrogel-based co-delivery of vaccine and immune checkpoint inhibitors improves tumor immunotherapy.

Immunotherapy, an attractive option for cancer treatment, necessitates the direct stimulation of immune cells in vivo and the simultaneous effective inhibition of immunosuppressive tumor microenvironments.Methods: In the present study, we developed an injectable PEG-b-poly(L-alanine) hydrogel for co-delivery of a tumor vaccine and dual immune checkpoint inhibitors to increase tumor immunotherapy efficacy. Tumor cell lysates, granulocyte-macrophage colony stimulating factor (GM-CSF), and immune checkpoint inhibitors (anti-CTLA-4/PD-1 antibody) were readily encapsulated in the porous hydrogel during the spontaneous self-assembly of polypeptide in aqueous solution.Results: Sustained release of tumor antigens and GM-CSF persistently recruited and activated dendritic cells (DCs) and induced a strong T-cell response in vivo, which was further enhanced by the immune checkpoint therapy. The hydrogel vaccine also upregulated the production of IgG and the secretion of cytokines including IFN-γ, IL-4, and TNF-α. Importantly, the hydrogel-based combination therapy had superior immunotherapy effects against melanoma and 4T-1 tumor in comparison with the vaccine alone or in addition with a single immune checkpoint blockade. In studying the underlying mechanism, we found that the hydrogel-based combinatorial immunotherapy not only significantly increased the activated effector CD8+ T cells within the spleens and tumors of vaccinated mice, but also reduced the ratio of Tregs.Conclusion: Our findings indicate that the polypeptide hydrogel can be used as an effective sustained delivery platform for vaccines and immune checkpoint inhibitors, providing an advanced combinatorial immunotherapy approach for cancer treatment.

Differential control of human Treg and effector T cells in tumor immunity by Fc-engineered anti–CTLA-4 antibody

Anti-CTLA-4 mAb is efficacious in enhancing tumor immunity in humans. CTLA-4 is expressed by conventional T cells upon activation and by naturally occurring FOXP3+CD4+ Treg cells constitutively, raising a question of how anti-CTLA-4 mAb can differentially control these functionally opposing T cell populations in tumor immunity. Here we show that FOXP3high potently suppressive effector Treg cells were abundant in melanoma tissues, expressing CTLA-4 at higher levels than tumor-infiltrating CD8+ T cells. Upon in vitro tumor-antigen stimulation of peripheral blood mononuclear cells from healthy individuals or melanoma patients, Fc-region-modified anti-CTLA-4 mAb with high antibody-dependent cell-mediated cytotoxicity (ADCC) and cellular phagocytosis (ADCP) activity selectively depleted CTLA-4+FOXP3+ Treg cells and consequently expanded tumor-antigen-specific CD8+T cells. Importantly, the expansion occurred only when antigen stimulation was delayed several days from the antibody treatment to spare CTLA-4+ activated effector CD8+T cells from mAb-mediated killing. Similarly, in tumor-bearing mice, high-ADCC/ADCP anti-CTLA-4 mAb treatment with delayed tumor-antigen vaccination significantly prolonged their survival and markedly elevated cytokine production by tumor-infiltrating CD8+ T cells, whereas antibody treatment concurrent with vaccination did not. Anti-CTLA-4 mAb modified to exhibit a lesser or no Fc-binding activity failed to show such timing-dependent in vitro and in vivo immune enhancement. Thus, high ADCC anti-CTLA-4 mAb is able to selectively deplete effector Treg cells and evoke tumor immunity depending on the CTLA-4-expressing status of effector CD8+ T cells. These findings are instrumental in designing cancer immunotherapy with mAbs targeting the molecules commonly expressed by FOXP3+ Treg cells and tumor-reactive effector T cells.

Involvement of CD8+ T cell subsets in early response to vascular injury in patients with peripheral artery disease in vivo

AimsAdaptive immunity is critical in vascular remodelling following arterial injury. We hypothesized that acute changes in T cells at a percutaneous transluminal angioplasty (PTA) site could serve as an index of their potential interaction with the injured vascular wall. Methods and ResultsT cell subsets were characterised in 45 patients with Rutherford 3–4 peripheral artery disease (PAD) undergoing PTA. Direct angioplasty catheter blood sampling was performed before and immediately after the procedure. PTA was associated with an acute reduction of α/β-TcR CD8+ T cells. Further characterisation revealed significant reduction in pro-atherosclerotic CD28nullCD57+ T cells, effector (CD45RA+CCR7-) and effector memory (CD45RA-CCR7-) cells, in addition to cells bearing activation (CD69, CD38) and tissue homing/adhesion markers (CD38, CCR5). ConclusionsThe acute reduction observed here is likely due to the adhesion of cells to the injured vascular wall, suggesting that immunosenescent, activated effector CD8+ cells have a role in the early vascular injury immune response following PTA in PAD patients.

Protective effect of Lactobacillus reuteri DSM 17938 against experimental necrotizing enterocolitis is mediated by Toll-like receptor 2.

Lactobacillus reuteri DSM 17938 (LR 17938) has been shown to reduce the incidence and severity of necrotizing enterocolitis (NEC). It is unclear if preventing NEC by LR 17938 is mediated by Toll-like receptor 2 (TLR2), which is known to mediate proinflammatory responses to bacterial cell wall components. NEC was induced in newborn TLR2-/- or wild-type (WT) mice by the combination of gavage-feeding cow milk-based formula and exposure to hypoxia and cold stress. Treatment groups were administered formula supplemented with LR 17938 or placebo (deMan-Rogosa-Sharpe media). We observed that LR 17938 significantly reduced the incidence of NEC and reduced the percentage of activated effector CD4+T cells, while increasing Foxp3+ regulatory T cells in the intestinal mucosa of WT mice with NEC, but not in TLR2-/- mice. Dendritic cell (DC) activation by LR 17938 was mediated by TLR2. The percentage of tolerogenic DC in the intestine of WT mice was increased by LR 17938 treatment during NEC, a finding not observed in TLR2-/- mice. Furthermore, gut levels of proinflammatory cytokines IL-1β and IFN-γ were decreased after treatment with LR 17938 in WT mice but not in TLR2-/- mice. In conclusion, the combined in vivo and in vitro findings suggest that TLR2 receptors are involved in DC recognition and DC-priming of T cells to protect against NEC after oral administration of LR 17938. Our studies further clarify a major mechanism of probiotic LR 17938 action in preventing NEC by showing that neonatal immune modulation of LR 17938 is mediated by a mechanism requiring TLR2. NEW & NOTEWORTHY Lactobacillus reuteri DSM 17938 (LR 17938) has been shown to protect against necrotizing enterocolitis (NEC) in neonates and in neonatal animal models. The role of Toll-like receptor 2 (TLR2) as a sensor for gram-positive probiotics, activating downstream anti-inflammatory responses is unclear. Our current studies examined TLR2 -/- mice subjected to experimental NEC and demonstrated that the anti-inflammatory effects of LR 17938 are mediated via a mechanism requiring TLR2.

Peripheral T-Cell Reactivity to Heat Shock Protein 70 and Its Cofactor GrpE from Tropheryma whipplei Is Reduced in Patients with Classical Whipple's Disease.

Classical Whipple's disease (CWD) is characterized by the lack of specific Th1 response toward Tropheryma whipplei in genetically predisposed individuals. The cofactor GrpE of heat shock protein 70 (Hsp70) from T. whipplei was previously identified as a B-cell antigen. We tested the capacity of Hsp70 and GrpE to elicit specific proinflammatory T-cell responses. Peripheral mononuclear cells from CWD patients and healthy donors were stimulated with T. whipplei lysate or recombinant GrpE or Hsp70 before levels of CD40L, CD69, perforin, granzyme B, CD107a, and gamma interferon (IFN-γ) were determined in T cells by flow cytometry. Upon stimulation with total bacterial lysate or recombinant GrpE or Hsp70 of T. whipplei, the proportions of activated effector CD4+ T cells, determined as CD40L+ IFN-γ+, were significantly lower in patients with CWD than in healthy controls; CD8+ T cells of untreated CWD patients revealed an enhanced activation toward unspecific stimulation and T. whipplei-specific degranulation, although CD69+ IFN-γ+ CD8+ T cells were reduced upon stimulation with T. whipplei lysate and recombinant T. whipplei-derived proteins. Hsp70 and its cofactor GrpE are immunogenic in healthy individuals, eliciting effective responses against T. whipplei to control bacterial spreading. The lack of specific T-cell responses against these T. whipplei-derived proteins may contribute to the pathogenesis of CWD.

Role of bone marrow-derived CD11c+ dendritic cells in systolic overload-induced left ventricular inflammation, fibrosis and hypertrophy.

Inflammatory responses play an important role in the development of left ventricular (LV) hypertrophy and dysfunction. Recent studies demonstrated that increased T-cell infiltration and T-cell activation contribute to LV hypertrophy and dysfunction. Dendritic cells (DCs) are professional antigen-presenting cells that orchestrate immune responses, especially by modulating T-cell function. In this study, we investigated the role of bone marrow-derived CD11c+ DCs in transverse aortic constriction (TAC)-induced LV fibrosis and hypertrophy in mice. We observed that TAC increased the number of CD11c+ cells and the percentage of CD11c+ MHCII+ (major histocompatibility complex class II molecule positive) DCs in the LV, spleen and peripheral blood in mice. Using bone marrow chimeras and an inducible CD11c+ DC ablation model, we found that depletion of bone marrow-derived CD11c+ DCs significantly attenuated LV fibrosis and hypertrophy in mice exposed to 24weeks of moderate TAC. CD11c+ DC ablation significantly reduced TAC-induced myocardial inflammation as indicated by reduced myocardial CD45+ cells, CD11b+ cells, CD8+ T cells and activated effector CD8+CD44+ T cells in LV tissues. Moreover, pulsing of autologous DCs with LV homogenates from TAC mice promoted T-cell proliferation. These data indicate that bone marrow-derived CD11c+ DCs play a maladaptive role in hemodynamic overload-induced cardiac inflammation, hypertrophy and fibrosis through the presentation of cardiac self-antigens to T cells.

Old Mice Accumulate Activated Effector CD4 T Cells Refractory to Regulatory T Cell-Induced Immunosuppression.

Chronic low-grade inflammation and reduced lymphocyte potency are implicated in the pathogenesis of major illnesses associated with aging. Whether this immune phenotype results from a loss of cell-mediated regulation or intrinsic dysregulated function of effector T cells (Teffs) requires further research. Here, we report that, as compared with young C57BL6 mice, old mice show an increased frequency of CD4+CD62L− Teffs with a dysregulated activated phenotype and markedly increased effector functions. Analysis of the frequency and suppressive function of CD4+FoxP3+ regulatory T cells (Tregs) indicates an increase in the frequency of FoxP3+ T cells with aging which, however, occurs within the CD4+CD25− T cells. Furthermore, whereas Tregs from young and old mice similarly suppress Teffs from young mice, both have a compromised suppressive capacity of Teffs from old mice, a phenomenon which is partially recovered in the presence of IL-2-producing CD4+CD62L+ non-Teffs. Finally, we observed that Teff subsets from old mice are enriched with IL-17A-producing T cells and exhibit intrinsically dysregulated expression of genes encoding cell-surface molecules and transcription factors, which play a key role in T-cell activation and regulation. We, thus, demonstrate an age-related impairment in the regulation of effector CD4 T cells, which may underlie the higher risk for destructive inflammation associated with aging.

Targeting a metabolic pathway to fight the flu

Our antiviral arsenal to fight influenza viruses is limited and we need novel anti-flu drugs. Recently, cellular drug targets came into focus and omics analysis were instrumental to suggest candidate factors. In this issue of The FEBS Journal, Kainov and colleagues used transcriptome data to investigate virus-induced changes in tryptophan metabolism that may serve as immunomodulatory approach against viruses.

Abstract B90: PFK-158 is a first-in-human inhibitor of PFKFB3 that selectively suppresses glucose metabolism of cancer cells and inhibits the immunosuppressive Th17 cells and MDSCs in advanced cancer patients

Abstract Background: 6-phosphofructo-2-kinase (PFKFB3) is an enzyme that controls the intracellular concentration of fructose-2,6-bisphosphate which is an allosteric activator of 6-phosphofructo-1-kinase (PFK-1), a key enzyme of glycolysis. PFK-1 is tightly controlled by multiple metabolic feedback mechanisms and dictates the overall rate of glycolytic flux to lactate and the TCA cycle. In human cancers, several oncogenic proteins (e.g. HIF-1α, PTEN, and AKT) converge to increase the expression and activity of PFKFB3, leading to the high glycolytic rates typically observed in cancer cells. In addition to being a promising cancer metabolism target, PFKFB3 is required for the differentiation and tumor-promoting functions of the immunomodulatory Th17 cells and myeloid derived suppressor cells (MDSCs), which are attractive cellular targets to induce tumor immunity and potentially mediate intrinsic resistance to immune checkpoint inhibitors. PFK-158 is a potent selective small molecule inhibitor of PFKFB3 that displays broad anti-tumor activity causing significant growth inhibition in human and syngeneic preclinical models. As resistance mechanisms frequently activate pathways that result in up-regulation of glycolysis and PFKFB3, combination treatments of PFK-158 with cytotoxic and targeted agents have resulted in increased efficacy and tumor regression. Results: PFK-158 is a potent selective small molecule inhibitor of PFKFB3 that displays broad anti-tumor activity and causes significant growth inhibition in multiple human and syngeneic preclinical models. The tolerability and potential clinical benefit of PFK-158 are being investigated in advanced cancer patients with solid malignancies in a Phase 1 dose-escalation, multi-center clinical trial (clinicaltrials.gov # NCT02044861). The final cohort (650 mg/m2) has been open for enrollment and PFK-158 has been well tolerated to date. Secondary end-points have been incorporated to assess peripheral F2,6BP levels and immunosuppressive and effector cells populations. Of the 15 patients evaluable for response assessment at the end of two months of treatment, 6 patients have experienced a clinical benefit associated with PFK-158 administration, including a late stage pancreatic cancer patient that had a 75% reduction in her CA19-9 levels after 1 month, a renal cell carcinoma patient currently in month 9 and an adenocystic carcinoma patient in month 12. In addition, we examined the immunomodulatory effects of PFK-158 on Th17 cells and MDSCs in vitro, in B16 melanoma-bearing mice and in advanced cancer patients and found that PFK-158: (i) suppresses human Th17 cell and MDSC differentiation in vitro; (ii) decreases splenic and tumor-infiltrating Th17 cells, γδ T17 cells and MDSCs, and increases CD4+ and CD8+ T cells in the tumors of B16-F10 melanoma-bearing mice; and (iii) decreases peripheral blood Th17 cells, γδ T17 cells and MDSCs and increases activated effector CD4+ and CD8+ T cells in advanced cancer patients. Interestingly, we are discerning a correlation between the initial level of circulating Th17 cells and clinical responses to PFK-158. Conclusion: PFK-158 is the first-in-human and first-in-class PFKFB3 inhibitor that is currently under clinical development. To date, PFK-158 has been well tolerated and shows signs of clinical activity. In addition to controlling glycolysis, over expression of PFKFB3 in key immunesuppressive cells also leads to an immunomodulatory mechanism of action, suggesting that additional clinical benefit could result from combining PFK-158 with targeted agents as well as with immunotherapeutic agents. Note: This abstract was not presented at the conference. Citation Format: Sucheta Telang, Kavitha Yaddanapudi, Jaspreet Grewal, Rebecca Redman, Siqing Fu, Paula Pohlmann, Devalingam Mahalingam, Michael Kurman, Gilles Tapolsky, Jason Chesney.{Authors}. PFK-158 is a first-in-human inhibitor of PFKFB3 that selectively suppresses glucose metabolism of cancer cells and inhibits the immunosuppressive Th17 cells and MDSCs in advanced cancer patients. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2016 May 12-15; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(24 Suppl):Abstract nr B90.

Characterization of Human CD8 T Cell Responses in Dengue Virus-Infected Patients from India.

ABSTRACTEpidemiological studies suggest that India has the largest number of dengue virus infection cases worldwide. However, there is minimal information about the immunological responses in these patients. CD8 T cells are important in dengue, because they have been implicated in both protection and immunopathology. Here, we provide a detailed analysis of HLA-DR+ CD38+ and HLA-DR− CD38+ effector CD8 T cell subsets in dengue patients from India and Thailand. Both CD8 T cell subsets expanded and expressed markers indicative of antigen-driven proliferation, tissue homing, and cytotoxic effector functions, with the HLA-DR+ CD38+ subset being the most striking in these effector qualities. The breadth of the dengue-specific CD8 T cell response was diverse, with NS3-specific cells being the most dominant. Interestingly, only a small fraction of these activated effector CD8 T cells produced gamma interferon (IFN-γ) when stimulated with dengue virus peptide pools. Transcriptomics revealed downregulation of key molecules involved in T cell receptor (TCR) signaling. Consistent with this, the majority of these CD8 T cells remained IFN-γ unresponsive even after TCR-dependent polyclonal stimulation (anti-CD3 plus anti-CD28) but produced IFN-γ by TCR-independent polyclonal stimulation (phorbol 12-myristate 13-acetate [PMA] plus ionomycin). Thus, the vast majority of these proliferating, highly differentiated effector CD8 T cells probably acquire TCR refractoriness at the time the patient is experiencing febrile illness that leads to IFN-γ unresponsiveness. Our studies open novel avenues for understanding the mechanisms that fine-tune the balance between CD8 T cell-mediated protective versus pathological effects in dengue.IMPORTANCE Dengue is becoming a global public health concern. Although CD8 T cells have been implicated both in protection and in the cytokine-mediated immunopathology of dengue, how the balance is maintained between these opposing functions remains unknown. We comprehensively characterized CD8 T cell subsets in dengue patients from India and Thailand and show that these cells expand massively and express phenotypes indicative of overwhelming antigenic stimulus and tissue homing/cytotoxic-effector functions but that a vast majority of them fail to produce IFN-γ in vitro. Interestingly, the cells were fully capable of producing the cytokine when stimulated in a T cell receptor (TCR)-independent manner but failed to do so in TCR-dependent stimulation. These results, together with transcriptomics, revealed that the vast majority of these CD8 T cells from dengue patients become cytokine unresponsive due to TCR signaling insufficiencies. These observations open novel avenues for understanding the mechanisms that fine-tune the balance between CD8-mediated protective versus pathological effects.

Adhesion Molecules Associated with Female Genital Tract Infection.

Efforts to develop vaccines that can elicit mucosal immune responses in the female genital tract against sexually transmitted infections have been hampered by an inability to measure immune responses in these tissues. The differential expression of adhesion molecules is known to confer site-dependent homing of circulating effector T cells to mucosal tissues. Specific homing molecules have been defined that can be measured in blood as surrogate markers of local immunity (e.g. α4β7 for gut). Here we analyzed the expression pattern of adhesion molecules by circulating effector T cells following mucosal infection of the female genital tract in mice and during a symptomatic episode of vaginosis in women. While CCR2, CCR5, CXCR6 and CD11c were preferentially expressed in a mouse model of Chlamydia infection, only CCR5 and CD11c were clearly expressed by effector T cells during bacterial vaginosis in women. Other homing molecules previously suggested as required for homing to the genital mucosa such as α4β1 and α4β7 were also differentially expressed in these patients. However, CD11c expression, an integrin chain rarely analyzed in the context of T cell immunity, was the most consistently elevated in all activated effector CD8+ T cell subsets analyzed. This molecule was also induced after systemic infection in mice, suggesting that CD11c is not exclusive of genital tract infection. Still, its increase in response to genital tract disorders may represent a novel surrogate marker of mucosal immunity in women, and warrants further exploration for diagnostic and therapeutic purposes.