Enhanced Effector Function Research Articles

Co-Expression of miR155 or LSD1 shRNA Increases the Anti-Tumor Functions of CD19 CAR-T Cells.

Chimeric antigen receptor (CAR) T cells targeting CD19 antigen have produced remarkable clinical outcomes for cancer patients. However, identifying measures to enhance effector function remains one of the most challenging issues in CD19-targeted immunotherapy. Here, we report a novel approach in which a microRNA (miRNA) or short-hairpin RNA (shRNA) cassette was integrated into CAR-expressing retroviral vectors. Using this system, we generated anti-CD19 CAR-T cells co-expressing miR155 or LSD1 shRNA and found that anti-CD19 CAR-T cells with miR155 upregulation or LSD1 downregulation exhibited increased anti-tumor functions in vitro and in vivo. Transcriptional profiling analysis by RNA sequencing revealed the targets of miR155 and LSD1 in anti-CD19 CAR-T cells. Our experiments indicated that introduction of miRNA or shRNA expression into anti-CD19 CAR T-cells might be an effective strategy to improve the anti-tumor effects of CAR-T cell therapy.

Chemical Synthesis of Antibody-Hapten Conjugates Capable of Recruiting the Endogenous Antibody to Magnify the Fc Effector Immunity of Antibody for Cancer Immunotherapy.

Monoclonal antibodies (mAbs) with enhanced effector functions in cancer immunotherapy, such as complement-dependent cytotoxicity (CDC) and antibody-dependent cell-mediated cytotoxicity (ADCC), could improve the clinical performance. Here, we develop an mAb-hapten conjugate strategy to augment the mAb effector functions with the engagement of endogenous antibodies. An "off-the-shelf" mAb, rituximab, is site-specifically conjugated with the rhamnose (Rha) hapten to generate rituximab-Rha conjugates. The octopus-like conjugates could recruit anti-Rha antibodies onto the cancer cell surface and further form an immune complex that is able to provide multivalent Fc domains to interact with immune cells or complement protein C1q, leading to magnified ADCC and CDC simultaneously. One optimal conjugate R2 with PEG2 as a linker exhibits the most potent in vitro cancer cell killing activity and significant in vivo antitumor efficacy in a xenograft model. This is a general and cost-effective approach to generate mAb with improved effector functions that may have broad applications.

Impact of acute exercise on peripheral blood mononuclear cells nutrient sensing and mitochondrial oxidative capacity in healthy young adults.

Regular exercise is associated with changes in peripheral blood mononuclear cell (PBMC) proportions that have enhanced effector functions in young and old adults; however, the effects of acute exercise on PBMC nutrient sensors and metabolic function in active young adults is unknown. To fill this gap, activation status and nutrient‐sensing mechanisms of PBMCs isolated from 21 healthy active adults (20–35 yr; 36.5 ± 6.3 V̇O2peak) were characterized before and after 30 min of moderate‐to‐vigorous cycling (65%–75% V̇O2peak). In addition, changes in PBMC mitochondrial respiratory function in response to exercise were assessed using high‐resolution respirometry. There was an increase in the number of activated CD69+/CD4 (79% increase) and CD69+/CD8 (166% increase) T‐cells in response to the acute bout of exercise, while the nutrient‐sensing mechanisms remained unchanged. PBMC mitochondrial respiration did not increase on a cell‐per‐cell basis, however, mitochondrial oxidative capacity (OXPHOS) increased at the tissue level (18.6 pmol/(s*ml blood) versus 29.3 pmol/(s*ml blood); p < 0.05) in response to acute exercise. Thus, this study shows that acute exercise preferentially mobilizes activated T‐cells while concomitantly increasing PBMC mitochondrial oxidative capacity at the tissue level, rather than acutely changing mitochondrial oxidative capacity at the cellular level in young adults.

127P Lactobacillus plantarum IMB19 exerts anti-tumor immunity by enhancing effector functions of tumor associated type 1 macrophage and CD8+ T-cells

Numerous studies suggest the potential application of commensal microbiota to treat cancers, autoimmunity, and diverse disorders. However, it is still unclear how these microbes exert therapeutic efficacies. Challenges in developing the microbiome-based therapies include identifying the most effective bacterial strains and defining microbial effector components, and the downstream mechanisms of action.

Cytokine-induced natural killer cell training is dependent on cellular metabolism and is defective in obesity

AbstractNatural killer (NK) cells are a population of innate immune cells that can rapidly kill cancer cells and produce cytokines such as interferon-γ. A key feature of NK cells is their ability to respond without prior sensitization; however, it is now well established that NK cells can possess memory-like features. After activation with cytokines, NK cells demonstrate enhanced effector functions upon restimulation days or weeks later. This demonstrates that NK cells may be trained to be more effective killers and harnessed as more potent cancer immunotherapy agents. We have previously demonstrated that cellular metabolism is essential for NK cell responses, with NK cells upregulating both glycolysis and oxidative phosphorylation upon cytokine stimulation. Limiting NK cell metabolism results in reduced cytotoxicity and cytokine production. We have also demonstrated that defective NK cell responses in obesity are linked to defective cellular metabolism. In the current study, we investigated if cellular metabolism is required during the initial period of NK cell cytokine training and if NK cells from people with obesity (PWO) can be effectively trained. We show that increased flux through glycolysis and oxidative phosphorylation during the initial cytokine activation period is essential for NK cell training, as is the metabolic signaling factor Srebp. We show that NK cells from PWO, which are metabolically defective, display impaired NK cell training, which may have implications for immunotherapy in this particularly vulnerable group.

Tumor-Experienced Human NK Cells Express High Levels of PD-L1 and Inhibit CD8+ T Cell Proliferation.

Natural Killer (NK) cells play a key role in cancer immunosurveillance. However, NK cells from cancer patients display an altered phenotype and impaired effector functions. In addition, evidence of a regulatory role for NK cells is emerging in diverse models of viral infection, transplantation, and autoimmunity. Here, we analyzed clear cell renal cell carcinoma (ccRCC) datasets from The Cancer Genome Atlas (TCGA) and observed that a higher expression of NK cell signature genes is associated with reduced survival. Analysis of fresh tumor samples from ccRCC patients unraveled the presence of a high frequency of tumor-infiltrating PD-L1+ NK cells, suggesting that these NK cells might exhibit immunoregulatory functions. In vitro, PD-L1 expression was induced on NK cells from healthy donors (HD) upon direct tumor cell recognition through NKG2D and was further up-regulated by monocyte-derived IL-18. Moreover, in vitro generated PD-L1hi NK cells displayed an activated phenotype and enhanced effector functions compared to PD-L1- NK cells, but simultaneously, they directly inhibited CD8+ T cell proliferation in a PD-L1-dependent manner. Our results suggest that tumors might drive the development of PD-L1-expressing NK cells that acquire immunoregulatory functions in humans. Hence, rational manipulation of these regulatory cells emerges as a possibility that may lead to improved anti-tumor immunity in cancer patients.

γδ T cells cultured with artificial antigen-presenting cells and IL-2 show long-term proliferation and enhanced effector functions compared with γδ T cells cultured with only IL-2 after stimulation with zoledronic acid

Background aimsImmunotherapeutic approaches using γδ T cells have emerged as the function of γδ T cells in tumor surveillance and clearance has been discovered. In vitro expansion methods of γ9δ2 T cells have been based on phosphoantigens and cytokines, but expansion methods using feeder cells to generate larger numbers of γδ T cells have also been studied recently. However, there are no studies that directly compare γδ T cells cultured with phosphoantigens with those cultured with feeder cells. Therefore, this study aimed to compare the expansion, characteristics and effector functions of γδ T cells stimulated with K562-based artificial antigen-presenting cells (aAPCs) (aAPC-γδ T cells) and γδ T cells stimulated with only zoledronic acid (ZA) (ZA-γδ T cells). MethodsPeripheral blood mononuclear cells were stimulated with ZA for 7 days, and aAPC-γδ T cells were stimulated weekly with K562-based aAPCs expressing CD32, CD80, CD83, 4-1BBL, CD40L and CD70, whereas ZA-γδ T cells were stimulated with only IL-2. Cultured γδ T cells were analyzed by flow cytometry for the expression of co-stimulatory molecules, activating receptors and checkpoint inhibitors. Differentially expressed gene (DEG) analysis was also performed to determine the difference in gene expression between aAPC-γδ T cells and ZA-γδ T cells. In vitro cytotoxicity assay was performed with calcein AM release assay, and in vivo anti-tumor effect was compared using a U937 xenograft model. ResultsFold expansion on day 21 was 690.7 ± 413.1 for ZA-γδ T cells and 1415.2 ± 1016.8 for aAPC- γδ T cells. Moreover, aAPC-γδ T cells showed continuous growth, whereas ZA-γδ T cells showed a decline in growth after day 21. The T-cell receptor Vγ9+δ2+ percentages (mean ± standard deviation) on day 21 were 90.0 ± 2.7% and 87.0 ± 4.5% for ZA-γδ T cells and aAPC-γδ T cells, respectively. CD25 and CD86 expression was significantly higher in aAPC-γδ T cells. In DEG analysis, aAPC-γδ T cells and ZA-γδ T cells formed distinct clusters, and aAPC-γδ T cells showed upregulation of genes associated with metabolism and cytokine pathways. In vitro cytotoxicity revealed superior anti-tumor effects of aAPC-γδ T cells compared with ZA-γδ T cells on Daudi, Raji and U937 cell lines. In addition, in the U937 xenograft model, aAPC-γδ T-cell treatment increased survival, and a higher frequency of aAPC-γδ T cells was shown in bone marrow compared with ZA-γδ T cells. ConclusionsOverall, this study demonstrates that aAPC-γδ T cells show long-term proliferation, enhanced activation and anti-tumor effects compared with ZA-γδ T cells and provides a basis for using aAPC-γδ T cells in further studies, including clinical applications and genetic engineering of γδ T cells.

Abstract LB165: Exercise training boosts CTL infiltration and sensitizes tumors to immune checkpoint blockade

Abstract Exercise training boosts CTL infiltration and sensitizes tumors to immune checkpoint blockade Igor L. Gomes-Santos, Zohreh Amoozgar, Ashwin S. Kumar, William W. Ho, Rakesh K. Jain, Dai Fukumura Edwin L. Steele Laboratories, Department of Radiation Oncology, Massachusetts General Hospital &amp; Harvard Medical School, Boston, MA, USA. Exercise can promote health and beneficial effects on cancer patients. However, how exercise training (ExTr) induces antitumor effects is not well understood. We investigated underlining mechanisms by which ExTr regulates the tumor microenvironment (TME) and therapeutic response to immunotherapy in murine breast cancer (BC) models – E0771 and MCa-M3C grown in syngeneic female mice (C57BL/6 and FVB, respectively). We started intensity-controlled ExTr (45 min daily sessions at 60% of maximal capacity, moderate-to-vigorous intensity on a running treadmill) when tumors reached 100 mm3. ExTr with this regimen certainly delayed tumor growth in both models. We then performed flow cytometry, RNA-seq, ELISA, and immunohistochemistry to characterize the effects of ExTr on TME. ExTr normalized abnormal tumor vasculature – indicated by the increased pericyte coverage and perfusion, reduced hypoxia in both models. Also, ExTr induced a shift in metabolic signature towards oxidative metabolism and enhanced antitumor immunity, accessed by RNA-seq. ExTr boosted CD8+ T cell infiltration, with enhanced effector function (higher IFNγ, Granzyme B, TNFα cytokine production) and proliferation (Ki67 expression). The therapeutic benefits of ExTr depend on CD8+ T cells, as its selective depletion with anti-CD8β antibody abrogates the exercise-mediated tumor control. Importantly, ExTr selectively boosted CD8+ T cell infiltration without affecting other T cells subsets or myeloid cells. Mechanistically, we found that ExTr enhances the CXCL9/11-CXCR3 chemokine system that mediates CD8+ T cell infiltration to tumors, as the recruitment of CD8+ T cells and antitumor effects of ExTr were abrogated in Cxcr3-/- mice (C57BL/6 background). We further tested treatments with immune checkpoint blockade (ICB) consisted of anti-PD-1 alone or in combination with anti-CTLA-4. Indeed, ExTr sensitized refractory BCs to the ICB therapies. Taken together, we established that ExTr induces vascular normalization in established BCs and improves perfusion, oxygenation, and oxidative metabolism. Importantly, ExTr boosts CD8+ T cell infiltration and function and induces CD8+ T cell-dependent antitumor effects and sensitization to ICB therapies. Since exercise has already been recommended to cancer patients as supportive care, implementing ExTr into a clinical investigation should be relatively straightforward. Our findings and mechanistic insights provide a strong rationale for the clinical translation of ExTr as a physiological intervention. Citation Format: Igor L. Gomes-Santos. Exercise training boosts CTL infiltration and sensitizes tumors to immune checkpoint blockade [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 LB165.

Abstract 1838: LBL-024, an anti-PD-L1 and 4-1BB bispecific antibody with highly differentiated binding affinity, shows anti-tumor efficacy in a mouse tumor model

Abstract Objective: 4-1BB is an inducible costimulatory receptor expressed on activated T and NK cells. 4-1BB ligation on T cells triggers a signaling cascade that results in upregulation of antiapoptotic molecules, cytokine secretion, and enhanced effector function. PD-L1, a ligand of PD-1, is mainly expressed on tumor cells, and a key checkpoint inhibitor implicated in tumor immune evasion. A tetravalent bispecific antibody LBL-024, targeting both PD-L1 and 4-1BB, was generated to maximize anti-tumor immunity. Methods: The bispecific antibody was designed as 2:2 IgG-scFv format with an affinity ratio of approximately 500:1 between two molecules. The binding affinity of LBL-024 to human PD-L1 and 4-1BB was determined by ForteBio. The activity of LBL-024 was determined using several in vitro assays, including PD-1/NFAT, 4-1BB/NF-κB reporter gene and PBMC activation assays. The in vivo anti-tumor activity of LBL-024 was investigated in the PD-L1/4-1BB double knock-in mouse model (Biocytogen) implanted with MC38 cells expressing PD-L1. Results: The binding affinity of LBL-024 to PD-L1 and 4-1BB was designed with affinity differentiation (~500-fold) between two molecules, with 0.29 nM to PD-L1 and 146 nM to 4-1BB, respectively, which may restrict activating 4-1BB signal on T cells only in PD-L1 expressing tumor microenvironment, rather activating T cells systemically. In vitro LBL-024 could block PD-1-PD-L1 interaction, recovering NFAT reporter signal comparable to anti-PD-L1 antibody. In 4-1BB/NF-κB reporter gene assay, LBL-024 activated the reporter gene system, only in the presence of CHO-K1-PD-L1 cells, suggesting possibly more restricted activation of 4-1BB within tumor microenvironment and potentially less toxicity in patients. The potency of LBL-024 on 4-1BB/NF-κB reporter gene assay was much stronger than Urelumab, a 4-1BB antibody. In pre-activated human PBMC, co-cultured with CHO-K1-PD-L1 cells, IL-2 level was significantly elevated more in LBL-024 treated group than using an anti-PD-L1, anti-4-1BB, or even the combination of anti-PD-L1 and anti-4-1BB was applied. In a model of huPD-L1/hu4-1BB double knock-in mice implanted with MC38-PD-L1 cells. LBL-024 was shown a significant anti-tumor activity (p&amp;lt;0.001), with 81.8% of tumor growth inhibition (TGI), more efficacious than mice dosed with anti-PD-L1 (70.5% of TGI) and anti-4-1BB (46.1% of TGI). Conclusions: LBL-024, an anti-PD-L1 and 4-1BB bispecific antibody was shown a great synergetic anti-tumor efficacy in a mouse tumor model, more effective than the antibodies targeting either PD-L1 or 4-1BB alone. With highly differentiated binding affinity between two molecules, the efficacy and toxicity could be well balanced, to avoid the potential toxicity of systemic activation of 4-1BB, hence a promising bispecific antibody for further clinical development. Citation Format: Xiao Huang, Jianming Sun, Yurong Qin, Huan Lin, Jing Guan, Shoupeng Lai, Xiaoqiang Kang, Hong Ling. LBL-024, an anti-PD-L1 and 4-1BB bispecific antibody with highly differentiated binding affinity, shows anti-tumor efficacy in a mouse tumor model [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 1838.

Δ133p53α enhances metabolic and cellular fitness of TCR-engineered T cells and promotes superior antitumor immunity

BackgroundTumor microenvironment-associated T cell senescence is a key limiting factor for durable effective cancer immunotherapy. A few studies have demonstrated the critical role of the tumor suppressor TP53-derived p53 isoforms...

SGNTGT-001: A phase 1 study of SEA-TGT, an effector-function enhanced monoclonal antibody (mAb), in advanced malignancies (trial in progress).

TPS2657 Background: T-cell immunoreceptor with Ig and ITIM domains (TIGIT) is an inhibitory immune checkpoint receptor expressed on subsets of T cells and NK cells. SEA-TGT is an effector-function enhanced human mAb that targets TIGIT with pico-molar affinity and blocks TIGIT’s interaction with CD155 and CD112. SEA-TGT was developed to have amplified binding to and engagement of Fcγ receptors. Enhanced effector function increases TIGIT+ T-regulatory cell depletion, enhances innate immune cell activation, and augments naïve and memory CD8+ T-cell responses. Preclinically, SEA-TGT elicits superior anti-tumor immune responses compared to other TIGIT mAbs without effector-enhanced backbones, with curative anti-tumor activity as monotherapy and in combination with other immune-modulators. Methods: This phase 1, open-label, multicenter, dose-escalation/expansion study (NCT04254107) is assessing the safety, tolerability and preliminary activity of SEA-TGT monotherapy in up to 205 adults (≥18 years) with histologically or cytologically confirmed relapsed, refractory, or progressive metastatic solid tumors (non-small cell lung, gastric/GE junction carcinomas, cutaneous melanoma, head and neck squamous cell carcinoma, bladder cancer, ovarian cancer or triple-negative breast cancer) or lymphomas (classical Hodgkin lymphoma, diffuse large B-cell lymphoma, or peripheral T-cell lymphoma, not otherwise specified). SEA-TGT will be infused on Day 1 of 21-day cycles. In Part A, the safety and tolerability of SEA-TGT will be assessed in ̃25 subjects to identify the maximum tolerated dose and recommended phase II dose (RP2D). In Part B, the safety and antitumor activity of the RP2D will be assessed in ̃180 subjects in disease-specific expansion cohorts. Primary endpoints are adverse events, laboratory abnormalities, dose-limiting toxicities, and dose-level safety and activity. Secondary endpoints are objective response (OR) rates, duration of OR, complete response, progression-free survival, overall survival, PK, and antidrug antibodies. Exploratory biomarkers of SEA-TGT-mediated pharmacodynamic (PD) effects, PK-PD correlations, and correlative analyses of predictive and PD measurements with response, toxicity, and resistance will be explored. The study was opened April 2020 and is enrolling across sites in North America and Europe. Clinical trial information: NCT04254107.

CD74+ macrophages are associated with favorable prognosis and immune contexture in hepatocellular carcinoma.

CD74 was initially thought to participate mainly in antigen presentation as an MHC class II chaperone. Recent studies have shown that CD74 plays an important role within the cell and throughout the immune system in a wide spectrum of neoplasms. However, the role of CD74 in hepatocellular carcinoma (HCC) remains elusive. In this study, HCC tissues from Zhongshan Hospital and data from The Cancer Genome Atlas (TCGA) were obtained and analyzed. Immunohistochemistry, flow cytometry, and single-cell RNA sequencing (scRNA-seq) were performed to detect the characteristics of CD74+ cells and explore their impact on the tumor microenvironment (TME) of HCC. Our data revealed that stromal CD74+ cell enrichment was associated with favorable prognosis in patients with HCC. CD74 was abundant in a large portion of HCC specimens and prominently distributed on stromal macrophages. scRNA-seq data also indicated that the pathways related to immune response were significantly upregulated in CD74+ macrophages. High infiltration of CD74+ macrophages was associated with increased infiltration of CD8+ cytotoxic T lymphocytes (CTLs) with enhanced effector functions in HCC. Besides, blocking CD74 weakened the antitumor activity and proliferation ability of CD8+ CTLs in HCC. Our findings highlight the critical role of CD74 in HCC. New drugs and antibodies targeting CD74 may be effective strategies for HCC therapy.

The responder/nonresponder phenomena: The immune system’s role in determining the outcome of radiation therapy in rectal cancer

Abstract While surgical resection is a viable option to treat patients with rectal cancer (RC), it is not without significantly reducing patients’ quality of life. In an effort to delay or even prevent surgery, patients are often administered pre-operative radiation therapy (RT). Although RT can significantly reduce tumor burden in a subset of patients, there are many patients whose tumors do not respond to RT. To study this, we developed an orthotopic model of RC that recapitulates human disease in which administration of clinically relevant RT results in mice bearing tumors that demonstrate a reduction of tumor burden (responders), and a group of animals whose tumors respond poorly (nonresponders). RNA-sequencing of intratumoral immune populations identified a unique gene signature associated with each group. Tumor-associated macrophages (TAMs) from nonresponding tumors were more similar to TAMs from untreated tumors when compared to TAMs from responding tumors. Interestingly, TAMs in responding tumors upregulate pathways associated with pattern recognition receptor signaling and damage associated molecular patterns. These data suggest that TAMs from responding tumors may be polarized to respond to the immunostimulatory damage induced by RT. While TAM polarization plays a large role in promoting either an immunostimulatory or immunosuppressive microenvironment, CD8+ T cells are directly responsible for killing tumor cells. We sequenced intratumoral CD8+ T cells as well and determined that T cells from responding tumors induced pathways consistent with enhanced effector function. These data suggest that a targeted therapy aimed at the TAM:CD8+ T cell interaction may promote a stronger RT response in the nonresponding tumors.

Signature of Enhanced Effector Function Defines Early Life Regulatory T Cells

Abstract The induction of self-tolerance and regulation of inflammatory immune responses is critical during early life when a myriad of primary antigenic challenges are encountered. Regulatory T Cells (Tregs) promote self-tolerance and function to modulate immune responses utilizing a variety of immunosuppressive mechanisms. Tregs are known to be disproportionately more abundant during early life, suggesting an essential role during immune development. We performed bulk RNA sequencing and multi-parameter flow cytometry on Tregs isolated from adult and pediatric (1 – 3 years of age) tissue (tonsils) and blood to discern the defining characteristics of early life Tregs. Early life Tregs were found to have an increased expression of genes associated with cell cycle (Ki67), effector and suppressive function (ICOS/CTLA4/IL10/GZMB/GZMA) and chemokine signaling (CCR5/CCR9) compared to adults. Importantly, distinctions in canonical effector function genes were apparent across all early life Tregs and not restricted to tissue site. Interestingly, CCR9, essential in gut-specific homing, was upregulated in both tissue and blood early life Tregs, suggesting a biasing towards intestinal immune homeostasis during childhood. We further explored the upregulation of genes related to cell cycle and found that early life Tregs exhibit higher proliferative capacity compared to both adult Tregs and early life CD4 conventional T cells, revealing a potential effector mechanism unique to early life. Early life Tregs are defined by a signature of augmented effector functionality enhancing their ability to respond to the abundant immunological challenges of infancy and childhood.

In Like a Lamb; Out Like a Lion: Marching CAR T Cells Toward Enhanced Efficacy in B-ALL.

Combining synthetic biology with adoptive T-cell transfer has led to promising advances in the treatment of relapsed/refractory B-cell acute lymphoblastic leukemia (R/R B-ALL), diffuse large B-cell lymphoma (DLBCL), and mantle cell lymphoma (MCL). Chimeric antigen receptors (CARs) are synthetic receptors that redirect T-cell specificity against cancer. CARs include "built-in" signaling domains that reprogram T-cell metabolism, enhance effector function, and support long-term persistence. Despite their success in blood-based malignancies, relapse can occur in CD19-redirected CAR T-cell therapies for several reasons, including poor engraftment, impaired in vivo proliferation, and T-cell senescence. Herein, we explain how subtle alterations in CAR design may overcome barriers to effective adoptive immunotherapy. We also discuss how the physiochemical properties of the single-chain variable fragment (scFv) affect differentiation and persistence. Moreover, we describe innovative advances in CAR engineering and provide insight into the development of humanized scFvs whose proposed benefits include increased persistence and improved clinical outcomes. Tumor cells can evade CAR T-cell-mediated detection and elimination due to the emergence or presence of CD19-negative leukemic cell subpopulations. We also discuss the opportunities and challenges in targeting other B-ALL-associated antigens. Identifying alternate targets is fundamentally necessary to restore the success of CAR T-cell therapies in CD19-negative patients with B-ALL.

Exercise Training Improves Tumor Control by Increasing CD8+ T-cell Infiltration via CXCR3 Signaling and Sensitizes Breast Cancer to Immune Checkpoint Blockade.

The mechanisms behind the antitumor effects of exercise training (ExTr) are not fully understood. Using mouse models of established breast cancer, we examined here the causal role of CD8+ T cells in the benefit acquired from ExTr in tumor control, as well as the ability of ExTr to improve immunotherapy responses. We implanted E0771, EMT6, MMTV-PyMT, and MCa-M3C breast cancer cells orthotopically in wild-type or Cxcr3-/- female mice and initiated intensity-controlled ExTr sessions when tumors reached approximately 100 mm3 We characterized the tumor microenvironment (TME) using flow cytometry, transcriptome analysis, proteome array, ELISA, and immunohistochemistry. We used antibodies against CD8+ T cells for cell depletion. Treatment with immune checkpoint blockade (ICB) consisted of anti-PD-1 alone or in combination with anti-CTLA-4. ExTr delayed tumor growth and induced vessel normalization, demonstrated by increased pericyte coverage and perfusion and by decreased hypoxia. ExTr boosted CD8+ T-cell infiltration, with enhanced effector function. CD8+ T-cell depletion prevented the antitumor effect of ExTr. The recruitment of CD8+ T cells and the antitumor effects of ExTr were abrogated in Cxcr3-/- mice, supporting the causal role of the CXCL9/CXCL11-CXCR3 pathway. ExTr also sensitized ICB-refractory breast cancers to treatment. Our results indicate that ExTr can normalize the tumor vasculature, reprogram the immune TME, and enhance the antitumor activity mediated by CD8+ T cells via CXCR3, boosting ICB responses. Our findings and mechanistic insights provide a rationale for the clinical translation of ExTr to improve immunotherapy of breast cancer.

Harnessing the IL-21-BATF Pathway in the CD8+ T Cell Anti-Tumor Response.

Simple SummaryIn cancer, CD8+ T cells enter a state of dysfunction within the tumor that prevents them from targeting and killing tumor cells. Our study aims to uncover how CD8+ T cells can be helped by CD4+ T cells or modified in order to improve their effector function against cancer. Thus, allowing them to better fight and control tumors. Our work shows that the protein Basic Leucine Zipper ATF-Like Transcription Factor (BATF) may be a key regulator of CD8+ T cells and their anti-tumor function. These findings can provide further insight for the development of novel therapeutic treatments for cancer patients.In cancer, CD8+ T cells enter a dysfunctional state which prevents them from effectively targeting and killing tumor cells. Tumor-infiltrating CD8+ T cells consist of a heterogeneous population of memory-like progenitor, effector, and terminally exhausted cells that exhibit differing functional and self-renewal capacities. Our recently published work has shown that interleukin (IL)-21-producing CD4+ T cells help to generate effector CD8+ T cells within the tumor, which results in enhanced tumor control. However, the molecular mechanisms by which CD4+ helper T cells regulate the differentiation of effector CD8+ T cells are not well understood. In this study, we found that Basic Leucine Zipper ATF-Like Transcription Factor (BATF), a transcription factor downstream of IL-21 signaling, is critical to maintain CD8+ T cell effector function within the tumor. Using mixed bone marrow chimeras, we demonstrated that CD8+ T cell-specific deletion of BATF resulted in impaired tumor control. In contrast, overexpressing BATF in CD8+ T cells enhanced effector function and resulted in improved tumor control, bypassing the need for CD4+ helper T cells. Transcriptomic analyses revealed that BATF-overexpressing CD8+ T cells had increased expression of costimulatory receptors, effector molecules, and transcriptional regulators, which may contribute to their enhanced activation and effector function. Taken together, our study unravels a previously unappreciated CD4+ T cell-derived IL-21–BATF axis that could provide therapeutic insights to enhance effector CD8+ T cell function to fight cancer.

GMP-Compliant Universal Antigen Presenting Cells (uAPC) Promote the Metabolic Fitness and Antitumor Activity of Armored Cord Blood CAR-NK Cells.

Natural killer (NK) cells are innate lymphocytes recognized for their important role against tumor cells. NK cells expressing chimeric antigen receptors (CARs) have enhanced effector function against various type of cancer and are attractive contenders for the next generation of cancer immunotherapies. However, a number of factors have hindered the application of NK cells for cellular therapy, including their poor in vitro growth kinetics and relatively low starting percentages within the mononuclear cell fraction of peripheral blood or cord blood (CB). To overcome these limitations, we genetically-engineered human leukocyte antigen (HLA)-A− and HLA-B− K562 cells to enforce the expression of CD48, 4-1BBL, and membrane-bound IL-21 (mbIL21), creating a universal antigen presenting cell (uAPC) capable of stimulating their cognate receptors on NK cells. We have shown that uAPC can drive the expansion of both non-transduced (NT) and CAR-transduced CB derived NK cells by >900-fold in 2 weeks of co-culture with excellent purity (>99.9%) and without indications of senescence/exhaustion. We confirmed that uAPC-expanded research- and clinical-grade NT and CAR-transduced NK cells have higher metabolic fitness and display enhanced effector function against tumor targets compared to the corresponding cell fractions cultured without uAPCs. This novel approach allowed the expansion of highly pure GMP-grade CAR NK cells at optimal cell numbers to be used for adoptive CAR NK cell-based cancer immunotherapy.

IL-10 Enhances Human Natural Killer Cell Effector Functions via Metabolic Reprogramming Regulated by mTORC1 Signaling.

Cell metabolism plays a pivotal role in regulating the effector functions of immune cells. Stimulatory cytokines, such as interleukin (IL)-2 or IL-12 and IL-15, activate glycolysis and oxidative phosphorylation in natural killer (NK) cells to support their enhanced effector functions. IL-10, a pleiotropic cytokine, is known to suppress macrophage activation but stimulate NK cells. However, it remains unclear if IL-10 has an effect on the metabolism of human NK cells and if so, what metabolic mechanisms are affected, and how these metabolic changes are regulated and contribute to the effector functions of NK cells. In this study, we demonstrate that IL-10 upregulates both glycolysis and oxidative phosphorylation in human NK cells, and these metabolic changes are crucial for the enhanced effector functions of NK cells. Mechanistically, we unravel that IL-10 activates the mammalian target of rapamycin complex 1 (mTORC1) to regulate metabolic reprogramming in human NK cells.

Expansion of circulating peripheral TIGIT+CD226+ CD4 T cells with enhanced effector functions in dermatomyositis

BackgroundT cell Ig and ITIM domain (TIGIT)/CD226 pathway has a critical role in regulating T cell responses and has come to the forefront in cancer as a promising immunotherapeutic target. However, its role in autoimmune diseases is just beginning to be elucidated. Dermatomyositis (DM) is an autoimmune disease, in which T cell dysregulation plays a pivotal role, and importantly, it is a common immune-related adverse event in response to treatment of cancers with immune checkpoint inhibitors, but no studies have implicated the TIGIT/CD226 axis in DM.MethodsWe recruited 30 treatment-naïve DM patients and 26 healthy controls. Flow cytometry analysis was used to investigate the co-expression of TIGIT and CD226 on T cells in blood samples. Magnetic bead or FACS-based cell isolation, T cell proliferation assay, and intracellular cytokine staining were performed to analyze the functions of different TIGIT/CD226 phenotypes. Recombinant proteins CD155, CD112, and anti-CD226 antibodies were used to suppress the function of TIGIT/CD226-expressing CD4 T cells.ResultsFour distinct subsets of T cells based on TIGIT/CD226 co-expression, TIGIT+CD226−, TIGIT+CD226+, TIGIT−CD226+, and TIGIT−CD226−, were identified and characterized in DM patients. Our data showed that the function of CD4 T cell subset varied by the TIGIT/CD226 phenotype. An elevated TIGIT+CD226+ CD4 subset with enhanced effector function was observed in patients with DM, especially the patients complicated with interstitial lung disease. This subpopulation was closely related to DM activity and decreased significantly in DM remission after treatment. Furthermore, the effector function of TIGIT+CD226+ CD4 subset could be suppressed by blocking CD226.ConclusionOur data revealed that the TIGIT and CD226 expression profiles could be used to identify functionally distinct subsets of CD4 T cells and TIGIT+CD226+ CD4 T cells is a significant subset in DM with enhanced frequency and effector function. This abnormal subset could be suppressed by blocking CD226, providing insight into the therapeutic target of the TIGIT/CD226 axis.