111 research outputs found
Use of genome wide expression profiles in analysis of T cell dysfunction in Hepatitis C virus infection
No full textDuring the course of infection with chronic pathogens such as Hepatitis C virus (HCV), Hepatitis B virus (HBV) and HIV, virus-specific CD8+ T cells differentiate into heterogeneous dysfunctional subpopulations. Advances in multi-parameter flow cytometry have allowed these subpopulations to be further classified into classes of exhausted T cells, primarily by their expression of multiple inhibitory receptors. However, the exact phenotype of CD8+ T cells during exhaustion is an area of great interest as many inhibitory receptors are also expressed on functional CD8+ T cells. Discovering novel and specific markers of T cell exhaustion is fundamental in developing strategies to restore CD8+ T cell function in chronic viral infections. Recently, genome wide expression profiles have identified broad molecular phenotypes in exhausted T cells that could not have been discovered by flow cytometry alone. I show how similar genomic approaches identify and further characterise the ectonucleotidase CD39 as a novel marker of CD8+ T cell exhaustion in chronic viral infection. I show that CD39 is highly expressed in HCV and HIV-specific CD8+ T cells and that CD39+ CD8+ T cells are enriched with gene signatures of exhaustion. CD39 is highly co-expressed with multiple inhibitory receptors including PD-1, enzymatically active on CD8+ T cells in HCV infection and positively correlated with viral load in both HCV and HIV. I also demonstrate the discovery of a novel CD39High population of cells in the mouse model of chronic Lymphocytic Choriomenigitis Virus (LCMV) infection, which express the highest degrees of PD-1, LAG3 and 2B4 in the CD39+ fraction. Thus, CD39 is a novel and specific marker of severe CD8+ T cell exhaustion in human and animal models of chronic viral infection
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Regulation of immune homeostasis, autoimmunity and protective immunity by the CD160 and PD-1 co-inhibitory molecules
No full textCo-stimulatory and co-inhibitory pathways are critical for immune homeostasis, immune tolerance and protective immunity. In this thesis, we analyzed how two co-inhibitory molecules, CD160 and programmed cell death-1 (PD-1), regulate immune homeostasis, autoimmunity and protective immunity.
The function of CD160 on T cells remains unclear with conflicting studies supporting both co-inhibitory and co-stimulatory roles. Here we demonstrate that CD160 has a co-stimulatory role in promoting CD8+ T cell effector functions needed for optimal clearance of oral Listeria monocytogenes (Lm) infection. CD160–/– mice did not clear oral Lm as efficiently as WT littermates. Defective bacterial clearance is due to compromised IEL and splenic CD8+ T cell functions – reduced granzyme B, TNF-α and IFN-γ production in CD160–/– IELs and splenic CD8+ T cells. Adoptive transfer studies showed that RAG–/– recipients receiving CD160–/– CD8+ T cells had a higher mortality and a higher bacteria burden compared to RAG–/– recipients receiving WT CD8+ T cells. These findings demonstrate that CD160 provides key co-stimulatory signals to CD8+ T cells for protective immunity during an acute mucosal bacteria infection.
Secondly, we studied the role of PD-1 on regulatory T cells (Tregs). The PD-1 co-inhibitory pathway is critical for immune tolerance. Studies of PD-1 function have focused primarily on effector T cells, and little is known about how PD-1 regulates Tregs, which are critical for maintaining the balance between tolerance and immunity. We demonstrated that PD-1 deficient Tregs have increased suppressive capacity in vitro and in vivo compared to WT Tregs. Mice lacking PD-1 selectively on Tregs had delayed onset and severity of experimental autoimmune encephalitis (EAE) and were protected against diabetes in non-obese diabetic (NOD) mice. The enhanced suppressive capacity in PD-1 deficient Tregs is due to down-regulation of the PI3K-AKT pathway in Tregs and PD-1 deficient Tregs have altered bioenergetics. Our findings demonstrate that signals through PD-1 in Treg cells restrain their activation and function and are vital for optimal homeostasis, protective immunity and tolerance.
These findings advance our mechanistic understanding of how co-inhibitory molecules on different immune cell types regulate immune homeostasis, autoimmunity and immunity.Biology, Molecular and Cellula
Abstract A16: Defining molecular mechanisms of resistance to tumor immunity
No full textAbstract
Recent success of immune checkpoint blockade solidifies the importance of the immune system in the defense against cancer. The clinical impact of the immune response is, however, very heterogeneous, with some patients achieving dramatic responses while others fail to respond. Known genomic correlates of response to immunotherapy are not perfectly predictive of clinical outcome, supporting the existence of unknown mechanisms of resistance to tumor immunity. I hypothesize somatic acquired mutations of individual tumors may account for heterogeneity in the spontaneous response to tumors and response to immunotherapy. I have undertaken a systematic in vivo screen to identify mechanisms of resistance to tumor immunity in order to discover new mechanisms of immune resistance, define a comprehensive set of therapeutic targets and provide biomarkers of sensitivity to immunotherapeutic strategies. Mouse tumor cell lines (MC38 colon carcinoma or B16 melanoma) were engineered to express a library of barcoded open reading frames (ORFs) mutagenized to encode known cancer-associated somatic mutations from the Pan Cancer analysis within The Cancer Genome Atlas (TCGA). These cell lines form tumors when implanted subcutaneously in immunocompetent animals. Tumor-bearing animals were then subjected to immunotherapy with either therapeutic vaccination or checkpoint blockade with anti-PD-1. Barcode relative representation was measured by next generation sequencing at the time of tumor implantation and at the time of tumor harvest post-immunotherapy. Barcoded mutant ORFs that confer immune resistance increased in representation under immune pressure in comparison to untreated or immunodeficient animals. A mutation in Phospho-Inositol 3 Kinase (PI3K), PIK3CA c.3140A>G, consistently increased in representation in both B16 and MC38 immunotherapy-treated tumors. This mutation encodes a constitutively active mutant catalytic domain of PI3K, PIK3CA H1047R. MC38 tumors homogenously expressing PIK3CA H1047R and implanted into wild type mice failed to respond to anti-PD-1 therapy, while tumors expressing a control gene or non-scoring mutation in PI3K regressed after treatment with anti-PD-1. Pharmacologic PI3K inhibition resensitized tumors to treatment with anti-PD-1. PD-1-treated PIK3CA H1047R tumors had fewer infiltrating CD8+ T cells as measured by immunohistochemistry and flow cytometry of tumor infiltrating lymphocytes. I conclude that PI3K has, in addition to its well-described oncogenic role, a role in tumor immune evasion. As such, activation of PI3K may be useful as a predictor of resistance to immunotherapy. Importantly, these findings also provide a rationale for therapeutic combination trials of immune checkpoint blockade and PI3K inhibition.
Citation Format: Natalie B. Collins, Robert Manguso, Hans Pope, W. Nicholas Haining. Defining molecular mechanisms of resistance to tumor immunity. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2016 Oct 20-23; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2017;5(3 Suppl):Abstract nr A16.</jats:p
The Numerology of T Cell Functional Diversity
No full textMemory T cells are heterogeneous in phenotype and function. In this issue of Immunity, Newell et al. (2012) use a new flow cytometry platform to show that the functional heterogeneity of the human T cell compartment is even greater than previously thought
Abstract 1019: <i>In vivo</i> CRISPR screening identifies Ptpn2 as a target for cancer immunotherapy
No full textAbstract
Despite the dramatic clinical success of cancer immunotherapy with PD-1 checkpoint blockade, most patients don’t experience sustained clinical benefit, suggesting that additional therapeutic strategies are needed. Functional genomic screens in cancer cells to discover new therapeutic targets are usually carried out in vitro where interaction with the immune system is absent. Here we report a pooled, loss-of-function genetic screening approach using CRISPR/Cas9 genome editing that is conducted in vivo in mouse transplantable tumors treated with vaccination and PD-1 checkpoint blockade. We tested 2,400 genes expressed by melanoma cells for those that synergize with or cause resistance to checkpoint blockade, and recovered the known immune evasion molecules, PD-L1 and CD47. Loss of function of multiple genes required to sense interferon-y caused resistance to immunotherapy. Deletion of Ptpn2, a pleotropic protein tyrosine phosphatase improved response to immunotherapy. In vivo, Ptpn2 deficient tumors showed increased infiltration of activated CD8+T cells. In vitro, Ptpn2 loss by tumor cells increased antigen presentation to T cells. Biochemical, transcriptional and genetic epistasis experiments demonstrated that loss of function of Ptpn2 sensitizes tumors to immunotherapy by enhancing interferon-y-mediated effects on the tumor cell. Thus, augmenting interferon-y signaling in tumor cells could increase the efficacy of immunotherapy. More generally, in vivo genetic screens in tumor models can identify new immunotherapy targets and rationally prioritize combination therapies.
Citation Format: Robert T. Manguso, Hans W. Pope, Margaret D. Zimmer, Flavian D. Brown, Kathleen B. Yates, Brian C. Miller, Natalie B. Collins, Kevin Bi, Martin W. Lafleur, Vikram R. Juneja, Sarah A. Weiss, David E. Fisher, David E. Root, Arlene H. Sharpe, John G. Doench, W Nicholas Haining. In vivo CRISPR screening identifies Ptpn2 as a target for cancer immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1019. doi:10.1158/1538-7445.AM2017-1019</jats:p
Abstract PR11: Dissecting mechanisms of PD-1 blockade with single-cell RNA-sequencing
No full textAbstract
Anti-PD-1 therapy is an important new treatment option for many different types of malignancies, but overall response rates are less than 40%. We do not yet understand which patients will benefit and what resistance mechanisms allow tumor escape. The goal of this work is to understand the mechanisms by which anti-PD-1 therapy augments the anti-tumor immune response at the cellular level. Given that anti-PD-1 therapy is thought to work by altering the immunosuppressive tumor microenvironment, efforts to improve its efficacy will require a deep understanding of this complicated milieu. This will require analysis of thousands of cells using methodology that avoids the pitfalls of current techniques that have either limited scope - flow cytometry, immunohistochemistry - or limited resolution - bulk RNA sequencing. To this end, we have developed a massively parallel single-cell RNA-sequencing platform (Seq-Well) that comprehensively defines the global expression profile of all major immune lineages in the tumor microenvironment. Seq-Well uses a fabricated chip with nearly 100,000 nanowells into which barcoded beads and individual cells are distributed prior to lysis and RNA capture. Mice were implanted with two different transplantable models of cancer (MC38 colon carcinoma or B16 melanoma) and treated with anti-PD-1 or control antibodies. Tumors were harvested and CD45+ tumor-infiltrating leukocytes isolated by FACS. Thousands of cells were sequenced using Seq-Well with a median recovery of approximately 1,000 genes/cell. This level of expression diversity allows us to clearly distinguish different cell populations within the tumor microenvironment. We detect two transcriptionally distinct populations of CD8+ T cells, one that is highly proliferative (as marked by Ki-67), and one that has higher expression of perforin and TIM-3. The Ki-67+ population is enriched for a gene expression signature characterized by effector CD8+ T cells early in viral infection, consistent with their more proliferative nature. We hypothesize that this cluster of CD8+ T cells is also more functional given this signature enrichment and its lower expression of TIM-3, a marker found on exhausted CD8+ T cells. Comparisons of anti-PD-1 treated and control treated tumors are ongoing. In conclusion, massively parallel single-cell RNA-sequencing is a promising technology for the analysis of tumor immune infiltrates that will allow us to address the mechanisms by which checkpoint blockade controls tumor growth. By advancing our knowledge of an important immune checkpoint therapy, we aim to better understand who will respond to therapy, what resistance mechanisms may develop, and how to augment therapeutic efficacy with additional treatments.
This abstract is also being presented as Poster A79.
Citation Format: Brian C. Miller, Marc H. Wadsworth 2nd, Kevin Bi, Travis K. Hughes, Arlene H. Sharpe, Alex K. Shalek, W. Nicholas Haining. Dissecting mechanisms of PD-1 blockade with single-cell RNA-sequencing. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2016 Oct 20-23; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2017;5(3 Suppl):Abstract nr PR11.</jats:p
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