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Supplementary Data from Olfactory Receptor OR2H1 Is an Effective Target for CAR T Cells in Human Epithelial Tumors

Supplementary Figure from Olfactory Receptor OR2H1 Is an Effective Target for CAR T Cells in Human Epithelial Tumors

Recent studies suggest that B cells could play an important role in the tumor microenvironment. However, the role of humoral responses in endometrial cancer remains insufficiently investigated. Using a cohort of 107 patients with different histological subtypes of endometrial carcinoma, we evaluated the role of coordinated humoral and cellular adaptive immune responses in endometrial cancer. Concomitant accumulation of T, B, and plasma cells at tumor beds predicted better survival. However, only B-cell markers corresponded with prolonged survival specifically in high-grade endometrioid type and serous tumors. Immune protection was associated with class-switched IgA and, to a lesser extent, IgG. Expressions of polymeric immunoglobulin receptor (pIgR) by tumor cells and its occupancy by IgA were superior predictors of outcome and correlated with defects in methyl-directed DNA mismatch repair. Mechanistically, pIgR-dependent, antigen-independent IgA occupancy drove activation of inflammatory pathways associated with IFN and TNF signaling in tumor cells, along with apoptotic and endoplasmic reticulum stress pathways, while thwarting DNA repair mechanisms. Together, these findings suggest that coordinated humoral and cellular immune responses, characterized by IgA:pIgR interactions in tumor cells, determine the progression of human endometrial cancer as well as the potential for effective immunotherapies.Significance:This study provides new insights into the crucial role of humoral immunity in human endometrial cancer, providing a rationale for designing novel immunotherapies against this prevalent malignancy.See related commentary by Osorio and Zamarin, p. 766

Supplementary Figure from IgA-Dominated Humoral Immune Responses Govern Patients' Outcome in Endometrial Cancer

Supplementary Data from Treatment of Chronic Lymphocytic Leukemia with a Hypomethylating Agent Induces Expression of NXF2, an Immunogenic Cancer Testis Antigen

Purpose: Critical to the success of active immunotherapy against cancer is the identification of immunologically recognized cancer-specific proteins with low tolerogenic potential. Cancer testis antigens (CTA), in particular, fulfill this requirement as a result of their aberrant expression restricted to cancer cells and lack of expression in normal tissues bypassing tolerogenic mechanisms against self. Although CTAs have been extensively studied in solid malignancies, little is known regarding their expression in chronic lymphocytic leukemia (CLL).Experimental Design: Using a two-pronged approach we evaluated the immunogenicity of 29 CTAs in 22 patients with CLL and correlated these results to reverse transcriptase PCR data from CLL cell lines and patient cells.Results: We identified IgG-specific antibodies for one antigen, NXF2, and confirmed this response by ELISA and Western blot. We found that treatment of CLL with 5-aza-2′-deoxycytidine can induce expression of NXF2 that lasted for several weeks after treatment. Treatment also increased levels of MHC and costimulatory molecules (CD80, CD86, and CD40) necessary for antigen presentation. In addition, we identified other promising antigens that may have potential immunotherapeutic application.Conclusions: Our findings suggest that NXF2 could be further pursued as an immunotherapeutic target in CLL, and that treatment with demethylating agents could be exploited to specifically modulate CTA expression and effective antigen presentation in malignant B cells.

Recent studies suggest that B cells could play an important role in the tumor microenvironment. However, the role of humoral responses in endometrial cancer remains insufficiently investigated. Using a cohort of 107 patients with different histological subtypes of endometrial carcinoma, we evaluated the role of coordinated humoral and cellular adaptive immune responses in endometrial cancer. Concomitant accumulation of T, B, and plasma cells at tumor beds predicted better survival. However, only B-cell markers corresponded with prolonged survival specifically in high-grade endometrioid type and serous tumors. Immune protection was associated with class-switched IgA and, to a lesser extent, IgG. Expressions of polymeric immunoglobulin receptor (pIgR) by tumor cells and its occupancy by IgA were superior predictors of outcome and correlated with defects in methyl-directed DNA mismatch repair. Mechanistically, pIgR-dependent, antigen-independent IgA occupancy drove activation of inflammatory pathways associated with IFN and TNF signaling in tumor cells, along with apoptotic and endoplasmic reticulum stress pathways, while thwarting DNA repair mechanisms. Together, these findings suggest that coordinated humoral and cellular immune responses, characterized by IgA:pIgR interactions in tumor cells, determine the progression of human endometrial cancer as well as the potential for effective immunotherapies. Significance: This study provides new insights into the crucial role of humoral immunity in human endometrial cancer, providing a rationale for designing novel immunotherapies against this prevalent malignancy. See related commentary by Osorio and Zamarin, p. 766

Although chimeric antigen receptor (CAR)-expressing T cells have proven success in hematologic malignancies, their effectiveness in solid tumors has been largely unsuccessful thus far. We found that some olfactory receptors are expressed in a variety of solid tumors of different histologic subtypes, with a limited pattern of expression in normal tissues. Quantification of OR2H1 expression by qRT-PCR and Western blot analysis of 17 normal tissues, 82 ovarian cancers of various histologies, eight non–small cell lung cancers (NSCLCs), and 17 breast cancers demonstrated widespread OR2H1 expression in solid epithelial tumors with expression in normal human tissues limited to the testis. CAR T cells recognizing the extracellular domain of the olfactory receptor OR2H1 were generated with a targeting motif identified through the screening of a phage display library and demonstrated OR2H1-specific cytotoxic killing in vitro and in vivo, using tumor cells with spontaneous expression of variable OR2H1 levels. Importantly, recombinant OR2H1 IgG generated with the VH/VL sequences of the CAR construct specifically detected OR2H1 protein signal in 60 human lung cancers, 40 ovarian carcinomas, and 73 cholangiocarcinomas, at positivity rates comparable with mRNA expression and without OR2H1 staining in 58 normal tissues. CRISPR/Cas9-mediated ablation of OR2H1 confirmed targeting specificity of the CAR and the tumor-promoting role of OR2H1 in glucose metabolism. Therefore, T cells redirected against OR2H1-expressing tumor cells represent a promising therapy against a broad range of epithelial cancers, likely with an admissible toxicity profile.

Despite repeated associations between T cell infiltration and outcome, human ovarian cancer remains poorly responsive to immunotherapy. We report that the hallmarks of tumor recognition in ovarian cancer-infiltrating T cells are primarily restricted to tissue-resident memory (TRM) cells. Single-cell RNA/TCR/ATAC sequencing of 83,454 CD3(+) CD8(+)CD103(+)CD69(+) TRM cells and immunohistochemistry of 122 high-grade serous ovarian cancers shows that only progenitor (TCF1(low)) tissue-resident T cells (TRMstem cells), but not recirculating TCF1(+) T cells, predict ovarian cancer outcome. TRMstem cells arise from transitional recirculating T cells, which depends on antigen affinity/persistence, resulting in oligoclonal, trogocytic, effector lymphocytes that eventually become exhausted. Therefore, ovarian cancer is indeed an immunogenic disease, but that depends on similar to 13% of CD8(+) tumor-infiltrating T cells (similar to 3% of CD8(+) clonotypes), which are primed against high-affinity antigens and maintain waves of effector TRM-like cells. Our results define the signature of relevant tumor-reactive T cells in human ovarian cancer, which could be applicable to other tumors with unideal mutational burden.

Phosphatidylinositol-3 kinase (PI3K) delta signaling is essential for CLL B cell survival and proliferation. Several PI3K inhibitors are approved for treatment of CLL, but PI3K inhibitor-induced alterations in T cell function have been correlated with immune-related adverse events (irAEs). Zandelisib is a novel selective PI3Kδ inhibitor in clinical development with an intermittent dosing schedule for treatment of B cell malignancies. Here, we investigated the dynamic immunomodulatory properties of zandelisib alone or in combination with BTK inhibitor ibrutinib in normal human T cells, as well as the impact of continuous dosing of zandelisib on effector and regulatory immune cells, irAEs, and survival in a preclinical CLL murine model. Normal human T cells treated ex vivo with zandelisib, ibrutinib, or both were assessed for phenotype and function. A CLL model was initiated by transferring leukemic EμTCL1 splenocytes into syngeneic wildtype recipients. CLL-bearing mice were treated with vehicle or zandelisib (50 mpk bid or 100 mpk qd) for 3 weeks (n=5 per group). Antitumor efficacy, T cell and myeloid subsets in blood, bone marrow and spleen were analyzed. Survival was also observed in a separate cohort of mice treated with zandelisib (100 mpk qd). T cell proliferation was impaired by zandelisib (250nM) and ibrutinib (100 nM) alone, with the combination further reducing T cell proliferation. Zandelisib decreased expression of suppressive markers PD-1, CTLA-4, GITR and CD39 on iTregs; however, no further decrease was observed when zandelisib was combined with ibrutinib. Zandelisib also reduced normal T cell activation and expression of immune checkpoints PD-1 and CTLA-4 on CD4+ T cells and dose dependently inhibited T cell memory differentiation ex vivo, increasing the naïve/central to effector memory ratio. Combination with ibrutinib did not further affect T cell activation, checkpoint expression or memory differentiation. In the CLL murine model, no meaningful difference was seen between bid or qd administration of zandelisib in the reduction of tumor burden (CD19+ CD5+ CLL cells). Zandelisib treatment reduced activated T cells, antigen-experienced T cells, and Tregs. Increased naïve/central to effector memory ratio was observed in treated mice, and PD-1 was downregulated on memory T cells, indicating less T cell exhaustion. Zandelisib decreased macrophages in the spleen but did not impact myeloid-derived suppressor cells. Zandelisib treatment improved survival of CLL-bearing mice significantly when compared to vehicle group (median survival of zandelisib group 140 days vs. 105 days vehicle). In conclusion, zandelisib treatment reduced Tregs, prevented terminal memory differentiation, and T-cell exhaustion—features that have been shown to permit CLL immune evasion—demonstrated antitumor efficacy, and improved overall survival in a preclinical CLL model. Citation Format: Kamira Maharaj, Melanie Mediavilla-Varela, John J. Powers, Sandra Wiley, Angimar Uriepero, Wael Gamal, Kun Jiang, Eva Sahakian, Javier Pinilla-Ibarz. Efficacy and immune profiling of PI3K delta inhibitor zandelisib (ME-401) in a preclinical chronic lymphocytic leukemia (CLL) model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5496.

Tertiary Lymphoid Structures (TLS) are commonly identified in human tumors with improved outcome, but how they are orchestrated remains elusive. We show that ablation of the genomic organizer SATB1 triggers the polarization of CD4+ T-cells into antigen-specific T Follicular Helper (TFH) cells. In contrast, SATB1 expression is required for conversion of CD4+ T-cells into induced regulatory T-cells (Treg), including T Follicular Regulatory (TFR) cells. Paradoxically, Tgf-β-driven Satb1 silencing causes Icos de-repression and prevents TFR differentiation, driving TFH polarization and increased isotype-switched B cell responses. Tumors in mice carrying Satb1-deficient CD4+ T-cells accumulate more tumor antigen-specific, Light/Cxcl13/Il-21-producing TFH cells, causing germinal center formation and spontaneous TLS assembly, which antagonizes tumor growth. Accordingly, TFH cell transfer induces large TLS within orthotopic ovarian tumors. Therefore, SATB1 repression in CD4+ T-cells licenses TFH cell differentiation while preventing Treg conversion, a central mechanism during TLS assembly that can be exploited to boost anti-tumor immunity.

Despite repeated associations between T-cell infiltration and patient outcome, human ovarian cancer remains poorly responsive to immunotherapy. We report that hallmarks of tumor recognition in ovarian cancer-infiltrating T-cells are primarily restricted to tissue-resident memory (TRM) cells. In mouse models we found that TRM T-cells were better than the re-circulating counterpart at controlling tumor growth. Single-cell RNA/TCR/ATAC sequencing of 83,454 CD3+CD8+CD103+CD69+ TRM cells and 24,175 CD3+CD8+CD103− re-circulating TILs showed that progenitor (TCF1low) tissue-resident memory T-cells (TRMstem cells) arise from transitional recirculating T-cells, which depends on antigen affinity/persistence, resulting in oligoclonal, trogocytic, effector lymphocytes. This effector population develops into proliferative lymphocytes that eventually become exhausted TRMs. Immunohistochemistry of 122 high-grade serous ovarian cancer tissues showed that only TRMstem cells, but not re-circulating TCF1+ T-cells, predict ovarian cancer outcome. Therefore, ovarian cancer is indeed an immunogenic disease that depends on ~13% of CD8+ tumor-infiltrating T-cells (~3% of CD8+ clonotypes), which are primed against high-affinity antigens and maintain waves of effector TRM cells. Support for Shared Resources was provided by Cancer Center Support Grant (CCSG) CA076292 to H. Lee Moffitt Cancer Center and by CCSG CA010815 to The Wistar Institute. This study was supported by grants from NIH (R01CA157664, R01CA124515, R01CA178687, R01CA211913 and U01CA232758 to JRCG; R01CA184185 and RO1CA262121 to PCR.)

Chimeric antigen receptor (CAR) T cells have been successful for hematological malignancies, but so far showed limited efficiency against solid tumors. Olfactory receptors are expressed in a variety of epithelial cancers, with a limited expression in healthy tissues. We quantified olfactory receptor OR2H1 expression in ovarian cancer, non-small cell lung cancer (NSCLC), breast cancer, and many normal tissues, and we found OR2H1 is expressed in multiple solid epithelial tumors but limited to testis among healthy human tissues. CAR T cells targeting OR2H1-extracellular domain were generated, and OR2H1-specific cytotoxic killing was confirmed, both in vitro and in vivo. Correspondingly, OR2H1-CAR T cells mediate significant therapeutic effects against OR2H1+ NSCLC and high-grade serous ovarian cancer. Interestingly, OR2H1 ablation significantly delays malignant progression of at least NSCLC, but OR2H1 expression is found to be essential for the cytotoxic effects of the OR2H1-CAR T cells. In summary, T cells directed against OR2H1-expressing tumor cells could be a potential therapeutic approach in future for treating OR2H1-expressing cancers. Supported by CA076292, R01CA157664, R01CA124515, R01CA178687, R01CA211913, U01CA232758

The role of humoral responses in endometrial cancer remains insufficiently investigated. Using a cohort of 107 patients with different histological subtypes of endometrial carcinoma, we report that concomitant accumulation of T, B and plasma cells at tumor beds predicts better survival. However, only B cell markers predict survival specifically in high-grade endometrioid type and serous tumors. Accordingly, immune protection is associated with class-switched IgA and, to a lesser extent, IgG. Notably, expression of polymeric immunoglobulin receptor (pIgR) by tumor cells and its occupancy by IgA are superior predictors of outcome, and correlate with defects in methyl mismatch repair. Mechanistically, pIgR-dependent, antigen-independent IgA occupancy drives inflammatory pathways associated with IFN and TNF signaling in tumor cells, along with apoptotic and ER stress pathways, while thwarting DNA repair mechanisms. Therefore, coordinated humoral and cellular immune responses, characterized by IgA:pIgR interactions in tumor cells, determine the progression of human endometrial cancer, and therefore the potential for effective immunotherapies. Supported by grants from NIH (R01CA157664, R01CA124515, R01CA178687 and R01CA211913), and from Cancer Center Support Grant (CCSG) CA076292

The immune checkpoint receptor PD-1 on T follicular helper (Tfh) cells promotes Tfh:B cell interactions and appropriate positioning within tissues. Here, we examined the impact of regulation of PD-1 expression by the genomic organizer SATB1 on Tfh cell differentiation. Vaccination of CD4

The object of this study is to relate qualitative and quantitative ratings as expressed by judges and to relate sensory responses to physical measurements and gas chromatograms. Judges were asked to rate acceptability, flavor, and mouthfeel of blueberries on a five-point scale in a darkened room. With another specimen, they were asked to judge appearance and color without, however, tasting these berries. The judges were also asked to complete a questionnaire describing, in qualitative terms, the characteristics of each specimen. Varieties were subjected to analysis by physical and chemical measurements, and gas chromatograms were prepared. Contingency analysis and analysis of variance were performed to relate qualitative and quantitative ratings. The relationship between physical measurements or chromatograms and ratings is displayed in tables of means; although these numbers can be expressed as single correlations, using a random model, a single measure based on just four products was deemed to be less informative than a tabulation of correspondence.

Analysis of variance was applied to 3,107 magnitude estimations (ME) obtained upon 173 panelists (16 panels) evaluating the areas of 18 geometric figures. As Butler et al. (1987) pointed out, if a full ANOVA is applied to the values transformed to logarithms, there is no need to re-scale, i.e., normalize the values as is usually done. In fact, even with re-scaling, total variability is not accounted for adequately unless all the sources of variation are partitioned out in the analysis. Under some circumstances, re-scaling is needed: some laboratories re-scale to the arithmetic mean, others to the geometric one. The error was somewhat greater when the data were re-scaled to the arithmetic mean. The residuals of the ME values transformed to logarithms conformed essentially to a normal distribution. Kurtosis was greater, 5.30; otherwise the distribution was close to a log normal one. The average coefficient of variation was

Sensory evaluation of isolated natural capsaicinoids showed that there was a significant linear relationship between pungency and concentration. Detection thresholds were evaluated using an ASTM method. Pungency thresholds were calculated using the established relationships between pungency and concentration of individual compounds. Total pungency of a mixture of capsaicinoids was highly correlated with the sum of pungencies of individual capsaicinoids. Determination of the total pungency of Capsicum extracts can be accomplished by adding the pungencies of individual capsaicinoids upon multiplication of their pungency threshold values by their respective concentrations.

Thesholds for vanillin dissolved in water were determined first by 32 panelists, then by 100 additional panelists. Extreme-value analysis was applied to the results from the first 32 determinations to estimate the likelihood of thresholds later encountered being even lower than those already detected. No threshold among the 100 additional determinations was below the 5% confidence limit for the regression curve based on the 32 determinations originally made. There was evidence that some panelists were ageusic to vanillin in aqueous solution. Although some investigators have suggested that the frequency distribution for threshold values of vanillin is bimodal, application of the Kolmogorov-smirnov test to the distribution of the 132 values, as natural logarithms, gave little cause to reject the hypothesis that the distribution was unimodal.

Sour rye breads containing varying acetic or lactic acid concentrations were used as samples in Experiment 1. Assessors (N=18) could detect difference between 0 to 2 g of added acetic acid (p< 0.02, triangle test), whereas difference between lactic acid concentrations at the same level was detected at p < 0.001. In Experiment 2 five commercial rye breads were profiled by 14 assessors on a 10-cm graphical scale using 12 flavor descriptors, including desirability. Chemical determinations of pH, titratable acidity, acetic and lactic acid and NaCl concentration were made. Acetic and lactic acid concentrations were strongly related to perceived sourness of odor and taste. The effect of lactic acid on pH and titrable acidity was more pronounced than that of lactic acid. Stepwise discriminant analysis of the sensory data resulted in a 57.1% success rate in discriminating among the samples (nine descriptors used), whereas the analysis of the chemical data discriminated among the samples with a 80% success rate, two variables were useful. Combination of the sensory and chemical data led to 83.3% success with twelve variables.

The British Standards Institution and the International Standards Organization specify that when mixtures of tea and milk are to be evaluated sensorially, the tea should be added to the milk. Four cups of tea prepared by pouring the tea into the cup first (TIF) and four cups prepared by placing the milk in the cup first (MIF) were presented to 155 panelists in the dark. They were instructed to select by taste the four cups containing the MIF tea. The frequency of correct selection was well above the chance level, indicating that the panelists had detected a difference in flavor. The same task was performed by 131 panelists except they evaluated the tea in light. The panelists were not highly trained tea tasters; in fact, most were not regular drinkers of hot tea. Notwithstanding that, their discriminating power was sufficient to resolve the subtle differences which did exist.

The review included a discussion of the reasons why acidification of canned tomatoes is sometimes desirable from a microbiological point of view, the possible Changes in the pH of tomatoes as a result of the breeding of new varieties, and the methods and suitable acids for acidification. Acidification does cause some minor differences in the chemical components of the canned product, but the differences appear to be inconsequential. Although can corrosion may be accelerated slightly, this again does not seem to be of practical importance. The can manufacturers recommend the same type of can whether or not tomatoes are acidified. Flavor studies almost invariably have indicated that tomatoes are as acceptable as nonacidified tomatoes at least down to pH 4.1 to 4.0. In fact, most studies show that acidified tomatoes are somewhat more acceptable. Reasons are suggested as to why outbreaks of botulism have occurred from home‐canned tomatoes, although the pH appears to have been below 4.5, and as to why outbreak...