Transcriptional and Epigenetic Regulation of T Cell States in Cancer

B cells constitute a major component of tumor-infiltrating leukocytes. However, the influence of these cells on malignancy is currently under debate, reflecting the heterogeneity of B cell subsets in tumors. With recent advances, it becomes apparent that this debate includes not only the evaluation of B cells themselves, but also the underlying immune microenvironment network, which scripts the highly heterogeneous B cell populations in tumors and directs the roles of those sub-populations in disease progression and clinical treatment. In this review, we summarize recent findings on the heterogeneous subset composition of B cells in both human and mouse tumor models and their different impacts on disease progression. We further describe the multidimensional interplays between B cells and other immune cells in the tumor microenvironment, which account for the regulation of B cell differentiation and function in situ. We also assess the potential influences of distinct sub-tumor locations on B cell function in primary tumors during development and those under immunotherapy treatment. Illuminating the heterogeneous nature of B cell subset composition, generation, localization, and related immune network in tumor is of immense significance for comprehensively understanding B cell response in tumor and designing more efficacious cancer immunotherapies.

Checkpoint blockade therapies that aim to reactivate anti-tumor immune responses have revolutionized cancer treatment, resulting in durable responses in a significant proportion of patients with advanced disease. Nevertheless, many patients fail to reach long-term clinical benefit, and therefore, a better insight into the mechanisms underlying response to immunotherapy is required to enhance its success rates. Recent findings suggest that immune cell infiltrates in cancer can be highly heterogeneous, potentially contributing to differential therapy outcomes. However, immune cell states have thus far been studied mainly in patients with end-stage melanoma and diverse treatment histories, whereas patients with early-stage metastatic disease are generally treatment naïve, allowing assessment of naturally evolved T cell functionality and immune cell composition. To characterize the heterogeneity in the tumor immune environment within and between patients, we profiled the tumor microenvironment of six stage III melanoma patients by single-cell RNA sequencing, generating an unbiased map of the expression signatures of immune cells as well as tumor and stromal cells. In parallel, we also derived the T cell receptor sequences for single T cells, and used them to determine intra- and inter-tumoral T cell clonality and infer the functionality of clonally expanded T cell populations in the tumor. A series of different cell types and cellular states that have been described previously in end-stage melanoma was also observed in this early-stage metastatic disease, including cytotoxic, exhausted, and regulatory T cells, as well as various myeloid subsets. Notably, despite identical disease stage and treatment background, the composition of the immune cell populations differed considerably between patients. In addition, while analysis of T cell states showed high variability between and within patients, clonally expanded T cells identified within patients predominantly adapted similar cell profiles. Currently we are assessing whether the occurrence of distinct T cell states can be coupled to other aspects of the immune infiltrate. In addition to describing T cell heterogeneity, our data suggest the existence of novel immune cell subsets within the tumor microenvironment. Our findings demonstrate that, using our single-cell RNA-sequencing approach, we can determine the immunological make-up in melanoma during early stages of metastatic disease, in an unbiased manner. Collectively, our approach should be helpful in determining the mechanisms underlying the development and effectiveness of tumor-specific immune responses in metastatic melanoma. Citation Format: Ido Yofe, Hanjie Li, Anne van der Leun, Lubling Yaniv, Assaf Weiner, Alexander van Akkooi, Amos Tanay, Ton Schumacher, Ido Amit. Dissecting immune cell heterogeneity in human cancer by single-cell RNA-sequencing [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2017 Oct 1-4; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2018;6(9 Suppl):Abstract nr B54.

Introduction: Functional heterogeneity in cancer may result in the metastasis of various types of tumour cells throughout the body. Attempting to explain functional heterogeneity in cancer cells has given rise to two models. The Cancer Stem Cell model proposes that a subset of tumour cells self-replicate and that heterogeneity is a progeny of various cancer stem cells (CSCs). The Clonal Evolution Model proposes heterogeneity as a product of mutations across tumour cells that accumulate and metastasize linearly or branching. Methods: Research was conducted through open-access journals and information was compiled surrounding CSC models using the Google Scholar and McMaster Library database search engines. Inclusions were sources that detailed the relationship between both models of functional heterogeneity and microenvironments and treatments. Literature that did not center around tumour microenvironments was not included in this literature review. Results: The two main models of tumour proliferation were explored and related to hypoxic tumour microenvironments. Various markers, etiologic agents and toxins were identified that contribute to tumour progression. Cell signalling and pathways that contribute to major cellular functions were identified, along with possible disruptions and epigenetic changes that lead to tumour and CSC proliferation. Discussion: This study reveals that the tumour microenvironment plays a large role in the proliferation of CSCs. Although the therapies targeting microenvironments are in early stages of development, focusing on these CSC targeted- therapies may lead to better treatments for cancer or more effective combination therapies. Strengths of the paper include the compilation of major contributing areas to CSC proliferation, whereas limitations encompass the high variability of tumour cells that are not all covered in this review. Conclusion: While no definitively eradicating treatment for CSCs currently exist, the recent developments in cancer research indicate promising new techniques for its management. Implications: By further studying malignant CSCs, highly effective cancer treatments may result, leading to the advancement of CSC recognition and combination therapy.

T cell dysfunction or ‘exhaustion’ is a hallmark of solid malignancies. Despite the critical importance of CD8 T lymphocytes in controlling tumor growth and immunotherapy efficacy, the mechanisms governing T cell exhaustion remain unclear. Here, we focused on resolving the heterogeneity and molecular programming of exhausted CD8 T cell states in the context of pancreatic cancer. Pancreatic ductal adenocarcinoma (PDAC) is an aggressive disease with a discouraging 5-year survival rate of approximately 8%. PDAC tumors commonly present as immunologically ‘cold’, devoid of protective T cell populations, and are typically resistant to promising immunotherapies such as immune checkpoint blockade or adoptive cell therapy. Therefore, understanding the unique microenvironmental and CD8 T cell-intrinsic signals governing T cell exhaustion and accumulation in PDAC tumors may reveal crucial insight for engineering CD8 T cells for optimal immunotherapy efficacy. Here, we developed preclinical PDAC models for profiling tumor-specific murine CD8 T cell responses. Through application of spectral flow cytometry and multimodal single-cell sequencing approaches, we have uncovered impressively diverse antigen-specific CD8 T cell states in PDAC tumors. Select CD8 T cell populations identified within the PDAC tumor microenvironment included canonical populations of terminally-exhausted and progenitor-exhausted CD8 T cells as well as unexpected and uncharacterized CD8 T cell populations. Accordingly, we also have identified critical transcription factors controlling key features of CD8 T cell heterogeneity and persistence in PDAC tumors. These findings may ultimately provide insight for tailoring CD8 T cell fate and function in PDAC. Supported by 2022 AAI Intersect Fellowship for Computational Scientists and Immunologists

Chapter 26 - Application of Theranostics to Measure and Treat Cell Heterogeneity in Cancer

DAP10 integration in CAR-T cells enhances the killing of heterogeneous tumors by harnessing endogenous NKG2D

Most diseases are accompanied by altered metabolism at the cellular level. In cancer, metabolic reprogramming that is, the regulated alteration of metabolism as a consequence of tumorigenic mutations and other factors is viewed as an essential component of malignant transformation. It is unknown to what extent cell-autonomous vs. non-cell autonomous factors influence metabolic pathway choice. We performed metabolic profiling in a large panel of molecularly annotated non-small cell lung cancer (NSCLC) cell lines grown in standard monolayer culture in order to define the scope of cell-autonomous metabolic variability in this disease and to understand the mechanisms by which metabolic phenotypes are established. We then identified a new pathway that allows cells to tolerate a cell-extrinsic stress: the loss of anchorage dependence. Regardless of the intrinsic metabolic preferences in monolayer culture, NSCLC cells activated an unconventional pathway allowing reducing equivalents to shuttle from the cytosol to the mitochondria to counteract oxidative stress during anchorage loss. Finally, we performed clinical studies involving a combination of pre-surgical imaging and intra-operative 13C infusions to assess metabolic heterogeneity between individual NSCLC tumors in human patients, and to characterize the extent of metabolic heterogeneity within individual tumors. These studies revealed enhanced glycolysis and glucose oxidation in tumors compared to adjacent lung, regardless of driver mutation. Surprisingly, we also found that regional metabolic heterogeneity could be mapped prior to surgery by examining tissue perfusion using contrast MRI, and that glucose’s contribution to central carbon metabolism is inversely proportional to tumor perfusion. Together, these efforts identify a complex but finite set of metabolic phenotypes that support cell growth and may predict therapeutic vulnerabilities. They also underscore the potential for both existing and novel imaging approaches to inform about metabolic phenotypes in vivo, including phenotypes specified by both cell-intrinsic and cell-extrinsic factors. Citation Format: Ralph J. DeBerardinis. Metabolic heterogeneity in cancer cells and tumors. [abstract]. In: Proceedings of the Fourth AACR International Conference on Frontiers in Basic Cancer Research; 2015 Oct 23-26; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2016;76(3 Suppl):Abstract nr IA07.

A View to Natural Killer Cells in Hepatitis C

Metabolic regulation of T cells in the tumor microenvironment by nutrient availability and diet

Editor's evaluation: Mesenchymal stem cell suppresses the efficacy of CAR-T toward killing lymphoma cells by modulating the microenvironment through stanniocalcin-1

Decision letter: Mesenchymal stem cell suppresses the efficacy of CAR-T toward killing lymphoma cells by modulating the microenvironment through stanniocalcin-1

Article Figures and data Abstract Editor's evaluation eLife digest Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract Stem cells play critical roles both in the development of cancer and therapy resistance. Although mesenchymal stem cells (MSCs) can actively migrate to tumor sites, their impact on chimeric antigen receptor modified T cell (CAR-T) immunotherapy has been little addressed. Using an in vitro cell co-culture model including lymphoma cells and macrophages, here we report that CAR-T cell-mediated cytotoxicity was significantly inhibited in the presence of MSCs. MSCs caused an increase of CD4+ T cells and Treg cells but a decrease of CD8+ T cells. In addition, MSCs stimulated the expression of indoleamine 2,3-dioxygenase and programmed cell death-ligand 1 which contributes to the immune-suppressive function of tumors. Moreover, MSCs suppressed key components of the NLRP3 inflammasome by modulating mitochondrial reactive oxygen species release. Interestingly, all these suppressive events hindering CAR-T efficacy could be abrogated if the stanniocalcin-1 (STC1) gene, which encodes the glycoprotein hormone STC-1, was knockdown in MSC. Using xenograft mice, we confirmed that CAR-T function could also be inhibited by MSC in vivo, and STC1 played a critical role. These data revealed a novel function of MSC and STC-1 in suppressing CAR-T efficacy, which should be considered in cancer therapy and may also have potential applications in controlling the toxicity arising from the excessive immune response. Editor's evaluation This study uncovers the contributions of MSC on modulating CAR T-cell behaviour. Based on the importance in basic biology and its immediate impact on translational potential, all reviewers are satisfied on the advances in this study. https://doi.org/10.7554/eLife.82934.sa0 Decision letter Reviews on Sciety eLife's review process eLife digest Immunotherapy is a type of cancer treatment that helps the immune system fight cancer. For example, chimeric antigen receptor T cell (CAR-T) therapy is used to target several types of blood cancer. It works by reprogramming patients’ immune cells to target specific tumor cells. In blood cancers, CAR-T therapy works very well, but it can cause extreme responses from the patient’s immune system, which can be life threatening. In solid tumors, CAR-T therapy is much less successful because the tumors secrete molecules into the space surrounding them, which weaken the immune processes that attack cancerous cells. Stem cells are the master cells of the body. Originating in the bone marrow, they can repair and regenerate the body’s cells. Cancer stem cells play a role in resistance to CAR-T therapy, due – in part – to their ability to renew themselves, but the role of another type of stem cell, called mesenchymal stem cells, was less clear. Mesenchymal stem cells develop into tissues that line organs and blood vessels. Although it is known that mesenchymal stem cells are present in most cancers and play a role in shaping and influencing the space around tumors, their impact on CAR-T therapy has not been studied in depth. To find out more, Zhang et al. looked at the influence of a protein, called staniocalcin-1 (STC1), on CAR-T therapy, by studying cells grown in the laboratory and human tumor cells that had been implanted in mice. Zhang et al. found that mesenchymal stem cells reduce the ability of CAR-T therapy to destroy cancer cells and that they needed STC1 to do this successfully. They also increased the expression of molecules that dampen the immune system, and suppressed molecules called inflammasomes, which are an important part of the way the immune system detects disease. Moreover, reducing the amount of STC1 that mesenchymal stem cells expressed restored the effectivity of CAR-T therapy. This study increases our understanding of the way that mesenchymal stem cells affect CAR-T therapy. It has the potential to open up a new way of improving the efficiency of this treatment and of reducing the harmful side effects that it can cause. Introduction Advances in chimeric antigen receptor modified T cell therapy (CAR-T) in recent years have shown enormous promise in cancer immunotherapy, which has produced unprecedented clinical outcomes, most notably for patients with hematologic malignancies (Singh et al., 2016; Park et al., 2018). Despite the striking achievements, CAR-T therapy is also facing many challenges such as the treatment-related severe toxicity and side effects, including cytokine release syndrome (CRS) and neurotoxicity (Hong et al., 2020; Freyer and Porter, 2020). CRS is the most common acute toxicity associated with an excessive immune response that causes fever, hypotension, and respiratory insufficiency. The neurotoxicity induced by CAR-T therapy exhibits a diverse array of neurologic symptoms such as tremors, expressive aphasia, and impaired attention. The precise mechanism that causes these life-threatening side effects remains unclear (Freyer and Porter, 2020; Jiang et al., 2019). On the other hand, the success of CAR-T therapy in treating solid tumors is still very limited (Martinez and Moon, 2019). Identifying hurdles and potential mechanisms that impede the function of CAR-T cells is of vital importance to expanding its use. The immunosuppressive tumor microenvironment (TME) is one of the obstacles that diminishes the efficacy of CAR-T therapy, especially for solid tumors. Among the many factors that can modulate TME and immune response, the impact of mesenchymal stem cell (MSC) on CAR-T therapy has been little studied. MSC is a type of adult stem cell with high proliferative activity and multidirectional differentiation capacity. However, MSCs have additional paracrine effects that are believed to underlie their therapeutic functions (Jiang and Xu, 2020). By secreting a variety of cytokines into the tissue microenvironment, it has been known that MSCs can modulate extracellular matrix, promote angiogenesis, and suppress inflammation and apoptosis (Keating, 2012; Wang et al., 2014; Regmi et al., 2019). Some MSC-secreted cytokines, such as stromal cell-derived factor 1 and stem cell factor, play important roles in hematopoietic and immune regulation (Kawaguchi et al., 2019; Markov et al., 2007). In addition, studies suggest that MSCs can modulate the function of monocytic lineages cells, especially macrophages (Németh et al., 2009; YlÖstalo et al., 2012; Choi et al., 2011). Some reports also showed that MSCs could directly affect the functionality and cellular responses of T cells, Tregs, and memory T cells (Cen et al., 2019; Tumangelova-Yuzeir et al., 2019; Luque-Campos et al., 2019). It was reported that human mesenchymal stem cells (hMSCs) could be activated by lipopolysaccharide (LPS)-stimulated macrophages to increase the expression and secretion of stanniocalcin-1 (STC1) (Oh et al., 2014). STC1 was a mitochondria-related glycoprotein originally identified as a calcium/phosphate regulating hormone in bony fishes, and later on, it was found to be a pleiotropic factor involved in various degenerative diseases such as ocular and renal disease, as well as idiopathic pulmonary fibrosis (Yeung et al., 2012; Ohkouchi et al., 2015). STC1 could improve the cell survival and regeneration of MSCs in a paracrine fashion (Ono et al., 2015). There was also evidence suggesting that STC1 played an oncogenic role in various types of tumors (Du et al., 2011; Liu et al., 2010). Based on a retrospective study of ~1500 clinical samples, it was concluded that high STC1 expression is associated with the poor clinical outcome of breast cancer (Chang et al., 2015). It was proved that STC1 is involved in several oxidative and cancer-related signaling pathways, such as NF-κB, extracellular-signal-regulated kinase (ERK), and c-Jun NH(2)-terminal kinase (JNK) pathways (Nguyen et al., 2009; Chan et al., 2017). The expression and secretion of STC1 in cancer tissue can be stimulated by external stimuli, including external cytokines and oxidative stress (Nguyen et al., 2009). Under hypoxia conditions, STC1 could be modulated by Hypoxia-inducible factor-1 (HIF-1) to facilitate the reprogramming of tumor metabolism from oxidative to glycolytic metabolism (Yeung et al., 2005). STC1 was also reported to participate in the process of epithelial-to-mesenchymal transition, which is associated with tumor invasion and the reshape of the tumor microenvironment, as well as increasing therapy resistance (Pastushenko and Blanpain, 2019). Considering the pleiotropic role of STC1, especially its intercellular linkage between MSCs, cancer cells, and macrophage stimulation, it is interesting to know what role it plays in connection to the functions of MSC in TME. Therefore, we generated a stable STC1 knockdown MSC cell line. With a cell co-culture model containing CAR-T cells, hMSCs, macrophages, and Pfeiffer lymphoma cells to partially mimic the tumor microenvironment together with a xenograft mice model, here we studied the impacts of MSC on CAR-T efficacy and the potential immune response change in the presence and absence of STC1. Results Stable knockdown of STC1 in hMSC-inhibited cell migration, slightly suppressed cell proliferation, but no increase in apoptosis To study the function of STC1, we first generated a stable knockdown cell line by lentivirus-based shRNA for the STC1 gene, and the expression of STC1 protein was evaluated by Western blot (Figure 1A). STC1 stable knockdown in hMSCs exhibited a minor effect in cell survival (Figure 1B) and slightly reduced proliferation rate based on the small increase in the proportion of cells in G0/G1 phases versus that in the S phase (Figure 1C) as determined by MTT (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide) and Fluorescence-activated Cell Sorting (FACS) analysis. To investigate whether knockdown of STC1 affects cell migration, wound healing and transwell chamber assays were performed. After creating a ‘scratch’ in a monolayer of hMSCs, the closure of the gap was determined after 24 hr. As shown in Figure 1D, compared to control hMSCs, the gap was less filled in hMSCshSTC1. The inhibitory effect on cell migration was further confirmed by a transwell assay. As shown in Figure 1E, there were significant migration and invasion observed in hMSCsshCtrl, whereas there was a >30% reduction in migration across the transwell chamber membrane in hMSCsshSTC1. To further determine whether knockdown of STC1 may have any lethal effect, apoptosis was determined by two different assays. To measure the early apoptosis, cells were stained with the Alexa Fluor 488 annexin V and the propidium iodide (PI) followed by flow cytometry to detect apoptosis-associated phosphatidylserine (PS) expression and membrane permeability (Figure 1F). Parallelly, no DNA fragmentation was detected as determined with the TUNEL assay (Figure 1G, the green dots were from the background due to overexposure). Both studies showed that knockdown of STC1 did not cause apoptosis of hMSCs. Figure 1 Download asset Open asset The impact of stanniocalcin-1 (STC1) knockdown on cell proliferation, migration, and apoptosis of hMSCs. (A) Western blot analysis of STC1 protein expression in hMSCs. (B) Cell viability determined by MTT, measurements are shown as the mean ± SD from three independent experiments. (C) FACS analysis of cell cycle progression on hMSCs w/o STC1 knockdown. (D, E) Knockdown of STC1 suppressed cell migration as determined by wound healing and transwell chamber assays. (F) Apoptosis determination by the Alexa Fluor 488 annexin V and PI detection. (G) DNA fragmentation determination by transferase-mediated dUTP nick-end labeling (TUNEL) assay. Figure 1—source data 1 Labeled original blots of Figure 1A. https://cdn.elifesciences.org/articles/82934/elife-82934-fig1-data1-v3.zip Download elife-82934-fig1-data1-v3.zip Figure 1—source data 2 Unlabeled original blots of Figure 1A. https://cdn.elifesciences.org/articles/82934/elife-82934-fig1-data2-v3.zip Download elife-82934-fig1-data2-v3.zip Figure 1—source data 3 Figure 1B in Excel file. https://cdn.elifesciences.org/articles/82934/elife-82934-fig1-data3-v3.xlsx Download elife-82934-fig1-data3-v3.xlsx The presence of hMSCs inhibited CAR-T cell killing activity, but knockdown of STC1 completely abrogated this inhibition To investigate the impact of hMSCs on CAR-T treatment, we used an in vitro cell co-culture model modified according to previous studies to mimic a simplified situation of tumor environment (Singh et al., 2017; Liu et al., 2021). The co-culture contained CD19 CAR-T cells, Pfeiffer cells that were from human diffuse large cell lymphoma, and M2 macrophages (derived from THP-1 cells by phorbol-12-myristate-13-acetate [PMA] polarization for 24 hr) at a cell number ratio of 1:3:1. The cell-killing activity of CAR-T cells toward Pfeiffer cells was determined by lactate dehydrogenase (LDH) cytotoxicity assay on total cell co-culture. As shown in Figure 2A, 67% of Pfeiffer cells were killed after being exposed to CAR-T cells for 24 hr, and 93% were killed at 48 hr as compared to mock-treated control. After adding hMSCs into the co-culture, the cell-killing activity of CAR-T was significantly inhibited (Figure 2A). The number of hMSC added was the same as the CAR-T cell. Interestingly, the inhibitory effect of hMSCs on CAR-T cytotoxicity could be completely abrogated if knockdown STC1 gene in hMSCs. These results for the first time revealed that CAR-T efficacy could be affected by the presence of MSCs, and the gene STC1 played a critical role. Figure 2 Download asset Open asset Analysis of cytotoxicity, T cell composition, and immune-suppressive markers. The cell co-culture contained chimeric antigen receptor modified T cell (CAR-T) cells, Pfeiffer cells, M2 macrophages, and control or stanniocalcin-1 (STC1) knockdown hMSCs in a ratio of 1:3:1:1. After 24 hr (or 48 hr for cytotoxicity) incubation, the following analysis was conducted: (A) The impact of hMSC (w/o STC1) on the cytotoxicity of CAR-T toward Pfeiffer cells; (B) FACS analysis of CD4+ and CD8+ composition. (C) Quantitation of the FACS data on CD4+ and CD8+; (D) FACS analysis of Treg+ cells (CD4+CD127+CD25+); (E) Quantitation of Treg+ cells. (F) Western blot analysis of indoleamine 2,3-dioxygenase (IDO) and programmed cell death-ligand 1 (PD-L1) expression in the cell co-culture. Data in bar graphs are presented as the mean ± SD from three independent experiments (p values are as indicated, n=3). Figure 2—source data 1 Figure 2A in Excel file. https://cdn.elifesciences.org/articles/82934/elife-82934-fig2-data1-v3.xlsx Download elife-82934-fig2-data1-v3.xlsx Figure 2—source data 2 Figure 2C in Excel file. https://cdn.elifesciences.org/articles/82934/elife-82934-fig2-data2-v3.xlsx Download elife-82934-fig2-data2-v3.xlsx Figure 2—source data 3 Figure 2E in Excel file. https://cdn.elifesciences.org/articles/82934/elife-82934-fig2-data3-v3.xlsx Download elife-82934-fig2-data3-v3.xlsx Figure 2—source data 4 Labeled original blots of Figure 2F. https://cdn.elifesciences.org/articles/82934/elife-82934-fig2-data4-v3.zip Download elife-82934-fig2-data4-v3.zip Figure 2—source data 5 Unlabeled original blots of Figure 2F. https://cdn.elifesciences.org/articles/82934/elife-82934-fig2-data5-v3.zip Download elife-82934-fig2-data5-v3.zip Co-culturing with hMSCs caused an increase of CD4+ T cells and Treg cells but a decrease of CD8+ T cells Previous studies have demonstrated that the composition of CD4+ and CD8+ T cell subsets was crucial for CAR-T cell efficacy (Sommermeyer et al., 2016; Turtle et al., 2016). To investigate the mechanism of how hMSC inhibited the cytotoxicity of CAR-T, the amount of CD4+ and CD8+ T cells were analyzed by flow cytometry 24 hr after co-culture. As shown in Figure 2B and C, the ratio between CD4+ and CD8+ was about 1:4 when there were no hMSCs in co-culture (Figure 2C). However, the addition of hMSC caused a significant increase of CD4+ and a decrease of CD8+ T cells (Figure 2B), resulting in a ratio change to 2:1. Similar to the change of CD4+ T cells, the percentage of regulatory T cells (Treg) was also significantly increased from ~3 to 12% when co-culture with hMSC (Figure 2D and E). When using hMSCshSTC1, all the changes were completely reversed back to the level similar to that of co-culture without hMSCs. This explains the reduced CAR-T cytotoxicity since CD8+ T cells are directly responsible for specific lytic activity against lymphoma (Sommermeyer et al., 2016). Tregs, which account for 5–10% of the total number of CD4+ T cells, are known to play a role in suppressing the function of T cells and other immune cells (Zhang et al., 2018). Therefore, the above results indicate that hMSCs’ inhibitory effect on CAR-T cytotoxicity was due to both suppression of CD8+ cells and the induction of Treg cells, and the presence of STC1 was indispensable for these impacts of hMSC. The presence of hMSC enhanced immune suppression and STC1 played a key role The immune-suppressive TME is the main cause of CAR-T cell exhaustion which attenuates its efficacy. To further investigate the function of STC1 and the molecular mechanism of hMSC on CAR-T resistance, some key regulators of TME were determined. As shown in Figure 2F, the addition of hMSC to the cell co-culture stimulated the expression of indoleamine 2,3-dioxygenase (IDO) and programmed cell death-ligand 1 (PD-L1). IDO and PD-L1 are two of the most important immunosuppressive proteins. IDO is an intracellular enzyme that converts tryptophan into inhibitory metabolites for T-cell activity (Ninomiya et al., 2015). PD-L1 is expressed in tumor cells and immune cells contributing to the immune-suppressive TME (Ribas and Hu-Lieskovan, 2016). When using hMSCshSTC1, the expression level of IDO and PD-L1 was both significantly reduced by more than 50%, though still higher than that without hMSC. These results indicated that the presence of hMSC can enhance the expression of immune suppressive proteins in Pfeiffer cells and macrophages, and the presence of STC1 is important for hMSC to exert these effects. hMSCs suppressed key components of the NLRP3 inflammasome by modulating mitochondrial ROS release In the co-culture model, M2 macrophages were included since a previous study showed that macrophages could activate hMSCs to secrete STC1 (Cen et al., 2019). In addition, the macrophage is a critical part of immune response and an important regulator of immunotherapy (DeNardo and Ruffell, 2019). To further identify the mechanisms mediating the inhibitory effects of hMSCs, the activation of the NLRP3 inflammasome was determined. The NLRP3 inflammasome is a critical component of the innate immune system mediating caspase-1 activation and proinflammatory cytokines secretion in response to harmful stimuli such as infection and endogenous stress (Menu and Vince, 2011). As shown in Figure 3A, the release of cleaved caspase-1 p20 in cell lysates, which is the indicator of caspase-1 activation, was detected after the PMA polarization of THP-1 cells to form the M1 macrophages (M-THP1). Following co-culture with CD19 CAR-T, the level of cleaved caspase-1 was significantly upregulated. The increase of active caspase-1 was abrogated when hMSCs were added into the co-culture. knock-down of STC1 led to another reverse and completely blocked the inhibitory function of hMSCs (Figure 3A). Concomitant with the reduction in active caspase-1, the cleaved IL-1β mature form and absent-in-melanoma 2 (AIM2), two key components of the inflammasome (Kelley et al., 2019), were both increasingly expressed following M-THP1 polarization and further incubation with CAR-T (Figure 3A). Compared to the partial inhibition of the active caspase-1 formation, the addition of hMSC in the cell co-culture showed a stronger inhibition of these two proteins, and their expression level was returned to the base level of Pfeiffer plus CAR-T (Figure 3A). This result suggests that the immune-suppressive effect of hMSC was through its impact on macrophages, not CAR-T or Pfeiffer cells. Knockdown of STC1 abrogated the inhibition of hMSC on IL-1β and AIM2 (Figure 3A). The levels of IL-1β in the supernatants by showed similar results as cell (Figure Figure 3 Download asset Open asset The impact of mesenchymal stem cells (MSCs) on the expression of key components involved in the of NLRP3 inflammasome and mitochondrial reactive oxygen species (A) The protein expression of caspase-1, and AIM2 in cell was analyzed by Western (B) Quantitation of IL-1β secretion in the supernatants by (C) FACS analysis of ROS level and with and (D) Quantitation of ROS level based on the percentage of cells that were both for and were 24 hr the co-culture of different cells. For the measurements of results are shown as the mean ± SD from three independent experiments (p values are as indicated, n=3). Figure data 1 Labeled original blots of Figure Download Figure data 2 Unlabeled original blots of Figure Download Figure data 3 Figure in Excel file. Download Figure data 4 Figure in Excel file. Download is one of the stimuli that the NLRP3 and it was reported that STC1 is by macrophages and to to suppress et al., 2009). Therefore, we determined the impact of hMSC on the intracellular level of reactive oxygen species and in macrophages by and As shown in Figure and the presence of suppressed both the cellular and mitochondrial ROS induced by the co-culture of CAR-T cells, tumor cells, and Knockdown of STC1 the function of hMSC in suppressing This result well with the expression of caspase-1, and suggesting that hMSCs inhibited NLRP3 inflammasome activation in macrophages was most by the oxidative hMSCs showed inhibition on CD19 CAR-T therapy in xenograft mice, which was abrogated by STC1 knockdown The immune-suppressive impact of hMSC on CAR-T therapy and the function of STC1 were further evaluated in a xenograft of Pfeiffer cells and of we hMSC into the tumor CAR-T treatment by As shown in Figure CD19 CAR-T treatment with the of a significant effect, and the tumors at However, the showed a increase in tumor and of Figure 4 Download asset Open asset The inhibition of hMSC on chimeric antigen receptor modified T cell (CAR-T) therapy in xenograft mice on (A) The and progression of tumor in three of mice with the control without any treatment, and was when the was confirmed after the Pfeiffer cells. (B) analysis of and Treg cells as the in tumor tissue at cells or (C) The tumor change with (D) The presented as the mean ± SD (p values are as indicated, n=3). Figure data 1 Figure in Excel file. Download Figure data 2 Figure in Excel file. Download Based on the analysis of IL-1β in tumor tissue on the number of cells from to in the it from 5 to in the that hMSC could suppress TME and STC1 knockdown significantly this impact (Figure with the results in a large amount of CD4+ T cells were detected in the but much less in the On the the amount of CD8+ T cells was significantly increased in the compared to that of the (Figure Based on the of a master regulator involved in the development of Treg cells, the amount of Treg cells was also increased in the compared to that of the (Figure These results further confirmed that knockdown of STC1 abrogated the immune-suppressive of MSC. The changes in the were with the changes in the tumor (Figure and The survival time of mice demonstrated that mice in CAR-T with the had the survival with no by (Figure Compared to the control with no CAR-T treatment, tumor in the was and all for These results confirmed the inhibitory effects of hMSC on CAR-T therapy in and demonstrated that STC1 is an important factor therapy efficacy. Discussion Stem cells are believed to play critical roles in resistance to cancer therapy, which is a to poor treatment responses and tumor Previous studies have been on the role of cancer stem cells. In the we presented that the presence of MSCs in TME may also be an important of cancer treatment resistance. By modulating MSCs showed a suppressive function on CAR-T efficacy toward lymphoma cells, and the presence of the STC1 gene played a critical role. The role of STC1 in cancer is Some reports showed that it an oncogenic whereas other studies the et al., 2019). The expression of STC1 has been reported to impact various types of such as tumor by the expression of in cancer cells et al., and poor clinical in and cancers (Yeung et al., 2012; et al., 2019). To the potential roles of STC1 in immunotherapy are still we demonstrated that the presence of STC1 is critical for MSC to exert its immunosuppressive role by T cell some key immune and with other immune cells. a significant of CD8+ T together with the of CD4+ T cell subsets and indicated that the suppressed CAR-T efficacy was at partially associated with function in modulating the proliferation of different T-cell the suppression of CD8+ T cells was completely abrogated if knockdown STC1 in MSCs, it is that STC1 played a key role Moreover, that STC1 is into the extracellular in a paracrine of the T cell subsets is most cytokine expression or other molecules activated by STC1. In line with our it was reported that STC-1 with immunotherapy and T cell activation by which membrane antigen function and et al., 2021). The presence of MSCs also stimulated the expression of IDO and two important immune-suppressive of IDO is an endogenous mechanism controlling excessive immune which can be produced both by tumor cells and macrophages et al., of immunosuppressive metabolites can T-cell proliferation and T-cell through the receptor et al., 2011; et al., PD-L1 is a of the mechanism of immunotherapy by T cell function and antigen (Ribas and Hu-Lieskovan, 2016). There have been studies the between MSCs and cancer cells, resulting in regulating the expression of PD-L1 on the of various cancer cells or TME and et al., 2019; et al., et al., 2018). here we demonstrated that the expression of both IDO and PD-L1 by MSCs was much reduced if the STC1 gene was

CD8 T cells play a critical role in antitumor immunity. However, over time, they often become dysfunctional or exhausted and ultimately fail to control tumor growth. To effectively harness CD8 T cells for cancer immunotherapy, a detailed understanding of the mechanisms that govern their differentiation and function is crucial. This review summarizes our current knowledge of the molecular pathways that regulate CD8 T cell heterogeneity and function in chronic infection and cancer and outlines how T cells respond to therapeutic checkpoint blockade. We explore how T cell-intrinsic and -extrinsic factors influence CD8 T cell differentiation, fate choices, and functional states and ultimately dictate their response to therapy. Identifying cells that orchestrate long-term antitumor immunity and understanding the mechanisms that govern their development and persistence are critical steps toward improving cancer immunotherapy.

Inhibitory receptors have been extensively described for their importance in regulating immune responses in chronic infections and cancers. Blocking the function of inhibitory receptors such as PD-1, CTLA-4, 2B4, Tim-3, and LAG-3 has shown promise for augmenting CD8 T cell activity and boosting pathogen-specific immunity. However, the prevalence of inhibitory receptors on CD4 T cells and their relative influence on CD4 T cell functionality in chronic HIV infection remains poorly described. We therefore determined and compared inhibitory receptor expression patterns of 2B4, CTLA-4, LAG-3, PD-1, and Tim-3 on virus-specific CD4 and CD8 T cells in relation to their functional T cell profile. In chronic HIV infection, inhibitory receptor distribution differed markedly between cytokine-producing T cell subsets with, gamma interferon (IFN-γ)- and tumor necrosis factor alpha (TNF-α)-producing cells displaying the highest and lowest prevalence of inhibitory receptors, respectively. Blockade of inhibitory receptors differentially affected cytokine production by cells in response to staphylococcal enterotoxin B stimulation. CTLA-4 blockade increased IFN-γ and CD40L production, while PD-1 blockade strongly augmented IFN-γ, interleukin-2 (IL-2), and TNF-α production. In a Friend retrovirus infection model, CTLA-4 blockade in particular was able to improve control of viral replication. Together, these results show that inhibitory receptor distribution on HIV-specific CD4 T cells varies markedly with respect to the functional subset of CD4 T cells being analyzed. Furthermore, the differential effects of receptor blockade suggest novel methods of immune response modulation, which could be important in the context of HIV vaccination or therapeutic strategies.IMPORTANCE Inhibitory receptors are important for limiting damage by the immune system during acute infections. In chronic infections, however, their expression limits immune system responsiveness. Studies have shown that blocking inhibitory receptors augments CD8 T cell functionality in HIV infection, but their influence on CD4 T cells remains unclear. We assessed the expression of inhibitory receptors on HIV-specific CD4 T cells and their relationship with T cell functionality. We uncovered differences in inhibitory receptor expression depending on the CD4 T cell function. We also found differences in functionality of CD4 T cells following blocking of different inhibitory receptors, and we confirmed our results in a Friend virus retroviral model of infection in mice. Our results show that inhibitory receptor expression on CD4 T cells is linked to CD4 T cell functionality and could be sculpted by blockade of specific inhibitory receptors. These data reveal exciting possibilities for the development of novel treatments and immunotherapeutics.

Using epigenetics to define vaccine-induced memory T cells