Kevin Bi

Normalizing the environment recapitulates adult human immune traits in laboratory mice.

Our current understanding of immunology was largely defined in laboratory mice, partly because they are inbred and genetically homogeneous, can be genetically manipulated, allow kinetic tissue analyses to be carried out from the onset of disease, and permit the use of tractable disease models. Comparably reductionist experiments are neither technically nor ethically possible in humans. However, there is growing concern that laboratory mice do not reflect relevant aspects of the human immune system, which may account for failures to translate disease treatments from bench to bedside. Laboratory mice live in abnormally hygienic specific pathogen free (SPF) barrier facilities. Here we show that standard laboratory mouse husbandry has profound effects on the immune system and that environmental changes produce mice with immune systems closer to those of adult humans. Laboratory mice—like newborn, but not adult, humans—lack effector-differentiated and mucosally distributed memory T cells. These cell populations were present in free-living barn populations of feral mice and pet store mice with diverse microbial experience, and were induced in laboratory mice after co-housing with pet store mice, suggesting that the environment is involved in the induction of these cells. Altering the living conditions of mice profoundly affected the cellular composition of the innate and adaptive immune systems, resulted in global changes in blood cell gene expression to patterns that more closely reflected the immune signatures of adult humans rather than neonates, altered resistance to infection, and influenced T-cell differentiation in response to a de novo viral infection. These data highlight the effects of environment on the basal immune state and response to infection and suggest that restoring physiological microbial exposure in laboratory mice could provide a relevant tool for modelling immunological events in free-living organisms, including humans.Our current understanding of immunology was largely defined in laboratory mice because of experimental advantages including inbred homogeneity, tools for genetic manipulation, the ability to perform kinetic tissue analyses starting with the onset of disease, and tractable models. Comparably reductionist experiments are neither technically nor ethically possible in humans. Despite revealing many fundamental principals of immunology, there is growing concern that mice fail to capture relevant aspects of the human immune system, which may account for failures to translate disease treatments from bench to bedside1–8. Laboratory mice live in abnormally hygienic “specific pathogen free” (SPF) barrier facilities. Here we show that the standard practice of laboratory mouse husbandry has profound effects on the immune system and that environmental changes result in better recapitulation of features of adult humans. Laboratory mice lack effector-differentiated and mucosally distributed memory T cells, which more closely resembles neonatal than adult humans. These cell populations were present in free-living barn populations of feral mice, pet store mice with diverse microbial experience, and were induced in laboratory mice after co-housing with pet store mice, suggesting a role for environment. Consequences of altering mouse housing profoundly impacted the cellular composition of the innate and adaptive immune system and resulted in global changes in blood cell gene expression patterns that more closely aligned with immune signatures of adult humans rather than neonates, altered the mouse’s resistance to infection, and impacted T cell differentiation to a de novo viral infection. These data highlight the impact of environment on the basal immune state and response to infection and suggest that restoring physiological microbial exposure in laboratory mice could provide a relevant tool for modeling immunological events in free-living organisms, including humans.

In vivo CRISPR screening identifies Ptpn2 as a cancer immunotherapy target

Immunotherapy with PD-1 checkpoint blockade is effective in only a minority of patients with cancer, suggesting that additional treatment strategies are needed. Here we use a pooled in vivo genetic screening approach using CRISPR–Cas9 genome editing in transplantable tumours in mice treated with immunotherapy to discover previously undescribed immunotherapy targets. We tested 2,368 genes expressed by melanoma cells to identify those that synergize with or cause resistance to checkpoint blockade. We recovered the known immune evasion molecules PD-L1 and CD47, and confirmed that defects in interferon-γ signalling caused resistance to immunotherapy. Tumours were sensitized to immunotherapy by deletion of genes involved in several diverse pathways, including NF-κB signalling, antigen presentation and the unfolded protein response. In addition, deletion of the protein tyrosine phosphatase PTPN2 in tumour cells increased the efficacy of immunotherapy by enhancing interferon-γ-mediated effects on antigen presentation and growth suppression. In vivo genetic screens in tumour models can identify new immunotherapy targets in unanticipated pathways.

Sequential Infection with Common Pathogens Promotes Human-like Immune Gene Expression and Altered Vaccine Response

Immune responses differ between laboratory mice and humans. Chronic infection with viruses and parasites are common in humans, but are absent in laboratory mice, and thus represent potential contributors to inter-species differences in immunity. To test this, we sequentially infected laboratory mice with herpesviruses, influenza, and an intestinal helminth and compared their blood immune signatures to mock-infected mice before and after vaccination against yellow fever virus (YFV-17D). Sequential infection altered pre- and post-vaccination gene expression, cytokines, and antibodies in blood. Sequential pathogen exposure induced gene signatures that recapitulated those seen in blood from pet store-raised versus laboratory mice, and adult versus cord blood in humans. Therefore, basal and vaccine-induced murine immune responses are altered by infection with agents common outside of barrier facilities. This raises the possibility that we can improve mouse models of vaccination and immunity by selective microbial exposure of laboratory animals to mimic that of humans.

Targeted reconstruction of T cell receptor sequence from single cell RNA-seq links CDR3 length to T cell differentiation state

Abstract The T cell compartment must contain diversity in both T cell receptor (TCR) repertoire and cell state to provide effective immunity against pathogens. However, it remains unclear how differences in the TCR contribute to heterogeneity in T cell state. Single cell RNA-sequencing (scRNA-seq) can allow simultaneous measurement of TCR sequence and global transcriptional profile from single cells. However, current methods for TCR inference from scRNA-seq are limited in their sensitivity and require long sequencing reads, thus increasing the cost and decreasing the number of cells that can be feasibly analyzed. Here we present TRAPeS, a publicly available tool that can efficiently extract TCR sequence information from short-read scRNA-seq libraries. We apply it to investigate heterogeneity in the CD8+ T cell response in humans and mice, and show that it is accurate and more sensitive than existing approaches. Coupling TRAPeS with transcriptome analysis of CD8+ T cells specific for a single epitope from Yellow Fever Virus (YFV), we show that the recently described ‘naive-like’ memory population have significantly longer CDR3 regions and greater divergence from germline sequence than do effector-memory phenotype cells. This suggests that TCR usage is associated with the differentiation state of the CD8+ T cell response to YFV.

Mixed signature of activation and dysfunction allows human decidual CD8+ T cells to provide both tolerance and immunity

Significance Successful pregnancy requires establishment of immune tolerance for invading fetal trophoblasts, as well as immunity to a variety of pathogens that cause placental and congenital infections. Decidual CD8+ T cells are key cells for recognition and response to foreign fetal, placental, and viral antigens at the maternal–fetal interface. Thus, regulation of decidual CD8+ T cell activation and cytotoxicity is crucial for a healthy pregnancy. Here, we demonstrate that decidual CD8+ T cells have a mixed profile of T cell dysfunction, activation, and effector function, which allows for both immune tolerance and immunity. This is of great relevance for understanding the development of pregnancy complications as well as prevention of congenital infections that occur as result of impaired placental immunity. Understanding how decidual CD8+ T cell (CD8+ dT) cytotoxicity is regulated and how these cells integrate the competing needs for maternal–fetal tolerance and immunity to infection is an important research and clinical goal. Gene-expression analysis of effector-memory CD8+ dT demonstrated a mixed transcriptional signature of T cell dysfunction, activation, and effector function. High protein expression of coinhibitory molecules PD1, CTLA4, and LAG3, accompanied by low expression of cytolytic molecules suggests that the decidual microenvironment reduces CD8+ dT effector responses to maintain tolerance to fetal antigens. However, CD8+ dT degranulated, proliferated, and produced IFN-γ, TNF-α, perforin, and granzymes upon in vitro stimulation, demonstrating that CD8+ dT are not permanently suppressed and retain the capacity to respond to proinflammatory events, such as infections. The balance between transient dysfunction of CD8+ dT that are permissive of placental and fetal development, and reversal of this dysfunctional state, is crucial in understanding the etiology of pregnancy complications and prevention of congenital infections.

TCR‐pMHC encounter differentially regulates transcriptomes of− tissue‐resident CD8 T cells

To investigate the role of TCR‐pMHC interaction in regulating lung CD8 tissue‐resident T cell (TR) differentiation, polyclonal responses were compared against NP366‐374/Db and PA224‐233/Db, two immunodominant epitopes that arise during influenza A infection in mice. Memory niches distinct from iBALTs develop within the lamina propria, supporting CD103+ and CD103− CD8 TR generation and intraepithelial translocation. Gene set enrichment analysis (GSEA) and weighted gene co‐expression network analysis (WGCNA) identify dominant TCR, adherens junction, RIG‐I‐like and NOD‐like pattern recognition receptor as well as TGF‐β signaling pathways and memory signatures among PA224‐233/Db T cells consistent with T resident memory (TRM) status. In contrast, NP366‐374/Db T cells exhibit enrichment of effector signatures, upregulating pro‐inflammatory mediators even among TRM. While NP366‐374/Db T cells manifest transcripts linked to canonical exhaustion pathways, PA224‐233/Db T cells exploit P2rx7 purinoreceptor attenuation. The NP366‐374/Db CD103+ subset expresses the antimicrobial lactotransferrin whereas PA224‐233/Db CD103+ utilizes pore‐forming mpeg‐1, with <22% of genes correspondingly upregulated in CD103+ (or CD103−) subsets of both specificities. Thus, TCR‐pMHC interactions among TR and antigen presenting cells in a tissue milieu strongly impact CD8 T cell biology.

Abstract 4242: Methylation, SNP and expression analysis of the Vitamin D receptor (VDR) gene in different tumor tissues.

The vitamin D receptor gene encodes the nuclear hormone receptor for vitamin D3 and belongs to the family of trans-acting transcriptional regulatory factors and shows sequence similarity to the steroid and thyroid hormone receptors. Downstream targets of this nuclear hormone receptor are principally involved in mineral metabolism though the receptor regulates a variety of other metabolic pathways, such as those involved in the immune response and cancer. In light of evidence for promoter methylation of the vitamin D receptor in the control of the expression of this gene, we have designed methylation assays that cover many regions of this gene in order to determine its methylation profile in normal individuals, ovarian tumors and ovarian cell lines. We have isolated the DNA from ten tumor tissues and their adjacent normal tissue from four different tumor types; ovarian, breast, colon and cervical. By utilizing a combination of bisulfite sequencing, SNP genotyping, and expression analysis, we show that there are distinct patterns of methylation levels between different regions of the VDR gene in each of the cancers observed. We have also analyzed a number of SNPs within these genes and show a possible correlation with the methylation and /or expression of the genes and the SNP genotypes. These data may provide information that could lead to the development of biomarkers for different cancers. Citation Format: Matthew L. Poulin, Ann Meyer, Gregory Gonzalez, Kevin Bi, Liying Yan. Methylation, SNP and expression analysis of the Vitamin D receptor (VDR) gene in different tumor tissues. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4242. doi:10.1158/1538-7445.AM2013-4242

Comparative transcriptome analysis reveals distinct genetic modules associated with Helios expression in intratumoral regulatory T cells

Significance Regulatory T cells (Tregs) play a critical role in inflammatory, autoimmune, and antitumor immune responses. Increased expression of transcription factor Helios by tumor-infiltrating Tregs can enhance immune-suppressive activity, while deletion of Helios promotes an effector T helper (Th) cell phenotype that can contribute to the host antitumor immune response. We report that chronic inflammatory conditions of tumors induce Helios-deficient Tregs to express increased levels of genes associated with T cell activation and Th cell differentiation. Helios-dependent changes in gene expression are restricted to tumor sites and not observed in peripheral lymphoid tissues. We suggest that Helios-deficient Tregs that recognize tumor-associated self-antigens may become unstable in the tumor microenvironment and undergo reprogramming into effector T cells that can inhibit tumor growth. Regulatory T cells (Tregs) are key modulators of immune tolerance, capable of suppressing inflammatory immune responses and promoting nonlymphoid tissue homeostasis. Helios, a transcription factor (TF) that is selectively expressed by Tregs, has been shown to be essential for the maintenance of Treg lineage stability in the face of inflammatory conditions that include autoimmune disease and cancer. Helios-deficient Tregs within tumors acquire effector T cell function and contribute to immune responses against cancer. However, the underlying genetic basis of this Treg reprogramming is not well understood. Here, we report that Helios-deficient Tregs within the chronic inflammatory tumor microenvironment (TME) derepress genetic programs associated with T helper (Th) cell differentiation by up-regulating Th cell-associated TFs and effector cytokines. These genetic changes of Helios-deficient Tregs are most apparent in a Treg subpopulation with high affinity for self-antigens, as detected by both increased GITR/PD-1 expression and increased responsiveness to self-antigens. Their combined effects may promote a phenotype conversion of Tregs into effector T cells within the TME, where TCR engagement and costimulatory receptor expression by Tregs are increased. These data provide a genetic basis for the unstable phenotype of Helios-deficient Tregs within the inflammatory environment of tumors and suggest that immune milieu-dependent alterations in gene expression are a central feature of Treg conversion.

Abstract PR11: Dissecting mechanisms of PD-1 blockade with single-cell RNA-sequencing

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.

Abstract 1019: In vivo CRISPR screening identifies Ptpn2 as a target for cancer immunotherapy

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