Prognostic Implications of Heterogeneity in Intra-tumoral Immune Composition for Recurrence in Early Stage Lung Cancer.

Background: Studies in the past have identified selected immune cells that associate with different clinical outcomes in non-small cell lung cancer (NSCLC). Considering the fact that immune responses are heterogenous and that the clinical outcome could be influenced by the interplay of various immune cell types, it is imperative to evaluate multiple intra-tumoral immune cell types in the same set of patients. Objective: To evaluate the individual and combined effects of diverse intra-tumoral immune cell types on recurrence after complete surgical resection in early stage lung adenocarcinoma. Methods: We obtained NCBI GEO datasets for lung adenocarcinoma, the most prevalent histological subtype of NSCLC and re-analyzed the gene expression data of 292 patients with early stage cancer (IA/IB). CIBERSORT was used to resolve 22 immune cell types from the tumor transcriptomes. Survival analysis was carried out to assess the effect of immune cell types and genes associated with recurrence. Results: Out of the 22 cell types, a high proportion of Tregs and monocyte-macrophages in the tumors were associated with significantly increased probability of recurrence. Conversely, increased proportion of non-Treg CD4+ T cells and plasma cells were associated with a lower probability of recurrence. The higher expression of CCL20 (which can direct the migration of cells of B cell lineage), XCL1 (associated with prototypical Th1 responses) and the immunoglobulin chains IGHV4.34 and IGLV6.57 were associated with a significantly lower probability of recurrence. Importantly, the intra-tumoral immune phenotype comprising these four cell types varied among patients and differentially associated with recurrence depending on net levels of positive and negative prognostic factors. Despite a high level of intra-tumoral plasma cells, a concomitant high level of monocyte-macrophages reduced the freedom from recurrence from ~80 to ~50% at 80 months (p < 0.05). Furthermore, stratification of the patients on the basis of a score estimated from the levels of four cell types enabled the identification of patients with significantly increased probability of recurrence (~50%) after surgery. Significance: Our analysis suggests that concomitant levels of macrophages and plasma cells, in addition to the T regs and non-TregCD4+ T cells in tumors can identify patients with early stage lung cancer at greater risk of recurrence.

Pathologic and Protective Roles for Microglial Subsets and Bone Marrow- and Blood-Derived Myeloid Cells in Central Nervous System Inflammation.

Inflammation is a series of processes designed for eventual clearance of pathogens and repair of damaged tissue. In the context of autoimmune recognition, inflammatory processes are usually considered to be pathological. This is also true for inflammatory responses in the central nervous system (CNS). However, as in other tissues, neuroinflammation can have beneficial as well as pathological outcomes. The complex role of encephalitogenic T cells in multiple sclerosis and its animal model experimental autoimmune encephalomyelitis (EAE) may derive from heterogeneity of the myeloid cells with which these T cells interact within the CNS. Myeloid cells, including resident microglia and infiltrating bone marrow-derived cells, such as dendritic cells (DC) and monocytes/macrophages [bone marrow-derived macrophages (BMDM)], are highly heterogeneous populations that may be involved in neurotoxicity and also immunoregulation and regenerative processes. Better understanding and characterization of myeloid cell heterogeneity is essential for future development of treatments controlling inflammation and inducing neuroprotection and neuroregeneration in diseased CNS. Here, we describe and compare three populations of myeloid cells: CD11c(+) microglia, CD11c(-) microglia, and CD11c(+) blood-derived cells in terms of their pathological versus protective functions in the CNS of mice with EAE. Our data show that CNS-resident microglia include functionally distinct subsets that can be distinguished by their expression of CD11c. These subsets differ in their expression of Arg-1, YM1, iNOS, IL-10, and IGF-1. Moreover, in contrast to BMDM/DC, both subsets of microglia express protective interferon-beta (IFNß), high levels of colony-stimulating factor-1 receptor, and do not express the Th1-associated transcription factor T-bet. Taken together, our data suggest that CD11c(+) microglia, CD11c(-) microglia, and infiltrating BMDM/DC represent separate and distinct populations and illustrate the heterogeneity of the CNS inflammatory environment.

Anti-PD-L1 prolongs survival and triggers T cell but not humoral anti-tumor immune responses in a human MUC1-expressing preclinical ovarian cancer model.

Monoclonal antibodies that block inhibitory immune checkpoint molecules and enhance anti-tumor responses show clinical promise in advanced solid tumors. Most of the preliminary evidence on therapeutic efficacy of immune checkpoint blockers comes from studies in melanoma, lung and renal cancer. To test the in vivo potential of programmed death-ligand 1 (PD-L1) blockade in ovarian cancer, we recently generated a new transplantable tumor model using human mucin 1 (MUC1)-expressing 2F8 cells. The MUC1 transgenic (MUC1.Tg) mice develop large number of intraperitoneal (IP) tumors following IP injection of 8 × 10(5) syngeneic 2F8 cells. The tumors are aggressive and display little T cell infiltration. Anti-PD-L1 antibody was administered IP every 2 weeks (200 µg/dose) for a total of three doses. Treatment was started 21 days post-tumor challenge, a time point which corresponds to late tumor stage. The anti-PD-L1 treatment led to substantial T cell infiltration within the tumor and significantly increased survival (p = 0.001) compared to isotype control-treated mice. When the same therapy was administered to wild-type mice challenged with 2F8 tumors, no survival benefit was observed, despite the presence of high titer anti-MUC1 antibodies. However, earlier treatment (day 11) and higher frequency of IP injections restored the T cell responses and led to prolonged survival. Splenocyte profiling via Nanostring using probes for 511 immune genes revealed a treatment-induced immune gene signature consistent with increased T cell-mediated immunity. These findings strongly support further preclinical and clinical strategies exploring PD-L1 blockade in ovarian cancer.

Chemokine receptor expression by inflammatory T cells in EAE.

Chemokines direct cellular infiltration to tissues, and their receptors and signaling pathways represent targets for therapy in diseases such as multiple sclerosis (MS). The chemokine CCL20 is expressed in choroid plexus, a site of entry of T cells to the central nervous system (CNS). The CCL20 receptor CCR6 has been reported to be selectively expressed by CD4(+) T cells that produce the cytokine IL-17 (Th17 cells). Th17 cells and interferon-gamma (IFNγ)-producing Th1 cells are implicated in induction of MS and its animal model experimental autoimmune encephalomyelitis (EAE). We have assessed whether CCR6 identifies specific inflammatory T cell subsets in EAE. Our approach was to induce EAE, and then examine chemokine receptor expression by cytokine-producing T cells sorted from CNS at peak disease. About 7% of CNS-infiltrating CD4(+) T cells produced IFNγ in flow cytometric cytokine assays, whereas less than 1% produced IL-17. About 1% of CD4(+) T cells produced both cytokines. CCR6 was expressed by Th1, Th1+17 and by Th17 cells, but not by CD8(+) T cells. CD8(+) T cells expressed CXCR3, which was also expressed by CD4(+) T cells, with no correlation to cytokine profile. Messenger RNA for IFNγ, IL-17A, and the Th1 and Th17-associated transcription factors T-bet and RORγt was detected in both CCR6(+) and CXCR3(+) CD4(+) T cells. IFNγ, but not IL-17A mRNA expression was detected in CD8(+) T cells in CNS. CCR6 and CD4 were co-localized in spinal cord infiltrates by double immunofluorescence. Consistent with flow cytometry data some but not all CD4(+) T cells expressed CCR6 within infiltrates. CD4-negative CCR6(+) cells included macrophage/microglial cells. Thus we have for the first time directly studied CD4(+) and CD8(+) T cells in the CNS of mice with peak EAE, and determined IFNγ and IL17 expression by cells expressing CCR6 and CXCR3. We show that neither CCR6 or CXCR3 align with CD4 T cell subsets, and Th1 or mixed Th1+17 predominate in EAE.