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BackgroundDespite striking successes, immunotherapies aimed at increasing cancer-specific T cell responses are unsuccessful in most patients with cancer. Inactivating regulatory T cells (Treg) by inhibiting the PI3Kδ signaling enzyme has shown promise in preclinical models of tumor immunity and is currently being tested in early phase clinical trials in solid tumors.MethodsMice bearing 4T1 mammary tumors were orally administered a PI3Kδ inhibitor (PI-3065) daily and tumor growth, survival and T cell infiltrate were analyzed in the tumor microenvironment. A second treatment schedule comprised PI3Kδ inhibitor with anti-LAG3 antibodies administered sequentially 10 days later.ResultsAs observed in human immunotherapy trials with other agents, immunomodulation by PI3Kδ-blockade led to 4T1 tumor regressor and non-regressor mice. Tumor infiltrating T cells in regressors were metabolically fitter than those in non-regressors, with significant enrichments of antigen-specific CD8+ T cells, T cell factor 1 (TCF1)+ T cells and CD69− T cells, compatible with induction of a sustained tumor-specific T cell response. Treg numbers were significantly reduced in both regressor and non-regressor tumors compared with untreated tumors. The remaining Treg in non-regressor tumors were however significantly enriched with cells expressing the coinhibitory receptor LAG3, compared with Treg in regressor and untreated tumors. This striking difference prompted us to sequentially block PI3Kδ and LAG3. This combination enabled successful therapy of all mice, demonstrating the functional importance of LAG3 in non-regression of tumors on PI3Kδ inhibition therapy. Follow-up studies, performed using additional cancer cell lines, namely MC38 and CT26, indicated that a partial initial response to PI3Kδ inhibition is an essential prerequisite to a sequential therapeutic benefit of anti-LAG3 antibodies.ConclusionsThese data indicate that LAG3 is a key bottleneck to successful PI3Kδ-targeted immunotherapy and provide a rationale for combining PI3Kδ/LAG3 blockade in future clinical studies.

The nature of the tumor microenvironment (TME) influences the ability of tumorspecific T cells to control tumor growth. In this study we performed an unbiased comparison of the TME of Treg-replete and Treg-depleted carcinogen-induced tumors, including Treg-depleted responding (regressing) and non-responding (growing) tumors. This analysis revealed an inverse relationship between extracellular matrix (ECM) and T cell infiltrates where responding tumors were T cell rich and ECM poor whereas the converse was observed in non-responder tumors. For this reason, we hypothesised that the ECM acted as a barrier to successful T cell infiltration and tumor rejection. However, further experiments revealed that this was not the case but instead showed that an effective T cell response dramatically altered the density of ECM in the TME. Along with loss of ECM and high numbers of infiltrating T cells, responder tumors were distinguished by the development of lymphatic and blood vessel networks with specialized immune function. ECM-rich tumors exhibited a stem cell-like gene expression profile and superior tumor-initiating capacity, whereas such features were absent in responder tumors. Overall, these findings define an extended role for an effective immune response, not just in direct killing of tumor cells, but in widescale remodelling of the TME to favor loss of ECM, elimination of cancer stem cells, and propagation of adaptive immunity.

High endothelial venules (HEV) are specialised post capillary venules that recruit naïve T-cells and B-cells into secondary lymphoid organs (SLOs) such as lymph nodes (LN). Expansion of HEV networks in SLOs occurs following immune activation in order to support development of an effective immune response. In this study, we used a carcinogen-induced model of fibrosarcoma to examine HEV remodelling after depletion of regulatory T cells (Treg). We used light sheet fluorescent microscopic imaging (LSFM) to visualise entire HEV networks, subsequently applying computational tools to enable topological mapping and extraction of numerical descriptors of the networks. Whilst these analyses revealed profound cancer- and immune-driven alterations to HEV networks within LNs, these changes did not identify successful responses to treatment. The presence of HEV networks within tumours did however clearly distinguish responders from non-responders. Finally, we show that a successful treatment response is dependent on coupling tumour-associated HEV (TA-HEV) development to T cell activation implying that T cell activation acts as the trigger for development of TA-HEVs which subsequently serve to amplify the immune response by facilitating extravasation of T cells into the tumour mass.