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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.

    

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2. 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)

   null (Ed.)

   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. 

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3. 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 are 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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4. Development and Optimization of a Novel Centrifugal Bioreactor with a Real-Time Monitoring Sensor for T Cell Exhaustion with Applications in Cancer Immunotherapy

   Brenden Fraser-Hevlin, Kitana M. ( November 2021 , Annual meeting American Institute of Chemical Engineers)

   Cancer has been one of the most significant and critical challenges in the field of medicine. It is a leading cause of death both in the United States and worldwide. Common cancer treatments such as radiation and chemotherapy can be effective in destroying cancerous tissue but cause many detrimental side effects. Thus, recent years have seen new treatment methods that do not harm healthy tissue, including immunotherapy. Adoptive cell therapy (ACT) is one form of immunotherapy in which patients’ immune cells are modified to target cancer cells and then reintroduced into the body. ACT is promising, but most current treatments are inefficient and costly. Widespread implementation of ACT has been a difficult task due to the high treatment cost and inefficient methods currently used to expand the cells. Additionally, if the manufacturing process is not carefully controlled, it can result in the cells losing their cancer-killing ability after expansion. To address the need for an economically feasible culture process to expand immune cells for immunotherapy, our laboratory has designed a centrifugal bioreactor (CBR) expansion system. The CBR uses a balance of centrifugal forces and fluid forces, as shown in Figure 1, to quickly expand infected CD8+ T-cells from a bovine model up to high population densities. With other applications, the CBR has achieved cell densities as high as 1.8 x 108 cells/mL over 7 days in an 11.4-mL chamber. For this study, our goal is to begin validating the CBR by optimizing the growth of CEM (human lymphoblastic leukemia) cells, which are similar cell to cytotoxic T lymphocytes (CTLs). This can be accomplished by measuring kinetic growth parameters based on the concentrations of glucose and inhibitory metabolites in the culture. We hypothesize that by designing a kinetic model from static culture experiments, we can predict the parameters necessary to achieve peak CEM and eventually CTL growth in the CBR. We will report on kinetic growth studies in which different glucose concentrations are tested, and a maximum specific growth rate and Monod constant determined, as well as studies where varying levels of the inhibitory growth byproducts, lactate and ammonium, are added to the culture and critical inhibitor concentrations are determined. Another recent conceptual development for the design of the CBR is a real-time monitoring and feedback control system to regulate the cellular environment, based on levels of surface co-receptors and mRNA signaling within the culture. Prior studies have pinpointed T cell exhaustion as a significant issue in achieving successful immunotherapy, particularly in treatments for solid tumors; T cell exhaustion occurs during a period of chronic antigen stimulation when the cells lose their ability to target and kill cancer cells, currently theorized to be associated with particular inhibitory receptors and cytokines in the immune system. Designing a system with a fiber optic sensor that can monitor the cell state and use feedback control to regulate the pathways involved in producing these receptors will ensure the cells maintain cytotoxic properties during the expansion process within a Centrifugal Fluidized Expansion we call the CentriFLEX. In this presentation, we will also report on early results from development of this exhaustion monitoring system. In brief, achieving optimal kinetic models for the CBR system and methods to prevent T cell exhaustion has the potential to significantly enhance culture efficiency and availability of immunotherapy treatments. 

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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 of 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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