IL-6 underlies microenvironment immunosuppression and resistance to therapy in glioblastoma.

The immunobiology of hepatocellular carcinoma in humans and mice: Basic concepts and therapeutic implications

TDI inhalation in guinea-pigs involves migration of dendritic cells

The immune tolerance of the tumor immune microenvironment (TIME) restricts the response to immune checkpoint inhibitors (ICIs). Targeted activation of dendritic cells (DCs) in the TIME seems to be a scheme for improving the therapeutic effect of ICIs treatment. The purpose of this study was to utilize nanotechnology to reprogram the immunosuppressive tumor immune microenvironment in situ, improving the response of ICIs to lung cancer. In this study, a folic acid (FA)-modified nanoassembly (NA) loaded with low-dose paclitaxel (PTX) (FA-PTX NA), self-assembled by distearoylphosphatidylethanolamine-methoxy polyethylene glycol 2000-folic acid (DSPE-mPEG2000-FA) and PTX, was designed to reprogram the DC function of the TIME to sensitize cells to cancer immunotherapy. The characteristics of FA-PTX NAs were studied, and the cytotoxicity, cellular uptake, and DC stimulation of FA-PTX NAs were evaluated in vitro using a Lewis lung carcinoma (LLC) cell line and bone marrow-derived cells (BMDCs). Following this, the effect of the reprogrammed TIME and on the sensitization to immunotherapy in vivo were examined in a C57BL/6 mouse LLC subcutaneous xenograft model. The prepared FA-PTX NAs exhibited a slightly negative surface charge, appropriate size and shape, good drug release profiles, and high drug encapsulation efficiency and blood compatibility. The FA-PTX NAs were effectively uptaken by bone BMDCs, increasing the activation and expression of the costimulatory factor of BMDCs in vitro. In the LLC xenograft model treated with intravenous injection of FA-PTX NAs, the numbers of CD4+ and CD8+ T cells in the TIME increased significantly, the killing activity of tumor-specific cytotoxic T lymphocytes (CTLs) was significantly enhanced, and at the same time, the concentration of transforming growth factor β (TGF-β) decreased significantly. Furthermore, the infiltrated CD8+ T cells in TIME were mainly distributed in the tumor parenchyma. The combination of FA-PTX NAs and ICIs effectively inhibited the growth of LLC xenograft tumor, demonstrating a greater effect than that of ICIs alone. Moreover, it was found that apoptosis induction, increase in CD4+ and CD8+ T-cell infiltration, and improvement in the distribution of CD8+ T cells were involved in the anticancer mechanism of this combination treatment. The NA loaded with low-dose PTX can reprogram the DC function in the TIME and exert a synergistic anticancer effect with ICIs in lung cancer treatment. Increased sensitization to ICI therapy as stimulated by PTX-enhanced NAs has potential applications in lung cancer immunotherapy.

Immune checkpoint inhibitors (ICI), including those targeting cytotoxic T-lymphocyte-associated-antigen-4 (CTLA-4) and programmed cell death receptor-1 (PD-1), have shown tremendous potential against solid tumor malignancies; however, response to ICI remains unpredictable with 60-90% of patients receiving minimal to no benefit. Lack of efficacy is commonly attributed to inadequate tumor-specific T cell generation and the immunosuppressive effects of the tumor immune microenvironment (TIME). Thus, we hypothesized that a combinatory treatment strategy aiming to enhance antigen presentation and revert the immunosuppressive TIME could improve response rates of ICI in established solid tumors. Using a syngeneic tumor model of HPV-associated head and neck cancer (mEER) established to 60-75 mm 2 in size, we found that CTLA-4 and/or PD-1 inhibition only minorly delayed tumor growth and flow cytometry profiling revealed that the TIME maintained a “cold” or immunosuppressed state similar to untreated tumors. When PD-1/CTLA-4 inhibition was combined with a weekly dose of tumor-directed radiation (10 Gy x 2), we observed upregulation of antigen presentation molecules in the draining lymph node but the combination remained incapable of generating long-term survival benefit. This lack of efficacy was attributed to the immunosuppressed and lymphodepleted TIME, a common consequence of radiation. Thus, to improve the TIME, we optimized an immune-stimulating drug combination previously developed in our lab combining cyclophosphamide (CTX) and a small molecule inducible nitric oxide synthase (iNOS) inhibitor L-n6-(1-iminoethyl)-lysine (L-NIL). When we combined CTX/L-NIL immunomodulation, PD-1/CTLA-4 checkpoint inhibition, and radiation (collectively called the “CPR” regimen), we observed complete rejection of approximately 70% of established tumors in a CD8 T-cell dependent manner and potent development of immunologic memory against tumor-associated antigens. Tumor immune profiling after treatment revealed a “cold to hot” transition of the TIME, including increased levels of myeloid and lymphoid subsets associated with anti-tumoral immune responses (i.e. CD8 T cells, dendritic cells, M1 macrophages) and downregulation of immunosuppressive cellular subsets (i.e. T regulatory cells, granulocytic myeloid derived suppressor cells). We observed strong lymphoproliferation effects in the tumor-draining lymph node which resulted in significant TIME improvements including a 15-fold increase in the CD8 to regulatory T cell ratio. Thus, we have demonstrated that the rational combination of TIME immunomodulation, localized radiation to enhance antigen presentation, and immune checkpoint inhibitors to prevent T-cell exhaustion can promote the immunologic rejection of established solid tumors. Citation Format: Jared M. Newton, Aurelie Hanoteau, Hsuan-Chen Liu, Angelina Gaspero, Robyn D. Gartrell, Thomas D. Hart, Damya Laoui, Falguni Parikh, Yvonne M. Saenger, Andrew G. Sikora. Radiation, immune checkpoint inhibition, and modulation of the tumor immune microenvironment promotes immunologic rejection of established HPV-associated tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4068.

Cancer cell intrinsic TIM-3 induces glioblastoma progression

Immune checkpoint inhibitors (ICI), including those targeting cytotoxic T-lymphocyte-associated-antigen-4 (CTLA-4) and programmed cell death receptor-1 (PD-1), have shown tremendous potential against solid tumor malignancies; however, response to ICI remains unpredictable with 60-90% of patients receiving minimal to no benefit. Lack of efficacy is commonly attributed to inadequate tumor-specific T cell generation and the immunosuppressive effects of the tumor immune microenvironment (TIME). Thus, we hypothesized that a combinatory treatment strategy aiming to enhance antigen presentation and revert the immunosuppressive TIME could improve response rates of ICI in established solid tumors. Using a syngeneic tumor model of HPV-associated head and neck cancer (mEER) established to 60-75 mm2 in size, we found that CTLA-4 and/or PD-1 inhibition only minorly delayed tumor growth and flow cytometry profiling revealed that the TIME maintained a “cold” or immunosuppressed state similar to untreated tumors. When PD-1/CTLA-4 inhibition was combined with a weekly dose of tumor-directed radiation (10 Gy x 2), we observed upregulation of antigen presentation molecules in the draining lymph node but the combination remained incapable of generating long-term survival benefit. This lack of efficacy was attributed to the immunosuppressed and lymphodepleted TIME, a common consequence of radiation. Thus, to improve the TIME, we optimized an immune-stimulating drug combination previously developed in our lab combining cyclophosphamide (CTX) and a small molecule inducible nitric oxide synthase (iNOS) inhibitor L-n6-(1-iminoethyl)-lysine (L-NIL). When we combined CTX/L-NIL immunomodulation, PD-1/CTLA-4 checkpoint inhibition, and radiation (collectively called the “CPR” regimen), we observed complete rejection of approximately 70% of established tumors in a CD8 T-cell dependent manner and potent development of immunologic memory against tumor-associated antigens. Tumor immune profiling after treatment revealed a “cold to hot” transition of the TIME, including increased levels of myeloid and lymphoid subsets associated with anti-tumoral immune responses (i.e. CD8 T cells, dendritic cells, M1 macrophages) and downregulation of immunosuppressive cellular subsets (i.e. T regulatory cells, granulocytic myeloid derived suppressor cells). We observed strong lymphoproliferation effects in the tumor-draining lymph node which resulted in significant TIME improvements including a 15-fold increase in the CD8 to regulatory T cell ratio. Thus, we have demonstrated that the rational combination of TIME immunomodulation, localized radiation to enhance antigen presentation, and immune checkpoint inhibitors to prevent T-cell exhaustion can promote the immunologic rejection of established solid tumors. Citation Format: Jared M. Newton, Aurelie Hanoteau, Hsuan-Chen Liu, Angelina Gaspero, Robyn D. Gartrell, Thomas D. Hart, Damya Laoui, Falguni Parikh, Yvonne M. Saenger, Andrew G. Sikora. Radiation, immune checkpoint inhibition, and modulation of the tumor immune microenvironment promotes immunologic rejection of established HPV-associated tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4068.

635 Background: IL-2 can be curative in a small subset of ccRCC patients (pts). Recent data suggest that ICIs may lead to long lasting responses, and ICI therapy following progression on IL-2 seems reasonable. However, data regarding the safety and efficacy of ICI following IL-2 is limited. Methods: ccRCC pts treated with ICI after first line were identified at UT Southwestern Medical Center from 2014 – 2018 and assessed by comparison to IL-2 naïve pts at the time of ICI initiation. Outcomes included objective response rates (ORR) determined by RECIST v1.1, time to next treatment (TNT) and overall survival (OS). Immune-related adverse events (irAEs) were graded per CTCAE v4.0. Comparisons were performed using Pearson Chi-square, independent t-tests, and Fischer’s exact tests as appropriate. The log-rank test was utilized for Kaplan-Meier survival analyses. Results: We identified 127 ccRCC pts treated with ICI after first line, 17.3% (22) who had received prior IL-2 and 83.7% (105) who were IL-2 naïve. Pts in the IL-2 group were younger (mean age, 56 vs. 63 years; p=0.003), and more heavily pretreated (median 2 prior lines vs. 1; p <0.0001). The distribution by IMDC criteria was similar with 86.4% (19) and 76.4% (81) in intermediate/ poor group in IL-2 vs. IL-2 naïve groups, respectively (p = 0.431). The ORR was 9.1% (2/22) vs. 21.2% (22/104) in the IL-2 vs. IL-2 naïve groups, respectively (p=0.244). The median TNT was 7.5 (95% CI: 1.8 to 13.2) vs. 9.2 (95% CI: 6.0 – 12.4; p= 0.667) months in the IL-2 vs. IL-2 naïve groups, respectively. The median OS was 26.4 (95% CI: 17.3 – 35.5) vs. 29.8 (23.1 to 36.4) months in the IL-2 vs. IL-2 naïve group, respectively. 18.2% (4) vs. 21.9% (23) pts developed a grade 3/4 irAE in the IL-2 vs. IL-2 naïve group, respectively (p=0.698). Conclusions: Our study suggests that ICI therapy following treatment with high-dose IL-2 remains efficacious and is not associated with an increased risk of irAEs.

Integrative genomics highlights opportunities for innovative therapies targeting the tumor microenvironment in gallbladder cancer

Improved Quality of Life with Interleukin-6 (IL-6) Blockade with Siltuximab Peri-Autologous Hematopoietic Stem Cell Transplantation (AHCT) in Older Patients with Multiple Myeloma (MM)

Dendritic cell (DC) migration from peripheral organs to lymph nodes plays a key role in initiating immune responses, whether migratory DCs bring antigen in tow to lymph nodes or position themselves to capture antigen that drains into the lymph node. CCR7 prominently controls DC migration into afferent lymphatic vessels and the positioning of DCs within the lymph node. Expression of CCR7 is not sufficient for function, as its function is positively regulated by a variety of other extracellular triggers. At least one of these triggers, synthesis and secretion of PGE(2), is brought on by the activation of p38 MAP kinase. The MAP kinase pathway has been well studied in DCs and exhibits a complex regulatory role in which the activation of different MAP kinase members leads to biologically distinct outcomes that are dependent upon stage of differentiation at the time of activation as well as the duration of signaling. Almost all of our knowledge of how DCs mature and ultimately mobilize to lymph nodes comes from studies in which DC migration is probed in the context of immune activation and priming. A reasonable body of evidence has gathered to suggest that many molecular events important for DC migration in this context do not affect accumulation of DCs in lymph nodes in the steady state, but mediators that interface with the signaling adaptor DAP-12 may play key roles in the steady state. It may thus become possible to devise approaches to modulate DC mobilization in the context of inflammation without affecting the traffic of DCs during more quiescent conditions. Considering the finely tuned regulation of DC maturation, migration, and cytokine production, with the realization that these phenotypes can be mutually exclusive, manipulation of DC migration in the clinic will be a challenging, albeit feasible, task.

Selective 5-Lipoxygenase Expression in Langerhans Cells and Impaired Dendritic Cell Migration in 5-LO-Deficient Mice Reveal Leukotriene Action in Skin

The FDA has recently approved a high tumor mutational burden (TMB-High, defined by ≥10 mutations/Mb) as a biomarker for the treatment of advanced solid tumors with pembrolizumab, an immune checkpoint inhibitor (ICI) that targets PD1. However, recent studies have shown that this TMB-high biomarker is only able to stratify ICI responders in a subset of cancer types, where the mechanisms underlying this observation have remained unknown. We hypothesized that the tumor immune microenvironment (TME) may determine the ability of high-TMB to stratify responders of ICI (termed TMB power) in each cancer type. To systematically study this hypothesis, we first inferred the levels of 31 immune-related factors characteristic of the TME of different cancer types in the TCGA. We next integrated this information with a cohort of 2,277 ICI-treated patients with TMB and response measures, to identify the key immune factors that can determine TMB power across 14 different cancer types. This cohort was created by collating the largest publicly available cohort comprising 1959 patients together with a new cohort of 318 patients, where TMB has been quantified using the MSK-IMPACT panel. We find that high levels of M1 macrophages and low levels of resting dendritic cells in the TME characterize cancer types with high TMB power. These findings are aligned with prior reports that M1 macrophages could be anti-tumorigenic by fostering an inflammation response and activating CD8 T cells against tumor, and that resting dendritic cells may induce tolerance to tumor antigens via inducing T cell death or their anergic state. A model based on these two immune factors strongly predicts TMB power across cancer types (Spearman Rho=0.76, P<3.6E-04). Using this model, we provide predictions of the TMB power in nine additional cancer types, including rare cancers, for which TMB and ICI response data are not yet publicly available on a large scale. These predictions can be used to prioritize the clinical trials testing the usage of TMB-high biomarker in new cancer types. In this line, our analysis indicates that TMB-High may be strongly predictive of ICI response in cervical squamous cell carcinoma, suggesting that such a study should be prioritized. Citation Format: Neelam Sinha, Sanju Sinha, Christina Valero, Alejandro A. Schaffer, Kenneth Aldape, Kevin Litchfield, Timothy A. Chan, Luc G. T. Morris, Eytan Ruppin. Immune determinants of the association between tumor mutational burden and immunotherapy response across cancer types [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 P054.

Dendritic cell (DC) migration is required for efficient presentation of antigen to T cells and the initiation of an adaptive immune response. In spite of its importance, many aspects of DC migration have not been characterized. DCs encounter a variety of environments with different stiffness and geometry, but the effect of these parameters on DC migration has not yet been determined. We addressed this question by comparing DC motility on standard migration surfaces (polydimethylsiloxane (PDMS)-coated coverslips) and micropost array detectors (mPADs). These two surfaces differ in both stiffness and geometry. We found that DC migration was affected by substrate type, with significant increases in speed and significant decreases in persistence time on mPADs made of PDMS as compared to spin-coated PDMS coverslips. To determine whether the geometry or compliance of the post arrays was responsible for these changes in DC migration, we quantified DC motility on mPADs of identical geometry but different stiffness. Migration was indistinguishable on these mPADs, suggesting that DCs are responsive to geometry of ligand presentation and not stiffness. Further, by micropatterning ligands on flat PDMS surfaces in similar geometries to the mPAD arrays, we determined that DCs respond to the geometry of printed ligand. Finally, we used a variety of small molecule inhibitors to identify pathways involved in geometry sensing. We saw a significant role for myosin contractility and α5β1 integrin engagement. We also noted significant reorganization of the actin cytoskeleton into dynamic actin rings when DCs were motile on posts. From these experiments, we conclude that DCs are insensitive to substrate compliance in the range tested but respond to changes in geometry via a mechanism that involves integrin function, myosin contractility, and remodeling of the actin cytoskeleton. As a possible explanation, we postulate a consistent role for filopodial extension and contraction as the driver of DC motility.

SAMP1/YitFc Mice Develop Ileitis via Loss of CCL21 and Defects in Dendritic Cell Migration

151 Background: Claudin-1 (CLDN1) is a tight junction protein overexpressed in several cancers, where it is involved in the tumor microenvironment remodeling and stiffening of the tumor-stroma interface. This potentially prevents penetration of tumor infiltrating immune cells, in turn hindering the efficacy of immune checkpoint inhibitors (ICI). Given recent evidence supporting ICI efficacy in nasopharyngeal carcinoma (NPC), we investigated whether CLDN1 is overexpressed in NPC and its association with the tumor immune microenvironment (TME). Methods: 131 NPC patients treated at a single institution were prospectively recruited. TME profiling was performed using deconvolutional methods (CIBERSORT, quanTIseq) on tumor transcriptomes to estimate proportions of immune cell subsets, and expression of immune-related genes and pathways. Transcriptome profiling was performed on tumors with >70% cellularity and normal controls using Illumina Truseq (CA). We stratified tumors by their median CLDN1 expression and compared the immune profile of CLDN1high versus CLDN1low tumors. Cox regression model was used to analyze disease-free survival (DFS) as the primary clinical endpoint. Results: We observed CLDN1 overexpression in NPC versus normal tissues ( P=0.0046) that was independent of known prognostic clinical parameters e.g., TNM-stage and EBV DNA levels. Consistent with our hypothesis, CLDN1 expression was negatively correlated with total immune cell fraction in the tumor ( P<0.001). We observed reduced abundance of most immune subsets in CLDN1high tumors, notably CD8 T cells ( P<0.001), activated CD4 memory T cells ( P<0.001), and M1 and M2 macrophages ( P=0.048 and P<0.001). There was an overall downregulation of both stimulatory and suppressive GO immune-related gene-sets and genes in CLDN1high versus CLDN1low tumors, thereby corroborating our preceding observation of reduced immune infiltration in the TME in CLDN1 overexpressing NPC. Our findings were validated in a published dataset of 113 NPC tumors (Zhang et al., 2017). Of clinical relevance, reduced immune infiltration in the TME was prognostic for DFS; HR of CLDN1high, CD8 T celllow (stratified by median) NPC was 2.1 (95% CI:0.9-5.0) when compared with CLDN1low, CD8 T cellhigh NPC. Conclusions: Here, we report the overexpression of CLDN1 in NPC that is ubiquitous across disease stages and associated with an immune-low TME. This influence on the TME is associated with an inferior outcome of NPC patients. Given the role of the tumor TME in ICI response, our findings provide a rationale for future trials investigating combination of CLDN1 targeting with ICI to reverse this immune exclusion effect, potentially leading to improved outcomes.