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The same types of cells can assume diverse states with varying functionalities. Effective cell therapy can be achieved by specifically driving a desirable cell state, which requires the elucidation of key transcription factors (TFs). Here, we integrated epigenomic and transcriptomic data at the systems level to identify TFs that define different CD8+T cell states in an unbiased manner. These TF profiles can be used for cell state programming that aims to maximize the therapeutic potential of T cells. For example, T cells can be programmed to avoid a terminal exhaustion state (TexTerm), a dysfunctional T cell state that is often found in tumors or chronic infections. However, TexTermexhibits high similarity with the beneficial tissue-resident memory T states (TRM) in terms of their locations and transcription profiles. Our bioinformatic analysis predictedZscan20, a novel TF, to be uniquely active in TexTerm. Consistently,Zscan20knock-out thwarted the differentiation of TexTermin vivo, but not that of TRM. Furthermore, perturbation ofZscan20programs T cells into an effector-like state that confers superior tumor and virus control and synergizes with immune checkpoint therapy. We also identifiedJdp2andNfil3as powerful TexTermdrivers. In short, our multiomics-based approach discovered novel TFs that enhance anti-tumor immunity, and enable highly effective cell state programming.One sentence summaryMultiomics atlas enables the systematic identification of cell-state specifying transcription factors for therapeutic cell state programming.

SummaryThe limited efficacy of immunotherapies against glioblastoma illustrates the urgent need to better understand the interactions between the central nervous system and the immune system. Here, we showed that a protective response to αCTLA-4 therapy depended on a mutualistic relationship between microglia and CD4+ T cells. Suppression of gliomas by CD4+ T cells did not require tumor-intrinsic MHC-II expression, but rather was dependent on the selective expression of MHC-II and antigen presentation by local microglia that in turn, sustained CD4+ T cell tumoricidal effector functions. CD4+ T cell secretion of IFNγ made the glioma cells vulnerable to enhanced tumor surveillance and phagocytosis by microglia via the AXL/MER tyrosine kinase receptors that were necessary for tumor suppression. This work illustrates a novel partnership between CD4+ T cells and microglia that unleashes the tumoricidal properties of microglia that can be harnessed to improve immunotherapies for glioblastoma.

SummaryT cell metabolic fitness plays a pivotal role in anti-tumor immunity and metabolic deregulation causes T cell dysfunction (i.e., ‘exhaustion’) in cancer. We identify that the scavenger receptor CD36 limits anti-tumor CD8+T cell effector functions through lipid peroxidation. In murine tumors, oxidized phospholipids (OxPLs) were highly abundant and CD8+TILs increased uptake and accumulation of lipids and lipid peroxidation. Functionally ‘exhausted’ CD8+TILs substantially increased CD36 expression and CD36-deficient CD8+TILs had more robust anti-tumor activity and cytokine production than wild-type cells. We further show that CD36 promotes uptake of oxidized low-density lipoproteins (OxLDL) and induces lipid peroxidation in CD8+TILs, and OxLDL inhibits CD8+T cell functions in a CD36-dependent manner. Moreover, glutathione peroxidase 4 (GPX4) over-expression lowers lipid peroxidation and restores functionalities in CD8+TILs. These results define a key role for an oxidized lipid-CD36 axis in promoting intratumoral CD8+T cell dysfunction.

The mammalian genome comprises nuclear DNA (nDNA) derived from both parents and mitochondrial DNA (mtDNA) that is maternally inherited and encodes essential proteins required for oxidative phosphorylation. Thousands of copies of the circular mtDNA are present in most cell types that are packaged by TFAM into higher-order structures called nucleoids1. Mitochondria are also platforms for antiviral signalling2 and, due to their bacterial origin, mtDNA and other mitochondrial components trigger innate immune responses and inflammatory pathology2,3. We showed previously that cytoplasmic release of mtDNA activates the cGAS–STING–TBK1 pathway resulting in interferon-stimulated gene (ISG) expression that promotes antiviral immunity4. Here, we find that persistent mtDNA stress is not associated with basally activated NF-κB signalling or interferon gene expression typical of an acute antiviral response. Instead, a specific subset of ISGs that includes Parp9 remains activated by the unphosphorylated form of ISGF3 that enhances nDNA damage and repair responses. In cultured primary fibroblasts and cancer cells, the chemotherapeutic drug doxorubicin causes mtDNA damage and release, which leads to cGAS–STING–dependent ISG activation. In addition, mtDNA stress in TFAM-deficient mouse melanoma cells produces tumours that are more resistant to doxorubicin in vivo. Finally, Tfam+/− mice exposed to ionizing radiation exhibit enhanced nDNA repair responses in spleen. Therefore, we propose that damage to and subsequent release of mtDNA elicits a protective signalling response that enhances nDNA repair in cells and tissues, suggesting that mtDNA is a genotoxic stress sentinel. Persistent mitochondrial DNA stress is shown to upregulate nuclear DNA damage and repair responses via activation of the cGAS–STING pathway and a subset of interferon-stimulated genes.

Although initially responsive to chemotherapy, patients with advanced breast cancer often relapse, generally with incurable metastatic disease. This may be due to a subpopulation of tumor cells, called cancer-initiating cells, or cancer stem cells (CSCs), which are relatively resistant to chemotherapy and have self-renewing and tumor-initiating capacities. Prior studies in our laboratory found that CSCs may express ROR1, an onco-embryonic, tyrosine-kinase-like orphan receptor, which we found could bind Wnt5a to activate non-canonical Wnt-signaling (Proc Nat Acad Sci USA 111:17266, 2014). Interrogation of the transcriptomes of breast-cancer cells obtained from patients before and after paclitaxel therapy revealed that chemotherapy treatment enhanced cancer-cell expression of ROR1, along with genes induced by activation of Rho-GTPases (e.g. RhoA, cdc42, and Rac1). We found that primary breast-cancer patient-derived xenografts with high-level expression of ROR1 were enriched for cells that had activated Rho-GTPases and stem-cell-like gene-expression signatures. Furthermore, we found that treatment of breast cancer cell lines with Wnt5a induced ROR1-dependent activation of Rho-GTPases andAKT and induced high-level protein expression of BMI1, also known as polycomb group RING finger protein 4 (PCGF4) or RING finger protein 51 (RNF51); Wnt5a also enhanced the capacity of breast cancer cell lines to form spheroids. All these effects could be inhibited by cirmtuzumab, a humanized high-affinity anti-ROR1 mAb, which can block Wnt5a signaling. We find that ROR1-positive breast cancer cells have a greater capacity to form spheroids or engraft immune-deficient mice than did ROR1-negative cancer cells isolated from the same PDX tumor. Treatment of immune-deficient mice bearing breast-cancer PDX with paclitaxel reduced tumor volumes but enhanced expression of ROR1 and other CSC markers, such as aldehyde dehydrogenase 1 (ALDH1). Moreover, the breast cancer cells surviving such paclitaxel treatment had increased activation of Rho-GTPases and AKT, and increased expression of BMI1, relative to that of breast cancer cells obtained from the same primary tumor prior to therapy. On the other hand, treatment of such mice with cirmtuzumab also reduced breast cancer PDX tumor volumes, but the remaining cells had reduced expression of ROR1 and CSC markers and had impaired capacity to re-engraft immune-deficient mice. Finally, therapy with cirmtuzumab and paclitaxel was more effective in eradicating breast-cancer PDX than treatment with either agent alone. Collectively, these findings support use of cirmtuzumab in combination with conventional anti-cancer drugs to improve the outcome of patients with advanced breast cancer. Citation Format: Zhang S, Zhang H, Ghia EM, Liu G, Tripple V, Xu S, Cui B, Widhopf G, Yu J, Schwab R, Messer K, Parker BA, Kipps TJ. Breast cancer initiating cells express functional ROR1, which can be targeted by cirmtuzumab to potentially mitigate the risk of relapse after therapy [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P1-09-07.

Summary A common metabolic alteration in the tumor microenvironment (TME) is lipid accumulation, a feature associated with immune dysfunction. Here, we examined how CD8+ tumor infiltrating lymphocytes (TILs) respond to lipids within the TME. We found elevated concentrations of several classes of lipids in the TME and accumulation of these in CD8+ TILs. Lipid accumulation was associated with increased expression of CD36, a scavenger receptor for oxidized lipids, on CD8+ TILs, which also correlated with progressive T cell dysfunction. Cd36−/− T cells retained effector functions in the TME, as compared to WT counterparts. Mechanistically, CD36 promoted uptake of oxidized low-density lipoproteins (OxLDL) into T cells, and this induced lipid peroxidation and downstream activation of p38 kinase. Inhibition of p38 restored effector T cell functions in vitro, and resolution of lipid peroxidation by overexpression of glutathione peroxidase 4 restored functionalities in CD8+ TILs in vivo. Thus, an oxidized lipid-CD36 axis promotes intratumoral CD8+ T cell dysfunction and serves as a therapeutic avenue for immunotherapies.

BACKGROUND: Allergen-specific immunotherapy is the only curative treatment for type I allergy. It can be administered subcutaneously (SCIT) or sublingually (SLIT). The clinical efficacy of these two treatment modalities appears to be similar, but potential differences in the immunological mechanisms involved have not been fully explored.OBJECTIVE: To compare changes in the allergen-specific T cell response induced by subcutaneous versus sublingual administration of allergen-specific immunotherapy (AIT).METHODS: Grass pollen allergic patients were randomized into groups receiving either SCIT injections, or SLIT tablets or neither. PBMC were tested for Timothy grass (TG)-specific cytokine production by ELISPOT after in vitro expansion with TG peptide pools. Phenotypic characterization of cytokine producing cells was performed by FACS.RESULTS: In the SCIT group, decreased IL-5 production was observed starting 10 months after treatment was commenced. At 24 months, T cell responses showed IL-5 levels significantly below the before treatment baseline. No significant reduction of IL-5 was observed in the SLIT or untreated group. However, a significant transient increase in IL-10 production after 10 months of treatment compared to baseline was detected in both treatment groups. FACS analysis revealed that IL-10 production was associated with CD4(+) T cells that also produced IFNγ, and therefore may be associated with an IL-10-secreting type 1 cell phenotype.CONCLUSION AND CLINICAL RELEVANCE: The most dominant immunological changes on a cellular level was a decrease in IL-5 in the SCIT group and a significant, transient increase of IL-10 observed after 10 months of treatment in both treated groups. The distinct routes of AIT administration may induce different immune-modulatory mechanisms at the cellular level. This article is protected by copyright. All rights reserved.

Summary Immunotherapy has revolutionized cancer treatment, yet most patients do not respond. Here, we investigated mechanisms of response by profiling the proteome of clinical samples from advanced stage melanoma patients undergoing either tumor infiltrating lymphocyte (TIL)-based or anti- programmed death 1 (PD1) immunotherapy. Using high-resolution mass spectrometry, we quantified over 10,300 proteins in total and ∼4,500 proteins across most samples in each dataset. Statistical analyses revealed higher oxidative phosphorylation and lipid metabolism in responders than in non-responders in both treatments. To elucidate the effects of the metabolic state on the immune response, we examined melanoma cells upon metabolic perturbations or CRISPR-Cas9 knockouts. These experiments indicated lipid metabolism as a regulatory mechanism that increases melanoma immunogenicity by elevating antigen presentation, thereby increasing sensitivity to T cell mediated killing both in vitro and in vivo. Altogether, our proteomic analyses revealed association between the melanoma metabolic state and the response to immunotherapy, which can be the basis for future improvement of therapeutic response.

A panel of 133 allergens derived from 28 different sources, including fungi, trees, grasses, weeds, and indoor allergens, was surveyed utilizing prediction of HLA class II-binding peptides and ELISPOT assays with PBMC from allergic donors, resulting in the identification of 257 T cell epitopes. More than 90% of the epitopes were novel, and for 14 allergen sources were the first ever identified to our knowledge. The epitopes identified in the different allergen sources summed up to a variable fraction of the total extract response. In cases of allergens in which the identified T cell epitopes accounted for a minor fraction of the extract response, fewer known protein sequences were available, suggesting that for low epitope coverage allergen sources, additional allergen proteins remain to be identified. IL-5 and IFN-γ responses were measured as prototype Th2 and Th1 responses, respectively. Whereas in some cases (e.g., orchard grass, Alternaria, cypress, and Russian thistle) IL-5 production greatly exceeded IFN-γ, in others (e.g., Aspergillus, Penicillum, and alder) the production of IFN-γ exceeded IL-5. Thus, different allergen sources are associated with variable polarization of the responding T cells. The present study represents the most comprehensive survey to date of human allergen-derived T cell epitopes. These epitopes might be used to characterize T cell phenotype/T cell plasticity as a function of seasonality, or as a result of specific immunotherapy treatment or varying disease severity (asthma or rhinitis).

Whole extract or allergen-specific IgE testing has become increasingly popular in the diagnosis of peanut allergy. However, much less is known about T cell responses in peanut allergy and how it relates to different clinical phenotypes. CD4+ T cells play a major role in the pathophysiology of peanut allergy as well as tolerance induction during oral desensitization regimens. We set out to characterize and phenotype the T cell responses and their targets in peanut sensitized patients. Using PBMC from peanut-allergic and non-allergic patients, we mapped T cell epitopes for three major peanut allergens, Ara h 1, 2 and 3 (27 from Ara h 1, 4 from Ara h 2 and 43 from Ara h 3) associated with release of IFNγ (representative Th1 cytokine) and IL5 (representative Th2 cytokine). A pool containing 19 immunodominant peptides, selected to account for 60% of the total Ara h 1-3-specific T cell response in allergics, but only 20% in non-allergics, was shown to discriminate T cell responses in peanut-sensitized, symptomatic vs non-symptomatic individuals more effectively than peanut extract. This pool elicited positive T cell responses above a defined threshold in 12/15 sensitized, symptomatic patients, whereas in the sensitized but non-symptomatic cohort only, 4/14 reacted. The reactivity against this peptide pool in symptomatic patients was dominated by IL-10, IL-17 and to a lesser extend IL-5. For four distinct epitopes, HLA class II restrictions were determined, enabling production of tetrameric reagents. Tetramer staining in four donors (2 symptomatic, 2 non-symptomatic) revealed a trend for increased numbers of peanut epitope-specific T cells in symptomatic patients compared to non-symptomatic patients, which was associated with elevated CRTh2 expression whereas cells from non-symptomatic patients exhibited higher levels of Integrin β7 expression. Our results demonstrate differences in T cell response magnitude, epitope specificity and phenotype between symptomatic and non-symptomatic peanut-sensitized patients. In addition to IgE reactivity, analysis of peanut-specific T cells may be useful to improve our understanding of different clinical manifestations in peanut allergy.

Background: ROR1is an evolutionarily conserved, oncoembryonic surface-antigen expressed in breast cancer. Previously, we found that ROR1 can serve as a receptor for Wnt5a, which can induce non-canonical Wnt signaling that enhances cancer-cell migration. Recently we found that Wnt5a could induce ROR1 to complex with a cytoskeletal protein designated HS1, which recruited ARHGEF1, enhanced activation of RhoA, and promoted leukemia-cell migration. These effects of Wnt5a on CLL cells could be inhibited by cirmtuzumab, a humanized, high-affinity monoclonal antibody (mAb) specific for ROR1. As expression of HS1 is limited to hematopoietic cells we examined breast cancer cell lines and primary breast cancer cells for expression of cortactin. Cortactin (also called EMS1), is a cytoskeletal protein, which is homologous in structure and function to HS1 and is broadly expressed in human cancers, including breast cancer. Methods: We performed immunoprecipitation studies using high-affinity mAb to ROR1 or cortactin and immunoblot analysis to examine for the association of ROR1 with cortactin and tyrosine phosphorylation of cortactin at Y421. We also assessed the expression levels of cortactin, ROR1, and ARHGEF1, in the MCF7 breast cancer cell line and in breast-cancer patient-derived xenografts (PDX). We also generated MCF7-ROR1 cells, which were stably transfected to express ROR1 or various mutant forms of ROR1 generated to study the structure-function requirements for effective ROR1-cortactin interactions. Results: We found that ROR1 associates with cortactin in freshly-isolated breast cancer PDX tumors or in PDX cells cultured with exogenous Wnt5a. We corroborated these results in breast cancer cell line MCF7-ROR1 cells, which were transfected to express ROR1 and found to migrate more effectively than the parental MCF7 lacking expression of ROR1. Wnt5a also induced cortactin tyrosine phosphorylation at Y421, recruitment of ARHGEF1, and activation of RhoA, which we found associated with enhanced breast-cancer cell migation. The capacity of Wnt5a to induce such changes could be blocked by treatment of the cells with cirmtuzumab. We generated truncated forms of ROR1 without a proline-rich domain (PRD) and found PRD was necessary for this association or Wnt5a-induced cortactin phosphorylation and enhanced cancer-cell migration. Accordingly, we introduced single amino-acid substitutions of proline (P) to alanine (A) in the ROR1-PRD at positions 784, 808, 826, or 841 in potential SH3-binding motifs. In contrast to wild-type ROR1, or other ROR1P→A mutants, ROR1P(841)A had impaired capacity to recruit cortactin and ARHGEF1 to ROR1 in response to Wnt5a. Moreover, Wnt5a could not induce cells expressing ROR1P(841)A to phosphorylate cortactin or activate ARHGEF1, and was unable to enhance the motility of the MCF7 cells transfected with this mutant form of ROR1. Conclusions: Collectively, these studies indicate the capacity of cortactin to complex with ROR1 plays an important role in ROR1-dependent Wnt5a-enhanced breast cancer cell migration. These studies also demonstrate that cirmtuzumab can inhibit the formation of cortactin-ROR1 complexes, cortactin phosphorylation, and cancer-cell migration, and metastasis. Citation Format: Hasan MK, Tripple V, Widhopf II GF, Yu J, Zhang S, Parker BA, Kipps TJ. Wnt5a induces ROR1 to associate with cortactin, which undergoes tyrosine phosphorylation, and enhances migration of breast cancer cells [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P1-01-05.

Follicular helper T cells (Tfh cells) are a CD4 T cell subset essential for germinal center formation and B cell responses, although their role in allergy and allergen-specific immunotherapy (AIT) is unclear. Here, we show that AIT-treated patients have a higher ratio of regulatory Tfh cells (Tfr) to follicular T cells (Tfh) and hypothesize an IL-2 dependent mechanism.

Background Different populations of T cells are involved in the pathogenesis of allergic diseases. Objective We investigated changes in T H -cell populations in patients with allergies after specific immunotherapy (SIT). Methods PBMCs were isolated from patients with allergies who received SIT and those who did not (controls). We tested the ability of peptides from 93 timothy grass (TG) proteins to induce T-cell responses (cytokine production). We used ELISPOT and staining assays for intracellular cytokines to measure the production of IL-4, IL-5, IL-13, IFN-γ, and IL-10. Results Compared with PBMCs from controls, PBMCs from patients who received SIT produced lower levels of T H 2 cytokines on incubation with several different TG peptides. These data were used to select 20 peptides to be tested in an independent cohort of 20 patients with allergies who received SIT and 20 controls. We again observed a significant decrease in the production of T H 2 cytokines, and an increase in the production of the T H 1 cytokine IFN-γ, in PBMCs from the validation groups. These changes correlated with improved symptoms after SIT. Immunization with this selected pool of peptides (or their associated antigens) could protect a substantial proportion of the population from TG allergy. Conclusions We observed a significant decrease in the production of T H 2 cytokines by PBMCs from patients who received SIT for TG allergy compared to those who did not. These changes might be used to monitor response to therapy. The decrease occurred in response to antigens that elicit little (if any) IgE responses; these antigens might be developed for use in immunotherapy.

Background Patients with pollen allergies are frequently polysensitized. Pollens contain epitopes that are conserved across multiple species. Objective We sought to demonstrate that cross-reactive T cells that recognize conserved epitopes show higher levels of expansion than T cells recognizing monospecific epitopes because of more frequent stimulation. Method RNA was sequenced from 9 pollens, and the reads were assembled de novo into more than 50,000 transcripts. T-cell epitopes from timothy grass (Phleum pratense) were examined for conservation in these transcripts, and this was correlated to their ability to induce T-cell responses. T cells were expanded in vitro with P pratense –derived peptides and tested for cross-reactivity to pollen extracts in ELISpot assays. Results We found that antigenic proteins are more conserved than nonimmunogenic proteins in P pratense pollen. Additionally, P pratense epitopes that were highly conserved across pollens elicited more T-cell responses in donors with grass allergy than less conserved epitopes. Moreover, conservation of a P pratense peptide at the transcriptomic level correlated with the ability of that peptide to trigger T cells that were cross-reactive with other non– P pratense pollen extracts. Conclusion We found a correlation between conservation of peptides in plant pollens and their T-cell immunogenicity within P pratense , as well as their ability to induce cross-reactive T-cell responses. T cells recognizing conserved epitopes might be more prominent because they can be stimulated by a broader range of pollens and thereby drive polysensitization in allergic donors. We propose that conserved peptides could potentially be used in diagnostic or immunomodulatory approaches that address the issue of polysensitization and target multiple pollen allergies.

Bla g allergens are major targets of IgE responses associated with cockroach allergies. However, little is known about corresponding T cell responses, despite their potential involvement in immunopathology and the clinical efficacy of specific immunotherapy. Bioinformatic predictions of the capacity of Bla g 1, 2, 4, 5, 6, and 7 peptides to bind HLA-DR, -DP, and -DQ molecules, and PBMC responses from 30 allergic donors, identified 25 T cell epitopes. Five immunodominant epitopes accounted for more than half of the response. Bla g 5, the most dominant allergen, accounted for 65% of the response, and Bla g 6 accounted for 20%. Bla g 5 induced both IL-5 and IFN-γ responses, whereas Bla g 6 induced mostly IL-5, and, conversely, Bla g 2 induced only IFN-γ. Thus, responses to allergens within a source are independently regulated, suggesting a critical role for the allergen itself, and not extraneous stimulation from other allergens or copresented immunomodulators. In comparing Ab with T cell responses for several donor/allergen combinations, we detected IgE titers in the absence of detectable T cell responses, suggesting that unlinked T cell–B cell help might support development of IgE responses. Finally, specific immunotherapy resulted in IL-5 downmodulation, which was not associated with development of IFN-γ or IL-10 responses to any of the Bla g-derived peptides. In summary, the characteristics of T cell responses to Bla g allergens appear uncorrelated with IgE responses. Monitoring these responses may therefore yield important information relevant to understanding cockroach allergies and their treatment.