Immunosuppressive Populations Research Articles

Modulation of the Human Pancreatic Ductal Adenocarcinoma Immune Microenvironment by Stereotactic Body Radiotherapy.

Stereotactic body radiotherapy (SBRT) is an emerging treatment modality for pancreatic ductal adenocarcinoma (PDAC), which can effectively prime cytotoxic T cells by inducing immunogenic tumor cell death in preclinical models. SBRT effects on human PDAC have yet to be thoroughly investigated; therefore, this study aimed to characterize immunomodulation in the human PDAC tumor microenvironment following therapy. Tumor samples were obtained from patients with resectable PDAC. Radiotherapy was delivered a median of 7 days prior to surgical resection, and sections were analyzed by multiplex IHC (mIHC), RNA sequencing, and T-cell receptor sequencing (TCR-seq). Analysis of SBRT-treated tumor tissue indicated reduced tumor cell density and increased immunogenic cell death relative to untreated controls. Radiotherapy promoted collagen deposition; however, vasculature was unaffected and spatial analyses lacked evidence of T-cell sequestration. Conversely, SBRT resulted in fewer tertiary lymphoid structures and failed to lessen or reprogram abundant immune suppressor populations. Higher percentages of PD-1+ T cells were observed following SBRT, and a subset of tumors displayed more clonal T-cell repertoires. These findings suggest that SBRT augmentation of antitumor immunogenicity may be dampened by an overabundance of refractory immunosuppressive populations, and support the continued development of SBRT/immunotherapy combination for human PDAC.

Abstract IA02: Tumor microenvironment metabolism in T cell differentiation and dysfunction

Abstract Cancer thwarts immune destruction using several adaptations to suppress cancer immunity: upregulation of ligands for coinhibitory receptors like PD-1, recruitment of immunosuppressive populations like Treg cells, and withstanding cytolysis to generate a source of chronic antigen stimulation to drive T cell exhaustion. However, behind the veil of these molecular and cellular interactions is a unique metabolic landscape, driven by the energetic deregulation of the cancer cells themselves, that is toxic to infiltrating immune cells. Indeed, we and others have shown T cells infiltrating tumors succumb to severe metabolic insufficiency, characterized by a progressive loss of functional mitochondria. These metabolic states occur concomitant with the progression to terminal exhaustion. We hypothesized metabolic stress not only characterizes T cell exhaustion but plays a dominant role in driving the fate. We found that while T cells could withstand mitochondrial stressors like hypoxia in isolation, if T cells were experiencing persistent stimulation, they became extremely sensitive to oxygen tension. Indeed, continuous stimulation through the TCR under hypoxic conditions rapidly generated exhausted-like T cells in vitro. We used our in vitro system to dissect molecular mechanisms for this fate change, revealing mitochondrial stress produces intolerable levels of reactive oxygen species, which played a major role in driving exhaustion. Antioxidant chemicals and proteins as well as hypoxia mitigation could prevent T cell exhaustion in vivo and synergize with checkpoint blockade immunotherapy. Exhausted T cells are a major population within solid tumors, but what is their true function in cancer? Our metabolic and transcriptomic studies found the most terminally exhausted T cells bear striking similarity to tumor-infiltrating regulatory T cells. We thus asked whether exhausted CD8+ T cells harbored regulatory function using miniaturized suppression assays. Strikingly, when sorted from the same tumor, exhausted T cells carried similar suppressive capacity as CD4+ Treg cells. Unlike Foxp3+ Treg cells, which suppress through a myriad of mechanisms, exhausted CD8+ T cells suppress solely using the ectonucleotidase CD39, previously shown to define the most terminally exhausted cells. We found hypoxia exposure and HIF1α expression drive CD39 expression, which generates an adenosinergic local environment in which CD73+ immune cells create immunosuppressive adenosine. Deletion of CD39 within the CD8 lineage resulted in slowed tumor growth and heightened sensitivity to immunotherapy, highlighting a significant contribution by exhausted T cells to the tolerogenic environment in cancer. Our data highlight an unappreciated function of this terminally exhausted subset and suggest exhausted T cells are not merely dysfunctional, but in cancer become actively anti-functional, working against the immune response. By targeting adenosine, CD39, or the hypoxic nature of the tumor microenvironment, these deleterious functions may be limited to enable immunotherapy for cancer. Citation Format: Greg M. Delgoffe. Tumor microenvironment metabolism in T cell differentiation and dysfunction [abstract]. In: Abstracts: AACR Virtual Special Conference: Tumor Immunology and Immunotherapy; 2021 Oct 5-6. Philadelphia (PA): AACR; Cancer Immunol Res 2022;10(1 Suppl):Abstract nr IA02.

Coming to the Rescue: Regulatory T Cells for Promoting Recovery After Ischemic Stroke.

Immune cell infiltration to the injured brain is a key component of the neuroinflammatory response after ischemic stroke. In contrast to the large amount of proinflammatory immune cells, regulatory T cells, are an important subgroup of T cells that are involved in maintaining immune homeostasis and suppress an overshooting immune reaction after stroke. Numerous previous reports have consistently demonstrated the beneficial role of this immunosuppressive immune cell population during the acute phase after experimental stroke by limiting inflammatory lesion progression. Two recent studies expanded now this concept and demonstrate that regulatory T cells-mediated effects also promote chronic recovery after stroke by promoting a proregenerative tissue environment. These recent findings suggest that boosting regulatory T cells could be beneficial beyond modulating the immediate neuroinflammatory response and improve chronic functional recovery.

Hypoxia as a driver of resistance to immunotherapy.

Hypoxia, a hallmark of solid tumors, determines the selection of invasive and aggressive malignant clones displaying resistance to radiotherapy, conventional chemotherapy or targeted therapy. The recent introduction of immunotherapy, based on immune checkpoint inhibitors (ICPIs) and chimeric antigen receptor (CAR) T-cells, has markedly transformed the prognosis in some tumors but also revealed the existence of intrinsic or acquired drug resistance. In the current review we highlight hypoxia as a culprit of immunotherapy failure. Indeed, multiple metabolic cross talks between tumor and stromal cells determine the prevalence of immunosuppressive populations within the hypoxic tumor microenvironment and confer upon tumor cells resistance to ICPIs and CAR T-cells. Notably, hypoxia-triggered angiogenesis causes immunosuppression, adding another piece to the puzzle of hypoxia-induced immunoresistance. If these factors concurrently contribute to the resistance to immunotherapy, they also unveil an unexpected Achille's heel of hypoxic tumors, providing the basis for innovative combination therapies that may rescue the efficacy of ICPIs and CAR T-cells. Although these treatments reveal both a bright side and a dark side in terms of efficacy and safety in clinical trials, they represent the future solution to enhance the efficacy of immunotherapy against hypoxic and therapy-resistant solid tumors.

The Advantages and Challenges of Anticancer Dendritic Cell Vaccines and NK Cells in Adoptive Cell Immunotherapy.

In the last decade, an impressive advance was achieved in adoptive cell therapy (ACT), which has improved therapeutic potential and significant value in promising cancer treatment for patients. The ACT is based on the cell transfer of dendritic cells (DCs) and/or immune effector cells. DCs are often used as vaccine carriers or antigen-presenting cells (APCs) to prime naive T cells ex vivo or in vivo. Cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells are used as major tool effector cells for ACT. Despite the fact that NK cell immunotherapy is highly effective and promising against many cancer types, there are still some limitations, including insignificant infiltration, adverse conditions of the microenvironment, the immunosuppressive cellular populations, and the low cytotoxic activity in solid tumors. To overcome these difficulties, novel methods of NK cell isolation, expansion, and stimulation of cytotoxic activity should be designed. In this review, we discuss the basic characteristics of DC vaccines and NK cells as potential adoptive cell preparations in cancer therapy.

Inhibition of DCLK1 with DCLK1-IN-1 Suppresses Renal Cell Carcinoma Invasion and Stemness and Promotes Cytotoxic T-Cell-Mediated Anti-Tumor Immunity.

Simple SummaryIn this study, we found that the novel small molecule kinase inhibitor DCLK1-IN-1 not only inhibited DCLK1 phosphorylation, stemness, and EMT-related properties of RCC cells but also revealed its potential as an immunotherapy agent and potential combination therapy with anti-PD1 against RCC in immune co-culture experiments.The approval of immune checkpoint inhibitors has expanded treatment options for renal cell carcinoma (RCC), but new therapies that target RCC stemness and promote anti-tumor immunity are needed. Previous findings demonstrate that doublecortin-like kinase 1 (DCLK1) regulates stemness and is associated with RCC disease progression. Herein, we demonstrate that small-molecule kinase inhibitor DCLK1-IN-1 strongly inhibits DCLK1 phosphorylation and downregulates pluripotency factors and cancer stem cell (CSC) or epithelial-mesenchymal transition (EMT)-associated markers including c-MET, c-MYC, and N-Cadherin in RCC cell lines. Functionally, DCLK1-IN-1 treatment resulted in significantly reduced colony formation, migration, and invasion. Additionally, assays using floating or Matrigel spheroid protocols demonstrated potent inhibition of stemness. An analysis of clinical populations showed that DCLK1 predicts RCC survival and that its expression is correlated with reduced CD8+ cytotoxic T-cell infiltration and increases in M2 immunosuppressive macrophage populations. The treatment of RCC cells with DCLK1-IN-1 significantly reduced the expression of immune checkpoint ligand PD-L1, and co-culture assays using peripheral blood monocytes (PBMCs) or T-cell expanded PBMCs demonstrated a significant increase in immune-mediated cytotoxicity alone or in combination with anti-PD1 therapy. Together, these findings demonstrate broad susceptibility to DCLK1 kinase inhibition in RCC using DCLK1-IN-1 and provide the first direct evidence for DCLK1-IN-1 as an immuno-oncology agent.

Abstract PO-114: STAT3 in cancer-associated fibroblasts promotes an immunosuppressive tumor microenvironment

Abstract One of the defining characteristics of pancreatic ductal adenocarcinoma (PDAC) is the formation of a dense stroma comprised of cancer associated fibroblasts (CAFs) and immune cell populations. This stroma is immunosuppressive and can act as a physical barrier against common therapeutic treatments. Attempts to therapeutically target the PDAC stroma have yielded contradictory results, suggesting both tumor promoting and tumor limiting roles for CAFs. These studies emphasize the need to understand important transsignaling pathways between CAFs, tumor cells, and the immune microenvironment. IL-6 is a pleiotropic cytokine involved in several physiological functions and its increased expression is strongly associated with poor survival rates in PDAC patients. STAT3 is a major downstream target of IL-6, and its aberrant activation has been implicated in PDAC tumor progression and immune evasion. IL-6 expression and the IL-6/STAT3 signaling axis in PDAC has been characterized in epithelial tumor cells, however its stromal-specific function on PDAC has yet to be elucidated. We hypothesized that the STAT3 signaling axis in pancreatic CAFs contributes to the immunosuppressive and fibrotic phenotype seen with disease progression. Employing CreLoxP technology, the fibroblast specific protein-1 (Fsp-Cre) transgene was used to conditionally delete STAT3 in fibroblasts in the PdxFlp; KrasG12D; p53frt/frt (KPF) PDAC mouse model developed by our lab. Deletion of STAT3 in fibroblasts significantly increased the survival in a cohort of KPF mice compared to those with intact STAT3. In preliminary investigations, we found an increase in CD8+ T cell infiltration but a decrease in regulatory T cells in the STAT3-deleted cohort. We also observed a decrease in immunosuppressive M2 macrophage populations and an increase in M1 macrophages in the STAT3-deleted cohort. These preliminary results demonstrate a previously unexplored role of IL-6/STAT3 signaling in fibroblasts during PDAC progression. Citation Format: Julia E. Lefler, Michael Ostrowski, Catherine MarElia-Bennett. STAT3 in cancer-associated fibroblasts promotes an immunosuppressive tumor microenvironment [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2021 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2021;81(22 Suppl):Abstract nr PO-114.

EXTH-13. LOCAL DELIVERY OF AN IL-15 SUPERAGONIST USING A REPLICATING RETROVIRUS SIGNIFICANTLY IMPROVES SURVIVAL AND LYMPHOCYTE INFILTRATION IN POORLY IMMUNOGENIC MURINE GLIOBLASTOMA MODELS

Abstract Glioblastoma (GBM) leads to severe systemic and local immunosuppression, and immunotherapies have had limited clinical success. Here, we evaluated the treatment efficacy of RLI, a superagonist of T-cell activator IL-15, delivered to tumor cells using a tumor-selective retroviral replicating vector (RRV) in the syngeneic murine SB28 and Tu2449 GBM models, which are both engineered to be poorly immunogenic with low-mutational burden and known resistance to immunotherapy, and hence more accurate biomimetic models of human GBM. RRV-RLI replicated and spread effectively in cultured murine GBM cells with robust production of functional RLI (165.4 ± 5.3 ng/mL). Stereotactic injection of RRV-RLI into pre-established intracerebral SB28 tumors significantly reduced tumor growth on bioluminescent imaging, and increased median survival compared to control mice (55 vs. 19 days, p=0.002), leading to long-term survival in 12% of treated mice. In the Tu2449 model, imaging results showed complete eradication of intracerebral tumors after RRV-RLI treatment, with long-term survival (median not reached) in > 85% of treated mice, compared to a median survival of 12.5 days in control mice (p=0.001). RRV-RLI treated tumors showed significantly increased CD8 T-cell infiltration, without altering immunosuppressive cell populations. Similarly, broad anti-tumor inflammatory changes, including increased expression of genes involved in T-cell activation and killing, were observed in the NanoString nCounter platform using a 770-gene panel representing various immune cell types. Notably, RLI was not detected in the blood of treated mice, and tumor-localized RRV-RLI gene delivery showed no adverse systemic immune effects in either model. In summary, RRV-mediated RLI immunotherapy results in immunostimulatory and pro-inflammatory changes to the tumor microenvironment and achieves a significant survival benefit in two poorly immunogenic syngeneic murine models of GBM. This tumor-localized immunomodulatory gene therapy has the potential to safely reverse the T-cell depleted immunophenotype of GBM.

Devimistat in Combination with Bendamustine Potently Inhibits Tumor Growth and Promotes CD8+ T Cell Tumor Infiltration in a Syngeneic Mouse Model of CD30+ T-Cell Lymphoma

Introduction: Chimeric antigen receptor-based T cell therapy (CAR-T) has shown great promise in B cell malignancies. CD30-targeted therapies like brentuximab vedotin have activity in T cell malignancies expressing CD30. There is interest in developing CD30-targeted CAR-T therapies for the treatment of relapsed T cell malignancies. As the process of generating autologous T cells takes several weeks, there is a need for active bridging therapeutic regimens that can reduce tumor burden to facilitate subsequent treatment with CAR-T cell therapy. We propose the use of devimistat, a TCA cycle inhibitor, in combination with bendamustine as bridging therapy for the treatment of CD30+ T cell lymphoma (TCL) prior to the use of CAR-T cells. We hypothesize that TCA cycle inhibition will increase the sensitivity of TCL cells to bendamustine, negatively impact the function of immunosuppressive cell populations that depend on mitochondrial metabolism, and create a conducive environment for enhanced CAR-T cell efficacy. To test our hypothesis, we developed a model of CD30+ TCL, evaluated the efficacy of the combination, and characterized its effects by transcriptome profiling of immune cell populations in tumors.Methods: Mouse TCL cell line EL4-LUC2 was engineered to express human CD30 by retroviral transduction. Single and combination in vitro efficacy was evaluated by viability assays. Combination indices (CI) were calculated using Compusyn. CD30+ EL4-LUC2 cells (EL4.CD30) were inoculated subcutaneously into the flanks of C57Bl6 mice, tumor volumes were calculated, and CD30 expression in tumors was evaluated by immunohistochemical staining and flow cytometry. Transcriptome profiling was performed on a representative subset of tumors by single cell RNA sequencing (scRNA-seq).Results: EL4.CD30 cells displayed comparable CD30 expression to the human TCL cell line Karpas 299. EC 50 values for Devimistat and bendamustine in EL4.CD30 cells were 64.9 mM (95% CI: 62.4-67.4) and 101.2 mM (96.2-106.4), respectively; in Karpas 299, EC 50 values for Devimistat and bendamustine were substantially higher: 117.2 mM (114.7-119.6) and 188.2 mM (180.0 - 196.8), respectively. When dosed in combination, devimistat and bendamustine (D/B) showed synergy (CI = 0.5-0.8) at effect levels > 0.9 in 8 of the 16 dose combinations tested. D/B synergy at an effect level > 0.9 was less overt in Karpas 299 with only 2 of 16 tested combination levels displaying CI values of 0.5-0.9. Efficacy studies with EL4.CD30 tumor-bearing mice revealed potent D/B anti-tumor activity relative to vehicle and single-agent treatment groups. Six complete regressions and 4 partial regressions were observed in the D/B group (Fig. 1A). Moreover, tumor growth rates and median survival values were significantly different (p<0.0001) in D/B treated mice (Fig 1B-C). Transcriptome profiling of harvested tumors by scRNA-seq revealed substantial infiltration of cytotoxic T lymphocytes (CTL) exclusively in a combination-treated mouse that responded to therapy (Fig. 2A-C). Direct interrogation of Treg-associated transcripts revealed very low Treg numbers and no clear association to any treatment.Conclusions: Our data demonstrate that D/B has potent pharmacological activity in vitro and in vivo and promotes CTL influx into tumors. This suggests that D/B creates an immunopermissive environment fit for CAR-T cell activity. Subsequent studies will investigate the efficacy of CD30.CAR-T cells following D/B treatment. A Phase II pilot study evaluating the feasibility, safety, and tolerability of D/B in patients with relapsed/refractory T-cell Non-Hodgkin Lymphoma is currently ongoing (NCT04217317). [Display omitted] DisclosuresPardee: Rafael Pharmaceuticals: Consultancy, Research Funding; Karyopharm Pharmaceuticals: Research Funding; AbbVie: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Genetech: Membership on an entity's Board of Directors or advisory committees; BMS: Speakers Bureau; Pharmacyclics: Speakers Bureau.

Tasquinimod Targets Immunosuppressive Myeloid Cells, Increases Osteogenesis and Has Direct Anti-Myeloma Effects By Inhibiting c-Myc Expression in Vitro and In Vivo

IntroductionImmunotherapy has revolutionized cancer treatment and significantly affected the management of Multiple Myeloma (MM) patients. Unfortunately, these immunotherapeutic approaches are hampered by the presence of a suppressive bone marrow microenvironment including myeloid derived suppressor cells and tumor associated macrophages. Tasquinimod (TasQ), an immunomodulatory compound, is currently in phase Ib/IIa for relapsed/refractory MM patients (NCT04405167). TasQ blocks the interaction between S100A9 and its receptors, which is associated with reduced MDSC accumulation. In this study, we investigated TasQ-mediated direct and indirect effects on MM cell growth, bone disease and immunomodulation in vitro and in vivo using human myeloma cell lines and the immunocompetent 5TMM models.Material and methodsIn vitro, murine (5T33vt, 5TGM1) and human (JJN3, LP1, OPM2, and RPMI8226) MM cell lines were cultured at different concentrations of TasQ. Cell proliferation was assessed by BrdU staining using flow cytometry. C-Myc and pSTAT3 expression were analyzed by western blot. In vitro T cell proliferation experiments were performed using MACS-sorted CD11b + cells and CFSE-labeled T cells from naïve mice. Cells were cocultured for 72h in the presence of MM conditioned medium (5T33MMvt CM) with CD3/CD28 microbeads, followed by flow cytometry to assess T cell proliferation. For in vivo experiments, we used the 5T33 (aggressive) and 5TGM1 (moderate) MM models. On the second day after tumor cell injection, the mice were randomly assigned to the treatment group and the control group. The treatment group received 30 mg/kg of TasQ in drinking water for 35 days (5TGM1) and 21 days (5T33). Anti-tumor and immunomodulating effects were analyzed by flow cytometry (e.g. tumor cells, myeloid subsets, CD4/CD8 + T cells), qRT-PCR, western blot and serum ELISA (interferon-gamma). Effects on osteogenesis in the 5TGM1 model was investigated by Micro-CT. Statistical differences were assessed by Mann-Whitney U test and One-way ANOVA with p<0.05 considered as statistically significant.ResultsTasQ-treatment of murine and human myeloma cell lines (HMCL), at concentrations of 10-25uM, significantly reduced MM cell proliferation after 24h and 48h in vitro (n=3, p<0.05). In addition, a downregulation in c-Myc expression could be observed 6h after treatment of human MM cell lines (n=3). In vitro, TasQ significantly increased T cell proliferation in co-culture experiments with T cells and myeloid cells in 5T33MMvt CM (n=3, p<0.05).Using the immunocompetent 5TGM1 and 5T33MM model, we investigated direct and indirect anti-tumor effects of TasQ. We found that TasQ significantly reduced tumor load in the bone marrow of 5TGM1 (n=10/group, p=0.0012) and 5T33MM mice (n=10/group, p=0.0106) compared to vehicle-treated control mice. Using flow cytometry, we could not observe a difference in the percentage of CD4 + and CD8 + T cells. However, a significant upregulation in serum interferon-gamma could be observed in the 5T33MM mice (p=0.0284). While the percentage of CD11b + cells in the TasQ-treated group was significantly increased (p<0.05), the percentage of monocytic myeloid cells (CD11b +Ly6G -) was significantly reduced in both models (p<0.05). qRT-PCR results showed that the expression of IL-10 was downregulated in purified CD11b + myeloid cells (p<0.05). Consistent with the in vitro data, we observed a decrease in the protein expression of c-Myc in purified MM cells obtained from TasQ-treated mice compared to control mice. Micro-CT analysis of femurs demonstrated a significant increase in the percentage BV/TV (ratio of bone material volume over tissue volume) and trabeculae number (p<0.0001) in TasQ-treated 5TGM1 mice compared to untreated mice.ConclusionTasQ has pleiotropic effects on the MM cells and its surrounding bone marrow microenvironment. It affects MM cell growth by decreasing c-Myc expression. In addition, TasQ targets the immunosuppressive monocytic myeloid cell population and increases serum interferon-gamma levels, indicative for immune cell activation. Moreover, it stimulates osteogenesis in vivo. Taken together, all these data provide evidence for the therapeutic benefits of TasQ as an anti-MM therapy for patients. DisclosuresTörngren: Active Biotech: Current Employment. Eriksson: Active Biotech: Current Employment. De Veirman: Active Biotech AB: Research Funding.

Overcoming Immunotherapy Resistance by Targeting the Tumor-Intrinsic NLRP3-HSP70 Signaling Axis.

Simple SummaryThe tumor-intrinsic NLRP3 inflammasome is a newly recognized player in the regulation of tumor-directed immune responses and promises to provide fresh insight into how tumors respond to immunotherapy. This brief review discusses recent data describing how activation of the tumor-intrinsic NLRP3 inflammasome contributes to immune evasion and what this pathway may provide to the field of immuno-oncology both in terms of pharmacologic targets capable of boosting responses to checkpoint inhibitor therapies and predictive biomarkers indicating which tumors may be most susceptible to these new therapeutic strategies.The tumor-intrinsic NOD-like receptor family, pyrin-domain-containing-3 (NLRP3) inflammasome, plays an important role in regulating immunosuppressive myeloid cell populations in the tumor microenvironment (TME). While prior studies have described the activation of this inflammasome in driving pro-tumorigenic mechanisms, emerging data is now revealing the tumor NLRP3 inflammasome and the downstream release of heat shock protein-70 (HSP70) to regulate anti-tumor immunity and contribute to the development of adaptive resistance to anti-PD-1 immunotherapy. Genetic alterations that influence the activity of the NLRP3 signaling axis are likely to impact T cell-mediated tumor cell killing and may indicate which tumors rely on this pathway for immune escape. These studies suggest that the NLRP3 inflammasome and its secreted product, HSP70, represent promising pharmacologic targets for manipulating innate immune cell populations in the TME while enhancing responses to anti-PD-1 immunotherapy. Additional studies are needed to better understand tumor-specific regulatory mechanisms of NLRP3 to enable the development of tumor-selective pharmacologic strategies capable of augmenting responses to checkpoint inhibitor immunotherapy while minimizing unwanted off-target effects. The execution of upcoming clinical trials investigating this strategy to overcome anti-PD-1 resistance promises to provide novel insight into the role of this pathway in immuno-oncology.

Neutrophil kinetics and function after major trauma: A systematic review.

BACKGROUNDImmune dysfunction following major traumatic injury is complex and strongly associated with significant morbidity and mortality through the development of multiple organ dysfunction syndrome (MODS), persistent inflammation, immunosuppression, and catabolism syndrome and sepsis. Neutrophils are thought to be a pivotal mediator in the development of immune dysfunction.AIMTo provide a review with a systematic approach of the recent literature describing neutrophil kinetics and functional changes after major trauma in humans and discuss hypotheses as to the mechanisms of the observed neutrophil dysfunction in this setting.METHODSMedline, Embase and PubMed were searched on January 15, 2021. Papers were screened by two reviewers and those included had their reference list hand searched for additional papers of interest. Inclusion criteria were adults > 18 years old, with an injury severity score > 12 requiring admission to an intensive care unit. Papers that analysed major trauma patients as a subgroup were included.RESULTSOf 107 papers screened, 48 were included in the review. Data were heterogeneous and most studies had a moderate to significant risk of bias owing to their observational nature and small sample sizes. Key findings included a persistently elevated neutrophil count, stereotyped alterations in cell-surface markers of activation, and the elaboration of heterogeneous and immunosuppressive populations of cells in the circulation. Some of these changes correlate with clinical outcomes such as MODS and secondary infection. Neutrophil phenotype remains a promising avenue for the development of predictive markers for immune dysfunction.CONCLUSIONUnderstanding of neutrophil phenotypes after traumatic injury is expanding. A greater emphasis on incorporating functional and clinically significant markers, greater uniformity in study design and assessment of extravasated neutrophils may facilitate risk stratification in patients affected by major trauma.

Suppressor CD4+ T cells expressing HLA-G are expanded in the peripheral blood from patients with acute decompensation of cirrhosis

ObjectiveIdentifying components of immuneparesis, a hallmark of chronic liver failure, is crucial for our understanding of complications in cirrhosis. Various suppressor CD4+ T cells have been established as potent inhibitors...

Quantitative Assessment and Prognostic Associations of the Immune Landscape in Ovarian Clear Cell Carcinoma.

Simple SummaryOvarian clear cell carcinoma (OCCC) is a rare subtype of epithelial ovarian cancer that has a poor response to chemotherapy. Here, we assessed the immunological features of a series of 33 OCCCs and identified an immune-related gene expression signature that correlated with a patient’s risk of recurrence. Additionally, using multiplex immunofluorescence, we assessed the spatial distribution and abundance of immune cell populations at the protein level and identified that tumour-associated macrophages (TAM) and regulatory T cells are excluded from the vicinity of tumour cells in low-risk patients, suggesting that high-risk patients have a more immunosuppressive microenvironment. We also found that TAMs and cytotoxic T cells were also excluded from the vicinity of tumour cells in ARID1A mutated OCCCs, suggesting that the exclusion of these immune effectors could determine the host response in ARID1A mutant OCCCs.Ovarian clear cell carcinoma (OCCC) is a rare subtype of epithelial ovarian cancer characterised by a high frequency of loss-of-function ARID1A mutations and a poor response to chemotherapy. Despite their generally low mutational burden, an intratumoural T cell response has been reported in a subset of OCCC, with ARID1A purported to be a biomarker for the response to the immune checkpoint blockade independent of micro-satellite instability (MSI). However, assessment of the different immune cell types and spatial distribution specifically within OCCC patients has not been described to date. Here, we characterised the immune landscape of OCCC by profiling a cohort of 33 microsatellite stable OCCCs at the genomic, gene expression and histological level using targeted sequencing, gene expression profiling using the NanoString targeted immune panel, and multiplex immunofluorescence to assess the spatial distribution and abundance of immune cell populations at the protein level. Analysis of these tumours and subsequent independent validation identified an immune-related gene expression signature associated with risk of recurrence of OCCC. Whilst histological quantification of tumour-infiltrating lymphocytes (TIL, Salgado scoring) showed no association with the risk of recurrence or ARID1A mutational status, the characterisation of TILs via multiplexed immunofluorescence identified spatial differences in immunosuppressive cell populations in OCCC. Tumour-associated macrophages (TAM) and regulatory T cells were excluded from the vicinity of tumour cells in low-risk patients, suggesting that high-risk patients have a more immunosuppressive microenvironment. We also found that TAMs and cytotoxic T cells were also excluded from the vicinity of tumour cells in ARID1A-mutated OCCCs compared to ARID1A wild-type tumours, suggesting that the exclusion of these immune effectors could determine the host response of ARID1A-mutant OCCCs to therapy. Overall, our study has provided new insights into the immune landscape and prognostic associations in OCCC and suggest that tailored immunotherapeutic approaches may be warranted for different subgroups of OCCC patients.

Tumor promoting capacity of polymorphonuclear myeloid-derived suppressor cells and their neutralization.

Myeloid-derived suppressor cells (MDSC) represent a highly immunosuppressive population that expands in tumor bearing hosts and inhibits both T and NK cell antitumor effector functions. Among MDSC subpopulations, the polymorphonuclear (PMN) one is gaining increasing interest since it is a predominant MDSC subset in most cancer entities and inherits unique properties to facilitate metastatic spread. In addition, further improvement in distinguishing PMN-MDSC from neutrophils has contributed to the design of novel therapeutic approaches. In this review, we summarize the current view on the origin of PMN-MDSC and their relation to classical neutrophils. Furthermore, we outline the metastasis promoting features of these cells and promising strategies of their targeting to improve the efficacy of cancer immunotherapy.

The Impact of NK Cell-Based Therapeutics for the Treatment of Lung Cancer for Biologics: Targets and Therapy.

Lung cancer has a dismal prognosis and novel targeted therapies leave still room for major improvements and better outcomes. Immunotherapy targeting immune checkpoint (IC) proteins, either as single agents or in combination with chemotherapy, is active but responders constitute only approximately 10–15% of non-small cell lung cancer (NSCLC) patients. Other effector immune cells such as CAR-T cells or NK cells may help to overcome the limitations of the IC inhibitor therapies for lung cancer. NK cells can kill tumor cells without previous priming and are present in the circulatory system and lymphoid organs. Tissue-residing NK cells differ from peripheral effector cells and, in case of the lung, comprise CD56bright CD16-negative populations showing high cytokine release but low cytotoxicity in contrast to the circulating CD56dim CD16-positive NK cells exhibiting high cytotoxic efficacy. This local attenuation of NK cell killing potency seems due to a specific stage of NK differentiation, immunosuppressive factors as well as presence of myeloid-derived suppressor cells (MDSCs) and regulatory T cells (TREGs). Improved NK cell-based immunotherapies involve IL-2-stimulated effector cells, NK cells expanded with the help of cytokines, permanent NK cell lines, induced pluripotent stem cell-derived NK cells and NK cells armed with chimeric antigen receptors. Compared to CAR T cell therapy, NK cells administration is devoid of graft-versus-host disease (GvHD) and cytokine-release syndrome. Although NK cells are clearly active against lung cancer cells, the low-cytotoxicity differentiation state in lung tumors, the presence of immunosuppressive leucocyte populations, limited infiltration and adverse conditions of the microenvironment need to be overcome. This goal may be achieved in the future using large numbers of activated and armed NK cells as provided by novel methods in NK cell isolation, expansion and stimulation of cytotoxic activity, including combinations with monoclonal antibodies in antibody-dependent cytotoxicity (ADCC). This review discusses the basic characteristics of NK cells and the potential of NK cell preparations in cancer therapy.

Abstract 118: Myeloid PKCd activation inhibits innate immune suppression and promotes antigen cross-presentation in triple negative breast cancer

Abstract Immunotherapy has revolutionized cancer treatment showing unprecedented long-term antitumor responses. However, most patients do not respond to immunotherapy. Increasing preclinical and clinical evidence suggests that high tumor mutational burden does not always correlate with immunotherapy efficacy. Interestingly, cancers that have been associated with viruses imply that the pre-existing presentation of viral antigens may confer an increased response rate to immunotherapy. This suggests an important role of antigen presentation, particularly by the myeloid compartment in predicting immunotherapy efficacy. On the other hand, immunotherapy non-responders have high levels of circulating myeloid-derived suppressor cells (MDSCs)- an immunosuppressive innate cell population that suppresses T cells. Triple negative breast cancer (TNBC) is the most aggressive subtype of breast cancers with poor responses to conventional therapies. TNBC patients harbor higher levels of MDSC populations compared to non-TNBC patients. Consequently, TNBC and other solid tumor patients who have high levels of circulating MDSCs respond poorly to immunotherapy. Hence, strategies that enhance antigen cross-presentation while altering MDSC's suppressive function represent a promising therapeutic approach to overcome immunotherapy resistance. Protein Kinase C (PKC) is a family of kinases composed of 11 isoforms that play a critical role in cell signaling. PKC delta (PKCδ) is the most abundant isoform in myeloid cells and plays an important role in dendritic cell (DC) function. To date, the role of PKCδ in myeloid cells in cancer is unknown. Using varied informatic approaches in patient databases, we found that BC patients with both high expression of PRKCD (PKCδ gene) and either high expression of CD8+ T cell or low expression of MDSC gene signatures in tumors had a significantly greater overall survival compared to other groups, suggesting support for activation of PKCδ. Novel preliminary data suggests that PKC agonism using FDA-approved PEP005 and prostratin reduced MDSC generation from BM progenitors specifically via activation of PKCδ isoform. PKC agonism induced MDSC differentiation to CD103+ DC-like cells both ex-vivo and in adoptive transfer experiment. Additionally, PEP005-treated MDSCs lost their suppressive capacity on CD8+ T cells in both in vitro and in vivo suppression assays. Importantly, PEP005 enhanced MDSC cross-priming capacity in both in vitro and in adoptive transfer experiments. Finally, treatment of TNBC-bearing C57BL/6J mice with PKC agonist PEP005 markedly reduced tumor burden by decreasing the frequencies of M-MDSCs in tumor, spleen, and bone marrow while increasing cDC1 frequencies in tumors. These findings propose PKCδ as a novel target in myeloid cells to tip the balance from immune suppression to effective antitumor immunity. Citation Format: Mehdi Chaib, Jeremiah Holt, Laura Sipe, Ajeeth Pingili, T.j. Hollingsworth, Deidre Daria, Neil Hayes, Liza Makowski. Myeloid PKCd activation inhibits innate immune suppression and promotes antigen cross-presentation in triple negative breast cancer [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 118.

Immunomodulatory Effects of HDACi in Combination with Checkpoint Blockade and Lenalidomide in the Immunosuppressive Multiple Myeloma Bone Marrow Microenvironment

Background: The interaction of myeloma (MM) cells with bone marrow (BM) accessory cells and/or the extracellular matrix induces genomic, epigenomic, and functional changes which promote tumor development, progression, cell adhesion mediated-drug resistance (CAM-DR), and immune suppression. To develop the most effective anti-MM treatment strategy and prevent tumor escape from immune recognition, both enhancing anti-MM effector immune response and overcoming MM-induced immune suppression is essential. Suppressive immune cells including myeloid derived suppressor cells (MDSC) and regulatory T cells (Treg) act as tumor promoters and suppressors of effector immune response, and therefore represent a significant barrier to current anti-tumor therapeutic strategies. We have recently shown that WT161, a novel small molecule inhibitor of HDAC6, alone or in combination with proteasome inhibitors induces MM cytotoxicity even in the bortezomib resistant MM cells. To target the MM BM immune suppressive microenvironment, we here have assessed the immunomodulatory effects of a novel triple-combination strategy using HDACi with checkpoint blockade and lenalidomide (Len) in MM BM ex vivo.Methods: Differential effects of Class I and Class II HDACi including pan-HDAC inhibitor Trichostatin A (TsA, 5-100nM), HDAC1 selective inhibitor Romidepsin (Rom, 1.5-15nM), HDAC3 inhibitor BG45 (10-40uM), HDAC6 inhibitor Tubastatin A HCl (TubA) and WT161 (0.5-10uM) alone or in combination with anti-PD1 (20ug/ml) and Len (0.5-1uM) were evaluated in MM-BM. HDACi direct effect on MM (MM1.S, H929, and RPMI8226) cell growth was measured by MTT. In the autologous coculture settings, BMMC from patients with newly diagnosed (ND)-MM and relapsed (R)-MM were cultured with HDACi alone or in combination with anti-PD1 and Len, with or without drug-wash-out for 1-5 days. Effector cell activation, proliferation, and cytotoxic activity was measured by flow cytometry analysis. Triple-combination effects on MDSC-mediated immune suppression was determined in cultures of autologous T cells with CD11b+CD14+HLA-DR- mMDSC, CD11b+CD14+HLA-DR-CD33+CD15+nMDSC, and CD11b+CD14+HLA-DR+APCs.Results: Healthy PBMC and MM cell line cultures demonstrated a dose-dependent toxicity and inhibition of proliferation in both CD4 and CD8 T cells induced by Rom; in contrast, TubA, WT161, and BG45 significantly induced T cell proliferation and cytotoxic activity even after wash out of HDACi. Additionally, HDAC6 inhibitors TubA and WT161 markedly increased expression of T cell exhaustion antigens TIM3 and LAG3, and these effects were partially reversed by drug washout. Moreover, triple-combination of HDACi with PD-1 blockade and Len significantly induced CD25+CD69+ activated T and NK cell mediated cytotoxicity of CD138+ MM cells. Interestingly, in the autologous co-cultures of MM-BMMC, TubA, BG45 and Rom induced CD25+CD69+ activated T cells, and the PD-1 blockade and Len further enhanced T cell activation. However, HDAC inhibitors, with the exception of HDAC3 inhibitor BG45, expanded immunosuppressive mMDSC populations within MM-BM myeloid compartment. Specifically, WT161 induced differentiation of myeloid cells by shifting CD14-HLA-DR-CD33+CD15+ nMDSCs to CD14+HLA-DR- mMDSCs and CD14+HLA-DR+APCs. Moreover, Rom significantly increased frequency of immunosuppressive IFNα producing plasmacytoid dendritic cells (pDCs), while decreasing Tregs, in MM-BM. Importantly, WT161 and TubA in combination with PD-1 blockade and Len reversed MDSC-mediated immunosuppressive activity, as measured by T cell proliferation. Finally, inhibition of HDAC6 induced MM cell cytotoxicity, and addition of PD-1 blockade and Len further enhanced TubA- and WT161 induced anti-MM immune responses.Conclusion: These data suggest that distinct classes of HDACi may induce differential immunomodulatory effects in the MM BM. Inhibition of HDAC6 induces changes in immune suppressor cells leading to enhanced anti-MM immune response, and addition of PD-1 blockade with Len further enhances HDAC6 inhibition-induced effector cell mediated anti-MM immune response. Ongoing analysis of triple combination of HDAC6 inhibition with checkpoint blockade and lenalidomide in the MM BM microenvironment will further define the role of HDAC6 in disease pathogenesis, and suggest novel immune-based epigenetic and targeted combination therapies for MM. DisclosuresHofmeister:Roche: Research Funding; Thrassos: Honoraria, Membership on an entity's Board of Directors or advisory committees; Adaptive Biotechnologies: Honoraria, Membership on an entity's Board of Directors or advisory committees; Karyopharm: Research Funding; Takeda: Research Funding; Bristol-Myers Squibb: Research Funding; Celgene: Research Funding; Janssen: Research Funding. Bradner:Acetylon: Other: Scientific Founder; Novartis: Employment. Hideshima:Acetylon: Consultancy; C4 Therapeutics: Equity Ownership. Anderson:Oncopep: Other: scientific founder; Millenium Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; C4 Therapeutics: Other: scientific founder; Gilead Sciences: Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Membership on an entity's Board of Directors or advisory committees; MedImmune: Membership on an entity's Board of Directors or advisory committees. Gorgun:Bristol Mayer Squibb: Research Funding; Janssen: Research Funding; Merck: Research Funding.

A Proteomic Atlas of Lineage and Cancer-Polarized Expression Modules in Myeloid Cells Modeling Immunosuppressive Tumor-Infiltrating Subsets.

Monocytic and granulocytic myeloid-derived suppressor cells together with tumor-infiltrating macrophages constitute the main tumor-infiltrating immunosuppressive myeloid populations. Due to the phenotypic resemblance to conventional myeloid cells, their identification and purification from within the tumors is technically difficult and makes their study a challenge. We differentiated myeloid cells modeling the three main tumor-infiltrating types together with uncommitted macrophages, using ex vivo differentiation methods resembling the tumor microenvironment. The phenotype and proteome of these cells was compared to identify linage-dependent relationships and cancer-specific interactome expression modules. The relationships between monocytic MDSCs and TAMs, monocytic MDSCs and granulocytic MDSCs, and hierarchical relationships of expression networks and transcription factors due to lineage and cancer polarization were mapped. Highly purified immunosuppressive myeloid cell populations that model tumor-infiltrating counterparts were systematically analyzed by quantitative proteomics. Full functional interactome maps have been generated to characterize at high resolution the relationships between the three main myeloid tumor-infiltrating cell types. Our data highlights the biological processes related to each cell type, and uncover novel shared and differential molecular targets. Moreover, the high numbers and fidelity of ex vivo-generated subsets to their natural tumor-shaped counterparts enable their use for validation of new treatments in high-throughput experiments.

Immunomodulatory Therapies for the Treatment of Graft-versus-host Disease.

Allogeneic hematopoietic stem cell transplantation (allo-HCT) is a potentially curative therapy for patients suffering from hematological malignancies, and its therapeutic success is based on the graft-versus-leukemia (GvL) effect. Severe acute and chronic graft-versus-host disease (GvHD) are life-threatening complications after allo-HCT. To date, most of the approved treatment strategies for GvHD rely on broadly immunosuppressive regimens, which limit the beneficial GvL effect by reducing the cytotoxicity of anti-leukemia donor T-cells. Therefore, novel therapeutic strategies that rely on immunomodulatory rather than only immunosuppressive effects could help to improve patient outcomes. Treatments should suppress severe GvHD while preserving anti-leukemia immunity. New treatment strategies include the blockade of T-cell activation via inhibition of dipeptidyl peptidase 4 and cluster of differentiation 28-mediated co-stimulation, reduction of proinflammatory interleukin (IL)-2, IL-6 and tumor necrosis factor-α signaling, as well as kinase inhibition. Janus kinase (JAK)1/2 inhibition acts directly on T-cells, but also renders antigen presenting cells more tolerogenic and blocks dendritic cell-mediated T-cell activation and proliferation. Extracorporeal photopheresis, hypomethylating agent application, and low-dose IL-2 are powerful approaches to render the immune response more tolerogenic by regulatory T-cell induction. The transfer of immunomodulatory and immunosuppressive cell populations, including mesenchymal stromal cells and regulatory T-cells, showed promising results in GvHD treatment. Novel experimental procedures are based on metabolic reprogramming of donor T-cells by reducing glycolysis, which is crucial for cytotoxic T-cell proliferation and activity.