Tumor-draining lymph node (TDLN) invasion by metastatic cells in breast cancer correlates with poor prognosis and is associated with local immunosuppression, which can be partly mediated by ...regulatory T cells (Tregs). Here, we study Tregs from matched tumor-invaded and non-invaded TDLNs, and breast tumors. We observe that Treg frequencies increase with nodal invasion, and that Tregs express higher levels of co-inhibitory/stimulatory receptors than effector cells. Also, while Tregs show conserved suppressive function in TDLN and tumor, conventional T cells (Tconvs) in TDLNs proliferate and produce Th1-inflammatory cytokines, but are dysfunctional in the tumor. We describe a common transcriptomic signature shared by Tregs from tumors and nodes, including CD80, which is significantly associated with poor patient survival. TCR RNA-sequencing analysis indicates trafficking between TDLNs and tumors and ongoing Tconv/Treg conversion. Overall, TDLN Tregs are functional and express a distinct pattern of druggable co-receptors, highlighting their potential as targets for cancer immunotherapy.
CD4
T cell antitumor responses have mostly been studied in transplanted tumors expressing secreted model antigens (Ags), while most mutated proteins in human cancers are not secreted. The fate of ...Ag-specific CD4
T cells recognizing a cytoplasmic Ag in mice bearing autochthonous tumors is still unclear. Here we show, using a genetically engineered lung adenocarcinoma mouse model, that naive tumor-specific CD4
T cells are activated and proliferate in the tumor-draining lymph node (TdLN) but do not differentiate into effectors or accumulate in tumors. Instead, these CD4
T cells are driven toward anergy or peripherally-induced Treg (pTreg) differentiation, from the early stage of tumor development. This bias toward immune suppression is restricted to the TdLN, and is maintained by Tregs enriched in the tumor Ag-specific cell population. Thus, tumors may enforce a dominant inhibition of the anti-tumor CD4 response in the TdLN by recapitulating peripheral self-tolerance mechanisms.
Modulating an immune response in opposite directions represents the holy grail in allogeneic hematopoietic stem cell transplantation (allo-HSCT) to avoid insufficient reactivity of donor T cells and ...hematologic malignancy relapse while controlling the potential development of graft-versus-host disease (GVHD), in which donor T cells attack the recipient's tissues. IL-2/anti-IL-2 complexes (IL-2Cx) represent a therapeutic option to selectively accentuate or dampen the immune response. In dedicated experimental models of allo-HSCT, including also human cells injected in immunodeficient NSG mice, we evaluated side-by-side the therapeutic effect of two IL-2Cx designed either to boost regulatory T cells (Treg) or alternatively to activate effector T cells (Teff), on GVHD occurrence and tumor relapse. We also evaluated the effect of the complexes on the phenotype and function of immune cells in vivo. Unexpectedly, both pro-Treg and pro-Teff IL-2Cx prevented GVHD development. They both induced Treg expansion and reduced CD8+ T-cell numbers, compared to untreated mice. However, only mice treated with the pro-Treg IL-2Cx, showed a dramatic reduction of exhausted CD8+ T cells, consistent with a potent anti-tumor effect. When evaluated on human cells, pro-Treg IL-2Cx also preferentially induced Treg expansion in vitro and in vivo, while allowing the development of a potent anti-tumor effect in NSG mice. Our results demonstrate the clinical relevance of using a pro-Treg, but not a pro-Teff IL2Cx to modulate alloreactivity after HSCT, while promoting a graft-versus-leukemia effect.
Tumor-infiltrating CD8 + T cells progressively lose functionality and fail to reject tumors. The underlying mechanism and re-programing induced by checkpoint blockers are incompletely understood. We ...show here that genetic ablation or pharmacological inhibition of histone lysine methyltransferase Suv39h1 delays tumor growth and potentiates tumor rejection by anti-PD-1. In the absence of Suv39h1, anti-PD-1 induces alternative activation pathways allowing survival and differentiation of IFNγ and Granzyme B producing effector cells that express negative checkpoint molecules, but do not reach final exhaustion. Their transcriptional program correlates with that of melanoma patients responding to immune-checkpoint blockade and identifies the emergence of cytolytic-effector tumor-infiltrating lymphocytes as a biomarker of clinical response. Anti-PD-1 favors chromatin opening in loci linked to T-cell activation, memory and pluripotency, but in the absence of Suv39h1, cells acquire accessibility in cytolytic effector loci. Overall, Suv39h1 inhibition enhances anti-tumor immune responses, alone or combined with anti-PD-1, suggesting that Suv39h1 is an "epigenetic checkpoint" for tumor immunity.
Although immune checkpoint inhibitors have shown improvement in survival in comparison to chemotherapy in urothelial bladder cancer, many patients still fail to respond to these treatments and actual ...efforts are made to identify predictive factors of response to immunotherapy. Understanding the tumor-intrinsic molecular basis, like oncogenic pathways conditioning the presence or absence of tumor-infiltrating T cells (TILs), should provide a new rationale for improved anti-tumor immune therapies.
In this study, we found that urothelial bladder cancer from human samples bearing PIK3CA gene mutations was significantly associated with lower expression of a defined immune gene signature, compared to unmutated ones. We identified a reduced 10-gene immune gene signature that discriminates muscle-invasive bladder cancer (MIBC) samples according to immune infiltration and PIK3CA mutation. Using a humanized mouse model, we observed that BKM120, a pan-PI3K inhibitor, significantly inhibited the growth of a human bladder cancer cell line bearing a PIK3CA mutation, associated to increased immune cell infiltration (hCD45+). Using qRT-PCR, we also found an increase in the expression of chemokines and immune genes in PIK3CA-mutated tumors from mice treated with BKM120, reflecting an active immune infiltrate in comparison to untreated ones. Moreover, the addition of BKM120 rendered PIK3CA-mutated tumors sensitive to PD-1 blockade. Our results provide a relevant rationale for combination strategies of PI3K inhibitors with immune checkpoint inhibitors to overcome resistance to immune checkpoint inhibitors.
Regulatory T cells (Tregs) are plastic cells playing a pivotal role in the maintenance of immune homeostasis. Tregs actively adapt to the microenvironment where they reside; as a consequence, their ...molecular and functional profiles differ among tissues and pathologies. In tumors, the features acquired by Tregs remains poorly characterized. Here, we observe that human tumor-infiltrating Tregs selectively overexpress CD74, the MHC class II invariant chain. CD74 has been previously described as a regulator of antigen-presenting cell biology, however its function in Tregs remains unknown. CD74 genetic deletion in human primary Tregs reveals that CD74KO Tregs exhibit major defects in the organization of their actin cytoskeleton and intracellular organelles. Additionally, intratumoral CD74KO Tregs show a decreased activation, a drop in Foxp3 expression, a low accumulation in the tumor, and consistently, they are associated with accelerated tumor rejection in preclinical models in female mice. These observations are unique to tumor conditions as, at steady state, CD74KO-Treg phenotype, survival, and suppressive capacity are unaffected in vitro and in vivo. CD74 therefore emerges as a specific regulator of tumor-infiltrating Tregs and as a target to interfere with Treg anti-tumor activity.
As an important immune stimulator and modulator, IFNγ is crucial for gut homeostasis and its dysregulation links to diverse colon pathologies, such as colitis and colorectal cancer (CRC). Here, we ...demonstrated that the epigenetic regulator, CBX3 (also known as HP1γ) antagonizes IFNγ signaling in the colon epithelium by transcriptionally repressing two critical IFNγ-responsive genes:
STAT1
and
CD274
(encoding Programmed death-ligand 1, PD-L1). Accordingly, CBX3 deletion resulted in chronic mouse colon inflammation, accompanied by upregulated
STAT1
and
CD274
expressions. Chromatin immunoprecipitation indicated that CBX3 tethers to
STAT1
and
CD274
promoters to inhibit their expression. Reversely, IFNγ significantly reduces CBX3 binding to these promoters and primes gene expression. This antagonist effect between CBX3 and IFNγ on STAT1/PD-L1 expression was also observed in CRC. Strikingly, CBX3 deletion heightened CRC cells sensitivity to IFNγ, which ultimately enhanced their chemosensitivity under IFNγ stimulation in vitro with CRC cells and in vivo with a syngeneic mouse tumor model. Overall, this work reveals that by negatively tuning IFNγ-stimulated immune genes’ transcription, CBX3 participates in modulating colon inflammatory response and CRC chemo-resistance.
Synopsis
The epigenetic regulator CBX3 (also known as HP1γ) antagonized IFNγ/STAT1/PD-L1 axis and thereby decreased IFNγ-stimulated immune genes transcription, which ultimately modulated colon inflammatory response and CRC chemo-resistance.
CBX3 deletion resulted in chronic mouse colon inflammation, accompanied by upregulated STAT1 and PD-L1 levels in the colon epithelium.
CBX3 deletion heightened CRC cells’ sensitivity to IFNγ stimulation and increased STAT1/PD-L1 expression.
CBX3 antagonized IFNγ signaling by tethering to the promotors of Stat1 and Cd274, and transcriptionally repressing their expression. IFNγ stimulation reduced CBX3 binding and primed gene expression.
Re-sensitizing CRC cells to IFNγ by deletion CBX3 enhanced their chemosensitivity under IFNγ stimulation in vitro as well as in vivo.
The epigenetic regulator CBX3 (also known as HP1γ) antagonized IFNγ/STAT1/PD-L1 axis and thereby decreased IFNγ-stimulated immune genes transcription, which ultimately modulated colon inflammatory response and CRC chemo-resistance.
Innate lymphoid cells (ILCs) - which include cytotoxic Natural Killer (NK) cells and helper-type ILC - are important regulators of tissue immune homeostasis, with possible roles in tumor ...surveillance. We analyzed ILC and their functionality in human lymph nodes (LN). In LN, NK cells and ILC3 were the prominent subpopulations. Among the ILC3s, we identified a CD56
+
/ILC3 subset with a phenotype close to ILC3 but also expressing cytotoxicity genes shared with NK. In tumor-draining LNs (TD-LNs) and tumor samples from breast cancer (BC) patients, NK cells were prominent, and proportions of ILC3 subsets were low. In tumors and TD-LN, NK cells display reduced levels of NCR (Natural cytotoxicity receptors), despite high transcript levels and included a small subset CD127
−
CD56
−
NK cells with reduced function. Activated by cytokines CD56
+
/ILC3 cells from donor and patients LN acquired cytotoxic capacity and produced IFNg. In TD-LN, all cytokine activated ILC populations produced TNFα in response to BC cell line. Analyses of cytotoxic and helper ILC indicate a switch toward NK cells in TD-LN. The local tumor microenvironment inhibited NK cell functions through downregulation of NCR, but cytokine stimulation restored their functionality.
An important challenge in cancer immunotherapy is to expand the number of patients that benefit from immune checkpoint inhibitors (CI), a fact that has been related to the pre-existence of an ...efficient anti-tumor immune response. Different strategies are being proposed to promote tumor immunity and to be used in combined therapies with CI. Recently, we reported that intratumoral administration of naked poly A:U, a dsRNA mimetic empirically used in early clinical trials with some success, delays tumor growth and prolongs mice survival in several murine cancer models. Here, we show that CD103
cDC1 and, to a much lesser extent CD11b
cDC2, are the only populations expressing TLR3 at the tumor site, and consequently could be potential targets of poly A:U. Upon poly A:U administration these cells become activated and elicit profound changes in the composition of the tumor immune infiltrate, switching the immune suppressive tumor environment to anti-tumor immunity. The sole administration of naked poly A:U promotes striking changes within the lymphoid compartment, with all the anti-tumoral parameters being enhanced: a higher frequency of CD8
Granzyme B
T cells, (lower Treg/CD8
ratio) and an important expansion of tumor-antigen specific CD8
T cells. Also, PD1/PDL1 showed an increased expression indicating that neutralization of this axis could be exploited in combination with poly A:U. Our results shed new light to promote further assays in this dsRNA mimetic to the clinical field.
Senescent T cells have been described during aging, chronic infections, and cancer; however, a comprehensive study of the phenotype, function, and transcriptional program of this T cell population in ...breast cancer (BC) patients is missing. Compared to healthy donors (HDs), BC patients exhibit an accumulation of KLRG-1
CD57
CD4
and CD8
T cells in peripheral blood. These T cells infiltrate tumors and tumor-draining lymph nodes. KLRG-1
CD57
CD4
and CD8
T cells from BC patients and HDs exhibit features of senescence, and despite their inhibitory receptor expression, they produce more effector cytokines and exhibit higher expression of Perforin, Granzyme B, and CD107a than non-senescent subsets. When compared to blood counterparts, tumor-infiltrating senescent CD4
T cells show similar surface phenotype but reduced cytokine production. Transcriptional profiling of senescent CD4
T cells from the peripheral blood of BC patients reveals enrichment in genes associated with NK or CD8
-mediated cytotoxicity, TCR-mediated stimulation, and cell exhaustion compared to non-senescent T cells. Comparison of the transcriptional profile of senescent CD4
T cells from peripheral blood of BC patients with those of HDs highlighted marked similarities but also relevant differences. Senescent CD4
T cells from BC patients show enrichment in T-cell signaling, processes involved in DNA replication, p53 pathways, oncogene-induced senescence, among others compared to their counterparts in HDs. High gene expression of CD4, KLRG-1, and B3GAT1 (CD57), which correlates with increased overall survival for BC patients, underscores the usefulness of the evaluation of the frequency of senescent CD4
T cells as a biomarker in the follow-up of patients.
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