Effector Function Of CD8 Research Articles

Boosting the synergism between cancer ferroptosis and immunotherapy via targeted stimuli-responsive liposomes

Both ferroptotic therapy and immunotherapy have been widely employed in cancer treatment. However, ferroptotic cell death fails to induce dendritic cells maturation, which limits the therapeutic outcome of ferroptotic cancer therapy. To address this, the current work reports a tailored liposome to establish a positive loop between ferroptotic therapy and immunotherapy. As the key component of liposome, a unique phospholipid is designed to bear two arachidonic acid tails. The liposome is further surface-engineered with fucose ligand and physically encapsulates immunostimulatory CpG oligodeoxynucleotides (ODNs). The tailored liposome shows enhanced cellular uptake in a model 4T1 cell line. Meanwhile, the high level of reactive oxygen species in cancer cells can induce ferroptosis-specific peroxidation of DAPC and trigger the release CpG ODNs. The CpG ODNs further enable the maturation of dendritic cells and enhance the effector function of CD8+ T cells. IFN-γ released from CD8+ T cells promotes cancer cell ferroptosis via inhibiting SLC7A11 and suppressing the biosynthesis of glutathione. The tailored liposome can also act in synergism with PD-L1 antibody, resulting in enhanced anti-cancer efficacy in a 4T1 tumor-bearing mice model. This work provides a promising strategy for cancer treatment through orchestrating ferroptotic therapy and immunotherapy.

201P Role of aerobic exercise training on the mitochondrial metabolism and effector function of CD8+ tumor-infiltrating lymphocytes

201P Role of aerobic exercise training on the mitochondrial metabolism and effector function of CD8+ tumor-infiltrating lymphocytes

Fructose sweetens the adipocyte-T cell alliance against tumors

Dietary fructose is implicated in tumorigenesis, but whether dietary fructose regulates antitumor immunity remains elusive. In this issue of Cell Metabolism, Zhang etal. show that dietary fructose promotes adipocyte-derived leptin production, which attenuates terminal exhaustion programming and boosts the effector function of CD8+ Tcells for improved tumor control.

Immunological link between periodontitis and type 2 diabetes deciphered by single-cell RNA analysis.

Type 2 diabetes mellitus (DM) is a complex metabolic disorder that causes various complications, including periodontitis (PD). Although a bidirectional relationship has been reported between DM and PD, their immunological relationship remains poorly understood. Therefore, this study aimed to compare the immune response in patients with PD alone and in those with both PD and DM (PDDM) to expand our knowledge of the complicated connection between PD and DM. Peripheral blood mononuclear cells were collected from 11 healthy controls, 10 patients with PD without DM, and six patients with PDDM, followed by analysis using single-cell RNA sequencing. The differences among groups were then compared based on intracellular and intercellular perspectives. Compared to the healthy state, classical monocytes exhibited the highest degree of transcriptional change, with elevated levels of pro-inflammatory cytokines in both PD and PDDM. DM diminished the effector function of CD8+ T and natural killer (NK) cells as well as completely modified the differentiation direction of these cells. Interestingly, a prominent pathway, RESISTIN, which is known to increase insulin resistance and susceptibility to diabetes, was found to be activated under both PD and PDDM conditions. In particular, CAP1+ classical monocytes from patients with PD and PDDM showed elevated nuclear factor kappa B-inducing kinase activity. Overall, this study elucidates how the presence of DM contributes to the deterioration of T/NK cell immunity and the immunological basis connecting PD to DM.

Abstract A005: Synergistically Engaging a b-Selective IL-2 Agonist with PD1/PDL-1 Blockade in a Bifunctional Superkine, MDNA223

Abstract Introduction: Combination treatment with a long-acting IL-2 superkine (MDNA109FEAA-Fc; aka MDNA19) and anti-PD1 or anti-CTLA4 has shown clear evidence of synergy in blunting tumor growth. To further leverage this synergism, we designed MDNA223, a BiSKIT (Bifunctional SuperKine for ImmunoTherapy) comprising MDNA109FEAA with enhanced affinity for IL-2Rb and no binding to IL-2Ra, fused to either human or mouse anti-PD1. We present in vitro and in vivo characterization of MDNA223 and demonstrate its enhanced IL-2R agonism and effective PD1/PDL-1 blockade in multiple “hot” and “cold” syngeneic tumor models. Experimental Procedure: In vitro studies included human PBMCs and cell-based reporter assays. In vivo studies were conducted in CT26, B16F10, E0771 and TRAMP-C1 tumor models by intraperiotonneal (IP) administration. Intratumoral treatment was performed in a CT26 bilateral tumor model with or without a STING agonist. Analysis of tumor infiltrating lymphocytes (TILs) was performed using flow cytometry. Results: In studies with human PBMCs, MDNA223 showed increased p-STAT5 signaling in CD8+ T cells and reduced activity in Tregs compared to rhIL-2, consistent with its enhanced receptor selectivity. MDNA223 and anti-PD1 were similarly potent at PD1/PDL-1 blockade in a cell-based reporter assay. In mice, MDNA223 induced proliferation (based on Ki67 expression) and expansion of peripheral CD8+ T cells for over 7 days. IP treatment with MDNA223 resulted in significantly greater inhibition of CT26, B16F10, E0771 and TRAMP-C1 tumor growth when compared to combination of MDNA19 with anti-PD1 at equimolar dosage. This observation is consistent with the proposed cis-binding mechanism of MDNA223 to both, IL-2R and PD1 on the same effector immune cell. In a bilateral CT26 tumor model, IT treatment with MDNA223 not only inhibited the treated tumor but also exhibited an abscopal effect by slowing growth of the untreated tumor implanted on the opposite flank. TILs analysis of B16F10 melanoma following a single dose of MDNA223 showed increased infiltration of CD8+ T cells over Tregs compared to single agent treatment with MDNA19 or anti-PD1 as well as their combination. Treatment with MDNA223 also resulted in a larger population of functional effector CD8+ T cells expressing low levels of the Tim-3 exhaustion marker and high levels of the cytotoxic Granzyme-B protease. Conclusion: MDNA223 demonstrated superior therapeutic activity over the combination of MDNA19 and anti-PD1 in both immunologically ‘hot’ (CT26) and ‘cold’ (B16F10, E0771 and TRAMP-C1) tumor models. Additional studies with variants of MDNA223 capable of tumor selective targeting and/or conditional activation within the tumor micro-environment are in progress to maximize activity at the tumor site with tunable peripheral stimulation. Citation Format: Aanchal Sharma, Minh D To, Hardeep Kataria, Qian Liu, Rosemina Merchant, Fahar Merchant. Synergistically Engaging a b-Selective IL-2 Agonist with PD1/PDL-1 Blockade in a Bifunctional Superkine, MDNA223 [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor Immunology and Immunotherapy; 2023 Oct 1-4; Toronto, Ontario, Canada. Philadelphia (PA): AACR; Cancer Immunol Res 2023;11(12 Suppl):Abstract nr A005.

Expansion of CD4+ cytotoxic T lymphocytes with specific gene expression patterns may contribute to suppression of tumor immunity in oral squamous cell carcinoma: single-cell analysis and in vitro experiments.

Cancer immunotherapy targeting CD8+ T cells has made remarkable progress, even for oral squamous cell carcinoma (OSCC), a heterogeneous epithelial tumor without a substantial increase in the overall survival rate over the past decade. However, the therapeutic effects remain limited due to therapy resistance. Thus, a more comprehensive understanding of the roles of CD4+ T cells and B cells is crucial for more robust development of cancer immunotherapy. In this study, we examined immune responses and effector functions of CD4+ T cells, CD8+ T cells and B cells infiltrating in OSCC lesions using single-cell RNA sequencing analysis, T cell receptor (TCR) and B cell receptor (BCR) repertoire sequencing analysis, and multi-color immunofluorescence staining. Finally, two Kaplan-Meier curves and several Cox proportional hazards models were constructed for the survival analysis. We observed expansion of CD4+ cytotoxic T lymphocytes (CTLs) expressing granzymes, which are reported to induce cell apoptosis, with a unique gene expression patterns. CD4+ CTLs also expressed CXCL13, which is a B cell chemoattractant. Cell-cell communication analysis and multi-color immunofluorescence staining demonstrated potential interactions between CD4+ CTLs and B cells, particularly IgD- CD27- double negative (DN) B cells. Expansion of CD4+ CTLs, DN B cells, and their contacts has been reported in T and B cell-activated diseases, including IgG4-related disease and COVID-19. Notably, we observed upregulation of several inhibitory receptor genes including CTLA-4 in CD4+ CTLs, which possibly dampened T and B cell activity. We next demonstrated comprehensive delineation of the potential for CD8+ T cell differentiation towards dysfunctional states. Furthermore, prognostic analysis revealed unfavorable outcomes of patients with a high proportion of CD4+ CTLs in OSCC lesions. Our study provides a dynamic landscape of lymphocytes and demonstrates a systemic investigation of CD4+ CTL effects infiltrating into OSCC lesions, which may share some pathogenesis reported in severe T and B cell-activated diseases such as autoimmune and infectious diseases.

STING-driven activation of T cells: relevance for the adoptive cell therapy of cancer

Adoptive cell therapy (ACT) can successfully treat hema-topoietic cancers but lacks efficacy against solid tumors. This is due to insufficient T cell infiltration, high tumor heterogeneity, frequent antigen loss with subsequent tumor escape, and the immunosup-pressive tumor microenvironment (TME). Alternative methods to boost the anticancer efficacy of adoptively transferred cells are ac-tively pursued. Among adjuvants that are utilized to stimulate anti-cancer immune responses, ligands of the stimulator of interferon genes (STING) pathway have received increasing attention. STING activation can trigger dendritic cell (DC) activation and endogenous immune responses, thereby preventing tumor escape. Activation of the STING pathway in the context of ACT was accordingly associated with improved T cell trafficking and persistence in the TME com-bined with the reduced presence of immunosuppressive cells. Re-cent findings also suggest cell-intrinsic effects of STING ligands on T cells. Activation of the STING signaling pathway was in this regard shown to enhance effector functions of CD4+ and CD8+ T cells, sug-gesting that the STING signaling could be exploited to harness T cell anticancer functions. In this review, we will discuss how the STING signaling can be used to enhance the anticancer efficacy of ACT.

T-Bet Overexpression Increases 4-1BB-Costimulated CAR T Cell Potency

Data from our lab and others has demonstrated that CAR T cells form a mixed Th1/Th2 population which, in contrast to canonical T cell differentiation, produce both interferon gamma (IFN-γ) and IL-4. Whereas a growing body of evidence has highlighted the importance of IFN-γ in the CAR T cell response, IL-4 can have immune-suppressive functions. IFN-γ from CD4+ CAR T cells (CAR4s), has been shown to promote cytotoxic effector functions of CD8+ CAR T cells (CAR8s), as well as bolster activation of endogenous immune cells including CD8 T cells and NK cells (Boulch et al Sci Immunol. 2021). Given the importance of IFN-γ to effective anti-tumor immune responses, we generated murine CAR T cells that target CD19 with a CD28 costimulatory domain (1928z) and compared them to CAR T cells that overexpress T-bet (T-bet-1928z) in an effort to skew CAR T cells away from IL-4 production and towards IFN-γ production. Upon stimulation of CAR T cells with the murine B-cell acute lymphoblastic leukemia (B-ALL) cell line E2A-PBX, fewer T-bet-1928z CAR4s produced IL-4 compared to 1928z CAR4s. This suggests T-bet overexpression is a viable strategy to reduce Th2-like programming of effector functions in CAR4s. To determine the impact of T-bet overexpression on early CAR T cell efficacy in vivo, we adoptively transferred 1928z or T-bet-1928z CAR T cells into leukemia-bearing C57Bl6/J recipients and analyzed CAR T cells from the bone marrow at peak expansion of CAR T cells as well as post-contraction. Both CARs eliminated CD19+ cells from the bone marrow and mediated long-term survival. T-bet-1928z CAR4s demonstrated increased expansion at Day 4 compared to 1928z CAR4s (p=0.0412), although there was no significant difference in CAR8 numbers at this timepoint. At Day 11 post-CAR, we observed an increase in the number of T-bet-1928z CAR T cells present in the bone marrow (p<0.0001), observed in both CAR4s (p<0.0001) and CAR8s (p=0.0074). While levels of memory-associated transcription factors TCF1 and FOXO1 were the same between CARs, fewer T-bet-1928z CAR T cells expressed IL7Rα (p=0.0002) and eomesodermin (p<0.0001), which are associated with the ability of T cells to self-renew and persist in the bone marrow. To determine the impact of T-bet on the persistence and renewal capacity of 1928z CAR T cells, we adoptively transferred 1928z or T-bet-1928z CAR T cells into leukemia-bearing RAG1-knockout recipients to eliminate endogenous T cell responses to E2A-PBX. After initial leukemia clearance, we rechallenged these mice with E2A-PBX and analyzed the bone marrow after 13 days. Both CARs were able to clear the leukemia rechallenge. While CAR4s were present in the bone marrow at similar numbers, the number of CAR8s was significantly higher among mice treated with T-bet-1928z CAR T cells (p=0.0317). Our results suggest that while T-bet may enhance in vivo expansion of CAR T cells, it provides no long-term benefit or deficit to the CAR T cell response. 4-1BB co-stimulated CAR T cells are less potent than CD28-costimulated CAR T cells and have reduced effector functions. We therefore hypothesized that T-bet overexpression would improve the efficacy of 4-1BB-costimulated CAR T cells by boosting effector functions and potency. To test this, we transduced T cells with a CAR targeting CD19 that contains a 4-1BB costimulatory domain (19BBz), or a 19BBz CAR and T-bet (19BBz-T-bet). We adoptively transferred 19BBz or 19BBz-T-bet CAR T cells into leukemia-bearing C57Bl6/J recipients. Despite no difference in CAR T cell numbers in the bone marrow, we observed fewer leukemia cells in mice treated with 19BBz-T-bet CAR T cells (p=0.0318) at Day 4 post-CAR. Treatment with 19BBz-T-bet CAR T cells also significantly improved survival of leukemia-bearing mice compared to 19BBz CAR T cells (p=0.0494). Our data demonstrate T-bet overexpression improves CD28-costimulated CAR T cell expansion but does little to augment long-term responses and diminishes features associated with capacity for self-renewal. In contrast, T-bet overexpression boosts the potency of 4-1BB-costimulated CAR T cells without impairing persistence, resulting in improved overall efficacy of 4-1BB-costimulated CAR T cell therapies.

Regulatory T cells in inflamed liver are dysfunctional in murine primary biliary cholangitis.

Primary biliary cholangitis (PBC) is a chronic autoimmune disease characterized by immune-mediated destruction of intrahepatic small bile ducts. CD8 T cells play a critical role in biliary destruction. However, regulatory T cells (Tregs) have also been identified in the portal tracts of PBC patients. This study tested the hypothesis that hepatic Tregs in PBC were dysfunctional in suppressing immune responses in disease by using our human PBC-like autoimmune cholangitis (AIC) mouse model induced by 2-octynoic acid-conjugated ovalbumin (2-OA-OVA). Our results showed that female and male mice immunized with 2-OA-OVA developed AIC; however, female AIC mice had more severe liver inflammation and fibrosis than male AIC mice. Levels of functional effector CD8 T cells and their chemoattractants, CXCL9 and CXCL10, in the liver were markedly elevated in female AIC mice than in male AIC mice. These results reinforce that CD8 T cells are the primary effector cells in PBC. The number of hepatic Tregs in AIC mice was also higher than in saline-treated mice, but there was no difference between male and female AIC mice. The suppressive function of AIC Tregs was evident despite a discrepancy in the changes in their co-inhibitory receptors and inhibitory cytokines. However, the expansion of hepatic Tregs by low-dose IL-2 treatment did not reduce immune responses to AIC, which may be due to the dysfunction of Tregs in inhibiting T cells. In conclusion, the function of Tregs in the inflamed liver of PBC was insufficient, and low-dose IL-2 treatment could not restore their function to suppress pathological immune responses. Transferring normal Tregs or directly targeting effector CD8 T cells may be beneficial for treating PBC.

SCD1 inhibition enhances the effector functions of CD8+ T cells via ACAT1-dependent reduction of esterified cholesterol.

We previously reported that the inhibition of stearoyl-CoA desaturase 1 (SCD1) enhances the antitumor function of CD8+ T cells indirectly via restoring production of DC recruiting chemokines by cancer cells and subsequent induction of antitumor CD8+ T cells. In this study, we investigated the molecular mechanism of direct enhancing effects of SCD1 inhibitors on CD8+ T cells. In vitro treatment of CD8+ T cells with SCD1 inhibitors enhanced IFN-γ production and cytotoxic activity of T cells along with decreased oleic acid and esterified cholesterol, which is generated by cholesterol esterase, acetyl-CoA acetyltransferase 1 (ACAT1), in CD8+ T cells. The addition of oleic acid or cholesteryl oleate reversed the enhanced functions of CD8+ T cells treated with SCD1 inhibitors. Systemic administration of SCD1 inhibitor to MCA205 tumor-bearing mice enhanced IFN-γ production of tumor-infiltrating CD8+ T cells, in which oleic acid and esterified cholesterol, but not cholesterol, were decreased. These results indicated that SCD1 suppressed effector functions of CD8+ T cells through the increased esterified cholesterol in an ACAT1-dependent manner, and SCD1 inhibition enhanced T cell activity directly through decreased esterified cholesterol. Finally, SCD1 inhibitors or ACAT1 inhibitors synergistically enhanced the antitumor effects of anti-PD-1 antibody therapy or CAR-T cell therapy in mouse tumor models. Therefore, the SCD1-ACAT1 axis is regulating effector functions of CD8+ T cells, and SCD1 inhibitors, and ACAT1 inhibitors are attractive drugs for cancer immunotherapy.

Identification and Characterization of CD8+ CD27+ CXCR3- T Cell Dysregulation and Progression-Associated Biomarkers in Systemic Lupus Erythematosus.

Systemic Lupus Erythematosus (SLE) etiopathogenesis highlights the contributions of overproduction of CD4+ T cells and loss of immune tolerance. However, the involvement of CD8+ T cells in SLE pathology and disease progression remains unclear. Here, the comprehensive immune cell dysregulation in total 263 clinical peripheral blood samples composed of active SLE (aSLE), remission SLE (rSLE) and healthy controls (HCs) is investigated via mass cytometry, flow cytometry and single-cell RNA sequencing. This is observed that CD8+ CD27+ CXCR3- T cells are increased in rSLE compare to aSLE. Meanwhile, the effector function of CD8+ CD27+ CXCR3- T cells are overactive in aSLE compare to HCs and rSLE, and are positively associated with clinical SLE activity. In addition, the response of peripheral blood mononuclear cells (PBMCs) is monitored to interleukin-2 stimulation in aSLE and rSLE to construct dynamic network biomarker (DNB) model. It is demonstrated that DNB score-related parameters can faithfully predict the remission of aSLE and the flares of rSLE. The abundance and functional dysregulation of CD8+ CD27+ CXCR3- T cells can be potential biomarkers for SLE prognosis and concomitant diagnosis. The DNB score with accurate prediction to SLE disease progression can provide clinical treatment suggestions especially for drug dosage determination.

Lactobacillus gallinarum-derived metabolites boost anti-PD1 efficacy in colorectal cancer by inhibiting regulatory T cells through modulating IDO1/Kyn/AHR axis

ObjectiveGut microbiota is a key player in dictating immunotherapy response. We aimed to explore the immunomodulatory effect of probiotic Lactobacillus gallinarum and its role in improving anti-programmed cell death protein...

ATM-AMPKα mediated LAG-3 expression suppresses T cell function in prostate cancer

Immunotherapy for prostate cancer (PCa) faces serious challenges. Therefore, the co-inhibitory receptors that regulate T cell function of PCa must be elucidated. Here we identified that the inhibitory receptor LAG3 was significantly induced in T cells from PCa patients. Gene array analysis revealed that insufficient ataxia telangiectasia mutated (ATM) gene expression in PCa T cells was responsible for the elevated LAG3 expression. Mechanistically, insufficient ATM expression impaired its ability to activate AMPKα signaling and CD4+ T cell functions, which further enhances the binding of the transcription factors XBP1 and EGR2 to LAG3 promoter. Reconstitution of ATM and inhibition of XBP1 or EGR2 in PCa T cells suppressed LAG3 expression and restored the effector function of CD4+ T cells from PCa. Our study revealed the mechanism of LAG3 upregulation in CD4+ T lymphocytes of PCa patients and may provide insights for the development of immunotherapeutic strategies for PCa treatment.

Erucic acid improves the progress of pregnancy complicated with systemic lupus erythematosus by inhibiting the effector function of CD8+ Tcells.

Pathogenic CD8+ Tcells are pivotal contributors to the onset of systemic lupus erythematosus (SLE). Erucic acid (EA) has been proven to have anti-inflammatory activity. However, the capacity of EA to regulate pathogenic CD8+ Tcells in the context of pregnancy complicated with SLE (pSLE) remains unclear. In our investigation, we observed augmented CD8+ T cell effector function juxtaposed with diminished EA levels in pSLE patients relative to healthy pregnant controls. Significantly, plasma EA levels exhibited a negative correlation with the severity of pSLE-associated complications. In blood from patients with pSLE, EA inhibited the effector function of CD8+ Tcells, concurrently dampening the maintenance of stem cell-like memory CD8+ Tcells. Mechanistically, EA orchestrated the inhibition of CD8+ T cell effector function by impeding signal transducer and activator of transcription 3 phosphorylation and promoting ferroptosis. Moreover, EA supplementation in pregnant MRL/lpr mice manifested as the attenuation of uterine CD8+ T cell effector function, culminating in the mitigation of placental pathological damage. Our findings uncover the immune response modulatory effects of EA upon pathogenic CD8+ cells, thereby unveiling new perspectives for therapeutic strategies targeting pSLE patients.

434. INCREASING TUMOUR INFILTRATING LYMPHOCYTES THROUGH EXERCISE ALONE—A PILOT RCT IN ESOPHAGEAL ADENOCARCINOMA

Abstract Background The immune system is highly responsive and positively adapts to exercise. A single bout of exercise results in the mobilisation of highly functional effector CD8+ T cells and NK-cells into the circulation. Murine cancer models have shown that exercise reduces tumour burden by increasing the frequency of tumour-infiltrating lymphocytes (TILs). There are no studies assessing the impact of an exercise programme on the levels of TILs in patients’ solid tumours in any cancer. Methods We recently completed a 16-week randomised prehabilitation exercise program (NCT02950324) in esophageal cancer patients before esophagogastric cancer resection. Exercise training was a low-to-moderate intensity twice supervised, thrice home-based weekly program. Tumour specimens obtained at the time of resection were formalin fixed paraffin embedded (FFPE) for multispectral immunohistochemical analysis. Tumour tissues were stained with primary antibodies for CD68, CD57, CD8, CD4, FoxP3, Granzyme B, PDL1 and pancytokeratin. Cell populations and spatial relationships were analysed using the Phenoimager HT (Akoya Biosciences) and QuPath. Results Although our exercise program was ~33% of the physical activity guidelines for cancer patients, physical fitness and well-being were maintained rather than significantly reduced in the intervention group compared to the control group. Multispectral analysis observed that 3.2% ± 1.1% of cells in the tumours were CD8+ T cells compared to 1.4% ± 0.5% in the control group (p < 0.001). Furthermore, we observed positive associations between increased frequencies of CD8 + TILs (Fig 1C: r = 0.562, p = 0.016), Granzyme B+/CD8 + TILs (r = 0.637, p = 0.003) and larger increases in exercise induced aerobic capacity. This data suggests that the more exercise can increase aerobic fitness, the greater the likelihood of increasing functional TILs. Conclusion New approaches to improve outcomes following surgery for esophageal adenocarcinoma are required. One such approach is immunotherapy. However, immunotherapy is relatively ineffective in esophageal adenocarcinoma due to the lack of CD8+ T cells and NK cells in the tumours. Increasing TILs through exercise programmes that are designed to focus on maintaining or improving aerobic capacity may improve patients’ response to immunotherapy and positively impact prognosis and survival.

213 Complement C3 deficiency enhances the effector function of CD8+ T cells and inhibits tumour growth

213 Complement C3 deficiency enhances the effector function of CD8+ T cells and inhibits tumour growth

Engineered soluble, trimerized 4-1BBL variants as potent immunomodulatory agents.

Targeting co-stimulatory receptors promotes the activation and effector functions of anti-tumor lymphocytes. 4-1BB (CD137/TNFSF9), a member of the tumor necrosis factor receptor superfamily (TNFR-SF), is a potent co-stimulatory receptor that plays a prominent role in augmenting effector functions of CD8+ T cells, but also CD4+ T cells and NK cells. Agonistic antibodies against 4-1BB have entered clinical trials and shown signs of therapeutic efficacy. Here, we have used a T cell reporter system to evaluate various formats of 4-1BBL regarding their capacity to functionally engage its receptor. We found that a secreted 4-1BBL ectodomain harboring a trimerization domain derived from human collagen (s4-1BBL-TriXVIII) is a strong inducer of 4-1BB co-stimulation. Similar to the 4-1BB agonistic antibody urelumab, s4-1BBL-TriXVIII is very potent in inducing CD8+ and CD4+ T cell proliferation. We provide first evidence that s4-1BBL-TriXVIII can be used as an effective immunomodulatory payload in therapeutic viral vectors. Oncolytic measles viruses encoding s4-1BBL-TriXVIII significantly reduced tumor burden in a CD34+ humanized mouse model, whereas measles viruses lacking s4-1BBL-TriXVIII were not effective. Natural soluble 4-1BB ligand harboring a trimerization domain might have utility in tumor therapy especially when delivered to tumor tissue as systemic administrationmight induce liver toxicity.

Tucatinib promotes immune activation and synergizes with programmed cell death-1 and programmed cell death-ligand 1 inhibition in HER2-positive breast cancer.

Programmed cell death-1 (PD-1) and programmed cell death-ligand 1 (PD-L1) inhibitors have poor efficacy in patients with trastuzumab-resistant advanced HER2-positive breast cancer. Tucatinib is a potent, selective anti-HER2 tyrosine kinase inhibitor with proven clinical benefit in the advanced setting in patients with trastuzumab resistance. We investigated if tucatinib can alter the tumor microenvironment and if this could be harnessed for therapeutic efficacy. We investigated the antitumor efficacy and contribution of the immune response of tucatinib using 2 immunocompetent, HER2-positive murine breast cancer models (trastuzumab-sensitive H2N113; trastuzumab-resistant Fo5) and the efficacy of tucatinib with trastuzumab and PD-1 or PD-L1 checkpoint inhibitors. In both models, tucatinib statistically significantly inhibited tumor growth and demonstrated dose-dependent efficacy. Ex vivo analysis by flow cytometry of tumor-infiltrating lymphocytes in mice treated with tucatinib showed increased frequency, higher proliferation, and enhanced effector function of CD8+ effector memory T cells. Tucatinib treatment also increased frequency of CD8+PD-1+ and CD8+TIM3+ T cells, CD49+ natural killer cells, monocytes, and major histocompatibility complex II expression on dendritic cells and macrophages and a decrease in myeloid-derived suppressor cells. Gene expression analysis revealed statistically significant enrichment in pathways associated with immune activation, type I and II interferon response, adaptive immune response, and antigen receptor signaling. In vivo, tucatinib and α-PD-L1 or α-PD-1 demonstrated statistically significantly increased efficacy and improved survival of mice compared with tucatinib alone. Tucatinib modulates the immune microenvironment favorably, and combination treatment with α-PD-L1 or α-PD-1 demonstrated increased efficacy in preclinical HER2-positive tumor models. These findings provide a rationale for investigation of tucatinib and immune checkpoint inhibition in the clinic.

Hypoxia promotes cytolytic activity of CD8 T cells and pathogenesis in cutaneous leishmaniasis

Abstract Leishmaniasis is a disease caused by protozoan parasites of the genus Leishmania and the most common form of the disease is cutaneous leishmaniasis (CL). A fundamental question in CL is what regulates the development of severe disease, which is critical to develop therapies to ameliorate pathology. In a series of studies, we demonstrated CD8 T cell-dependent cytotoxicity as the main inducer of immunopathology in CL. This result was unexpected since IFN-γ production by CD8 T cells plays a protective role by promoting pathogen elimination. To resolve this paradox, we studied the CD8 T cells in different anatomic sites and found that the effector function of CD8 T cells in CL depends on their location: while CD8 T cells are cytotoxic (GzmB+) and produce little IFN-γ in the skin lesions, CD8 T cells in the draining lymph nodes (dLN) have the opposite profile. Importantly, GzmB-CD8 T cells from dLN quickly upregulate GzmB after injection into CL lesions. By transcriptional profiling, we found that CD8 T cells in lesions but not dLN have a hypoxic signature. In vivo, we observed that CL lesions are hypoxic using the Oxyphor G4 oxygen probe and pimonidazole staining. In vitro, we found that induction of hypoxia was sufficient to convert GzmB- into GzmB+ CD8 T cells, and significantly decreased CD8 T cell production of IFN-γ. In vivo, blocking hypoxia-inducible factor (HIF), a master regulator of hypoxia, with acriflavine decreased lesion development in mice. Mice with CD8 T cell deficient in HIF-1α produced significantly less GzmB in the skin lesions compared to wildtype mice. Together, our results suggest that the hypoxic signaling through the transcription factor HIF in CL lesions is necessary to convert CD8 T cells into pathogenic CD8 T cells in the skin lesion. Supported by grants from NIH (1R01AI162711)

PIM kinases are essential for CD8 T cell effector function and metabolism during chronic viral infection

Abstract The production of IL-21 by CD4 T cells is one mechanism by which CD8 T cells can sustain their effector functions, including increased metabolic demand, during chronic viral infection. However, signaling cascades downstream of IL-21 that control effector function in CD8 T cells remain incompletely understood. One such set of targets are the PIM kinases. This family of serine/threonine kinases functions downstream of IL-21 via JAK-STAT signaling and are involved in diverse cellular processes. Treatment of activated CD8 T cells with IL-21 induces expression of PIM1 kinase and phosphorylation of its targets S6 T372, 4EBP T37/42, and BAD S112, which promote transcription and inhibit apoptosis. Using the lymphocytic choriomeningitis virus (LCMV) Clone 13 mouse model of chronic infection, we show that virus-specific effector CD8 T cells display the highest expression of Pim1compared to exhausted and progenitor CD8 T cells at day 21 post-infection (p.i.). Infection of Pim1−/−mice with LCMV Clone 13 results in reduced virus-specific effector CD8 T cells and increased exhausted CD8 T cells at days 21–23 p.i. compared to wildtype controls. In addition, pharmacological inhibition of PIM kinases with AZD1208, a pan-PIM kinase inhibitor, in activated CD8 T cells results in compromised mitochondrial metabolism, which is essential for effector CD8 T cell function. Overall, our results suggest that PIM kinases are critical for effector CD8 T cell differentiation, function, and metabolism in the control of chronic viral infection. Supported by grants from NIH (R01 AI148403) and by an award from the Medical College of Wisconsin Center for Immunology.