br response which requires antigen recognition is composed o
response, which requires antigen recognition, is composed of both cy-totoxic CD8+T Iberiotoxin and CD4+T cells . Animal models have shown that in vivo eradication of tumors is for the most part mediated by cy-totoxic T-cells. The presence of intratumoral T-cells is an independent predictor of improved survival and has also been associated with in-creased secretion of interferon-gamma (IFNγ), interleukin-2 (IL-2) and TNFα [16,58,59]. As in Fig. 8C, groups included mice treated with control vehicle, anti-PD-L1 antibody, fulvestrant, JD128 or the combi-nation of fulvestrant with anti-PD-L1 antibody or JD128 and anti-PD-L1 antibody (Fig. 10).
A sequential gating strategy to analyze tumor CD3+ cell subsets is shown in Fig. 10A, while the normalized median intensity of distinct protein markers are show in a heatmap for all clusters analyzed by Cytofkit in Fig. 10B [45,46]. tSNE scatter plots for visualization of CD3+ cells that show clusters of CD8+, CD4+ and Tregs cells are presented in Fig. 10C. Importantly, the results show that both eﬀector and eﬀector memory CD8+ and CD4 + T-cells in tumors are several-fold higher in mice treated with either fulvestrant or JD128 antiestro-gens when combined with PD-L1 antibody as compared to controls (P < 0.05) (Fig. 10D). In addition, we find increased expression of known activation cytokines IFNγ, IL-2 and TNFα in CD8+ and CD4 + TIL subpopulations (Fig. 10E). These data appear to comple-ment reports on estrogen-specific alterations of these cytokines in in-dependent murine models . Furthermore, antiestrogen treatments evoke a significant reduction of T-regulatory T-cells (Tregs) (Fig. 10F) which are known to play an important role in the maintenance of tumor immune tolerance . In the process of tumor progression, Treg cells tend to accumulate in tumors and suppress T-cell responses at the tumor site. The number of tumor-infiltrating CD25+FoxP3+ Tregs is asso-ciated with poor prognosis and is identified as a significant predictor of poor outcome . In addition, to corroborate mass cytometry data we performed immunohistochemistry to detect CD8 + TILs presence in the tumor bed. IHC results shown in Fig. 8G confirmed data obtained by cyTOF, with antiestrogens and combination treatment significantly in-creasing CD8 + TIL infiltration (P < 0.0001).
3.8. Eﬀects of antiestrogens combined with ICIs on macrophage and dendritic cell subsets in 4T1 tumors in vivo
Since recent findings suggest that cells of the innate immune system play an important role in the decision between an eﬀective immune response versus induction of immune tolerance, we also investigated levels of dendritic cells (DC) that have a special function linking the innate immune response with the induction of adaptive immunity. These cells play a major role by processing and presenting antigens to T and B cells to generate an immune response. Stimulatory DCs promote eﬀective immune responses by stimulating T-cell proliferation and shaping specific T-cell response phenotypes . Importantly, treat-ment with both antiestrogens fulvestrant and JD128 alone as well as combined with anti-PD-L1 antibody (as in Fig. 8C) increased the po-pulation of DCs in 4T1 tumors (Fig. 11A).
Further, it is well documented that the highly inflammatory mi-croenvironment of tumors tends to recruit macrophages and peripheral blood monocytes . These myeloid cells receive tumor-derived sig-nals that alter gene expression and phenotype. A prominent myeloid cell subset that develops in the breast TME is the tumor-associated macrophage (TAM). Macrophages are key modulators and eﬀector cells in the immune response that exhibit high plasticity in response to various external signals (61). Depending on TME signals, macrophages occur as M1 macrophages associated with ‘tumoricidal’ activity with high production of reactive nitrogen and oxygen intermediates and pro-inflammatory cytokines or M2 macrophages involved in tumor pro-gression and immunoregulatory functions . The M2 phenotype predominates among TAMs, and a high density of TAMs correlates with poor prognosis in BC . CyTOF analyses based on experiments noted in Fig. 8C reveal that therapy with SERD128 combined with anti-PD-L1
Fig. 9. High-dimensional analysis of mass cytometry data shows antiestrogens decrease the amount of myeloid derived suppressor cells present in 4T1 tumors. Single cells were purified from 4T1 tumors grown in BALB/c mice, stained with a panel of 28 markers by mass cytometry. A) Sequential gating strategy to analyze tumor CD45+ cell subsets present in the TME. B) Phenograph example of diﬀerent cell populations identified by single cell analysis using Cytofkit. C) tSNE plots show cluster expression of markers for both populations of myeloid cells G-MDSC and M-MDSC. D) Representative plots of G-MDSC (CD11b+Ly6Ghi, Ly6Clo) and M-MDSC (CD11b+Ly6Chi, Ly6Glo) as percentage of CD45+ cells. E) Quantification of G-MDSC and M-MDSC present in the tumor bed of BALB/c mice with 4T1 tumors. *P < 0.05, ** P < 0.01. n = 6–11. F) ERα expression in total MDSC, G-MDSC and M-MDSC.