Density Of Tregs Research Articles

CD8+ T cells infiltrating into tumors were controlled by immune status of pulmonary lymph nodes and correlated with non-small cell lung cancer (NSCLC) patients’ prognosis treated with chemoimmunotherapy

PurposeNeoadjuvant chemoimmunotherapy has the potential to reduce tumor burden, improve the pathological complete response (pCR) rate, and significantly prolong patients’ disease-free survival (DFS). However, the treatment’s effectiveness varies among NSCLC patients. The immunological mechanisms contributing to tumor regression still require further exploration and elucidation. MethodsThe immune status of patients’ local tumor microenvironment (TME) before and after neoadjuvant chemoimmunotherapy, their paired pulmonary lymph nodes (11th LNs) after therapy, including infiltrating immune cell densities and their correlations, were analyzed using multiplex immunofluorescence. ResultsFifty-six NSCLC patients undergoing neoadjuvant chemoimmunotherapy were enrolled and subsequently underwent surgical resection and pathological evaluation. Among these, 19 patients achieved a pCR, 6 patients exhibited a major pathological response (MPR), and 31 patients did not achieve MPR. There were no significant difference in the densities of CD8+ T cell, Treg and Dendritic cell (DC) in patients’ TME before neoadjuvant therapy (n = 26, P = 0.091, P = 0.753, P = 0.905, respectively), but after treament, these immune cells’ dynamics were significantly different between different response group. CD8+ T cell densities were increased in pCR gourp (P = 0.006), but not in non-pCR group (P = 0.389); the densities of Treg were increased in non-pCR gourp (P = 0.0004), but DC were significantly decreased in non-pCR gourp (P = 0.005). After surgery, the TME were also significantly different: patients achieving pCR typically demonstrated high densities of CD8+ T cell, DC and low densities of Tregs (P = 0.0001, P < 0.0001 and P = 0.0004). The immune status of 11th LNs also exhibited significant differences. DC densities were much higher in pCR patients, whereas Treg in the pCR group were significantly lower than those in the non-pCR group (P = 0.0008 and P = 0.003). Furthermore, the densities of DC in the TME showed a moderate positive correlation with DC in 11th LNs (P = 0.0002), while the densities of Tregs in the TME exhibited a moderate negative correlation with DC densities in 11th LNs (P = 0.03). Patients who had high densities of CD8+ T cell in the resection tissues and DC in the LNs, experienced longer DFS (P = 0.048 and P = 0.024). ConclusionImmune cells in both pulmonary LNs and the TME collectively influence the remodeling of the NSCLC patient’s TME, thus impacting treatment response and prognosis.

Immune cell topography of head and neck cancer

BackgroundApproximately 50% of head and neck squamous cell carcinomas (HNSCC) recur after treatment with curative intent. Immune checkpoint inhibitors are treatment options for recurrent/metastatic HNSCC; however, less than 20% of...

Modified spatial architecture of regulatory T cells after neoadjuvant chemotherapy in non-small cell lung cancer patients

It is crucial to decipher the modulation of regulatory T cells (Tregs) in tumor microenvironment (TME) induced by chemotherapy, which may contribute to improving the efficacy of neoadjuvant chemoimmunotherapy in resectable non-small cell lung cancer (NSCLC). We retrospectively collected specimens from patients with II-III NSCLC, constituting two cohorts: a neoadjuvant chemotherapy (NAC) cohort (N = 141) with biopsy (N = 58) and postoperative specimens (N = 141), and a surgery-only cohort (N = 122) as the control group. Then, the cell density (Dens), infiltration score (InS), and Treg-cell proximity score (TrPS) were conducted using a panel of multiplex fluorescence staining (Foxp3, CD4, CD8, CK, CD31, ɑSMA). Subsequently, the association of Tregs with cancer microvessels (CMVs) and cancer-associated fibroblasts (CAFs) was analyzed. Patients with NAC treatment have a higher density of Tregs in both paired (P < 0.001) and unpaired analysis (P = 0.022). Additionally, patients with NAC treatment showed higher infiltration score (paired, P < 0.001; unpaired, P = 0.014) and more CD8+T cells around Tregs (paired/unpaired, both P < 0.001). Subgroup analysis indicated that tumors with a diameter of ≤ 5 cm exhibited increase in both Dens(Treg) and InS(Treg), and gemcitabine, pemetrexed and taxel enhanced Dens(Treg) and TrPS(CD8) following NAC. Multivariate analysis identified that the Dens(Tregs), InS(Tregs) and TrPS(CD8) were significantly associated with better chemotherapy response [OR = 8.54, 95%CI (1.69, 43.14), P = 0.009; OR = 7.14, 95%CI (1.70, 30.08), P = 0.024; OR = 5.50, 95%CI (1.09, 27.75), P = 0.039, respectively] and positive recurrence-free survival [HR = 3.23, 95%CI (1.47, 7.10), P = 0.004; HR = 2.70; 95%CI (1.27, 5.72); P = 0.010; HR = 2.55, 95%CI (1.21, 5.39), P = 0.014, respectively]. Moreover, TrPS(CD8) and TrPS(CD4) were negatively correlated with the CMVs and CAFs. These discoveries have deepened our comprehension of the immune-modulating impact of chemotherapy and underscored that the modified spatial landscape of Tregs after chemotherapy should be taken into account for personalized immunotherapy, aiming to ultimately improve clinical outcomes in patients with NSCLC.

Regulatory and memory T lymphocytes infiltrating prostate tumors predict long term clinical outcomes.

The localization, density but mostly the phenotype of tumor infiltrating lymphocytes (TIL) provide important information on the initial interaction between the host immune system and the tumor. Our objective was to assess the prognostic significance of T (CD3+), T regulatory (Treg) (FoxP3+) and T memory (Tmem) (CD45RO+) infiltrating lymphocytes and of genes associated with TIL in prostate cancer (PCa). Immunohistochemistry (IHC) was used to assess the infiltration of CD3+, FoxP3+ and CD45RO+ cells in the tumor area, tumor margin and adjacent normal-like epithelium of a series of 98 PCa samples with long clinical follow-up. Expression of a panel of 31 TIL-associated genes was analyzed by Taqman Low-Density Array (TLDA) technology in another series of 50 tumors with long clinical follow-up. Kaplan-Meier and Cox proportional hazards regression analyses were performed to determine association of these markers with biochemical recurrence (BCR), need for definitive androgen deprivation therapy (ADT) or lethal PCa. TIL subtypes were present at different densities in the tumor, tumor margin and adjacent normal-like epithelium, but their density and phenotype in the tumor area were the most predictive of clinical outcomes. In multivariate analyses, a high density of Treg (high FoxP3+/CD3+ cell ratio) predicted a higher risk for need of definitive ADT (HR=7.69, p=0.001) and lethal PCa (HR=4.37, p=0.04). Conversely, a high density of Tmem (high CD45RO+/CD3+ cell ratio) predicted a reduced risk of lethal PCa (HR=0.06, p=0.04). TLDA analyses showed that a high expression of FoxP3 was associated with a higher risk of lethal PCa (HR=5.26, p=0.02). Expression of CTLA-4, PD-1, TIM-3 and LAG-3 were correlated with that of FoxP3. Amongst these, only a high expression of TIM-3 was associated with a significant higher risk for definitive ADT in univariate Cox regression analysis (HR=3.11, p=0.01). These results show that the proportion of Treg and Tmem found within the tumor area is a strong and independent predictor of late systemic progression of PCa. Our results also suggest that inhibition of TIM-3 might be a potential approach to counter the immunosuppressive functions of Treg in order to improve the anti-tumor immune response against PCa.

Abstract 6970: identification of spatially enriched cancer cell- and immune cell-regulatory multi-marker-defined prognostic cancer associated fibroblast subsets of human colon cancer

Abstract Introduction: Subsets of cancer-associated fibroblasts (CAFs) in colorectal cancer remain poorly characterized preventing their use as biomarkers and drug targets. Results: Integrated scRNA seq profiling and multiplex staining identified four human CAF subsets These were A1 (PDGFRA+/FAP-/TF), PDGFRAhighA2 (PDGFRA+/FAP+/TF-), PDGFRAlowA2 (PDGFRA-/FAP+/TF-) and A3 (PDGFRA+/FAP-/TF+) (TF: Tissue Factor).Spatial analyses identified spatial associations indicative of specific functional interactions of fibroblast subsets with cancer cells or immune cells. Explorative analyses in six CRC cases suggested, in general, favorable effects of FAP-negative subsets (A1 and A3) on immune features and cancer cell proliferation, whereas FAP-positive subsets PDGFRAhighA2 and PDGFRAlowA2 overall showed pro-tumoral associations. To extend findings, niches were quantitated in U-CAN cohort of approximately 200 stage I-IV cases with around 80% stage II/III. Spatial screening generated more than 1600 different multi-marker-defined tissue metrics. Analyses identified a higher average of A1 density in areas surrounding proliferating vs non-proliferating T-cells, and a higher average PDGFRAlowA2 peri-T-cell density in the low proliferating T-cell group. Regarding cancer cell proliferation, high proliferating cancer islands showed lower peri-epithelial density of A3 and PDGFRAlowA2 cells as compared to low proliferating cancer islands. As for T-cell cancer island infiltration, high peri-cancer island density of A1 and PDGFRAhighA2 was associated with high T-cell cancer island infiltration. Additional analyses related CAF metrics to stage and survival. Density of PDGFRAlow A2 cells increased with stage (I-III), whereas density of A1 showed an opposite pattern with an inverse association with stage (I-III). Regarding survival associations, these were in general stronger for metrics collected in peripheral regions of tumors. Among prognosis signals related to niches referred to above, it was noted that A1 density in peri-cancer regions was significantly associated with good prognosis. Interestingly, this association was not detected when A1 density was scored in total stroma. Among all metrics, the strongest metrics were A1 density in 20μm A1 expansion areas (good prognosis) and T-reg density in the 10μm A3 expansion areas (bad prognosis) (P&amp;lt;0.001; univariate Cox-regression); both in peripheral regions. Conclusion: Four novel CRC CAF subsets, defined by PDGFRalpha, FAP and TF, was identified by integrated single cell RNA seq analyses and multiplex staining with associated digital image analyses. Non-random spatial distribution of these subsets and compartment-specific prognosis associations are compatible with distinct and different functions of these subsets. Citation Format: Linglong Huang, Mercedes Herrera, Jonas Sjölund, Vladimir Chocoloff, Simon Joost, Rasul M. Tabiev, Lina Wik Leiss, Carina Strell, Luis Nunes, Artur Mezheyeuski, David Edler, Anna Martling, Fredrik Pontén, Bengt Glimelius, Tobias Sjöblom, Maria Kasper, Kristian Pietras, Arne Östman. identification of spatially enriched cancer cell- and immune cell-regulatory multi-marker-defined prognostic cancer associated fibroblast subsets of human colon cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6970.

Abstract 7630: Therapeutic relevance of systemic immune status to treatment effects of abemaciclib

Abstract Background: The addition of a CDK4/6 inhibitor (CDK4/6i) on endocrine therapies (ET) has been currently a standard treatment for hormone receptor (HR)-positive HER2-negative metastatic breast cancers (MBCs). Especially abemaciclib has been implicated in multiple mechanisms of action including cell cycle arrest, senescence. In addition, immunomodulatory effects have been suggested but are not yet clear. The aim of the study was to investigate the clinical relevance of abemaciclib to systemic immune status by examining the status of peripheral immune cells in patients with MBC who received abemaciclib plus ET. Methods: Patients with MBC who were treated with abemaciclib plus ET in our hospital were included in this study. Response was evaluated by RECIST 1.1. Peripheral blood at the initiation of abemaciclib treatment, 4 weeks, 12 weeks and 24weeks after abemaciclib treatment initiation were analyzed using mass cytometry by time of flight (CyTOF). Changes of cellular densities of each immune cell and survival by immune cell density were analyzed without calculating p values because of small sample number. Results: Twenty patients were enrolled, among whom 14 and 6 received an aromatase inhibitor and fulvestrant, respectively, as concomitant ET. 7 premenopausal patients received an LH-RH analogue. The analysis of serial blood collection showed an increase in CD8-positive T cell density by 12.5% and conversely a decrease in monocyte density by 52.3% at 4 weeks after treatment initiation. In responders (N = 10), an increase of the ratio between CD8-positive T cell density and Treg density (CD8/Treg) at 4 weeks after the treatment initiation were observed (65.2%), while not in non-responders (7.3%). In the analysis of the blood samples at the treatment initiation, lower Treg density and lower NK density at treatment initiation indicated longer PFS than the counterparts (median PFS for Treg: low, not reached; high, 8.8 months; for NK: low, not reached; high, 8.8 months). Conclusion: An increase of CD8/Treg at 4 weeks after the treatment initiation was associated with a good response in patients with HR-positive HER2-negative MBC treated with abemaciclib plus ET. Our results suggest an association between systemic immune status and clinical outcome in MBC patients who received abemaciclib plus ET treatment. A larger study is needed to validate the results. Citation Format: Takayuki Ueno, Shigehisa Kitano, Takayuki Kobayashi, Makiko Yamashita, Fumikata Hara, Yukinori Ozaki, Meiko Nishimura, Toshimi Takano, Naoko Toriguchi, Tsutomu Kawaguchi. Therapeutic relevance of systemic immune status to treatment effects of abemaciclib [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7630.

Abstract 1517: Tc1 cytotoxic T-cells and Th1 helper T-cell rich tumor microenvironment is a hallmark of MSI colorectal cancers

Abstract Background: Microsatellite instability (MSI-h) is a strong biomarker to predict response to immune checkpoint therapy and patient’s outcome in colorectal cancer. Although enrichment of distinct T-cell subpopulations, such as type 1 cytotoxic T-cells (Tc1) or type 1 helper T-cells (Th1), have been identified to impact patient’s outcome and response to immune checkpoint therapy, only little is known about the underlying changes in the composition of the immune tumor microenvironment. Material and Methods: To assess the density, composition, degree of functional marker expression, and spatial interplay of T-cell subpopulations in 93 MSI and 1153 microsatellite stable (MSS) colorectal cancers, a tissue microarray as well as large sections were stained with antibodies directed against CD3, CD4, CD8, FOXP3, T-bet, GATA3, RORyT, BCL6, CD27, CD56, TIM3, PD-1, CTLA-4 Granzym B, and Ki67 using our BLEACH&amp;STAIN multiplex fluorescence immunohistochemistry approach. A deep learning-based framework comprising two different convolutional neuronal networks (U-Net and DeepLabv3+) was used for image analysis. Results: The composition of Type 1 (T-bet+), Type 2 (GATA3+), Type 17 (RORyT+), NKT-like (CD56+), regulatory (FOXP3+), and follicular (BCL6+) cytotoxic (CD3+CD8+) or helper (CD3+CD4+) T-cells showed marked differences between MSI and MSS patients. For instance, the density and proportion of Tc1 as well as Th1 was significantly higher (p&amp;lt;0.001 each) in MSI compared to MSS patients. In contrast, the density and composition of Tregs, Th2, Th17 T-cells was significantly lower in MSI compared to MSS patients in the intraepithelial tumor compartment. In addition, among the MSI colorectal cancers, NKT-cells showed the highest level of Granzyme B that was significantly higher than in MSS patients (p&amp;lt;0.05). The degree of immune checkpoint expression (i.e., TIM3, PD-1, CTLA-4) was significantly higher in MSI compared to MSS patients for most T-cell subpopulations (p&amp;lt;0.05 each). The additional analysis of 10 large sections confirmed the observations from the TMA analysis and revealed that the alterations in the T-cell composition seen in the center of the tumor largely reflect the T-cell composition at the invasive margin in both MSI and MSS patients. Conclusion: This study identified a higher proportion of Tc1 cytotoxic T-cells and Th1 helper T-cells accompanied by a paucity of regulatory T-cell subpopulations, Th17 and Th2 T- helper cells in MSI colorectal cancers compared to MSS patients. Citation Format: Tim Mandelkow, Zhihao Huang, Elena Bady, Jan H. Müller, Guido Sauter, Julia Ebner, Maximilian Lennartz, Natalia Gorbokon, Ronald Simon, Martina Kluth, Claudia Hube-Magg, Sarah Minner, Niclas C. Blessin. Tc1 cytotoxic T-cells and Th1 helper T-cell rich tumor microenvironment is a hallmark of MSI colorectal cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1517.

P379 PD-1-positive cells contribute to the diagnosis of Inflammatory Bowel Disease and can aid in predicting response to vedolizumab

Abstract Background Differentiating inflammatory bowel disease (IBD) from other inflammatory diseases is often challenging. Programmed cell death protein-1 (PD-1) is expressed in T cells and is an indicator of their exhaustion. The role of PD-1 expression in diagnosing IBD and predicting the response of biologic agents remains inconclusive. Methods In this study, endoscopic biopsy samples of 19 patients diagnosed with IBD and other inflammatory diseases were analysed using multiplexed immunohistochemistry . Other inflammatory diseases comprised five patients with intestinal tuberculosis, and five with intestinal Behçet’s disease. Additionally, a separate prospectively maintained "vedolizumab (VDZ) cohort", established in 12 ulcerative colitis(UC) patients who underwent endoscopic biopsy before VDZ administration was analysed to predict response to VDZ. Results In the immunohistochemistry analysis, the cell density of T cell subsets, including helper T cell (Th), cytotoxic T cell (Tc), regulatory T cell (Treg), PD-1+ cell, PD-1+ Th, PD-1+ Tc, and PD-1+ Treg was investigated and compared between IBD and other inflammatory disease. Cell densities of PD-1+ cells (p=0.028), PD-1+ Th (p=0.008), and PD-1+ Treg (p=0.024) were higher in IBD compared with other inflammatory disease.(Figure 1) In addition, the proportion of PD-1+ cells in Th and Treg was higher in the IBD group than in the other inflammatory disease group (median 7% vs. 3%, p=0.008; median 6% vs. 2%, p=0.004, respectively). Comparing within IBD, the cell density of Treg was higher in Crohn’s disease(CD) than in UC (p=0.042), whilst the cell density of Tc was higher in CD than in UC with marginal statistical significance (p=0.066). In the VDZ cohort, patients with a 14-week steroid-free clinical response had higher levels of PD-1+ cells (p=0.026), PD-1+ Th (p=0.026), and PD-1+ Treg (p=0.041) than the no response group.(Table 1) Conclusion We explored the relationship between PD-1 expression and IBD using multiplexed immunohistochemistry. We compared IBD and other inflammatory disease in their expression of various immune cells including PD-1+ cells, and found IBD had higher PD-1 expression, as well as higher PD-1+ Th and PD-1+ Treg compared with other inflammatory diseases. In addition, VDZ-responsive patients had higher PD-1 expression. Our study confirmed the difference in PD-1 expression in IBD and other inflammatory diseases and revealed that PD-1 expression could be a predictive marker for VDZ responsiveness.

Prognostic Impact of the Immune-Cell Infiltrate in N1-Positive Non–Small-Cell Lung Cancer

IntroductionThe tumoral immune milieu plays a crucial role for the development of non–small-cell lung cancer (NSCLC) and may influence individual prognosis. We analyzed the predictive role of immune cell infiltrates after curative lung cancer surgery. Materials and MethodsThe tumoral immune-cell infiltrate from 174 patients with pN1 NSCLC and adjuvant chemotherapy was characterized using immunofluorescence staining. The density and distribution of specific immune cells in tumor center (TU), invasive front (IF) and normal tissue (NORM) were correlated with clinical parameters and survival data. ResultsTumor specific survival (TSS) of all patients was 69.9% at 5 years. The density of tumor infiltrating lymphocytes (TIL) was higher in TU and IF than in NORM. High TIL density in TU (low vs. high: 62.0% vs. 86.7%; p = .011) and the presence of cytotoxic T-Lymphocytes (CTLs) in TU and IF were associated with improved TSS (positive vs. negative: 90.6% vs. 64.7% p = .024). High TIL-density correlated with programmed death-ligand 1 expression levels ≥50% (p < .001). Multivariate analysis identified accumulation of TIL (p = .016) and low Treg density (p = .003) in TU as negative prognostic predictors in squamous cell carcinoma (p = .025), whereas M1-like tumor- associated macrophages (p = .019) and high programmed death-ligand 1 status (p = .038) were associated with better survival in adenocarcinoma. ConclusionThe assessment of specific intratumoral immune cells may serve as a prognostic predictor in pN1 NSCLC. However differences were observed related to adenocarcinoma or squamous cell carcinoma histology. Prospective assessment of the immune-cell infiltrate and further clarification of its prognostic relevance could assist patient selection for upcoming perioperative immunotherapies.

49P Metformin is associated with increased intratumoural immune infiltrates in patients with primary invasive breast cancer

Obesity has been associated with poor clinical outcomes and chronic systemic inflammation in patients with breast cancer (BC). Metformin has demonstrated antitumorigenic effects both in pre-clinical and clinical studies. We hypothesize that chronic inflammation may lead to immune cell dysfunction in BC that can be restored via the use of metformin. The effect of metformin on intratumoural (IT) immune cells was evaluated in two independent peri-surgical window studies (Dundee, n=29; Oxford, n=31) using immunohistochemistry for selected immune cell markers, pre- and post-metformin. Immune cell infiltrates were digitally quantified using Definiens Architect software and correlated to clinical parameters. Descriptive and linear regression techniques were applied using STATA software. Median densities and 95% confidence intervals were reported. In the Dundee cohort, metformin was associated with a 2.5 (1.8-3.1) fold increase in IT CD68+ macrophages, 2.7 (2.1-9.8) fold increase in IT CD8+ T-cells and 0.5 (0.4-0.6) fold decrease in regulatory T-cells (Tregs) in patients with primary BC. These findings were validated in the Oxford cohort where metformin was significantly correlated with higher density of IT CD8+ T-cells and reduced Treg density (adj P<0.05). These changes were confined to the tumour islands rather than in the stroma. Stratified analysis by BMI in both cohorts showed an increase IT CD68+ macrophages and reduction in Tregs post metformin, in patients with high BMI (adj P<0.05) whereas there was no difference in patients with healthy BMI (adj P>0.05). In the Dundee cohort, linear regression showed that metformin was significantly associated with increased IT CD8+ independently of the baseline pathological or metabolic parameters [co-efficient 7.2, 95%CI (3.2-11.2)]. In the Oxford cohort, systemic inflammation (baseline serum leptin of >17 pg/ml) was associated with lack of correlation between CD8+ and PD1+ T-cells as well as between CD16+ and CD32B+ macrophages that was restored after the administration of metformin (adj P<0.05). Metformin is associated with changes in the IT immune cells. Further work to understand potential combination with immunotherapy is required.

Association of self-identified race and genetic ancestry with the immunogenomic landscape of primary prostate cancer.

The genomic and immune landscapes of prostate cancer differ by self-identified race. However, few studies have examined the genome-wide copy number landscape and immune content of matched cohorts with genetic ancestry data and clinical outcomes. Here, we assessed prostate cancer somatic copy number alterations (sCNA) and tumor immune content of a grade-matched, surgically treated cohort of 145 self-identified Black (BL) and 145 self-identified White (WH) patients with genetic ancestry estimation. A generalized linear model adjusted with age, preoperative prostate-specific antigen (PSA), and Gleason Grade Group and filtered for germline copy number variations (gCNV) identified 143 loci where copy number varied significantly by percent African ancestry, clustering on chromosomes 6p, 10q, 11p, 12p, and 17p. Multivariable Cox regression models adjusted for age, preoperative PSA levels, and Gleason Grade Group revealed that chromosome 8q gains (including MYC) were significantly associated with biochemical recurrence and metastasis, independent of genetic ancestry. Finally, Treg density in BL and WH patients was significantly correlated with percent genome altered, and these findings were validated in the TCGA cohort. Taken together, our findings identify specific sCNA linked to genetic ancestry and outcome in primary prostate cancer and demonstrate that Treg infiltration varies by global sCNA burden in primary disease.

EOMES+ Tr1-like Cells Are Enriched in Areas of NLPHL Group II (Patterns D to F) Supporting the Role of Tregs in Disease Recurrence/Progression

Nodular lymphocyte predominant Hodgkin lymphoma (NLPHL) is a rare lymphoma subtype characterized by proliferation of lymphocyte-predominant (LP) cells associated with a distinct tumor microenvironment. Six major histopathological growth patterns (A to F) have been described by Fan and colls., based on the cellular composition of the tumor microenvironment and its relationship with the LP cells. Group I (patterns A to C) is comprised of nodules rich in small B-cells and follicular dendritic cells and have been associated with low-risk of disease recurrence/progression, while group II (patterns D to F) is rich in small T-cells, lack follicular dendritic cells and have a higher risk of disease recurrence/progression. Although multiple studies have characterized the tumor microenvironment and its relationship with tumor biology in classic Hodgkin lymphoma and other lymphoma types, these studies are limited for NLPHL. Regulatory T-cells (Tregs), a highly immunosuppressive subset of CD4+ T cells, represent a key cellular player in the tumor microenvironment by limiting antitumor immune responses. Emerging studies suggest substantial heterogeneity in the immunophenotype and suppressive capacities of Tregs. For example, classic Tregs are positive for CD4, CD25 and FOXP3; however, a highly immunosuppressive subset of Tregs, named Tr1-like cells has been recently identified and characterized by a distinctive immunophenotype (CD4+, EOMES+, GZMK+ but Foxp3- CD25-) and distinct cytokine expression. Tr1-like are highly relevant in cancer because they seem to be clonally expanded in solid tumors and its presence and number correlates with disease progression (Bonnal et al. Nat Immunol, 2021). Here, we aimed to investigate the Tregs composition (classic and EOMES+ Tr1-like) in the tumor microenvironment of NLPHL and more specifically, to determine cell densities of Tregs in relation with the different histopathological growth patterns of NLPHL. We hypothesize that the number of Tregs will be higher in those immunoarchitectural patterns with the higher risk of disease recurrence/progression. Patients with a diagnosis of NLPHL between 2017 and 2021 with formalin-fixed paraffin embedded tissue biopsies available in our institution were included in the study. All cases were submitted to a previously optimized and validated multiplex IF panel which was specifically developed to assess classic and EOMES+ Tr-1 like Tregs. The panel included CD3, CD4, CD8, CD20, CD25, EOMES, Foxp3, and Granzyme K. Reactive tonsils were used as a positive control. The slides were imaged using Vectra Polaris system (Akoya Biosciences, Marlborough, MA) and up to 10 representative regions of interest (ROI) were selected from each case. The data was consolidated using Spotfire software. The nonparametric Mann-Whitney test was used to compare ROI representing group I and group II histologic areas. Eleven patients with available pretreatment samples were identified. The majority were male (n=7, 63%), with advanced stage of disease (n=7, 63% Stage III/IV); all patients were alive at last follow-up (median: 42 months). A total of 109 ROIs were analyzed (n=54 in group I and n=55 in group II) with a mean number of 22,723 cells/case, and a mean analyzed area of 1.6 mm2/case. The population of CD4+ T-cells was significantly increased in group I (patterns A-C) when compared to group II (patterns D to F) (median, 640 vs 2100, p=0.0013). Interestingly, the group II was associated with a significantly higher cell density of CD4+ EOMES+ Tr1-like cells when compared to group I (median, 0 vs 1010, p<0.0001) (Figure 1). Although not statistically significant, classical Tregs (CD3+CD4+Foxp3+) were also increased in the group II (median, 24.64 vs 67.77, p=0.094). In summary, increased cell densities of Tregs and specifically EOMES+ Tr1-like are strongly associated with architectural patterns of high-risk for disease recurrence and progression in NLPHL. Our data provide a biological rational to focus on these subset populations in efforts to decrease disease recurrence and progression in NLPHL. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

An Exploration of Oral-Gut Pathogens Mediating Immune Escape of Pancreatic Cancer via miR-21/PTEN Axis.

Oral-gut pathogens are closely associated with pancreatic cancer, such as Campylobacter jejuni, Clostridium difficile, Enterococcus faecalis, Escherichia coli, Fusobacterium nucleatum, Helicobacter pylori, Porphyromonas gingivalis, and Vibrio cholera, but the related mechanisms remain not well understood. Phosphatase and tensin homolog (PTEN, a widely known tumor suppressor) play a key role in the anti-cancer immune system. Pancreatic cancer cells with PTEN loss are often in the immunosuppressive tumor microenvironment regulated by myeloid-derived suppressor cells (MDSCs), regulatory T cells (Tregs), and M2 macrophages, which are regarded as the mechanism in the immune escape of cancers. The miR-21, as an oncogene in human cancers, plays an important role in pancreatic cancer progression, downregulates the levels of PTEN, and may promote cancer to evade host immune surveillance. Some oral-gut pathogens have been found to promote miR-21 expression and reduce PTEN expression. On the other hand, most gut pathogens infection is thought to produce reactive oxygen species (ROS) or activate inflammatory cytokines, which may also induce ROS-mediated miR-21 expression. These pathogens' infection is involved with the cell density of MDSCs, Tregs, and M2 macrophages. Therefore, it is quite reasonable to propose that oral-gut pathogens possibly promote pancreatic cancer escaping from host immune surveillance by activating the miR-21/PTEN axis and immune-suppressive cells. The present exploration suggests that an increased understanding of the pattern of the effects of gut pathogens on the miR-21/PTEN axis will lead to better insights into the specific mechanisms associated with the immune escape of pancreatic cancer caused by oral-gut microbiota.

Abstract 6112: Clinical relevance of intra-tumoral density of regulatory T cells as a predictive factor for chemotherapy response in metastatic colorectal cancer

Abstract Background: Within the tumor microenvironment, regulatory T cells (Tregs) are potent facilitators of immune tolerance, and in most solid tumors a higher proportion of Tregs compared to cytotoxic T cells predicts a worse outcome. However, the role of Tregs in colorectal cancer (CRC) is more controversial, possibly due in part to the effect of the gut microbiome on colorectal tumors. We explore the association between colorectal cancer (CRC) Treg density, cancer biology, and clinical outcome. The aim of the study is to investigate the clinical relevance of the fraction of Tregs within the colorectal cancer TME using xCell score with mRNA expression data across several CRC cohorts. Methods: We used xCell to estimate intra-tumoral Tregs in 898 CRC patients in the Cancer Genome Atlas (TCGA) and GCE39582 cohorts. into two groups based on high versus low proportions of Tregs. We studied the correlation between intra-tumoral Treg density and cancer biology including host immune response, ICM expression, and the tumor microbiome by comparing gene ex-pression between the two groups. We further examined two CRC cohorts which contained data on response to neoadjuvant chemotherapy using mFOLFOX6 and bevacizumab, in order to explore the association between Treg infiltration and response to treatment. Results: High-Treg CRCs enriched immune response-related gene sets; inflammatory response, IFN-γ and IFN-α response, IL2/IL6 signaling, and allograft rejection, and had significantly high infiltration of CD8, CD4, M1/M2 macrophage, and dendritic cells in both cohorts. While high-Treg CRCs enriched multiple pro-cancer signaling pathways compared to low-Treg CRCs, such as Epithelial Mesenchymal Transition, K-ras, Hypoxia, TGF-β, TNF-α, and angiogenesis, Treg infiltration was surprisingly associated with earlier CRC stage in TCGA. Metastatic CRCs with more Tregs showed a significantly better response to mFOLFOX6 versus low-Treg CRC metastases (88.9% response vs. 16.7%, p &amp;lt; 0.001). Finally, high-Treg CRCs were associated with increased expression of immune check-point molecules PD-L1/PD-L2, CTLA4, TIGIT and BTLA, implying susceptibility to immunotherapy. Conclusion: We show that unlike in other cancers, Tregs in CRC are not necessarily a marker of poor prognosis. High-Treg CRC enriched not only cancer aggravating gene sets, such as EMT, KRAS, TGF-β and angiogenesis, but also immune response-related gene sets, which is consistent with high infiltration of anti-cancerous immune cells. High Treg density was a predictor of response to chemotherapy in metastatic CRC. Citation Format: Masanori Oshi, Joy Sarkar, Rongrong Wu, Yoshihisa Tokumaru, Li Yan, Ryusei Matsuyama, Itaru Endo, Kazuaki Takabe. Clinical relevance of intra-tumoral density of regulatory T cells as a predictive factor for chemotherapy response in metastatic colorectal cancer [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 6112.

Abstract 2578: High regulatory T cells infiltrate within tertiary lymphoid structure restricts response to immune checkpoint blockers in sarcomas

Abstract Background: While composition of the tumor microenvironment (TME) is a prerequisite for an effective antitumor immunity, infiltration of organized B- and T-cells aggregates called tertiary lymphoid structures (TLS) has been recently demonstrated to predict response to immune checkpoint blockers (ICB) in sarcomas. However, only a minority of patient derive benefit, suggesting the implication of additional key determinants of ICB-mediated response in TLS-positive sarcomas, such as TLS composition. Using high-throughput spatial transcriptomics and multiplex immunofluorescence (IHF), we aimed at investigating the association between TLS composition and clinical outcome to ICB. Methods: In an exploratory cohort, we spatially profiled the expression of more than 18000-protein encoding genes from responders (R) and non-responders (NR) using Nanostring’s GeoMx Digital Spatial Profiler (DSP) Whole Transcriptome Atlas (WTA) assay. A first set of regions of interest (ROI) was selected in the TLS and further segmented in “B-cells” vs “no B-cells” areas according to CD20+ staining; a second set of ROI was selected in the tumor tissue and further segmented into “tumor” vs “stroma” areas according to CD45+ staining. Deconvolution of data was performed using SpatialDecon algorithm to estimate cell population within TLS. We then evaluated the association between immune cell composition and response to ICB in each segment. In a validation cohort, we performed multiplexed-IHF assay enabling detection of T cells (CD8/GzmA/CD4/FoxP3/CD56) and B cells. These panel was applied to whole sections baseline sarcoma samples. We investigated the association between immune cell composition and clinical benefit in term of progression-free survival (PFS) and overall survival (OS). Results: Six patients were selected for the exploratory cohort, including 3 R and 3 NR. Among the top immune cell infiltrate within TLS segment, NR demonstrated higher Treg infiltrate versus R in “no B-cells” compartment (3.4% vs 2.0%, respectively; p=0.010), whereas no association was observed between Treg infiltration and response to ICB in both stromal (p=0.67) or tumor cells (p=0.36) compartments from tumor area. In the validation cohort (N=16), we observed that Treg density within TLS was higher in NR versus R (p=0.0059). Patients with Treg-enriched TLS had shorter PFS (2.6 vs 11.1 months, p=0.042) and OS (9.0 vs 18.3 months, p=0.12) compared to those with Treg-low TLS infiltration. Concordantly, the CTLA-4 key Treg regulator gene was upregulated in the TLS regions from NR. Conclusions: Altogether, our findings suggest that the presence of Treg within TLS may exert a negative influence on the capacity of TLS to generate an effective antitumor immune response in sarcoma patients treated with ICB, providing new insights in understanding role of TLS in antitumor immunotherapy. Citation Format: Florent Peyraud, Jean-Philippe Guegan, Christophe Rey, Marina Pulido, Emmanuelle Bompas, Sophie Piperno-Neumann, Christine Chevreau, Nicolas Penel, François Bertucci, Maud Toulmonde, Carine Bellera, Catherine Sautès-Fridman, Antoine Bougoüin, Coralie Cantarel, Michèle Kind, Mariella Spalato-Ceruso, Bérengère Dadone-Montaudie, Jean-Yves Blay, Wolf Herman Fridman, François Le Loarer, Alban Bessede, Antoine Italiano. High regulatory T cells infiltrate within tertiary lymphoid structure restricts response to immune checkpoint blockers in sarcomas [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 2578.

Thrombopoietin receptor agonists increase splenic regulatory T-cell numbers in immune thrombocytopenia.

Thrombopoietin receptor agonists (TPO-RA) are a valid therapy for immune thrombocytopenia (ITP), due to megakaryocyte stimulation and (poorly characterised) immune-modulatory effects. The spleen is pivotal in the pathogenesis of ITP, yet little is known on its immune microenvironment and on effects of TPO-RA on this organ. To address these topics, we analysed 35 spleens removed for primary refractory ITP. Pre-splenectomy TPO-RA administration correlated with increased splenic regulatory T cells (Tregs), type 2 T-helper cells and histiocyte density and with reduced red pulp sinusoids. Surgical outcome was not associated with TPO-RA administration, other pre-splenectomy therapies and/or Treg density. In conclusion, TPO-RA affect the splenic microenvironment, but this has no impact on splenectomy outcome.

Tumour-draining lymph nodes in head and neck cancer are characterized by accumulation of CTLA-4 and PD-1 expressing Treg cells

IntroductionHigh Tregs infiltration within the tumour microenvironment (TME) of various cancers shows a positive correlation with poor prognosis. Despite the fact that tumour draining lymph nodes (TDLNs) are recognized as key organs playing a crucial role in response to immunotherapy and modulating anti-cancer immunity, the distribution of Tregs and their role in TDLNs remain uncertain thus far. The purpose of this project is to investigate the density of Tregs in TDLNs and non-TDLNs and their expression of immune checkpoint molecules – PD-1 and CTLA-4. MethodsSamples including TDLNs, non-TDLNs and metastatic lymph nodes (LNs) from 23 patients with oral squamous cell carcinoma (OSCC) were analyzed by multicolour flow cytometry with a focus on Tregs population and expression of CTLA-4 and PD-1. ResultsTDLNs and metastatic LNs were characterized by a significantly higher infiltration of Tregs defined as CD4+FoxP3+CD25highCD127low cells and significantly higher expression of CTLA-4 and PD-1 on Tregs compared with non-TDLNs. Tregs in TDLNs and metastatic LNs co-expressed CTLA-4 and PD-1 abundantly. High expression of these immune check-point molecules correlated with positive N-stage but not with T-stage. ConclusionTDLNs and metastatic LNs are characterized by a high accumulation of Tregs expressing high levels of CTLA-4 and PD-1. High infiltration of Tregs can be a potential driver of an immunosuppressive milieu in TDLNs that can, in turn, favour cancer progression. High accumulation of Tregs expressing CTLA-4 and PD-1 in TDLNs is associated with lymph node involvement, but not with the size of the primary tumour.

Differences in tumor microenvironment between HER2-positive and HER2-negative breast cancer.

e12562 Background: The tumor microenvironment of breast cancer is extremely complex, which containing different types of lymphocytes, macrophages, NK cells and other immune cell subtypes. At present, the relationship between immune cell subtypes and molecular subtypes of breast cancer is still unclear. Methods: Tumor tissue samples of 20 breast cancer patients were collected from January 2020. Then the density (number of positive cells/mm2) and ratio of immune cells in intratumoral area and stromal area were detected by multiple fluorescence immunohistochemistry (mIHC), the detection range was included CD3+T cells, CD4+T cells,CD8+T cells,PD-1+CD8+T cells,Tregs,M1/M2 tumor associated macrophages (TAM), PD-L1+CD68+TAM,B cells,CD56bright NK cells and CD56dim NK cells and so on. The immune cell subtypes of all area were included both intratumoral area and stromal area. Graphpad prism (version 7.01) was used for t test and the statistical significance was set at p=0.05. Results: These 20 patients were divided into two groups, HER2 positive (n=8) and HER2 negative group (n=12). The density of CD3+T cells in all area (including intratumoral area and stromal area) of tumor tissue in HER2 positive breast cancer patients were significantly higher than that of HER2 negative breast cancer patients (median density, HER2 positive group vs. HER2 negative group=2998 vs. 1520/mm², p=0.031) as well as the CD8+T cells (median density, HER2 positive group vs. HER2 negative group=536 vs. 175/mm², p=0.015). Meanwhile, the density of Tregs in all area of tumor tissue in HER2 positive breast cancer patients was also significantly higher (median density, HER2 positive group vs. HER2 negative group=119 vs. 27/mm², p=0.022).However, there was no significant difference in the other subtypes of immune cells infiltrated in tumor tissue between HER2 positive and HER2 negative samples, such as CD4+T cells, M1/M2 macrophages, B cells and so on (p＞0.05). Conclusions: Our research indicates that the patterns of immune cell subtypes in different types of early breast cancer are different. Further exploration of the immune microenvironment in different molecular types of breast cancer may provide additional targets for immunotherapy.

Holistic Characterization of Tumor Monocyte-to-Macrophage Differentiation Integrates Distinct Immune Phenotypes in Kidney Cancer.

The tumor immune microenvironment (TIME) is commonly infiltrated by diverse collections of myeloid cells. Yet, the complexity of myeloid-cell identity and plasticity has challenged efforts to define bona fide populations and determine their connections to T-cell function and their relationship to patient outcome. Here, we have leveraged single-cell RNA-sequencing analysis of several mouse and human tumors and found that monocyte-macrophage diversity is characterized by a combination of conserved lineage states as well as transcriptional programs accessed along the differentiation trajectory. We also found in mouse models that tumor monocyte-to-macrophage progression was profoundly tied to regulatory T cell (Treg) abundance. In human kidney cancer, heterogeneity in macrophage accumulation and myeloid composition corresponded to variance in, not only Treg density, but also the quality of infiltrating CD8+ T cells. In this way, holistic analysis of monocyte-to-macrophage differentiation creates a framework for critically different immune states.

IMMUNOlogical microenvironment in REctal Adenocarcinoma Treatment (IMMUNOREACT 2; NCT04917263): preliminary results on prediction of complete response to neoadjuvant therapy in rectal cancer

Background: After pathologic complete response (pCR) to neoadjuvant chemoradiotherapy (nCRT) for locally advanced rectal cancer (LARC) patients might be eligible for less radical surgery or non-operative management. A low stromal Treg density was observed more frequently in patients who had a pCR. Field cancerization concept means that some molecular events in carcinogenesis might occur even in normal tissues surrounding tumors. Our aim was to identify, among LARCs, those with complete response to nCRT analyzing the immune microenvironment in healthy mucosa.