Immune checkpoint inhibitor (ICI) treatment has recently become a first-line therapy for many non–small cell lung cancer (NSCLC) patients. Unfortunately, most NSCLC patients are refractory to ICI monotherapy, and initial attempts to address this issue with secondary therapeutics have proven unsuccessful. To identify entities precluding CD8+ T cell accumulation in this process, we performed unbiased analyses on flow cytometry, gene expression, and multiplexed immunohistochemical data from a NSCLC patient cohort. The results revealed the presence of a myeloid-rich subgroup, which was devoid of CD4+ and CD8+ T cells. Of all myeloid cell types assessed, neutrophils were the most highly associated with the myeloid phenotype. Additionally, the ratio of CD8+ T cells to neutrophils (CD8/PMN) within the tumor mass optimally distinguished between active and myeloid cases. This ratio was also capable of showing the separation of patients responsive to ICI therapy from those with stable or progressive disease in 2 independent cohorts. Tumor-bearing mice treated with a combination of anti-PD1 and SX-682 (CXCR1/2 inhibitor) displayed relocation of lymphocytes from the tumor periphery into a malignant tumor, which was associated with induction of IFN-γ–responsive genes. These results suggest that neutrophil antagonism may represent a viable secondary therapeutic strategy to enhance ICI treatment outcomes.
Julia Kargl, Xiaodong Zhu, Huajia Zhang, Grace H. Y. Yang, Travis J. Friesen, Melissa Shipley, Dean Y. Maeda, John A. Zebala, Jill McKay-Fleisch, Gavin Meredith, Afshin Mashadi-Hossein, Christina Baik, Robert H. Pierce, Mary W. Redman, Jeffrey C. Thompson, Steven M. Albelda, Hamid Bolouri, A. McGarry Houghton
BACKGROUND. The cytokine IL-7 is critical for T cell development and function. We performed a Phase Ib study in patients with type 1 diabetes (T1D) to evaluate how blockade of IL-7 would affect immune cells and relevant clinical responses. METHODS. Thirty-seven subjects with T1D received s.c. RN168, a monoclonal antibody that blocks the IL -7 receptor α (IL7Rα) in a dose-escalating study. RESULTS. Between 90% and 100% IL-7R occupancy and near-complete inhibition of pSTAT5 was observed at doses of RN168 1 mg/kg every other week (Q2wk) and greater. There was a significant decline in CD4+ and CD8+ effector and central memory T cells and CD4+ naive cells, but there were fewer effects on CD8+ naive T cells. The ratios of Tregs to CD4+ or CD8+ effector and central memory T cells versus baseline were increased. RNA sequencing analysis showed downmodulation of genes associated with activation, survival, and differentiation of T cells. Expression of the antiapoptotic protein Bcl-2 was reduced. The majority of treatment-emergent adverse events (TEAEs) were mild and not treatment related. Four subjects became anti–EBV IgG+ after RN168, and 2 had symptoms of active infection. The immunologic response to tetanus toxoid was preserved at doses of 1 and 3 mg/kg Q2wk but reduced at higher doses. CONCLUSIONS. This trial shows that, at dosages of 1–3 mg/kg, RN168 selectively inhibits the survival and activity of memory T cells while preserving naive T cells and Tregs. These immunologic effects may serve to eliminate pathologic T cells in autoimmune diseases. TRIAL REGISTRATION. NCT02038764. FUNDING. Pfizer Inc.
Kevan C. Herold, Samantha L. Bucktrout, Xiao Wang, Bruce W. Bode, Stephen E. Gitelman, Peter A. Gottlieb, Jing Hughes, Tenshang Joh, Janet B. McGill, Jeremy H. Pettus, Shobha Potluri, Desmond Schatz, Megan Shannon, Chandrasekhar Udata, Gilbert Wong, Matteo Levisetti, Bishu J. Ganguly, Pamela D. Garzone, the RN168 Working Group
Distinct subsets of Tregs reside in nonlymphoid tissues where they mediate unique functions. To interrogate the biology of tissue Tregs in human health and disease, we phenotypically and functionally compared healthy skin Tregs with those in peripheral blood, inflamed psoriatic skin, and metastatic melanoma. The mitochondrial enzyme, arginase 2 (ARG2), was preferentially expressed in Tregs in healthy skin, increased in Tregs in metastatic melanoma, and reduced in Tregs from psoriatic skin. ARG2 enhanced Treg suppressive capacity in vitro and conferred a selective advantage for accumulation in inflamed tissues in vivo. CRISPR-mediated deletion of this gene in primary human Tregs was sufficient to skew away from a tissue Treg transcriptional signature. Notably, the inhibition of ARG2 increased mTOR signaling, whereas the overexpression of this enzyme suppressed it. Taken together, our results suggest that Tregs express ARG2 in human tissues to both regulate inflammation and enhance their metabolic fitness.
Margaret M. Lowe, Ian Boothby, Sean Clancy, Richard S. Ahn, Wilson Liao, David N. Nguyen, Kathrin Schumann, Alexander Marson, Kelly M. Mahuron, Gillian A. Kingsbury, Zheng Liu, Priscila Munoz Sandoval, Robert Sanchez Rodriguez, Mariela L. Pauli, Keyon Taravati, Sarah T. Arron, Isaac M. Neuhaus, Hobart W. Harris, Esther A. Kim, Uk Sok Shin, Matthew F. Krummel, Adil Daud, Tiffany C. Scharschmidt, Michael D. Rosenblum
Pancreatic ductal adenocarcinoma (PDAC) has dismal five-year survival (<9%). We hypothesize that exposure of tumors to conventional therapies may preferentially modulate immune biomarkers in the tumor microenvironment in PDAC. PDAC patients who underwent upfront surgical resection or who received neoadjuvant FOLFIRINOX with or without neoadjuvant radiotherapy followed by surgical resection were selected for study. Total expression of immunologically relevant transcripts and spatially resolved expression of immunologically relevant proteins was quantitated using multiplexed methods (Nanostring nCounter and GeoMX platforms). This analysis identified numerous differentially expressed transcripts associated with the type of neoadjuvant therapy received. Moreover, we identified significant alterations in the expression and/or spatial distribution of immunologically relevant proteins in different regions (tumor cell rich, immune cell rich, stromal cell rich) of the TME. These data provide insight into the immunological effects of clinically relevant neoadjuvant therapy for resectable/borderline-resectable PDAC, by describing significant differences in the expression of key immunologic biomarkers within the PDAC microenvironment that were associated with the type of treatment patients received prior to surgical resection. This represents a comprehensive analysis of numerous biomarkers conducted on the PDAC microenvironment. This work may guide strategic new combination therapies for pancreatic cancer.
Matthew R. Farren, Layal Sayegh, Michael Brandon Ware, Hsiao-Rong Chen, Jingjing Gong, Yan Liang, Alyssa Krasinskas, Shishir K. Maithel, Mohammad Zaidi, Juan M. Sarmiento, David Kooby, Pretesh Patel, Bassel El-Rayes, Walid Shaib, Gregory B. Lesinski
Subpial demyelination is a specific hallmark of multiple sclerosis (MS) and a correlate of disease progression. Although the mechanism(s) that mediate pathogenesis in the subpial compartment remain unclear, it has been speculated that inflammation in the overlying meninges may be associated with subpial injury. Here we show that adoptive transfer of proteolipid protein-primed Th17 cells into SJL/J recipient mice induces subpial demyelination associated with microglial/macrophage activation, disruption of the glial limitans and evidence of an oxidative stress response. This pathology was topologically associated with foci of immune cells in the meninges and occurred in the absence of measurable anti-MOG IgM or IgG antibodies. To test the role of brain-infiltrating leukocytes on subpial injury, we modulated sphingosine 1-phosphate (S1P) receptor1,5 activity with BAF312 (siponimod) treatment. Administration of BAF312, even after adoptively transferred T cells had entered the brain, significantly ameliorated clinical EAE and diminished subpial pathology, concomitant with a selective reduction in the capacity of transferred T cells to make Th17 cytokines. We conclude that sustained subpial cortical injury is associated with the capacity for brain-resident T cells to produce Th17 cytokines, and this pathological process occurs in an S1P receptor1,5-dependent manner.
Lesley A. Ward, Dennis S.W. Lee, Anshu Sharma, Angela Wang, Ikbel Naouar, Xianjie I. Ma, Natalia Pikor, Barbara Nuesslein-Hildesheim, Valeria Ramaglia, Jennifer L. Gommerman
Overexpression and long terminal repeat (LTR) polymorphism of the HRES-1/Rab4 human endogenous retrovirus locus have been associated with T-cell activation and disease manifestations in systemic lupus erythematosus (SLE). Although genomic DNA methylation is overall diminished in SLE, its role in HRES-1/Rab4 expression is unknown. Therefore, we determined how lupus-associated polymorphic rs451401 alleles of the LTR regulate transcription from the HRES-1/Rab4 promoter and thus impact T-cell activation. The results show that cytosine-119 is hypermethylated while cytosine-51 of the promoter and the LTR-enhancer are hypomethylated in SLE. Pharmacological or genetic inactivation of DNA-methyltransferase-1 augmented the expression of HRES-1/Rab4. The minimal promoter was selectively recognized by metabolic stress sensor NRF1 when cytosine-119 but not cytosine-51 was methylated, and NRF1 stimulated HRES-1/Rab4 expression in human T cells. In turn, IRF2 and PSIP1 bound to the LTR-enhancer and exerted control over HRES-1/Rab4 expression in rs451401-genotype and methylation-dependent manners. The LTR-enhancer conferred markedly greater expression of HRES-1/Rab4 in subjects with rs451401CC over rs451401GG alleles that in turn promoted mechanistic target of rapamycin (mTOR) activation upon T-cell receptor stimulation. HRES-1/Rab4 alone robustly activated mTOR in human T cells. These findings identify HRES-1/Rab4 as a methylation and rs451401 allele-dependent transducer of environmental stress and controller of T-cell activation.
Aparna Godavarthy, Ryan Kelly, John Jimah, Miguel Beckford, Tiffany Caza, David Fernandez, Nick Huang, Manuel Duarte, Joshua Lewis, Hind J. Fadel, Eric M. Poeschla, Katalin Banki, Andras Perl
NK cells contribute to protective antitumor immunity, but little is known about the functional states of NK cells in human solid tumors. To address this issue, we performed single-cell RNA-seq analysis of NK cells isolated from human melanoma metastases, including lesions from patients who had progressed following checkpoint blockade. This analysis identified major differences in the transcriptional programs of tumor-infiltrating compared with circulating NK cells. Tumor-infiltrating NK cells represented 7 clusters with distinct gene expression programs indicative of significant functional specialization, including cytotoxicity and chemokine synthesis programs. In particular, NK cells from 3 clusters expressed high levels of XCL1 and XCL2, which encode 2 chemokines known to recruit XCR1+ cross-presenting DCs into tumors. In contrast, NK cells from 2 other clusters showed a higher level of expression of cytotoxicity genes. These data reveal key features of NK cells in human tumors and identify NK cell populations with specialized gene expression programs.
Lucas Ferrari de Andrade, Yuheng Lu, Adrienne Luoma, Yoshinaga Ito, Deng Pan, Jason W. Pyrdol, Charles H. Yoon, Guo-Cheng Yuan, Kai W. Wucherpfennig
Background: Serological tools for the accurate detection of recent malaria exposure are needed to guide and monitor malaria control efforts. IgG responses against P. vivax and P. falciparum merozoite surface protein-10 (MSP10) were measured as a potential way to identify recent malaria exposure in the Peruvian Amazon. Methods: A field-based study included 470 participants in a longitudinal cohort who completed a comprehensive evaluation: light microscopy and PCR on enrollment; at least one monthly follow-up by light microscopy; a second PCR; and serum and dried blood spots for serological analysis at the end of the follow-up. IgG titers against novel mammalian cell-produced recombinant PvMSP10 and PfMSP10 were determined by ELISA. Results: During the follow-up period, 205 participants were infected, including 171 with P. vivax, 26 with P. falciparum, 6 with infections by both species but at different times, and 2 with mixed infections. Exposure to P. vivax was more accurately identified when serological responses to PvMSP10 were obtained from serum (sensitivity, 58.1%; specificity, 81.8%; AUC: 0.76) than from dried blood spots (sensitivity, 35.2; specificity, 83.5%; AUC: 0.64) (PAUC < 0.001). Sensitivity was highest (serum, 82.9%; dried blood spot, 45.7%) with confirmed P. vivax infections occurring 7-30 days before sample collection; sensitivity decreased significantly in relation to time since last documented infection. PvMSP10 serological data did not show evidence of inter-species cross-reactivity. Anti-PfMSP10 responses poorly discriminated between P. falciparum exposed- and non-exposed individuals (AUC = 0.59, P > 0.05). Conclusions: Anti-PvMSP10 IgG indicates recent exposure to P. vivax at the population level in the Amazon region. Serum, not dried blood spots, should be used for such serological tests.
Angel Rosas-Aguirre, Kailash P. Patra, Maritza Calderón, Katherine Torres, Dionicia Gamboa, Edith Arocutipa, Edith Málaga, Katherine Garro, Carlos Fernández, Grace Trompeter, Yossef Alnasser, Alejandro Llanos-Cuentas, Robert H. Gilman, Joseph M. Vinetz
As sufficient extracellular arginine is crucial for T cell function, depletion of extracellular arginine by elevated Arginase 1 (Arg1) activity has emerged as a hallmark immunosuppressive mechanism. However, the potential cell-autonomous roles of arginases in T cells have remained unexplored. Here we show that the arginase isoform expressed by T cells, the mitochondrial Arginase 2 (Arg2), is a cell-intrinsic regulator of CD8+ T cell activity. Both germ-line Arg2 deletion and adoptive transfer of Arg2-/- CD8+ T cells significantly reduced tumor growth in preclinical cancer models by enhancing CD8+ T cell activation, effector function and persistence. Transcriptomic, proteomic and high-dimensional flow cytometry characterization revealed a CD8+ T cell-intrinsic role of Arg2 in modulating T cell activation, anti-tumor cytoxicity and memory formation, independently of extracellular arginine availability. Furthermore, specific deletion of Arg2 in CD8+ T cells strongly synergized with PD-1 blockade for the control of tumor growth and animal survival. These observations coupled with the finding that pharmacologic arginase inhibition accelerates activation of ex vivo human T cells unveil Arg2 as a new therapeutic target for T cell-based cancer therapies.
Adrià-Arnau Martí i Líndez, Isabelle Dunand-Sauthier, Mark Conti, Florian Gobet, Nicolás Núñez, J. Thomas Hannich, Howard Riezman, Geiger Roger, Alessandra Piersigilli, Kerstin Hahn, Sylvain Lemeille, Burkhard Becher, Thibaut De Smedt, Stéphanie Hugues, Walter Reith
Background: Bacille Calmette-Guérin (BCG) vaccine is protective in children but its efficacy wanes with age. Consequently, determining if BCG revaccination augments anti-TB immunity in young adults in TB endemic regions is vital. Methods: 200 healthy adults, BCG vaccinated at birth were tested for their IGRA status. Of these, 28 IGRA+ and 30 IGRA- were BCG revaccinated and 24 IGRA+ and 23 IGRA- subjects served as unvaccinated controls. T and innate cell responses to mycobacterial antigens were analyzed by 14-colour flow cytometry over 34 weeks. Results: IFN-γ and/or IL-2 Ag85A and BCG-specific CD4+ and CD8+ T-cell responses were boosted by revacciantion at 4 and 34 weeks respectively and were >2-fold higher in IGRA+ compared to IGRA- vaccinees. Polyfunctional Ag85A, BCG and Mtb latency Ag (LTAg)-specific CD4+ T-cells expressing up to 8 cytokines were also significantly enhanced in both IGRA+ and IGRA- vaccinees relative to unvaccinated controls, most markedly in IGRA+ vaccinees. A focussed analysis of Th17 responses revealed expansion of Ag85A, BCG and LTAg-specific total IL-17A+IL-17F+IL-22+ and IL-10+ CD4+ T-cell effectors in both IGRA+ and IGRA- subjects. Also, innate IFN-γ+ NK/γδ/NKT responses were higher in both IGRA+ and IGRA- vaccinees compared to controls. This is the first evidence that BCG revaccination significantly boosts anti-mycobacterial Th1/Th17 responses in IGRA+ and IGRA- subjects. Summary: These data show that BCG revaccination is immunogenic in IGRA- and IGRA+ subjects implying that Mtb pre-infection in IGRA+ subjects does not impact immunogenicity. This has implications for public health and vaccine development strategies. Funding: This work was funded principally by DBT-NIH (BT/MB/Indo-US/HIPC/2013).
Srabanti Rakshit, Asma Ahmed, Vasista Adiga, Bharath K. Sundararaj, Pravat Nalini Sahoo, John Kenneth, George D'Souza, Wesley Bonam, Christina Johnson, Kees L.M.C. Franken, Tom H.M. Ottenhoff, Greg Finak, Raphael Gottardo, Kenneth D. Stuart, Stephen C. De Rosa, M. Juliana McElrath, Annapurna Vyakarnam
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