TYPE Original Research PUBLISHED 11 October 2022 DOI 10.3389/fimmu.2022.993401 Remodeling the bladder tumor OPEN ACCESS immune microenvironment EDITED BY Sergei Kusmartsev, University of Florida, United States by mycobacterial species REVIEWED BY Guru P. Sonpavde, with changes in their cell Dana–Farber Cancer Institute, United States Chien-Feng Li, envelope composition National Health Research Institutes, Taiwan Jordi Senserrich 1†, Sandra Guallar-Garrido 2†, *CORRESPONDENCE Cecilia Cabrera Elisabet Gomez-Mora 1, Victor Urrea 1, Bonaventura Clotet 1,3,4,5, ccabrera@irsicaixa.es Esther Julián 2‡ and Cecilia Cabrera 1*‡ Esther Julián esther.julian@uab.cat 1 AIDS Research Institute IrsiCaixa, Institut de Recerca en Ciències de la Salut Germans Trias i Pujol † These authors have contributed (IGTP), Hospital Germans Trias i Pujol, Universitat Autònoma de Barcelona, Barcelona, Spain, equally to this work and share 2 Departament de Genètica i de Microbiologia, Facultat de Biociències, Universitat Autònoma de first authorship Barcelona, Barcelona, Spain, 3 Fundació lluita contra la SIDA, Infectious Diseases Department, Hospital Germans Trias i Pujol, Catalonia, Spain, 4 University of Vic-Central University of Catalonia ‡ These authors have contributed (UVic - UCC), Vic, Spain, 5 Consorcio Centro de Investigación Biomédica en Red de Enfermedades equally to this work and share Infecciosas (CIBERINFEC), Instituto de Salud Carlos III, Madrid, Spain last authorship SPECIALTY SECTION This article was submitted to Cancer Immunity and Immunotherapy, a section of the journal Intravesical BCG instillation after bladder tumor resection is the standard Frontiers in Immunology treatment for non-muscle invasive bladder cancer; however, it is not always RECEIVED 13 July 2022 effective and frequently has undesirable side effects. Therefore, new strategies ACCEPTED 20 September 2022 that improve the clinical management of patients are urgently needed. This PUBLISHED 11 October 2022 study aimed to comprehensively evaluate the bladder tumor immune CITATION microenvironment profile after intravesical treatment with a panel of Senserrich J, Guallar-Garrido S, Gomez-Mora E, Urrea V, Clotet B, mycobacteria with variation in their cell envelope composition and its impact Julián E and Cabrera C (2022) on survival using an orthotopic murine model to identify more effective and Remodeling the bladder tumor immune microenvironment by safer therapeutic strategies. tumor-bearing mice were intravesically treated mycobacterial species with changes in with a panel of BCG and M. brumae cultured under different conditions. their cell envelope composition. Untreated tumor-bearing mice and healthy mice were also included as Front. Immunol. 13:993401. doi: 10.3389/fimmu.2022.993401 controls. After mycobacterial treatments, the infiltrating immune cell COPYRIGHT populations in the bladder were analysed by flow cytometry. We provide © 2022 Senserrich, Guallar-Garrido, evidence that mycobacterial treatment triggered a strong immune infiltration Gomez-Mora, Urrea, Clotet, Julián and into the bladder, with BCG inducing higher global absolute infiltration than M. Cabrera. This is an open-access article distributed under the terms of the brumae. The induced global immune microenvironment was strikingly different Creative Commons Attribution License between the two mycobacterial species, affecting both innate and adaptive (CC BY). The use, distribution or immunity. Compared with M. brumae, BCG treated mice exhibited a more reproduction in other forums is permitted, provided the original robust infiltration of CD4+ and CD8+ T-cells skewed toward an effector author(s) and the copyright owner(s) memory phenotype, with higher frequencies of NKT cells, neutrophils/ are credited and that the original publication in this journal is cited, in gMDSCs and monocytes, especially the inflammatory subset, and higher accordance with accepted academic CD4+ TEM/CD4+ Treg and CD8+ TEM/CD4+ Treg ratios. Conversely, M. brumae practice. No use, distribution or treatment triggered higher proportions of total activated immune cells and reproduction is permitted which does not comply with these terms. activated CD4+ and CD8+ TEM cells and lower ratios of CD4+ TEM cells/CD4+ Tregs, CD8+ TEM cells/CD4+ Tregs and inflammatory/reparative monocytes. Frontiers in Immunology 01 frontiersin.org Senserrich et al. 10.3389/fimmu.2022.993401 Notably, the mycobacterial cell envelope composition in M. brumae had a strong impact on the immune microenvironment, shaping the B and myeloid cell compartment and T-cell maturation profile and thus improving survival. Overall, we demonstrate that the bladder immune microenvironment induced by mycobacterial treatment is species specific and shaped by mycobacterial cell envelope composition. Therefore, the global bladder immune microenvironment can be remodelled, improving the quality of infiltrating immune cells, the balance between inflammatory and regulatory/suppressive responses and increasing survival. KEYWORDS non-muscle invasive bladder cancer, BCG, Mycobacterium brumae, immunotherapy, intravesical mycobacterial treatment, tumor immune microenvironment GRAPHICAL ABSTRACT Introduction Intravesical BCG treatment is generally well tolerated, although both local and systemic side effects have been Bladder cancer (BC) is the fourth most common cancer in reported (6). To maximize the antitumor effect of BCG and men, and approximately 75% of BC cases are classified as non- reduce toxicity, several other alternatives, such as the use of muscle invasive bladder cancer (NMIBC) at initial diagnosis (1). genetically modified BCG strains or the combination of BCG The current gold standard for treatment of high-risk NMIBC is with other immunotherapeutic agents, have been widely transurethral resection of bladder tumor followed by intravesical explored (4, 5, 7), although none have been approved for bacillus Calmette–Gué rin (BCG) instillations as an clinical use. One promising alternative is the use of immunotherapeutic strategy to prevent recurrence and reduce mycobacteria other than BCG, such as Mycobacterium brumae the risk of progression (2). Unfortunately, despite treatment, 30- (M. brumae), a safe nontuberculous mycobacterium (8) that has 50% of patients receiving BCG fail to respond, and 10-15% been shown to have a potential role in NMIBC treatment in in experience progression to muscle-invasive bladder cancer (3). vitro and in vivo preclinical studies since it inhibits tumor To solve this difficult-to-treat disease many clinical trials are proliferation and triggers an effective antitumor immune testing the efficacy of innovative immunological and target response (9–11). agents in both BCG unresponsive and naïve patients, such as It is well recognized that the tumor immune microenvironment checkpoint inhibitors, oncolytic viruses, cancer vaccines, tyrosine not only is a key regulator of cancer progression but also plays a kinase receptor inhibitors, or other immunostimulatory agents as crucial role in cancer treatment response; therefore, strategies to the IL-15 superagonist N-803 (4, 5). optimize the tumor immune microenvironment are being Frontiers in Immunology 02 frontiersin.org Senserrich et al. 10.3389/fimmu.2022.993401 investigated (12). In BC, BCG treatment has been associated with differing on the amino acid source and glycerol concentration: L- nonspecific antitumor immune response in the bladder mucosa, but asparagine plus 6% glycerol (A60), L-glutamate plus 1.5% glycerol despite its long-term use, their exact mechanism of action is not (G15) and L-glutamate plus 6% glycerol (G60) (22). completely understood (13). Several studies have shown that the The murine BC cell line MB49 was maintained in Dulbecco’s pretreatment tumor immune microenvironment plays an modified Eagle’s medium (Gibco, BRL), with 10% fetal bovine important role in determining the BCG response in patients (14– serum (FBS) (Lonza, Switzerland), 100 U/mL penicillin G 18). Reports have also shown that post-BCG tissues are infiltrated (Laboratorios, Spain) and 100 mg/ml streptomycin (Laboratorio with increased numbers of different subpopulations of CD4+ and Reig Jofré, Spain). CD8+ T-cells and, to a minor extent, other immune populations (17, 18). However, our understanding of the BCG-induced immune landscape remains incomplete, mainly due to the difficulty Orthotopic model of BC and associated with obtaining tissue samples from patients during intravesical treatment treatment. Using animal models, it has been shown that CD4+ and CD8+ T-cells play a critical role in BCG- and M. brumae- The mouse orthotopic model of BC was generated as induced antitumor activity (10, 19–21), although comprehensive described previously (11). Briefly, 8 randomized C57BL/6 immune profiling has not been reported. female mice (6 to 8-week-old, Charles River Laboratories, Given the very few advances in therapeutic strategies for early- Spain) per group were used. Tumors were induced by initial stage disease over the past two decades, there is a major unmet need intravesical administration of poly-L-lysine (Sigma-Aldrich) for improved intravesical therapies for NMIBC. We very recently followed by MB49 cells instillation. 24 hours after tumor showed that the mycobacterial cell-surface is modified by culture implantation, mice were intravesically treated with BCG or M. conditions, which impacts antitumor immune activity (21, 22). This brumae grown in different conditions (21). Mycobacterial is relevant since commercially available BCG sub-strains are treatment was performed weekly for four weeks. Mice not currently cultured on different medium compositions, which is inoculated with tumor cells nor with mycobacteria were used one of the plausible reasons for the different treatment outcomes as controls. Tumor implantation was ensured due to the observed in NMIBC patients after BCG treatment. The aim of this presence of blood in urine between days 7 to 10 after study was to provide comprehensive immune profiling of the induction. Animal behavior and well-being was evaluated daily bladder in an orthotopic murine BC model upon mycobacterial to avoid unnecessary suffering, and animals were euthanized treatment using a panel of mycobacteria that displayed changes in when it was required. Animals were sacrificed at day 29 ± 1, and their cell envelope due to the culture conditions. Overall, we bladders were removed in aseptic conditions for immunological demonstrated that the global bladder tumor immune characterization. For survival analyses, another set of microenvironment could be remodelled by improving the balance experiments were performed following the same scheduled as between inflammatory and regulatory/suppressive responses after before, but analyzing clinical symptoms until day 60 after tumor mycobacterial treatment. This knowledge may provide clues for the induction when surviving animals were sacrificed (21). mechanism of action of mycobacteria as a cancer immunotherapy, how to fine-tune the tumor microenvironment to fight cancer more efficiently, and the identification of more effective and less toxic Bladder tissue processing and staining therapeutic strategies. The immune infiltrate present into the bladder was analyzed by flow cytometry as previously described (10). Briefly, bladders Materials and methods were minced using a scalpel followed by digestion with 0.5 mg/ mL collagenase II (Sigma, Spain) in RPMI-5% FBS 1U/mL Bacterial Strains and BC Cell Line DNAse I medium at 37°C for two-three successive 30 min- cycles, with continuous shaking. The cell suspension obtained M. brumae (American Type Culture Collection, ATCC 51384), was filtered through a 40-mm disposable plastic strainer (Becton and M. bovis BCG Connaught strain (ATCC 35745) were grown in & Dickinson) and pelleted for staining. Cells were labeled using Middlebrook 7H10 agar (Difco Laboratories, UK) supplemented two antibody panels: Panel 1: CD45, CD3, CD4, CD8, CD62L, with 10% oleic-albumin-dextrose-catalase enrichment medium for NK1.1, CD127, CD44, CD25 and TCRgd; Panel 2: CD45, CD3, 1-4 weeks, respectively. Mycobacteria grown in solid media were CD45R/B220, CD11b, CD11c, Ly6G, Ly6C and F4/80. LIVE/ used to inoculate three compositions of Sauton culture medium DEAD Fixable Aqua Dead Cell Stain Kit (Invitrogen) was used Frontiers in Immunology 03 frontiersin.org Senserrich et al. 10.3389/fimmu.2022.993401 to determine cell viability. The antibodies used are shown in fluorescence intensities of each marker across all populations Supplementary Table 1. were plotted on a hierarchically cluster heat map. Flow cytometry analysis Statistical analysis After gating on single-cell population, dead cells were GraphPad Prism 8.0 software (San Diego, USA) was used for excluded and immune cell were selected by morphological statistical analyses. Immune infiltration data were analyzed using parameters and expression of CD45. Absolute cell numbers Mann-Whitney tests. Statistical significance was assumed at p were obtained by using Perfect-Count Microspheres values below 0.05. (Cytognos). Samples were acquired in a Fortessa flow cytometer (Becton & Dickinson), and data were analyzed using FlowJo software (v10.7.1; TreeStar). Boolean gating was Results used to generate different population subsets analyzed. Analysis and presentation were performed using SPICE version 6.0 Absolute immune infiltration is species (https://niaid.github.io/spice). Global immune profile was done dependent and modulated by cell using OMIQ data analysis software (www.omiq.ai). Cells were envelope composition but is not a gated on CD45+ singlets and individual files were concatenated predictor of survival and clustered as a whole in each group of animals using the optimized t-distributed stochastic neighbor embedding (opt- A murine orthotopic model of BC was used to deeply SNE) algorithm for dimensional reduction and visualization. characterize the immune infiltration in bladder tumors after The opt-SNE plot of each panel was subdivided into 20 spatially intravesical treatment with a panel of mycobacteria that distinct subpopulations using a manual gating strategy. Median displayed changes in their cell envelope lipidomic profile A B C FIGURE 1 Robust infiltration of immune cells into the bladder after mycobacteria treatment. Absolute counts of (A) total live CD45+ cells, CD3+, CD4+ and CD8+ T-cells, and NK+ cells (CD3-NK1.1+); and (B) CD3- cells, CD11b+ myeloid cells (CD3-B220-CD11b+), neutrophils (CD3-B220-Ly6C+Ly6G+), monocytes (CD3-B220-Ly6G-CD11b+), dendritic cells (CD3-B220-Ly6G-CD11c+) and B-cells (CD3-B220+) were quantified by flow cytometry after bladder digestion of healthy mice (white bars and dots), untreated tumor-bearing mice (grey bars and dots) and mycobacteria-treated mice, either BCG (blue bars and dots) or M. brumae (red bars and dots) regardless the medium used to growth mycobacteria. (C) Absolute infiltration of total CD3+ T-cells, CD4+ T-cells, total CD3- cells and neutrophils of mice which received BCG (blue bars) or M. brumae (red bars) grown in different culture media. Each dot represents an individual mouse. Data represent the mean (bars) and ± SEM (error bars) and each dot represent an individual mouse. Statistically significant differences between the different BCG and M. brumae mycobacteria (intrastrain) are shown as blue and red lines, respectively. Interstrain differences or with the untreated groups (healthy and tumor) are shown in black. Differences were tested using Mann-Whitney U nonparametric test. *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001; ****p ≤ 0.0001. Frontiers in Immunology 04 frontiersin.org Frontiers in Immunology Senserrich et al. A B C D F G E 05 H I FIGURE 2 Global profile of bladder infiltrating immune cells. opt-SNE was used for the analysis of the immune infiltration into the bladder of healthy, non-treated and mycobacteria-treated tumor-bearing mice. Cells were stained using two panels of antibodies (Supplementary Table 1), measured by flow cytometry and analyzed using OMIQ software. (A) opt-SNE maps showing expression intensity of all markers from both antibody panels of concatenated files for all healthy animals, non-treated and all BCG- or M. brumae-treated mice, in multidimensional space. (B) The same opt-SNE maps with the expression intensity using panel 1 antibodies and (C) using panel 2 antibodies in bladders from mice treated with BCG or M. brumae grown in different culture media (D) Overlay of 20 gated cell 10.3389/fimmu.2022.993401 populations on to opt-SNE plots for both antibody panels. (E) Heatmaps showing expression intensity in all the populations gated in D) for all the markers using both antibody panels. Color-coded with dark green for higher expression and light green for lower expression. (F) Heatmaps displaying frequency for all the populations gated in D) among bladders from all animal groups using panel 1 and (G) using panel 2 antibodies. (H) Volcano plots showing adjusted p values versus log (Fold Change; FC) for immune populations in different groups of animals using panel 1 and (I) panel 2 antibodies. Populations whose expression is significantly different between group of animals with a adjusted p < 0.05 or p < 0.00 are shown in green. The red line denotes a p value of 0.001 or 0.05. frontiersin.org Senserrich et al. 10.3389/fimmu.2022.993401 induced by growth in different conditions (21). In comparison different culture media (Figures 2B, C, antibody panels 1 and 2, with no treatment, BCG and M. brumae treatments resulted in a respectively). A more marked effect was observed when robust and significant increase in the absolute number of infiltrating non-T-cells were analysed (Figure 2C). To immune cells infiltrating the bladder, including both T-cells provide more specificity to our analysis, we subdivided the and non-T-cells, with significantly higher infiltration in BCG- opt-SNE plot of each panel into 20 spatially distinct treated animals than in M. brumae-treated animals (Figures 1A, subpopulations (Figure 2D). Heatmaps revealed the B; gating strategy Supplementary Figures 1, 2). No correlation d i ff e r e n t i a l e x p r e s s i o n o f a l l m a r k e r s i n t h e 2 0 was found between total immune infiltration and survival, with subpopulations (Figure 2E). In panel 1, ten populations were longer survival in M. brumae- than in BCG-treated animals (21) CD3+ T-cells, among which six were CD4+ T-cells. Population (Supplementary Figure 3). 18 (P18) corresponded to naïve cells (CD62L+CD44-), P13 There were no statistically significant differences in the total represented natural killer T (NKT) gd cells (NK1+gd TCR+), infiltrating cells in different immune populations between the and the other populations, which were all CD44+, could be untreated groups (healthy and tumor), except for higher CD8+ considered different subsets of memory CD4+ T-cells. Within T-cell infiltration observed after tumor induction (Figure 1A). the identified CD8+ T-cells, memory and NKT CD8+ T-cells Several other populations were increased in the tumor group, but (P12 and P17, respectively) contributed to the tumor no significant differences were reached, most likely because of microenvironment. The CD3- compartment was composed of the low number of animals in the untreated tumor group that several populations with variable expression of different survived to the end of the experiment (Figures 1A, B). markers, including natural killer (NK) cells (NK1+, P20) and Between BCG and M. brumae grown in different culture innate lymphoid cells (ILCs, expressing CD127 and high levels media, we observed that treatment with M. brumae-A60 (M. of CD25; P1). In panel 2, nine CD3 + populations were brumae grown in A60 medium) induced significantly lower identified, three of which had high expression of Ly6C (P3, infiltration of most immune populations than all BCG and the P15 and P19). Within lymphoid non-T-cells (CD3-), two B-cell rest of the M. brumae treatments (Figure 1C and Supplementary populations (B220 + ) were identified (P16 and P1). Nine Figure 4). We have previously shown that M. brumae-A60 populations were assigned as myeloid cells (CD11b + ), treated mice have a prolonged survival than the rest of M. including dendritic cells (DCs, CD11c+; P9, P12 and P18), brumae and BCG treated mice, although no significant polymorphonuclear cells/granulocytic myeloid-derived differences were reached (Reference 21 and Supplementary suppressor cells (PMNs/gMDSCs, Ly6C + Ly6G + ; P5), Figure 3). This data further confirms the lack of association macrophages (F4/80 h i ; P10) and different monocyte between total immune infiltration and survival. When BCG- populations (P2, P4, P5 and P11). These data highlight the induced immune infiltration was analysed, there were significant high heterogeneity in the bladder tumor microenvironment decreases in the numbers of infiltrating CD4+ T-cells and B-cells after mycobacterial treatment. In concordance with the in the BCG-A60 group in comparison with the group BCG-G60 different spatial cell distributions observed in BCG- or M. and BCG-G15 groups, respectively (Figure 1C and brumae-treated mice (Figure 2A), clear differences were Supplementary Figure 3). observed when the frequencies of the different populations were plotted as a heatmap (Figures 2F, G, upper panels; corresponding to antibody panels 1 and 2, respectively). Mycobacterial cell envelope composition Seven and four populations identified with panels 1 and 2, lead to global changes in the immune respectively, were significantly enriched in BCG-treated mice microenvironment in bladder tumors compared with M. brumae-treated mice, with most of them being CD3+ populations (Figures 2H, I, left part of volcano To further explore the global immune profile of the bladder plots). In contrast, seven and five populations were tumor microenvironment after intravesical mycobacterial significantly enriched in M. brumae-treated mice (in panels 1 treatment, computational flow cytometric analysis was and 2, respectively), with most of them being CD3- populations performed. We generated a two-dimensional opt-SNE plot to (Figures 2H, I, right part of volcano plots). The heatmaps also dimensionally reduce the complete multiparametric dataset. showed different global immune profiles for animals treated Unlike conventional biaxial plots and sequential analysis of with BCG or M. brumae grown in different culture media pairs of markers, the opt-SNE map captured and summarized showing variations in the cell envelope lipid composition. M. the interrelationship between all the markers in an unbiased, brumae-A60 produced a more differential composition of data-driven manner. Using two antibody panels (described in infiltrated populations (Figure 2F, G, lower panels, panels 1 Supplementary Table 1) and this method, we observed marked and 2, respectively), with a lower proportion of naïve CD4+ T- differences in the spatial cell distribution, visualized as different cells (P18; Figure 2H, right panel, and Supplementary Figure 5, patterns of colours, among all groups of animals (Figure 2A) top panels) and significant enrichments or reductions in other and in animals treated with BCG or M. brumae grown in populations (Figure 2I, left panel, Supplementary Figure 5, Frontiers in Immunology 06 frontiersin.org Senserrich et al. 10.3389/fimmu.2022.993401 A B C D E F FIGURE 3 T- and non-T infiltrating cells into the bladder in different groups of mice. Frequencies of bladder infiltrating (A) CD3+ and CD3- cells; (B) CD4+ and CD8+ T-cells; (C) total CD25-expresing cells, CD3+CD25+ and CD3-CD25+ cells; (D) NK, NKT, TCRgd+ T-cells and ILC; (E) B-cells; and (F) B1 cells in different groups of mice. Frequencies are given in healthy mice (white bars and dots), untreated tumor-bearing mice (grey bars and dots) and mycobacteria treated-tumor bearing mice, either using BCG (blue bars and dots) or M. brumae (red bars and dots) as percentages of the total live CD45+ infiltrating immune cells. Data represent the mean (bars) and ± SEM (error bars) and each dot represent an individual mouse. Statistically significant differences between the different BCG and M. brumae mycobacteria (intrastrain) are shown as blue and red lines, respectively. Interstrain differences or with the untreated groups (healthy and tumor) are shown in black. Differences were tested using Mann- Whitney U nonparametric test. *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001; ****p ≤ 0.0001. bottom panels). Of note, two populations that were surface markers were used. Compared to healthy and consistently and significantly reduced with M. brumae-A60 untreated tumor-bearing mice, mice treated with instillation treatment compared to the other M. brumae treatments were of mycobacteria showed strong infiltration of CD3+ cells into P5 (PMNs/gMDSCs, Ly6C+Ly6G+) and P4 (monocytes, Ly6C+ the bladder. In parallel with the absolute infiltration results, cells) (Figure 2I, Supplementary Figure 5, bottom panels). BCG treatment recruited larger percentages than M. brumae These data suggest that modulation of the global immune treatment that, on the contrary, had an enriched CD3- cell infiltration, rather than the quantity of infiltrated cells, could infiltration (Figure 3A). Bladders from mycobacterium- play a critical role in improving intravesical treatment efficacy treated mice showed increased percentages of both CD4+ T- and survival. cells and CD8+ T-cells compared to bladders from healthy mice (Figure 3B). The CD4+ T-cell proportions were similar between BCG- and M. brumae-treated mice, although the BCG and M. brumae treatments induce BCG-treated tumors were significantly enriched in CD8+ T- differential lymphoid T and non-T-cell cells compared to the M. brumae-treated tumors (Figure 3B). bladder immune cell infiltration profiles, These data corroborated the results obtained with the whereas mycobacterial culture computational unsupervised opt-SNE analysis (Figures 2F, conditions have a major impact on the H, P17 population). Considering the expression of the tumor B-cell composition activation marker CD25, bladder tumors from M. brumae- treated mice were enriched in CD25+ populations, both CD3+ To further elucidate the heterogeneity of the immune cells and CD3- cells, compared to those from BCG-treated microenvironment in bladder tumors after different animals (Figure 3C). No significant differences were observed treatments, traditional biaxial gating strategies based on 1-3 between the groups treated with each mycobacterium grown Frontiers in Immunology 07 frontiersin.org Frontiers in Immunology Senserrich et al. A C B 08 FIGURE 4 CD4+ and CD8+ T -cell maturation profiles of bladder infiltrating cells differ after different mycobacteria treatments. Immune maturation profile in BCG- and M. brumae-treated mice in (A) CD4+ and CD8+ T- cells and in (B) CD4+ and CD8+ T-cells expressing CD25. The differential marker expression was analyzed by boolean gating. (C) CD4+ and CD8+ T-cell maturation profile in bladder tumors from mice treated with BCG and M. brumae cultured in different media. Pie charts illustrate relative proportions of each of the different subsets in the different group of animals. Arcs show population makeup and overlap within pie slice. Statistical analyses of the global maturation profiles were performed by partial permutation tests using the SPICE software (*p < 0.05, **p < 0.01, ***p < 0.001****p < 0.0001). Bar graphs indicate percentages of the different populations within CD4+ or CD8+ T-cells. Data represent the mean (bars) and ± SEM (error bars) and each dot represent an individual mouse treated with BCG (blue dots) or M. 10.3389/fimmu.2022.993401 brumae (red dots). Statistically significant differences between the different M. brumae mycobacteria are shown as red lines. Interstrain differences are shown in black. Differences were tested using Mann-Whitney U nonparametric test. *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001. N: naïve cells (CD62L+CD44-); CM: central memory cells (CD62L+CD44+) and EM: effector memory cells (CD62L-CD44+). frontiersin.org Senserrich et al. 10.3389/fimmu.2022.993401 A B C D FIGURE 5 Changes in the infiltration of CD4+ Tregs after BCG and M. brumae treatment. Absolute counts (A) and percentages (B) of infiltrating CD4+ Tregs (CD3+CD4+CD25+CD127-). (C) ratio of CD4+ TEM and (D) CD8+ TEM to CD4+ Treg cells. Data are shown in BCG- and M. brumae-treated tumor- bearing mice pooled or divided by groups according to the medium used to growth mycobacteria. BCG (blue bars and dots). M. brumae (red bars and dots). CD4+ TEM: CD3+CD4+CD44+CD62L-. CD8+ TEM: CD3+CD8+CD44+CD62L-. Statistically significant differences between the different M. brumae mycobacteria are shown as red lines. Interstrain differences are shown in black.Data represent the mean (bars) and ± SEM (error bars). Each dot represent an individual mouse. Differences were tested using Mann-Whitney U nonparametric test. *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001. in different culture media (data not shown). The infiltration mycobacteria were grown. The B-cell and B1 populations of NK and TCRgd+ T-cells remained constant regardless of were found to be significantly reduced in animals treated with the mycobacterial treatment administered (Figure 3D). both mycobacteria grown in A60 medium (Figures 3E, F). However, NKT cells were enriched in BCG-treated mice, whereas tumors from M. brumae-treated mice contained more infiltrating ILCs (Figure 3D). No significant BCG and M. brumae as well as their cell differences in infiltrated immune cells were observed among envelope composition alter the each mycobacterium grown in different culture media (data maturation profile of tumor-infiltrating not shown). The B-cell lineage contribution was also CD4+ and CD8+ T-cells evaluated. The total B-cell (CD3-B220+) and peritoneal B1 subset (CD3-B220+CD11b+) frequencies did not show any The maturation profile of tumor-infiltrating lymphocytes significant difference when comparing BCG- or M. brumae- after treatment was evaluated (gating strategy Supplementary treated mice (Figures 3E, F, gating strategy Supplementary Figure 1). Cells were phenotypically identified as naïve (TN, Figure 2). Nevertheless, the B-cell response in the tumor CD62L+CD44-), central memory (TCM, CD62L+CD44+) and microenvironment was strongly influenced by the antigenic effector memory T-cells (TEM, CD62L-CD44+). Infiltrating changes induced by the culture conditions in which the CD4+ and CD8+ lymphocytes were primarily composed of TEM Frontiers in Immunology 09 frontiersin.org Senserrich et al. 10.3389/fimmu.2022.993401 FIGURE 6 Changes in the tumor myeloid compartment after mycobacteria intravesical treatment. Pie chart representing proportional subpopulations of myeloid cells. CD3-CD11b+ tumor infiltrating myeloid cells were gated and the relative proportion of neutrophils, macrophages, monocytes and dendritic cells were represented and analyzed using SPICE software. Statistical analyses of the global maturation profiles were performed by partial permutation tests. Bar graphs indicate percentages of the different populations within the total CD11b+ cells. Data represent the mean (bars) and ± SEM (error bars) and each dot represent an individual mouse treated with BCG (blue dots) or M. brumae (red dots). Statistically significant differences between the different M. brumae mycobacteria are shown as red lines. Interstrain differences are shown in black. Differences were tested using Mann-Whitney U nonparametric test. *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001; ****p ≤ 0.0001. Neutrophils (red; CD11b+CD11c-Ly6G+), macrophages (yellow, CD11b+CD11c-F4/80hi), monocytes (green, CD11b+CD11c-F4/80lo/int) and dendritic cells (blue, CD11b+CD11c+). cells in all treated animals (Figure 4A). No significant differences left bar graph). A similar tendency was observed for the CD8+ T- were observed in the CD4+ T-cell maturation distribution of cell profile, although no significant differences were reached infiltrating cells when all the animals treated with BCG were (Figure 4C, lower pie charts and right bar graph). compared with those treated with M. brumae (Figure 4A, upper panel). However, the maturation profile of infiltrating CD8+ T- cells in the bladder was significantly different between animals Immunosuppressive tumor treated with each mycobacteria species, with larger frequencies of microenvironment is modulated TN and TCM cells observed after M. brumae treatment compared by changes in mycobacterial to BCG treatment (Figure 4A, lower panel). When the expression culture conditions of CD25 was included in the analysis by using a Boolean gating strategy, striking differences in the maturation profiles of both Regulatory T-cells (Treg) play crucial roles in the regulation CD4+ and CD8+ T-cell populations were found (Figure 4B). An of antitumor immunity (23) The absolute cell number of CD4+ enrichment in CD25+ TEM cells with an accumulation of CD25- Treg (defined as CD3+CD4+CD25+CD127-, gating strategy TN and TCM cells was observed in M. brumae-treated mice Supplementary Figure 1) was significantly higher in bladders compared with BCG-treated mice (Figure 4B). Regarding the from BCG-treated mice than in those from M. brumae-treated effect of culture conditions, M. brumae-A60-treated animals mice (Figure 5A), which was in line with the higher absolute showed a different maturation profile for infiltrating CD4+ T- infiltration of most cell populations observed after BCG cells than the rest of the animals, where the naïve/memory treatment. In this case, a sharp decrease in the total infiltration phenotype distribution of CD4+ T-cells was sharply skewed of these cells was observed in M. brumae-A60-treated mice. On towards an effector phenotype (Figure 4C, upper pie charts and the other hand, when Treg frequencies were analysed, a higher Frontiers in Immunology 10 frontiersin.org Senserrich et al. 10.3389/fimmu.2022.993401 A B FIGURE 7 M. brumae induce a less inflammatory monocyte phenotype than BCG in bladder tumor tissue and can be modulated by culture conditions. (A) FACS gating strategy used to determine inflammatory and reparative monocytes. Doublets were excluded, leucocytes were gated by forward and side scatter and live CD45+ tumor infiltrating leukocytes were selected. CD3- cells were gated and the B population (B220+ cells) and the neutrophils (Ly6C+Ly6G+) were excluded. Myeloid CD11b+ were selected and the percentage of inflammatory (F4/80lo/intLy6C+) and reparative monocytes (F4/80int/highLy6C-) were obtained. (B) Frequencies of inflammatory monocytes and reparative monocytes within total myeloid cells (CD11b+), and inflammatory/reparative ratio from mycobacteria-treated tumor-bearing mice. Data represent the mean (bars) and ± SEM (error bars) and each dot represent a separate mice treated with BCG (blue dots) or M. brumae (red dots). Statistically significant differences between the different M. brumae mycobacteria are shown as red lines. Interstrain differences are shown in black. Differences were tested using Mann- Whitney U nonparametric test. *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001; ****p ≤ 0.0001. relative CD4+ Treg frequency was found in M. brumae-treated monocytes were observed in tumors from M. brumae-treated mice than in BCG-treated mice, with the lowest levels observed mice compared with those from BCG-treated mice (Figure 6, bar in M. brumae-A60-treated mice (Figure 5B). When the balance graph). Moreover, the culture conditions for M. brumae had a between effector T-cells and Treg was analysed, both the CD4+ significant impact on the composition of myeloid cells in the T EM /CD4 + T reg and CD8 + T EM /CD4 + T reg ratios were tumor microenvironment. M. brumae-A60-treated mice showed a significantly higher in BCG-treated mice than in M. brumae- skewed myeloid tumor microenvironment in comparison with the treated mice (Figures 5C, D). rest of the M. brumae-treated mice, with the highest decreases in neutrophils and monocytes. DC infiltration was shown to be unaltered by the mycobacterium species or the culture medium Mycobacterial treatment shapes used for bacterial growth. the tumor-infiltrating myeloid compartment Monocyte compartment is skewed We next evaluated the effects of mycobacterial treatments on towards a reparative phenotype the composition of myeloid cells in the tumor microenvironment after M. brumae treatment (CD11b+ cells) (Figure 6, gating strategy Supplementary Figure 2). The total distribution of this compartment, composed of Mouse monocytes can be subdivided into two major subsets, neutrophils/gMDSCs, macrophages, monocytes and DCs, was which are ontologically related but functionally distinct, based on significantly different between BCG- and M. brumae-treated the expression of the antigen Ly6C: classical or inflammatory mice (Figure 6, pie charts). A relative enrichment in monocytes (Ly6C+) and non-classical resident or reparative macrophages and decreases in neutrophils/gMDSCs and monocytes (Ly6C-) (24). Therefore, we next evaluated whether Frontiers in Immunology 11 frontiersin.org Senserrich et al. 10.3389/fimmu.2022.993401 the different mycobacterial treatments had an effect on the through distinct lipid immune receptors (25). Therefore, the infiltration of these cells (CD11b+ Ly6G - F4/80 lo/int Ly6C+ lipid profile present in a mycobacterium may have an impact on inflammatory monocytes and CD11b+Ly6G- F4/80int/highLy6C- the global immune response. In our model, a multidimensional reparative monocytes, gating strategy in Figure 7A). In tumors immune profiling analysis revealed the high degree of from BCG-treated animals, the percentages of inflammatory heterogeneity and complexity of the phenotypes of immune monocytes were higher than those of reparative monocytes, cells mobilized into the bladder after intravesical treatment, and resulting in inflammatory/reparative ratios >1 (Figure 7B). In notably, the bladder immune landscape induced by the two contrast, a significantly lower proportion of inflammatory mycobacterial species or the different antigenic variants differed. monocytes and a higher proportion of reparative monocytes These data suggest that the tumor immune microenvironment were observed in M. brumae-treated animals compared to those in BC patients treated with intravesical therapy can be treated with BCG, with ratios below 1 (Figure 7B). When the effect remodelled to improve clinical benefit. The differential global of culture conditions was taken into account, no significant immune infiltration observed in the bladder could be explained differences were observed in BCG-treated mice, while M. by the differential cytokine profiles and levels previously brumae had a major impact on the tumor microenvironment. reported to be triggered in vitro (11, 22) and in vivo (10) by Remarkably, M. brumae-A60-treated mice showed monocyte each mycobacteria in the panel; very recently, this infiltration infiltration mainly limited to the reparative subset, with minimal was shown to be influenced by the mycobacterial surface lipid infiltration of inflammatory monocytes (Figure 7B). These data composition (21). indicate that monocyte phenotype and balance, which involve BCG treatment has been associated with an infiltration different functional activities, are strongly dependent on the dominated by CD3+ cells, particularly memory (CD44+) CD4+ Mycobacterium species and, in the case of M. brumae, are and CD8+ T-cells (19, 20). In our study, we also observed further modulated by mycobacterial cell envelope composition. elevated infiltration of CD3+ cells, mainly memory CD4+ T- cells, in the bladder of BCG-treated mice. Infiltrating CD4+ and CD8+ T-cells mostly displayed an effector memory phenotype, Discussion and a high proportion of these cells presented an activated phenotype (CD25 + ). These data agree with the immune In the current study, we showed that in an orthotopic activation described after in vitro exposure of human blood murine model of BC, intravesical treatment with BCG or M. cells to BCG or after immunological evaluation of tissues from brumae altered the bladder tissue microenvironment. The patients with NMIBC (17, 26, 27). Notably, compared with BCG induced immune landscape was species specific and shaped by treatment, M. brumae treatment triggered a more immune- the mycobacterial cell envelope composition modulated by the active tumor microenvironment, increasing the proportion of culture conditions. In addition, we demonstrated that survival is total activated immune cells and producing enrichment of more dependent upon the balance and quality of the infiltrating activated CD4+ and CD8+ TEM cells. Among the mice treated immune cells than on the absolute numbers. with M. brumae variants, M. brumae-A60-treated mice Both mycobacterial species were able to induce strong displayed a maturation profile distribution more sharply immune infiltration of the bladder, although the immune skewed towards an effector phenotype, with a greater impact profiles triggered by the two species differed both on the CD4+ T-cell population. This increase in the proportion quantitatively and qualitatively. The absolute quantification of of CD4+ TEM cells is relevant, since CD4+ T-cells have been infiltrating immune cells in the bladder showed that intravesical described as potent cytotoxic cells (28) and very recently, in BCG treatment induced strong immune infiltration, in line with bladder tumors, have been linked to the efficacy of previous reports (10, 19, 20). We observed increases in all the immunotherapy (29). immune cell populations tested, including both T-cells and non- Treg have been associated with a poor prognosis in many T-cells, innate and adaptive cells, and effector and regulatory cancers, including BC, due to their immunosuppressive functions cells. This broad immune infiltration triggered by BCG was within the tumor microenvironment (14, 30). In NMIBC, baseline massive and did not appear to be significantly modulated by the Treg in the tumor prior to BCG treatment have been previously cell envelope composition selected by the culture conditions for shown to correlate with treatment failure (14). However, in a the mycobacteria. Conversely, M. brumae treatment, which recent study, Lim et al. reported that although BCG treatment produced more moderate infiltration than BCG, resulted in enhanced Treg infiltration, the presence of these cells was not longer survival. In this case the infiltration was clearly associated with the response to treatment (18). Increasing modulated by changes in the cell envelope lipid composition. evidence indicates that improved clinical outcomes and The human immune system has developed powerful treatment responses are dependent on the intratumoral balance mechanisms to respond to mycobacterial antigens, among between regulatory and effector cells (29, 31). After mycobacterial which lipids are known to be potent immune inducers that treatment, in agreement with previous reports (18, 32), we can modulate both innate and adaptive immune responses observed the presence of CD4+ Treg in the bladder, with a Frontiers in Immunology 12 frontiersin.org Senserrich et al. 10.3389/fimmu.2022.993401 higher absolute infiltration but lower proportions observed after relevance of the B-cell subtypes present in our model remain BGG treatment than after M. brumae treatment. Higher CD4+ to be determined, as although tumoral B-cell populations have TEM cell/CD4+ Treg and CD8+ TEM cell/CD4+ Treg ratios were also been correlated with a good prognosis in BC (38), high levels of observed in bladders from BCG-treated mice than in those from either B-cells or plasma cells infiltration into tumors have also M. brumae-treated mice. These data show that M. brumae been associated with increased invasiveness (42), significantly establishes a more immunosuppressive microenvironment than worsening patient prognosis. BCG; however, the antigenic changes in M. brumae-A60 are able Different cell types within the myeloid cell lineage have also to equilibrate the balance between effector and regulatory cells in been shown to be involved in the regulation of immune activity in the tumor microenvironment, increasing the percentage of cancer (43). In agreement with previous reports (17, 28, 44), we infiltrating TEM cells and decreasing the percentage of Treg. found that mycobacterial treatment enhanced the infiltration of One limitation of our study was that CD4+ Treg were myeloid cells compared with no treatment. BCG and M. brumae identified by the expression of CD25 and the absence of treatments produced markedly different myeloid cell infiltration CD127 (32) but no other commonly used markers, such as the profiles, which were influenced by the cell envelope composition. described “specific” molecular marker FOXP3, were used (33). M. brumae induced a significantly lower proportion of the The identification of Treg following immune cell activation, as neutrophil/gMDSC population than BCG, with M. brumae-A60 during an immune response to a pathogen, is challenging since treatment inducing the lowest levels of these infiltrating cells. This conventional T-cells also express CD25 and lose CD127 population may actually contain both neutrophils and gMDSCs, expression (34). Moreover, accumulating evidence has as these cell types are phenotypically indistinguishable by means suggested that FOXP3 expression may not always specifically of the markers used in our study. Therefore, both cell types must identify Treg, as activated T-cells transiently upregulate FOXP3 be considered in the analysis and conclusions for tumor without acquiring a Treg phenotype or functions (35). Therefore, infiltration and their immune activities. On the one hand, in the highly inflammatory tumor microenvironment myeloid-derived suppressor cells (MDSCs) are well known to be established after mycobacterial treatment, the quantitative recruited, activated and induced by tumor-derived factors and to identification of Treg using phenotypic markers could produce directly accelerate tumor development, metastasis and an overestimation that does not represent the cells that actually neovascularization (45). Furthermore, following migration into exert a real immunosuppressive function. the tumor microenvironment, MDSCs attenuate the antitumor In addition to the classical CD3+ T-cell response, which reactivity of T-cells and NK cells via various mechanisms (46). forms the core of the adaptive immune system, other lymphoid Recently, one of the antitumoral mechanisms of cisplatin in BC populations have been described to play important roles in the was reported to be dependent on the depletion of gMDSCs. On regulation of antitumor immunity (36). In the context of BC, the other hand, neutrophils found in tumor tissue have been innate immune recognition of live BCG contributes to described to be capable of promoting tumor growth and immunogenicity, and there are reports indicating the progression, and their presence is often associated with a poor involvement of NK cells, NKT cells, TCRgd cells and ILCs in clinical outcome. However, other data point in the opposite antitumor immune responses and associations with a good direction, indicating that neutrophils can act as effector cells and response to immunotherapy (37). In our model, we found combat cancer, leading to the eradication of tumor cells (47). BCG evidence of significant infiltration of the bladder by all these instillation results in an early influx of neutrophils that is believed populations, but only the proportions of NKT cells and ILCs to orchestrate macrophage and T-cell recruitment through the were influenced by the mycobacterial species, with a lower release of chemokines, which is essential for achieving a positive proportion of NKT cells and a higher frequency of ILCs in M. outcome with BCG therapy (48). The low infiltration of brumae-treated mice than in BCG-treated mice. neutrophils observed after M. brumae, particularly after M. Tumor-infiltrating B-cells are another intratumoral non-T brumae-A60, treatment could be one of the causes of the lower lymphoid population that has been linked to survival in different total infiltrating immune cells observed in mice treated with these malignancies, including urothelial BC (38), with both positive bacteria. Nevertheless, a low number of neutrophils could be and negative outcomes reported depending on the cancer type beneficial, as antibody-mediated depletion of neutrophils has been and the specific B-cell population (39). Here, we showed that reported to result in decreased metastasis in mouse models of liver total B- and innate-like B1-cells remained unaffected by the and breast cancer metastasis and to inhibit cancer progression in a Mycobacterium species instilled in the bladder but that pancreatic ductal adenocarcinoma model (47). interestingly their proportions in immune infiltrates were In addition to neutrophils/gMDSCs, tumor-infiltrating strongly dependent on the mycobacterial cell envelope lipid macrophages or monocytes can be found in the urine and composition. This finding agrees with the participation of B- bladder wall of BCG-treated patients (17, 26, 27), and their cells in the lipid immune response (40), as demonstrated by the presence in bladder tumors prior to BCG therapy has been activation of B and B1 cells by lipids from Mycobacterium correlated with an increased recurrence risk (14, 49). In our tuberculosis (41). The precise identification and functional model, we observed that M. brumae, especially M. brumae-A60, Frontiers in Immunology 13 frontiersin.org Senserrich et al. 10.3389/fimmu.2022.993401 treatment shaped monocyte infiltration by decreasing classical Author contributions or inflammatory monocytes with antimicrobial functions (Ly6C+) and increasing nonclassical resident or reparative Conceptualization: SG-G, EJ. Performed experiments: SG-G, monocytes (Ly6C-) with anti-inflammatory and tissue-repairing EG-M. Analyzed the data: JS, VU, CC. Writing–Original Draft functions. In in vivo wound repair occurring during skeletal Preparation: JS, CC. Funding acquisition: EJ, CC, BC. Review & muscle repair or after myocardial infarction, there is a biphasic Editing Preparation: all authors. Visualization Preparation: all process that first involves the accumulation of inflammatory authors. All authors have read and agreed to the published monocytes and then transitions into reparative monocytes version of the manuscript. (50). Therefore, after treatment with M. brumae, bladder tumor inflammatory monocytes may be converted into anti- inflammatory reparative monocytes that contribute to Funding regulating the balance of inflammatory responses in the tumor microenvironment. This work was funded by the Spanish Ministry of Science, Another limitation of our study is the validation of our Innovation and Universities grant RTI2018-098777-B-I00 (EJ) results as a predictive power for BC patients receiving and FEDER Funds (EJ), Generalitat of Catalunya grant mycobacteria treatment. To compare the administration of 2017SGR-229 (EJ), Generalitat de Catalunya PhD contracts BCG strains grown in different culture media together with FI (SG-G). CC is a researcher at the Fundació Institut de infiltration of different immune populations into bladder tumor, Recerca en Ciències de la Salut Germans Trias i Pujol and the treatment outcome will be the basis for next studies. On supported by the Health Department of the Catalan the other hand, M. brumae treatment need to be further Government/Generalitat de Catalunya and ISCIII. This study evaluated in clinical trials. was partially funded by funds generated at the AIDS Research Overall, we demonstrate that the global bladder tumor Institute-IrsiCaixa. immune microenvironment can be remodelled by changes in both the bacterial species and the mycobacterial cell envelope composition, improving immune infiltration quality rather than quantity, enhancing survival and increasing safety. Our findings Acknowledgments indicate the importance of the balance between inflammatory and regulatory/suppressive activity and the ability to effectively We are grateful to the technical staff from the Estabulari manipulate this balance with therapeutic approaches to enhance UAB for their outstanding animal management. We thank the tumor control. IGTP Cytometry Core Facility and Marco Antonio Fernandez for his contribution to this publication. We are grateful to OMIQ for giving us the opportunity to use their platform. Graphical Data availability statement Abstract was created with Biorender.com. The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation. Conflict of interest The authors declare that the research was conducted in the Ethics statement absence of any commercial or financial relationships that could be construed as a potential conflict of interest. In vivo experiments were performed in accordance with national and European Union legislation regarding the protection of experimental animals, and were reviewed by the Publisher’s note Animal Care Committee at the Autonomous University of Barcelona and the Catalonia government (project 9171) and All claims expressed in this article are solely those of the approved by the Departament d'Agricultura, Ramaderia, Pesca, authors and do not necessarily represent those of their affiliated Alimentació i Medi Natural of the Catalan Regional organizations, or those of the publisher, the editors and the Government. Mice were supervised following a strict reviewers. Any product that may be evaluated in this article, or monitoring protocol in order to ensure animal welfare, and claim that may be made by its manufacturer, is not guaranteed euthanized, if required. Consent to participate: Not applicable. or endorsed by the publisher. Frontiers in Immunology 14 frontiersin.org Senserrich et al. 10.3389/fimmu.2022.993401 neutrophils and DCs were analyzed as CD11c-Ly6G+and CD11c+Ly6G-, Supplementary material respectively. Finally, CD11c-Ly6G- cells were gated into monocytes (F4/ 80lo/int) and macrophages (F4/80hi). The Supplementary Material for this article can be found SUPPLEMENTARY FIGURE 3 online at: https://www.frontiersin.org/articles/10.3389/ Survival curves after intravesical mycobacterial treatment. Kaplan-Meier fimmu.2022.993401/full#supplementary-material analysis survival of healthy mice, tumor-bearing mice treated with PBS. pooled BCG grown in different culture media, A-60-M. brumae and the SUPPLEMENTARY FIGURE 1 rest of M. brumae. ****p ≤ 0.0001 versus PBS group, log-rank (Mantel- Gating strategy to analyze the phenotype (percentage and absolute Cox) test. number) of leukocytes infiltrating the bladder. Doublets were excluded, lymphocytes were gated by forward and side scatter and live CD45+ SUPPLEMENTARY FIGURE 4 tumor infiltrating leukoytes were selected for further gating. ILCs were Bladder infiltration of immune cells after different mycobacteria defined as CD3 - CD127 + cells. NK as NK1.1 + CD3 - , NKT cells as treatments. Absolute counts of different cell populations were NK1.1+CD3+, and T-cells as NK1.1-CD3+ populations. From the CD3+ quantified by flow cytometry after bladder digestion of mice treated gate CD4+, CD8+ and TCRgd+ were defined as CD3+CD4+, CD3+CD8+ with BCG (blue bars) or M. brumae (red bars) grown in different culture and CD3+TCRgd+ cells, respectively. Regulatory CD4+ T-cells were media. Data represent the mean (bars) and ± SEM (error bars) and each selected from CD4 T-cells as CD25+CD127-. The different CD4+ and dot represent an individual mouse. Statistically significant differences CD8+ T-cell maturation phenotypes were analyzed using CD62L and between the different BCG and M. brumae mycobacteria (intrastrain) CD44 markers as follows: Naïve (N) were defined as CD62L+CD44-, are shown as blue and red lines, respectively. Interstrain differences are central memory (CM) as CD62L+CD44+, and effector memory (EM) as shown in black. Differences were tested using Mann-Whitney U CD62L-CD44+ cells. nonparametric test. *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001. SUPPLEMENTARY FIGURE 2 Gating strategy to analyze the phenotype (percentage and absolute SUPPLEMENTARY FIGURE 5 number) of non-T immune cells infiltrating the bladder. Doublets were Enriched immune populations into the bladder after M. brumae-A60 excluded, leukocytes were gated by forward and side scatter and live treatment. Volcano plots showing adjusted p values versus log (FC) for CD45+ tumor infiltrating leukocytes were selected for further gating. 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