PD-1 Dynamically Regulates Inflammation and Development of Brain-Resident Memory CD8 T Cells During Persistent Viral Encephalitis

Programmed cell death-1 (PD-1) receptor signaling dampens the functionality of T cells faced with repetitive antigenic stimulation from chronic infections or tumors. Using intracerebral (i.c.) inoculation with mouse polyomavirus (MuPyV), we have shown that CD8 T cells establish a PD-1hi, tissue-resident memory population in the brains (bTRM) of mice with a low-level persistent infection. In MuPyV encephalitis, PD-L1 was expressed on infiltrating myeloid cells, microglia and astrocytes, but not on oligodendrocytes. Engagement of PD-1 on anti-MuPyV CD8 T cells limited their effector activity. NanoString gene expression analysis showed that neuroinflammation was higher in PD-L1−/− than wild type mice at day 8 post-infection, the peak of the MuPyV-specific CD8 response. During the persistent phase of infection, however, the absence of PD-1 signaling was found to be associated with a lower inflammatory response than in wild type mice. Genetic disruption and intracerebroventricular blockade of PD-1 signaling resulted in an increase in number of MuPyV-specific CD8 bTRM and the fraction of these cells expressing CD103, the αE integrin commonly used to define tissue-resident T cells. However, PD-L1−/− mice persistently infected with MuPyV showed impaired virus control upon i.c. re-infection with MuPyV. Collectively, these data reveal a temporal duality in PD-1-mediated regulation of MuPyV-associated neuroinflammation. PD-1 signaling limited the severity of neuroinflammation during acute infection but sustained a level of inflammation during persistent infection for maintaining control of virus re-infection

Regulation of transcription factor IRF8 in myeloid progenitors is a critical checkpoint for formation of defective myeloid cells in cancer.

Abstract Defectivemyeloid cells are commonly observed across a large range of solid cancers. These defects contribute to immune suppression and tumor progression tometastatic disease. Early studies focused on characterizing such myeloid-derivedsuppressor cells (MDSC) in peripheral tissues and the tumor microenvironment, but molecular processes underlying their development remained unclear. Bone marrowis the major site for normal myeloid production and is known to respond to signalsfrom the periphery. Thus, we hypothesizedthat tumors perturb bone marrow myelopoiesis, resulting in production of MDSC. During myelopoiesis, transcription factor IRF8 expression within granulocyte/monocyteprogenitors (GMP) determines differentiation such that high IRF8 favorsmonocytes while low IRF8 favors granulocytes. We previously revealed that IRF8 suppressionby tumor-derived factors leads to accumulation of MDSC, but exactly when andwhere IRF8 is compromised was unknown. Here we utilize a novel mouse model expressingan IRF8-EGFP fusion protein to investigate specific changes in IRF8 during myelopoiesisin both orthotopic and spontaneous mammary tumor progression. Our results show that:1) GMP can be divided into IRF8hi and IRF8lo expressingpopulations corresponding to monocytic and granulocytic progenitor phenotypes,2) IRF8lo GMP preferentially expand with increasing tumor size andincreasing G-CSF serum concentration, and 3) enforced IRF8 expression restrainsthis expansion and reduces the frequency of myeloid suppressors in theperiphery. Altogether, these data identify modulation of IRF8 in myeloidprogenitors as a consequence of tumor development and potential target fortherapeutic intervention.</jats:p

Abstract 3249: Regulation of interferon regulatory factor-8 (IRF8) during myelopoiesis is a critical checkpoint for the formation of defective myeloid cells in cancer

Abstract Aberrant myelopoiesis is commonly observed in patients with solid cancers. This results in the production of myeloid-derived suppressive cells (MDSCs) which promote tumor growth and metastasis. However, the molecular mechanisms underlying MDSC development have remained poorly understood. The transcription factor IRF8 is integral for overseeing myelopoietic fate; high IRF8 expression during normal myelopoiesis favors monocytic differentiation and directly inhibits granulocytic differentiation. We recently showed that tumor-derived STAT3/5-activating cytokines downregulate the expression of IRF8 in myeloid cells leading to the accumulation of MDSCs in the periphery, which are primarily granulocytic. However it is unclear if MDSC production is a consequence of IRF8 downregulation in the periphery or upstream in the bone marrow during myelopoiesis. To that end, we utilized a novel mouse model expressing an IRF8-EGFP fusion protein which allowed us to investigate changes in IRF8 expression during both orthotopic and spontaneous mammary tumor growth and progression. Our results showed that: 1) the total granulocyte-monocyte progenitor (GMP) fraction, as with the peripheral MDSC population, was responsive to tumor burden and greatly expanded with increasing tumor size, and 2) IRF8 expression within the total GMPs significantly decreased during tumor growth, reflecting the expansion of a newly defined primarily IRF8lo granulocyte progenitor (GP) population, and 3) tumor-induced GPs were hyper-responsive not only to G-CSF, but also M-CSF to generate a ‘biased’ granulocytic MDSC-like phenotype. Expression of IRF8 in other progenitor populations was not affected, suggesting that IRF8lo GMPs/GPs are a key source of the peripheral MDSC response. Lastly, genetically enforced IRF8 expression in myeloid progenitors of the bone marrow constrained aberrant myelopoiesis during tumor growth, resulting in delayed autochthonous tumor growth and reduced spontaneous lung metastasis. Altogether, these data reinforce the notion that modulation of IRF8 in myeloid progenitors is an early consequence of the neoplastic process and a potential target for therapeutic intervention. NIH R01CA140622 NIH T32CA085183 Citation Format: Colleen S. Netherby, Michelle N. Messmer, Scott I. Abrams. Regulation of interferon regulatory factor-8 (IRF8) during myelopoiesis is a critical checkpoint for the formation of defective myeloid cells in cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3249.</jats:p

Balancing Inflammation and Central Nervous System Homeostasis: T Cell Receptor Signaling in Antiviral Brain TRM Formation and Function

Tissue-resident memory (TRM) CD8 T cells provide early frontline defense against regional pathogen reencounter. CD8 TRM are predominantly parked in nonlymphoid tissues and do not circulate. In addition to this anatomic difference, TRM are transcriptionally and phenotypically distinct from central-memory T cells (TCM) and effector-memory T cells (TEM). Moreover, TRM differ phenotypically, functionally, and transcriptionally across barrier tissues (e.g., gastrointestinal tract, respiratory tract, urogenital tract, and skin) and in non-barrier organs (e.g., brain, liver, kidney). In the brain, TRM are governed by a contextual milieu that balances TRM activation and preservation of essential post-mitotic neurons. Factors contributing to the development and maintenance of brain TRM, of which T cell receptor (TCR) signal strength and duration is a central determinant, vary depending on the infectious agent and modulation of TCR signaling by inhibitory markers that quell potentially pathogenic inflammation. This review will explore our current understanding of the context-dependent factors that drive the acquisition of brain (b)TRM phenotype and function, and discuss the contribution of TRM to promoting protective immune responses in situ while maintaining tissue homeostasis.</jats:p

Tumor-induced myeloid dysfunction and its implications for cancer immunotherapy

Immune function relies on an appropriate balance of the lymphoid and myeloid responses. In the case of neoplasia, this balance is readily perturbed by the dramatic expansion of immature or dysfunctional myeloid cells accompanied by a reciprocal decline in the quantity/quality of the lymphoid response. In this review, we seek to: 1) define the nature of the atypical myelopoiesis observed in cancer patients and the impact of this perturbation on clinical outcomes; 2) examine the potential mechanisms underlying these clinical manifestations; and 3) explore potential strategies to restore normal myeloid cell differentiation to improve activation of the host antitumor immune response. We posit that fundamental alterations in myeloid homeostasis triggered by the neoplastic process represent critical checkpoints that govern therapeutic efficacy, as well as offer novel cellular-based biomarkers for tracking changes in disease status or relapse

Tumor-induced STAT3 hyperactivation creates a dendritic cell differentiation block by significantly downregulating protein kinase C βII expression (P2003)

Abstract Tumor mediated blockade of dendritic cell (DC) differentiation is a large component of cancer induced immunosuppression, contributing to tumor outgrowth. Tumors impair DC differentiation via factors that hyperactivate STAT3 in DC progenitors, though the mechanisms by which this occurs are largely unknown. PKCβII is essential in DC differentiation, and we test here if tumor driven STAT3 hyperactivation downregulates PKCβII expression, and if this is the mechanism underlying impaired DC differentiation. Myeloid cells from tumor bearing mice have significantly decreased PKCβII expression. Culture in tumor conditioned media (TCM) significantly decreased PKCβII expression and significantly impaired phorbol ester driven DC differentiation in human monocytes and in a human cell line model (KG1). In KG1, this was dependent on PKCβII downregulation: enforced PKCβII expression preserved phorbol ester driven DC differentiation in the face of TCM. Interestingly, PKCβII overexpression and/or activation also antagonized TCM-driven STAT3 activation. STAT3 drives this decreased PKCβII expression: TCM induced STAT3 activation and drove 4.5 fold higher STAT3 binding to the PKCβ promoter (compared to media control, p&amp;lt;0.001). Mutation of a STAT3 consensus binding site in the promoter eliminates the ability of STAT3 to impair PKCβ promoter activity. Together, these observations argue that tumor driven STAT3 hyperactivation inhibits DC differentiation by downregulating PKCβII.</jats:p

IRF-8 levels constrain aberrant myelopoiesis and improve immune control in the metastatic tumor microenvironment (TUM4P.901)

Abstract Abnormal myelopoiesis is common in patients with diverse malignancies, including breast cancer. The myeloid compartment is essential for the induction of host immunity, thus alterations in myelopoiesis may help explain the failure to control neoplastic disease. Therefore, understanding the molecular basis of myeloid dysfunction may improve the efficacy of cancer immunotherapies. Given that interferon regulatory factor-8 (IRF-8) is crucial for myeloid differentiation, we hypothesized that changes in this master regulator underlie a novel mechanism for the altered myelopoiesis and myeloproliferative phenotypes observed in cancer, such as the accumulation of myeloid-derived suppressor cells (MDSC). We recently showed that tumors downregulate IRF-8 expression in myeloid progenitors in a cytokine-driven STAT3 or STAT5-dependent manner, resulting in MDSC generation. This suggested that IRF-8 expression functions as a ‘rheostat’ which directs the fate of the tumor-induced myeloid response. To test this, we examined the impact of modulating IRF-8 levels in two mouse mammary tumor models of lung metastasis using a newly designed genetic gain-of-function approach. In both models, we observed a significant reduction in lung metastases and the frequency of MDSC in IRF-8 transgenic mice compared to wild-type controls. Altogether, our results reveal an unrecognized role for IRF-8 in the host response against metastasis, which may lead to new therapeutic targets in cancer immunotherapy.</jats:p

IL-21 produced by CD4+ T cells drives differentiation of CD8+ T cells during polyomavirus encephalitis

Abstract The demyelinating brain disease Progressive Multifocal Leukoencephalopathy (PML), caused by JC polyomavirus (JCV), is a life-threatening complication for patients with HIV/AIDS, hematologic malignancies, rheumatologic diseases, and those receiving long-term immunomodulating therapies. Using mouse polyomavirus (MuPyV), our laboratory developed a robust model of polyomavirus-associated demyelinating leukoencephalitis, complete with viral infection and T cell infiltration. Increasing evidence supports the importance of CD4+ T cells in controlling JCV in the brain and thereby preventing PML. Recent studies in our laboratory show that CD4+ T cells are essential for differentiation and maintenance of MuPyV-specific brain-resident memory CD8+ T cells (bTRM). We find that the help CD4+ T cells are provide to virus-specific CD8+ T cells for their appropriate bTRM differentiation is IL-21. PD-1hiCXCR5hi MuPyV-specific CD4+ T cells are the primary sources of brain IL-21 and co-localize with CD8+ T cells in the brain. Brain MuPyV-specific CD8+ T cells have increased expression of the IL-21 receptor (IL21R) 15 days post-infection, which correlates to the time-point when they begin to express CD103, a canonical TRM marker. TRM maintenance is independent of circulating cells. In line with this property, we show that MuPyV-specific CD8+ T cells are not depleted by parentally administered anti-CD8. In addition, brain CD8+ T cells from IL21R−/ − mice have decreased expression of CD103 and are susceptible to depletion from peripheral anti-CD8. These data indicate that during polyomavirus infection, IL-21 by CD4+ T cells drives CD8+ T cells to differentiate into tissue resident memory in the brain.</jats:p

IL-21 from CD4 T cells drives differentiation of brain-resident CD8 T cells during polyomavirus CNS infection

Abstract CD4 T cells help CD8 T cells differentiate into competent effector and memory cells. We recently showed that CD4 T cells are essential for CD8 T cell differentiation into brain-resident memory (bTRM) during polyomavirus (PyV) infection. Here we identify IL-21 as the CD4 T cell help. Using MHC-II tetramers for two epitopes in mouse PyV, we show brain PyV-specific CD4 T cells express PD-1 and CXCR5. IL21-VFP reporter mice revealed CD4 T cells as the only cellular source of IL-21 in the brain. 2D micropipette adhesion assays, measuring TCR affinity, showed brain IL-21-producing CD4s have higher affinity TCRs than IL-21− cells. RNAseq analysis of IL-21+ vs. IL-21− brain CD4s corroborates that IL-21 production is positively associated with TCR affinity and that IL-21 producers are positively enriched for TFH signature genes. We further found that IL-21 receptor (IL21R) is required for CD8 TRM differentiation. Brain CD8 T cells upregulated IL21R at 15 days post-infection, coincident with expression of the TRM marker CD103. Few CD8 T cells in brains of IL-21R−/− mice expressed CD103, a finding confirmed using donor anti-PyV CD8 T cells lacking IL-21R in infected wild type (WT) recipients. Unlike IL21R-sufficient CD8s, IL21R−/− CD8 T cells are not maintained in the brain upon systemic CD8 antibody depletion. IL21R−/− CD8s also have a diminished response to a rechallenge in the brain than what we observed with WT CD8 T cells. RNAseq analysis revealed IL21R−/− CD8 T cells were negatively enriched for TRM core signature genes and oxidative metabolism genes compared to IL-21R-sufficient CD8 T cells. Thus, we conclude that IL-21 produced by high-affinity, PyV-specific CD4 T cells in the brain is required for differentiation of CD8 bTRM during PyV CNS infection.</jats:p