Biology subjects

Cillo, A. R.

Publications and source records attributed to Cillo, A. R..

8 recordsLinked to original sources

bioRxiv2025

SARC028 samples reveal an interplay between TGF-beta, interferon signaling and low HLA class I expression as contributors to Ewing sarcoma checkpoint blockade resistance

PurposeEwing sarcoma, in contrast to some adult sarcoma subtypes, generally does not respond to single agent immunotherapy targeting PD1. The features of Ewing sarcoma that preclude the effectiveness of immunotherapy remain largely unknown. To address this question, we utilized biopsies from patients with Ewing sarcoma obtained pre- and post-pembrolizumab (anti-PD1) therapy from the phase 2 clinical trial SARC028 to interrogate the Ewing tumor microenvironment and features associated with resistance to checkpoint inhibition. Experimental DesignWe utilize multiplexed immunofluorescence, spatial proteomics and spatial transcriptomics to analyze paired pre- and 8 weeks post-treatment biopsy specimens from patients with Ewing sarcoma enrolled on SARC028. ResultsPembrolizumab therapy did not alter the quantity of immune cell infiltration in Ewing tumor biopsies. Analysis of tumor-associated protein markers revealed increased immunoregulatory markers after pembrolizumab. Spatial transcriptomics identified ten cellular neighborhoods (CN) across patients consisting of specific cell subsets. CN10 was consistently observed across patients with poor response. This cellular neighborhood was enriched for a tumor subpopulation with high TGF-{beta} response, low interferon (IFN) response, and low HLA class I expression. IFN response, HLA class I expression, and overall immune infiltration were correlated. ConclusionsAnalyses of the tumor microenvironment from Ewing sarcoma biopsies reveals an immunosuppressive triad, the disruption of which should be pursued to improve antitumor immunity. This work highlights the unique insight that can be gained from the analysis of paired patient Ewing sarcoma tumor biopsy samples from clinical trials. Statement of translational relevanceSingle agent checkpoint inhibitors have demonstrated limited clinical benefit in bone sarcomas, including Ewing sarcoma (EwS). While in vitro and limited patient tumor analyses have suggested mechanisms of immunotherapy resistance in EwS, tumor biopsies following checkpoint inhibitor administration have not been examined. SARC028 was a phase II study investigating blockade of programmed cell death-1 (PD-1) in patients with relapsed/refractory sarcomas, including EwS. Tumor biopsies were collected prior to and 8 weeks after anti-PD1 therapy. Here, we leverage a unique opportunity to understand EwS immunotherapy resistance through analysis of paired tumor samples from SARC028. Using a multi-omics approach, we identify features of EwS that contribute to primary resistance to anti-PD1 including TGF-{beta}, reduced interferon signaling and low HLA class I expression. This study provides an improved understanding of tumor characteristics driving poor response to immunotherapy in relapsed EwS and will guide the development of future trials to modulate this immunosuppressive axis.

cancer biology↗

bioRxiv2024

HIV-1 control in vivo is related to the number but not the fraction of infected cells with viral unspliced RNA

In the absence of antiretroviral therapy (ART), a subset of individuals, termed HIV controllers, have levels of plasma viremia that are orders of magnitude lower than non-controllers who are at higher risk for HIV disease progression. In addition to having fewer infected cells resulting in fewer cells with HIV RNA, it is possible that lower levels of plasma viremia in controllers is due to a lower fraction of the infected cells having HIV-1 unspliced RNA (HIV usRNA) compared with non-controllers. To directly test this possibility, we used sensitive and quantitative single cell sequencing methods to compare the fraction of infected cells that contain one or more copies of HIV usRNA in peripheral blood mononuclear cells (PBMC) obtained from controllers and non-controllers. The fraction of infected cells containing HIV usRNA did not differ between the two groups. Rather, the levels of viremia were strongly associated with the total number of infected cells that had HIV usRNA, as reported by others, with controllers having 34-fold fewer infected cells per million PBMC. These results reveal for the first time that viremic control is not associated with a lower fraction of proviruses expressing HIV usRNA, unlike what is reported for elite controllers, but is only related to having fewer infected cells overall, maybe reflecting greater immune clearance of infected cells. Our findings show that proviral silencing is not a key mechanism for viremic control and will help to refine strategies towards achieving HIV remission without ART.

microbiology↗

bioRxiv2024

Immunocompetent murine model of Ewing sarcoma reveals role for TGFβ inhibition to enhance immune infiltrates in Ewing tumors during radiation

Ewing sarcoma (ES) is an aggressive cancer diagnosed in adolescents and young adults. The fusion oncoprotein (EWSR1::FLI1) that drives Ewing sarcoma is known to downregulate TGFBR2 expression (part of the TGF{beta} receptor). Because TGFBR2 is downregulated, it was thought that TGF{beta} likely plays an inconsequential role in Ewing biology. However, the expression of TGF{beta} in the Ewing tumor immune microenvironment (TIME) and functional impact of TGF{beta} in the TIME remains largely unknown given the historical lack of immunocompetent preclinical models. Here, we use single-cell RNAseq analysis of human Ewing tumors to show that immune cells, such as NK cells, are the largest source of TGF{beta} production in human Ewing tumors. We develop a humanized (immunocompetent) mouse model of ES and demonstrate distinct TME signatures and metastatic potential in these models as compared to tumors developed in immunodeficient mice. Using this humanized model, we study the effect of TGF{beta} inhibition on the Ewing TME during radiation therapy, a treatment that both enhances TGF{beta} activation and is used to treat aggressive ES. Utilizing a trivalent ligand TGF{beta} TRAP to inhibit TGF{beta}, we demonstrate that in combination with radiation, TGF{beta} inhibition both increases ES immune cell infiltration and decreases lung metastatic burden in vivo. The culmination of these data demonstrates the value of humanized models to address immunobiologic preclinical questions in Ewing sarcoma and suggests TGF{beta} inhibition as a promising intervention during radiation therapy to promote metastatic tumor control.

cancer biology↗

bioRxiv2022

Antibodies targeting conserved non-canonical antigens and endemic coronaviruses associate with favorable outcomes in severe COVID-19

While there have been extensive analyses characterizing cellular and humoral responses across the severity spectrum in COVID-19, predictors of outcomes within severe COVID-19 remain to be comprehensively elucidated. Recently, we identified divergent monocyte states as predictors of outcomes within severe COVID-19, but corresponding humoral profiles of risk have not been delineated. Furthermore, the nature of antibodies (Abs) directed against viral antigens beyond the spike protein or endemic coronavirus antigens and their associations with disease severity and outcomes remain poorly defined. We performed deep molecular profiling of Abs directed against a wide range of antigenic specificities in severe COVID-19 patients admitted to the ICU. The profiles consisted of canonical (S, RBD, N) and non-canonical (orf3a, orf8, nsp3, nps13 and M) antigenic specificities. Notably, multivariate machine learning (ML) models, generated using profiles of Abs directed against canonical or non-canonical antigens, were equally discriminative of recovery and mortality COVID-19 outcomes. In both ML models, survivors were associated with increased virus-specific IgA and IgG3 antibodies and with higher antigen-specific antibody galactosylation. Intriguingly, pre-pandemic healthy controls had cross-reactive Abs directed against nsp13 which is a conserved protein in other alpha and beta coronaviruses. Notably, higher levels of nsp13-specific IgA antibodies were associated with recovery in severe COVID-19. In keeping with these findings, a model built on Ab profiles for endemic coronavirus antigens was also predictive of COVID-19 outcome bifurcation, with higher levels of IgA and IgG3 antibodies against OC43 S and NL63 S being associated with survival. Our results suggest the importance of Abs targeting non-canonical SARS-CoV-2 antigens as well as those directed against endemic coronaviruses in favorable outcomes of severe COVID-19.

immunology↗

bioRxiv2021

Bifurcated monocyte states are predictive of mortality in severe COVID-19

Coronavirus disease 2019 (COVID-19) caused by SARS-CoV-2 infection presents with varied clinical manifestations1, ranging from mild symptoms to acute respiratory distress syndrome (ARDS) with high mortality2,3. Despite extensive analyses, there remains an urgent need to delineate immune cell states that contribute to mortality in severe COVID-19. We performed high-dimensional cellular and molecular profiling of blood and respiratory samples from critically ill COVID-19 patients to define immune cell genomic states that are predictive of outcome in severe COVID-19 disease. Critically ill patients admitted to the intensive care unit (ICU) manifested increased frequencies of inflammatory monocytes and plasmablasts that were also associated with ARDS not due to COVID-19. Single-cell RNAseq (scRNAseq)-based deconvolution of genomic states of peripheral immune cells revealed distinct gene modules that were associated with COVID-19 outcome. Notably, monocytes exhibited bifurcated genomic states, with expression of a cytokine gene module exemplified by CCL4 (MIP-1{beta}) associated with survival and an interferon signaling module associated with death. These gene modules were correlated with higher levels of MIP-1{beta} and CXCL10 levels in plasma, respectively. Monocytes expressing genes reflective of these divergent modules were also detectable in endotracheal aspirates. Machine learning algorithms identified the distinctive monocyte modules as part of a multivariate peripheral immune system state that was predictive of COVID-19 mortality. Follow-up analysis of the monocyte modules on ICU day 5 was consistent with bifurcated states that correlated with distinct inflammatory cytokines. Our data suggests a pivotal role for monocytes and their specific inflammatory genomic states in contributing to mortality in life-threatening COVID-19 disease and may facilitate discovery of new diagnostics and therapeutics.

immunology↗

bioRxiv2021

Microbiome-specific T follicular helper cells drive tertiary lymphoid structures and anti-tumor immunity against colorectal cancer.

Colorectal cancer (CRC) is a common and deadly disease, and patients with metastatic tumors often fail to respond to therapy. While select members of the microbiome are associated with improved anti-tumor immunity, mechanistic understanding of how the microbiome provides a benefit is lacking. We show that modification of the CRC-associated microbiome with a single immunogenic commensal bacteria can alter T cell differentiation, inhibit tumor growth, and increase survival. Microbiome-driven control of CRC required the formation of colonic tertiary lymphoid structures (TLS) and increased infiltration of the tumor with cytotoxic immune cells. In the context of CRC, CD4+ T cells specific to the newly introduced commensals differentiated into T follicular helper cells and were necessary for the formation of TLS, immune infiltration of the tumor, and control over CRC. Thus, modification of the intestinal T cell response by the microbiome can be used to augment anti-tumor immunity in colorectal cancer.

immunology↗

bioRxiv2020

Divergent cancer etiologies drive distinct B cell signatures and tertiary lymphoid structures

Current immunotherapy paradigms aim to reinvigorate CD8+ T cells, but the contribution of humoral immunity to antitumor immunity remains understudied1,2. Head and neck squamous cell carcinoma (HNSCC) is caused by either human papillomavirus (HPV+) or environmental carcinogens (i.e. tobacco and alcohol; HPV-)3,4. Here, we demonstrate that HPV+ HNSCC patients have transcriptional signatures of germinal center (GC) tumor infiltrating B cells (TIL-Bs) and spatial organization of immune cells consistent with GC-like tertiary lymphoid structures (TLS), both of which correlate with favorable outcomes in HNSCC patients. Further, our single-cell RNAseq data also indicate that GC TIL-Bs are characterized by distinct waves of gene expression consistent with dark zone, light zone and a transitional state of GC B cells. High-dimensional spectral flow cytometry permitted in depth characterization of activated, memory and GC TIL-Bs. Further, single cell RNAseq analysis and subsequent protein validation identified a role for semaphorin 4a (Sema4a) in the differentiation of GC TIL-Bs and indicated that expression of Sema4a was enhanced on GC TIL-Bs and within GC-like TLS in the TME. Thus, in contrast to some reports on the detrimental role of TIL-Bs in human tumors, our findings suggest that TIL-Bs play an instrumental role in antitumor immunity5,6. Novel therapeutics to enhance TIL-B responses in HNSCC should be prioritized as a compliment to current T-cell mediated immunotherapies.

immunology↗

bioRxiv2020

Systemic immune dysfunction in cancer patients driven by IL6 and IL8 induction of an inhibitory receptor module in peripheral CD8+ T cells

Many cancer patients do not develop a durable response to the current standard of care immunotherapies despite substantial advances in targeting immune inhibitory receptors1-5. A potential compounding issue, which may serve as an unappreciated, dominant resistance mechanism, is an inherent systemic immune dysfunction that is often associated with advanced cancer6-12. Minimal response to inhibitory receptor (IR) blockade therapy and increased disease burden have been associated with peripheral CD8+ T cell dysfunction, characterized by suboptimal T cell proliferation and chronic expression of IRs (eg. Programmed Death 1 [PD1] and Lymphocyte Activation Gene 3 [LAG3])13, 14. Here, we demonstrate that up to a third of cancer patients express robust intracellular LAG3 (LAG3IC), but not surface LAG3 (LAG3SUR), in peripheral CD8+ T cells compared to CD4+ T cells and regulatory T cells (Tregs). LAG3IC is associated with: (i) expression of a LAG3IC-dominant IR module that includes PD1IC, NRP1IC, CD39IC, and TIGITIC; (ii) decreased CD8+ but not CD4+ T cell function that can be reversed by anti-LAG3 (and/or anti-PD1), despite limited constitutive surface IR expression; and (iii) poor disease prognosis. Systemic immune dysfunction is restricted to CD8+ T cells, including a high percentage of peripheral naive CD8+ T cells, indicating a TCR-independent mechanism that is driven by the cytokine IL6 and the chemokine IL8. Thus, the combination of an increased LAG3-dominant IR module and elevated systemic IL6 and/or IL8 may serve as predictive biomarkers and increase the possibility that cancer patients will benefit from therapeutic combinations targeting these systemic cytokines in the setting of PD1 and/or LAG3 blockade.

cancer biology↗