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Carcinoembryonic antigen-related cell adhesion molecule 6 (CEACAM6), a cell surface receptor, is expressed on normal epithelial tissue and highly expressed in cancers of high unmet medical need, such as non-small cell lung, pancreatic, and colorectal cancer. CEACAM receptors undergo homo- and heterophilic interactions thereby regulating normal tissue homeostasis and angiogenesis, and in cancer, tumor invasion and metastasis. CEACAM6 expression on malignant plasma cells inhibits antitumor activity of T cells, and we hypothesize a similar function on epithelial cancer cells. The interactions between CEACAM6 and its suggested partner CEACAM1 on T cells were studied. A humanized CEACAM6-blocking antibody, BAY 1834942, was developed and characterized for its immunomodulating effects in co-culture experiments with T cells and solid cancer cells and in comparison to antibodies targeting the immune checkpoints programmed cell death protein 1 (PD-1), programmed death-ligand 1 (PD-L1), and T cell immunoglobulin mucin-3 (TIM-3). The immunosuppressive activity of CEACAM6 was mediated by binding to CEACAM1 expressed by activated tumor-specific T cells. BAY 1834942 increased cytokine secretion by T cells and T cell-mediated killing of cancer cells. The in vitro efficacy of BAY 1834942 correlated with the degree of CEACAM6 expression on cancer cells, suggesting potential in guiding patient selection. BAY 1834942 was equally or more efficacious compared to blockade of PD-L1, and at least an additive efficacy was observed in combination with anti-PD-1 or anti-TIM-3 antibodies, suggesting an efficacy independent of the PD-1/PD-L1 axis. In summary, CEACAM6 blockade by BAY 1834942 reactivates the antitumor response of T cells. This warrants clinical evaluation.

Regulatory CD4+ T cells (Treg) prevent tumor clearance by conventional T cells (Tconv) comprising a major obstacle of cancer immune-surveillance. Hitherto, the mechanisms of Treg repertoire formation in human cancers remain largely unclear. Here, we analyze Treg clonal origin in breast cancer patients using T-Cell Receptor and single-cell transcriptome sequencing. While Treg in peripheral blood and breast tumors are clonally distinct, Tconv clones, including tumor-antigen reactive effectors (Teff), are detected in both compartments. Tumor-infiltrating CD4+ cells accumulate into distinct transcriptome clusters, including early activated Tconv, uncommitted Teff, Th1 Teff, suppressive Treg and pro-tumorigenic Treg. Trajectory analysis suggests early activated Tconv differentiation either into Th1 Teff or into suppressive and pro-tumorigenic Treg. Importantly, Tconv, activated Tconv and Treg share highly-expanded clones contributing up to 65% of intratumoral Treg. Here we show that Treg in human breast cancer may considerably stem from antigen-experienced Tconv converting into secondary induced Treg through intratumoral activation., The mechanisms that shape the regulatory T cell repertoire in patients with cancer are not completely understood. Here, the authors observe that, in breast cancer patients, tumor-resident regulatory T cells do not show clonal relationship with their circulating counterpart, but share a common origin with intratumoral antigen-experienced conventional T cells.

BackgroundCancer immunotherapeutic strategies showed unprecedented results in the clinic. However, many patients do not respond to immuno-oncological treatments due to the occurrence of a plethora of immunological obstacles, including tumor intrinsic mechanisms of resistance to cytotoxic T-cell (TC) attack. Thus, a deeper understanding of these mechanisms is needed to develop successful immunotherapies.MethodsTo identify novel genes that protect tumor cells from effective TC-mediated cytotoxicity, we performed a genetic screening in pancreatic cancer cells challenged with tumor-infiltrating lymphocytes and antigen-specific TCs.ResultsThe screening revealed 108 potential genes that protected tumor cells from TC attack. Among them, salt-inducible kinase 3 (SIK3) was one of the strongest hits identified in the screening. Both genetic and pharmacological inhibitions of SIK3 in tumor cells dramatically increased TC-mediated cytotoxicity in several in vitro coculture models, using different sources of tumor and TCs. Consistently, adoptive TC transfer of TILs led to tumor growth inhibition of SIK3-depleted cancer cells in vivo. Mechanistic analysis revealed that SIK3 rendered tumor cells susceptible to tumor necrosis factor (TNF) secreted by tumor-activated TCs. SIK3 promoted nuclear factor kappa B (NF-κB)nuclear translocation and inhibited caspase-8 and caspase-9 after TNF stimulation. Chromatin accessibility and transcriptome analyses showed that SIK3 knockdown profoundly impaired the expression of prosurvival genes under the TNF–NF-κBaxis. TNF stimulation led to SIK3-dependent phosphorylation of the NF-κB upstream regulators inhibitory-κB kinase and NF-kappa-B inhibitor alpha on the one side, and to inhibition of histone deacetylase 4 on the other side, thus sustaining NF-κB activation and nuclear stabilization. A SIK3-dependent gene signature of TNF-mediated NF-κB activation was found in a majority of pancreatic cancers where it correlated with increased cytotoxic TC activity and poor prognosis.ConclusionOur data reveal an abundant molecular mechanism that protects tumor cells from cytotoxic TC attack and demonstrate that pharmacological inhibition of this pathway is feasible.

Endogenous antitumor effector T-cell responses and immune-suppressive regulatory T cells (Treg) critically influence the prognosis of patients with cancer, yet many of the mechanisms of how this occurs remain unresolved. On the basis of an analysis of the function, antigen specificity, and distribution of tumor antigen–reactive T cells and Tregs in patients with breast cancer and transgenic mouse tumor models, we showed that tumor-specific Tregs were selectively activated in the bone marrow (BM) and egressed into the peripheral blood. The BM was constantly depleted of tumor-specific Tregs and was instead a site of increased induction and activity of tumor-reactive effector/memory T cells. Treg egress from the BM was associated with activation-induced expression of peripheral homing receptors such as CCR2. Because breast cancer tissues express the CCR2 ligand CCL2, the activation and egress of tumor antigen–specific Tregs in the BM resulted in the accumulation of Tregs in breast tumor tissue. Such immune compartmentalization and redistribution of T-cell subpopulations between the BM and peripheral tissues were achieved by vaccination with adenoviral vector–encoded TRP-2 tumor antigen in a RET transgenic mouse model of spontaneous malignant melanoma. Thus, the BM simultaneously represented a source of tumor-infiltrating Tregs and a site for the induction of endogenous tumor-specific effector T-cell responses, suggesting that both antitumor immunity and local immune suppression are orchestrated in the BM.

Effector memory T cells (T(EM)) have an important role in immunity against infection. However, little is known about the factors regulating T(EM) maintenance and proliferation. In this study, we investigated the role of direct interactions between CD4(+) and CD8(+) T cells (TC) for human T(EM) expansion. Proliferation of separated or mixed CD4(+) and CD8(+)T(EM) populations was analyzed after polyclonal stimulation in vitro. Compared to each isolated subset mixed T(EM) populations showed increased proliferation and expansion of both CD4(+) and CD8(+)T(EM) subpopulations. Combined activation of CD4(+) and CD8(+) memory T cells (Tmem) induced an increased expression of CD40L and CD40 on both populations. Subsequently, CD40/CD40L caused a bi-directional stimulation of CD40(+)CD4(+)T(EM) by CD40L(+)CD8(+)T(EM) and of CD40(+)CD8(+)T(EM) by CD40L(+)CD4(+)T(EM). Blocking of CD40L on activated CD8(+)T(EM) selectively inhibited proliferation of CD4(+)T(EM), while blocking of CD40L on CD4(+)T(EM) abrogated proliferation of CD8(+)T(EM). Taken together, we demonstrate for the first time that the expression of CD40L is exploited on the one hand by CD8(+)T(EM) to increase the proliferation of activated CD4(+)T(EM) and on the other hand by CD4(+)T(EM) to support the expansion of activated CD8(+)T(EM). Thus, efficient T(EM) expansion requires bi-directional interactions between CD4(+) and CD8(+)T(EM) cells.