The small GTPase RhoA and its downstream effectors are critical regulators in the pathophysiological pro¬cesses of asthma. The underlying mechanism, however, remains undetermined. Here, we generated asthma mouse model with RhoA conditional knockout mice (Sftpc-cre;RhoAf/f) in type II alveolar epithelial cells (AT2) and demonstrated that AT2 cell specific deletion of RhoA leads to exacerbation of allergen-induced airway hyper-responsiveness and airway inflammation with elevated Th2 cytokines in bronchoalveolar lavage fluid (BALF). Notably, Sftpc-cre;RhoAf/f mice showed a significant reduction in TGF-β1 levels in BALFs and lung tissues, and administration of recombinant TGF-β1 to the mice rescued TGF-β1 and alleviated the increased allergic airway inflammation observed in Sftpc-cre;RhoAf/f mice. Using RNA-seq technology, we identified Slc26a4 (pendrin), a transmembrane anion exchange, as the most up-regulated gene in RhoA-deficient AT2 cells. The up-regulation of SLC26A4 was further confirmed in AT2 cells of asthmatic patients and mouse model and in human airway epithelial cells expressing dominant- negative RhoA (RhoA-N19). SLA26A4 was also elevated in serum from asthmatic patients and negatively associated with FEV1%. Furthermore, SLC26A4 inhibitor promoted epithelial TGF-β1 release and attenuated allergic airway inflammation. Our study reveals a previously undefined RhoA-SLC26A4 axis in AT2 cells that functions as a protective mechanism against allergic airway inflammation.
Danh C. Do, Yan Zhang, Wei Tu, Xinyue Hu, Xiaojun Xiao, Jingsi Chen, Haiping Hao, Zhigang Liu, Jing Li, Shau-Ku Huang, Mei Wan, Peisong Gao
Agonist CD40 antibodies are under clinical development in combination with chemotherapy as an approach to prime for anti-tumor T cell immunity. However, treatment with anti-CD40 is commonly accompanied by both systemic cytokine release and liver transaminase elevations which together account for the most common dose-limiting toxicities. Moreover, anti-CD40 treatment increases the potential for chemotherapy-induced hepatotoxicity. Here, we report a mechanistic link between cytokine release and hepatotoxicity induced by anti-CD40 when combined with chemotherapy and show that toxicity can be suppressed without impairing therapeutic efficacy. We demonstrate in mice and humans that anti-CD40 triggers transient hepatotoxicity marked by increased serum transaminase levels. In doing so, anti-CD40 sensitizes the liver to drug-induced toxicity. Unexpectedly, this biology is not blocked by depletion of multiple myeloid cell subsets, including macrophages, inflammatory monocytes, and granulocytes. Transcriptional profiling of the liver after anti-CD40 revealed activation of multiple cytokine pathways including TNF and interleukin (IL)-6. Neutralization of TNF, but not IL-6, prevented sensitization of the liver to hepatotoxicity induced with anti-CD40 in combination with chemotherapy without impacting anti-tumor efficacy. Our findings reveal a clinically feasible approach to mitigate toxicity without impairing efficacy in the use of agonist CD40 antibodies for cancer immunotherapy.
Meredith L. Stone, Jesse Lee, Veronica M. Herrera, Kathleen Graham, Jae W. Lee, Austin Huffman, Heather Coho, Evan Tooker, Max I. Myers, Michael Giannone, Yan Li, Thomas H. Buckingham, Kristen B. Long, Gregory L. Beatty
Existing patient-derived-xenograft (PDX) mouse models of solid tumors lack a fully tumor-donor matched, syngeneic, and functional immune system. We developed such a model by engrafting lymphopenic recipient mice with a fresh, undisrupted piece of solid tumor, whereby tumor-infiltrating lymphocytes (TILs) persisted in the recipient mice for several weeks. Successful tumor engraftment was achieved in eighty-three to eighty-nine percent of tumor-infiltrating-lymphocytes-PDX (TIL-PDX) mice, and these were seen to harbor exhausted immuno-effector as well as functional immuno-regulatory cells persisting for at least six months post-engraftment. Combined treatment with interleukin-15 (IL-15) stimulation and immune checkpoint inhibition (ICI) resulted in complete or partial tumor response in this model. Further, depletion of Cytotoxic T-lymphocytes (CTLs) and/or Natural Killer (NK) cells before combined immunotherapy revealed that both cell types were required for maximal tumor regression. Our novel TIL-PDX model provides a valuable resource for powerful mechanistic and therapeutic studies in solid tumors.
Duy T. Le, Tridu R. Huynh, Bryan M. Burt, George Van Buren, Shawn A. Abeynaike, Cristina Zalfa, Rana Nikzad, Farrah Kheradmand, John J. Tyner, Silke Paust
The skin lesion erythema migrans (EM) is an initial sign of the Ixodes-tick transmitted Borreliella spirochetal infection known as Lyme disease. T cells and innate immune cells have previously been shown to predominate the EM lesion and promote the reaction. Despite the established importance of B cells and antibodies in preventing infection, the role of B cells in the skin immune response to Borreliella is unknown. Here, we used single-cell RNA-Seq in conjunction with B cell receptor (BCR) sequencing to immunophenotype EM lesions and their associated B cells and BCR repertoires. We found that B cells were more abundant in EM in comparison to autologous uninvolved skin; many were clonally expanded and had circulating relatives. EM-associated B cells upregulated expression of MHC class II genes and exhibited preferential IgM isotype usage. A subset also exhibited low levels of somatic hypermutation despite a gene expression profile consistent with memory B cells. Our study demonstrates that single-cell gene expression with paired BCR sequencing can be used to interrogate the sparse B cell populations in human skin and reveals that B cells in the skin infection site in early Lyme disease express a phenotype consistent with local antigen presentation and antibody production.
Ruoyi Jiang, Hailong Meng, Khadir Raddassi, Ira Fleming, Kenneth B. Hoehn, Kenneth R. Dardick, Alexia A. Belperron, Ruth R. Montgomery, Alex K. Shalek, David A. Hafler, Steven H. Kleinstein, Linda K. Bockenstedt
The presence of an immunosuppressive tumor microenvironment is a major obstacle in the success of cancer immunotherapies. Because extracellular matrix components can shape the microenvironment, we investigated the role of matrix metalloproteinase 2 (MMP2) in melanoma tumorigenesis. Significantly, we found that MMP2 signals pro-inflammatory pathways on antigen presenting cells which requires both toll-like receptor (TLR) 2 and TLR4. B16 melanoma cells that express MMP2 at baseline have slower kinetics in Tlr2-/-Tlr4-/- mice, implicating MMP2 in promoting tumor growth. Indeed, Mmp2 overexpression in B16 cells potentiated rapid tumor growth which was accompanied by reduced intra-tumoral cytolytic cells and increased M2 macrophages. In contrast, knockdown of Mmp2 slowed tumor growth, and enhanced T cell proliferation and NK cell recruitment. Finally we found that these effects of MMP2 are mediated through dysfunctional dendritic cell (DC) - T cell cross-talk as they are lost in Batf3-/- and Rag2-/- mice, respectively. These findings provide insights into the detrimental role of endogenous alarmins like MMP2 in modulating immune responses in the tumor microenvironment.
Luciana R. Muniz-Bongers, Christopher B. McClain, Mansi Saxena, Gerold Bongers, Miriam Merad, Nina Bhardwaj
Recent advances in high-throughput T cell receptor (TCR) sequencing have allowed for new insights into the human TCR repertoire. However, methods for capturing antigen-specific repertoires remain an area of development. Here, we describe a potentially novel approach that utilizes both a biological and statistical enrichment to define putatively antigen-specific complementarity-determining region 3 (CDR3) repertoires in unselected individuals. The biological enrichment entails fluorescence-activated cell sorting of in vitro antigen-activated memory CD4+ T cells, followed by TCRβ sequencing. The resulting TCRβ sequences are then filtered by selecting those that are statistically enriched when compared to their frequency in the autologous resting T cell compartment. Applying this method to define putatively peanut protein-specific repertoires in 27 peanut-allergic individuals resulted in a library of 7345 unique CDR3β amino acid sequences that had similar characteristics to other validated antigen-specific repertoires in terms of homology and diversity. In-depth analysis of these CDR3βs revealed 36 public sequences that demonstrated high levels of convergent recombination. In a network analysis, the public CDR3βs unveiled themselves as core sequences with more edges than their private counterparts. This method has the potential to be applied to a wide range of T cell-mediated disorders, and to yield new biomarkers and biological insights.
Neal P. Smith, Bert Ruiter, Yamini V. Virkud, Ang A. Tu, Brinda Monian, James J. Moon, J. Christopher Love, Wayne G. Shreffler
T cell receptor (TCR) stimulation leads to expression of the transcription factor TOX. Prolonged TCR signaling, such as encountered during chronic infections or in tumors, leads to sustained TOX expression, which is required for the induction of a state of exhaustion or dysfunction. While CD8 memory T cells (Tmem) in mice typically do not express TOX at steady state, some human Tmem express TOX, but appear fully functional. This seeming discrepancy between mouse and human T cells has led to the speculation that TOX is differentially regulated between these species, which could complicate the interpretation of pre-clinical mouse model studies. We report here that similarly to TCR-mediated signals, inflammatory cytokines are also sufficient to increase TOX expression in human and mouse Tmem. Thus, TOX expression is controlled by the environment, which provides an explanation for the different TOX expression patterns encountered in T cells isolated from specific pathogen free laboratory mice versus humans. Finally, we report that TOX is not necessary for cytokine-driven expression of PD-1. Overall, our data highlight that the mechanisms regulating TOX expression are conserved across species and indicate that TOX expression reflects a T cell’s activation state, and does not necessarily correlate with T cell dysfunction.
Nicholas J. Maurice, Jacqueline Berner, Alexis K. Taber, Dietmar Zehn, Martin Prlic
Alloimmune responses driven by donor-specific antibodies (DSAs) can lead to antibody-mediated rejection (ABMR) in organ transplantation. Yet, the cellular states underlying alloreactive B cell responses and the molecular components controlling them remain unclear. Using high dimensional profiling of B cells in a cohort of 96 kidney transplant recipients, we identified expanded numbers of CD27+CD21- activated memory (AM) B cells that expressed the transcription factor T-bet in patients who developed DSAs and progressed to ABMR. Notably, AM cells were less frequent in DSA+ABMR- patients and at baseline levels in DSA- patients. RNA-seq analysis of AM cells in patients undergoing ABMR revealed these cells to be poised for plasma cell differentiation and to express restricted IGHV sequences reflective of clonal expansion. In addition to T-bet, AM cells manifested elevated expression of IRF4 and Blimp1, and upon co-culture with autologous T follicular helper cells, differentiated into DSA-producing plasma cells in an IL-21 dependent manner. The frequency of AM cells was correlated with the timing and severity of ABMR manifestations. Importantly, T-bet+ AM cells were detected within kidney allografts along with their restricted IGHV sequences. This study delineates a pivotal role for AM cells in promoting humoral responses and ABMR in organ transplantation and highlights them as important therapeutic targets.
Kevin Louis, Elodie Bailly, Camila Macedo, Louis Lau, Bala Ramaswami, Alexander Chang, Uma Chandran, Douglas Landsittel, Xinyan Gu, Geetha Chalasani, Adriana Zeevi, Parmjeet Randhawa, Harinder Singh, Carmen Lefaucheur, Diana Metes
Tissue-resident memory T cells (TRM) provide frontline defense against infectious diseases and contribute to antitumor immunity; however, aside from the necessity of TGF-β, knowledge regarding TRM-inductive cues remains incomplete, particularly for human cells. Oxygen tension is an environmental cue that distinguishes peripheral tissues from the circulation, and here, we demonstrate that differentiation of human CD8+ T cells in the presence of hypoxia and TGF-β1 led to the development of a TRM phenotype, characterized by a greater than 5-fold increase in CD69+CD103+ cells expressing human TRM hallmarks and enrichment for endogenous human TRM gene signatures, including increased adhesion molecule expression and decreased expression of genes involved in recirculation. Hypoxia and TGF-β1 synergized to produce a significantly larger population of TRM phenotype cells than either condition alone, and comparison of these cells from the individual and combination conditions revealed distinct phenotypic and transcriptional profiles, indicating a programming response to milieu rather than a mere expansion. Our findings identify a likely previously unreported cue for the TRM differentiation program and can enable facile generation of human TRM phenotype cells in vitro for basic studies and translational applications such as adoptive cellular therapy.
Farah Hasan, Yulun Chiu, Rebecca M. Shaw, Junmei Wang, Cassian Yee
Haploidentical hematopoietic stem cell transplantation (h-HSCT) represents an efficient curative approach for patients affected by hematologic malignancies in which the reduced intensity conditioning induces a state of immunologic tolerance between donor and recipient. However, opportunistic viral infections greatly affect h-HSCT clinical outcomes. Natural Killer (NK) cells are the first lymphocytes recovering after transplant and provide a prompt defense against human Cytomegalovirus (HCMV) infection/reactivation. By undertaking a longitudinal single cell computational profiling of multiparametric flow cytometry, we show that HCMV accelerates NK cell immune-reconstitution together with the expansion of CD158b1b2jpos/NKG2Aneg/NKG2Cpos/NKp30low NK cells. The frequency of this subset correlates with HCMV viremia, further increases in recipients experiencing multiple episodes of viral reactivations and persists for months after the infection. The transcriptional profile of FACS-sorted CD158b1b2jpos NK cells confirmed the ability of HCMV to de-regulate NKG2C, NKG2A and NKp30 gene expression, thus inducing the expansion of NK cells with adaptive traits. These NK cells are characterized by the down-modulation of several gene pathways associated with cell migration, cell-cycle, effector-functions and by a state of metabolic/cellular exhaustion. This profile reflects the functional impairments of adaptive NK cells to produce IFN-γ, a phenomenon also due to the viral-induced expression of LAG-3 and PD-1 checkpoint-inhibitors.
Elisa Zaghi, Michela Calvi, Simone Puccio, Gianmarco Spata, Sara Terzoli, Clelia Peano, Alessandra Roberto, Federica De Paoli, Jasper J.P. van Beek, Jacopo Mariotti, Chiara De Philippis, Barbara Sarina, Rossana Mineri, Stefania Bramanti, Armando Santoro, Vu Thuy Khanh Le-Trilling, Mirko Trilling, Emanuela Marcenaro, Luca Castagna, Clara Di Vito, Enrico Lugli, Domenico Mavilio
No posts were found with this tag.