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1. IL-10 producing CD8+ CD122+ PD-1+ regulatory T cells are expanded by dendritic cells silenced for Allograft Inflammatory Factor-1

   https://doi.org/10.1002/JLB.1A0118-010RR  

   Elizondo, Diana M. ; Andargie, Temesgen E. ; Haddock, Naomi L. ; da Silva, Ricardo L. Louzada ; de Moura, Tatiana Rodrigues ; Lipscomb, Michael W. ( December 2018 , Journal of Leukocyte Biology)

   Allograft Inflammatory Factor-1 (AIF1) is a cytoplasmic scaffold protein that contains Ca2+ binding EF-hand and PDZ interaction domains important for mediating intracellular signaling complexes in immune cells. The protein plays a dominant role in both macrophage- and dendritic cell (DC)-mediated inflammatory responses. This study now reports that AIF1 expression in DC is important in directing CD8+ T cell effector responses. Silencing AIF1 expression in murine CD11c+ DC suppressed antigen-specific CD8+ T cell activation, marked by reduced CXCR3, IFNγ and Granzyme B expression, and restrained proliferation. These primed CD8+ T cells had impaired cytotoxic killing of target cells in vitro. In turn, studies identified that AIF1 silencing in DC robustly expanded IL-10 producing CD8+ CD122+ PD-1+ regulatory T cells that suppressed neighboring immune effector responses through both IL-10 and PD-1-dependent mechanisms. In vivo studies recapitulated bystander suppression of antigen-responsive CD4+ T cells by the CD8+ Tregs expanded from the AIF1 silenced DC. These studies further demonstrate that AIF1 expression in DC serves as a potent governor of cognate T cell responses and present a novel target for engineering tolerogenic DC-based immunotherapies.

   Adaptive immune responses are impaired in CD8+ T cells primed by DC silenced for AIF1.
2. Spatial distribution of B cells and lymphocyte clusters as a predictor of triple-negative breast cancer outcome

   https://doi.org/10.1038/s41523-021-00291-z  

   Wortman, Juliana C. ; He, Ting-Fang ; Solomon, Shawn ; Zhang, Robert Z. ; Rosario, Anthony ; Wang, Roger ; Tu, Travis Y. ; Schmolze, Daniel ; Yuan, Yuan ; Yost, Susan E. ; et al ( July 2021 , npj Breast Cancer)

   While tumor infiltration by CD8⁺T cells is now widely accepted to predict outcomes, the clinical significance of intratumoral B cells is less clear. We hypothesized that spatial distribution rather than density of B cells within tumors may provide prognostic significance. We developed statistical techniques (fractal dimension differences and a box-counting method ‘occupancy’) to analyze the spatial distribution of tumor-infiltrating lymphocytes (TILs) in human triple-negative breast cancer (TNBC). Our results indicate that B cells in good outcome tumors (no recurrence within 5 years) are spatially dispersed, while B cells in poor outcome tumors (recurrence within 3 years) are more confined. While most TILs are located within the stroma, increased numbers of spatially dispersed lymphocytes within cancer cell islands are associated with a good prognosis. B cells and T cells often form lymphocyte clusters (LCs) identified via density-based clustering. LCs consist either of T cells only or heterotypic mixtures of B and T cells. Pure B cell LCs were negligible in number. Compared to tertiary lymphoid structures (TLS), LCs have fewer lymphocytes at lower densities. Both types of LCs are more abundant and more spatially dispersed in good outcomes compared to poor outcome tumors. Heterotypic LCs in good outcome tumors aremore »smaller and more numerous compared to poor outcome. Heterotypic LCs are also closer to cancer islands in a good outcome, with LC size decreasing as they get closer to cancer cell islands. These results illuminate the significance of the spatial distribution of B cells and LCs within tumors.

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3. Dissecting the immunosuppressive tumor microenvironments in Glioblastoma-on-a-Chip for optimized PD-1 immunotherapy

   https://doi.org/10.7554/eLife.52253  

   Cui, Xin ; Ma, Chao ; Vasudevaraja, Varshini ; Serrano, Jonathan ; Tong, Jie ; Peng, Yansong ; Delorenzo, Michael ; Shen, Guomiao ; Frenster, Joshua ; Morales, Renee-Tyler Tan ; et al ( September 2020 , eLife)

   Programmed cell death protein-1 (PD-1) checkpoint immunotherapy efficacy remains unpredictable in glioblastoma (GBM) patients due to the genetic heterogeneity and immunosuppressive tumor microenvironments. Here, we report a microfluidics-based, patient-specific ‘GBM-on-a-Chip’ microphysiological system to dissect the heterogeneity of immunosuppressive tumor microenvironments and optimize anti-PD-1 immunotherapy for different GBM subtypes. Our clinical and experimental analyses demonstrated that molecularly distinct GBM subtypes have distinct epigenetic and immune signatures that may lead to different immunosuppressive mechanisms. The real-time analysis in GBM-on-a-Chip showed that mesenchymal GBM niche attracted low number of allogeneic CD154+CD8+ T-cells but abundant CD163+ tumor-associated macrophages (TAMs), and expressed elevated PD-1/PD-L1 immune checkpoints and TGF-β1, IL-10, and CSF-1 cytokines compared to proneural GBM. To enhance PD-1 inhibitor nivolumab efficacy, we co-administered a CSF-1R inhibitor BLZ945 to ablate CD163+ M2-TAMs and strengthened CD154+CD8+ T-cell functionality and GBM apoptosis on-chip. Our ex vivo patient-specific GBM-on-a-Chip provides an avenue for a personalized screening of immunotherapies for GBM patients.
4. Experimental colitis promotes sustained, sex-dependent, T-cell-associated neuroinflammation and parkinsonian neuropathology

   https://doi.org/10.1186/s40478-021-01240-4  

   Houser, Madelyn C. ; Caudle, W. Michael ; Chang, Jianjun ; Kannarkat, George T. ; Yang, Yuan ; Kelly, Sean D. ; Oliver, Danielle ; Joers, Valerie ; Shannon, Kathleen M. ; Keshavarzian, Ali ; et al ( August 2021 , Acta Neuropathologica Communications)

   The etiology of sporadic Parkinson’s disease (PD) remains uncertain, but genetic, epidemiological, and physiological overlap between PD and inflammatory bowel disease suggests that gut inflammation could promote dysfunction of dopamine-producing neurons in the brain. Mechanisms behind this pathological gut-brain effect and their interactions with sex and with environmental factors are not well understood but may represent targets for therapeutic intervention.

   We sought to identify active inflammatory mechanisms which could potentially contribute to neuroinflammation and neurological disease in colon biopsies and peripheral blood immune cells from PD patients. Then, in mouse models, we assessed whether dextran sodium sulfate-mediated colitis could exert lingering effects on dopaminergic pathways in the brain and whether colitis increased vulnerability to a subsequent exposure to the dopaminergic neurotoxicant 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). We assessed the involvement of inflammatory mechanisms identified in the PD patients in colitis-related neurological dysfunction in male and female mice, utilizing mice lacking the Regulator of G-Protein Signaling 10 (RGS10)—an inhibitor of nuclear factor kappa B (NFκB)—to model enhanced NFκB activity, and mice in which CD8⁺T-cells were depleted.

   High levels of inflammatory markers includingCD8Band NFκB p65 were found in colon biopsies from PD patients, and reduced levels of RGS10 were found in immune cells inmore »the blood. Male mice that experienced colitis exhibited sustained reductions in tyrosine hydroxylase but not in dopamine as well as sustained CD8⁺T-cell infiltration and elevatedIfngexpression in the brain. CD8⁺T-cell depletion prevented colitis-associated reductions in dopaminergic markers in males. In both sexes, colitis potentiated the effects of MPTP. RGS10 deficiency increased baseline intestinal inflammation, colitis severity, and neuropathology.

   This study identifies peripheral inflammatory mechanisms in PD patients and explores their potential to impact central dopaminergic pathways in mice. Our findings implicate a sex-specific interaction between gastrointestinal inflammation and neurologic vulnerability that could contribute to PD pathogenesis, and they establish the importance of CD8⁺T-cells in this process in male mice.

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5. Quantifying the efficacy of checkpoint inhibitors on CD8+ cytotoxic T cells for immunotherapeutic applications via single-cell interaction

   https://doi.org/10.1038/s41419-020-03173-7  

   Sullivan, Matthew Ryan ; Ugolini, Giovanni Stefano ; Sarkar, Saheli ; Kang, Wenjing ; Smith, Evan Carlton ; Mckenney, Seamus ; Konry, Tania ( November 2020 , Cell Death & Disease)

   The inhibition of the PD1/PDL1 pathway has led to remarkable clinical success for cancer treatment in some patients. Many, however, exhibit little to no response to this treatment. To increase the efficacy of PD1 inhibition, additional checkpoint inhibitors are being explored as combination therapy options. TSR-042 and TSR-033 are novel antibodies for the inhibition of the PD1 and LAG3 pathways, respectively, and are intended for combination therapy. Here, we explore the effect on cellular interactions of TSR-042 and TSR-033 alone and in combination at the single-cell level. Utilizing our droplet microfluidic platform, we use time-lapse microscopy to observe the effects of these antibodies on calcium flux in CD8⁺T cells upon antigen presentation, as well as their effect on the cytotoxic potential of CD8⁺T cells on human breast cancer cells. This platform allowed us to investigate the interactions between these treatments and their impacts on T-cell activity in greater detail than previously applied in vitro tests. The novel parameters we were able to observe included effects on the exact time to target cell killing, contact times, and potential for serial-killing by CD8⁺T cells. We found that inhibition of LAG3 with TSR-033 resulted in a significant increase in calcium fluctuations ofmore »CD8⁺T cells in contact with dendritic cells. We also found that the combination of TSR-042 and TSR-033 appears to synergistically increase tumor cell killing and the single-cell level. This study provides a novel single-cell-based assessment of the impact these checkpoint inhibitors have on cellular interactions with CD8⁺T cells.

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