Molecular subtypes of breast cancer and presence of tumor-infiltrating immune cells have both been implicated as important predictive and prognostic factors for improved risk stratification and treatment individualization of breast cancer patients. Their association, however, has not been studied in detail. The aim of this study was to evaluate the expression of the T cell markers CD8, FoxP3, CD3 and ζ-chain in molecular subtypes of the invasive margin and tumor center of breast cancer and corresponding sentinel nodes and to deduct prognostic information from these findings. Tumor and sentinel node sections from 177 patients with primary, invasive, unilateral early-stage breast cancer were stained by immunohistochemistry and T-cell phenotypes quantified manually. Clinical data were collected from medical records. The degree of T-cell infiltration and expression of all markers differed significantly among the molecular subtypes, being highest in non-luminal, more aggressive tumors: more T-cell infiltration and higher expression of all markers were associated with hormone receptor negativity, higher proliferation and higher histological grades, but also with larger tumor size. Basal-like tumors, and most remarkably their tumor centers, hosted the highest number of FoxP3+ T-cells with an unfavorable ratio to cytotoxic CD8+ T-cells. T-cell infiltration was generally higher in the invasive margin than the tumor center. A scoring system based on densities of CD3 and CD8 could significantly separate molecular subtypes (p < 0.001). Thus, immunological patterns with functional implications within each subtype are associated with prognostic factors. These findings should be further validated in studies using larger patient populations and longer follow-up.

The success of anti-CTLA4 or anti-PD1 immune checkpoint inhibitors varies widely in melanoma, with the individual modes of action in tumors and treatment-specific biomarkers still being explored. We analyzed pre- and on-treatment tumor samples from melanoma patients treated with anti-CTLA4 or anti-PD1 to characterize immune cell infiltration and molecular pathways in the tumor microenvironment and correlate these findings with treatment response and survival. We observed a prominent role for immune cell infiltration in the tumor microenvironment (TME) for clinical benefit from anti-PD1 therapy, in contrast to anti-CTLA4 therapy. A higher degree of infiltration with CD3+ T cells (p = 0.046), PD1+ (p = 0.002), and INOS+ (p = 0.042) cells was significantly associated with disease control on anti-PD1 treatment. Whereas, a higher degree of infiltration with CD138+ B cells was significantly associated with longer progression-free survival (p = 0.048, HR: 0.301 (95% CI: 0.092–0.989)). The association between immune cell infiltration, TCR richness in tumor samples, and treatment outcomes was even more striking in on-treatment biopsies from patients receiving anti-PD1 treatment. An enrichment of the immunoglobulin signature in on-treatment tumor samples was observed in responding patients. The study highlights the importance of pre-existing immune infiltration in the TME, particularly B cell infiltration, for anti-PD1 success in melanoma patients.

Summary: The in-depth analysis of costimulatory signaling enhancing the activity of cytotoxic T cells (CTLs) represents a major approach towards immunotherapy development. Here we report that CD2 costimulation plays a critical role in killing by freshly isolated human CTLs, which represent a challenging but valuable study model to gain insight into CTL biology. We show that CD2 triggering critically aids signaling by the T cell receptor in the formation of functional immune synapses by promoting the polarization of lytic granules towards the microtubule-organizing center (MTOC). To gain insight into the underlying elusive mechanism, we explored the CD2 signaling network by phosphoproteomics, which revealed 616 CD2-regulated phosphorylation events in 373 proteins implicated in the regulation of vesicular trafficking, cytoskeleton organization, autophagy and metabolism. Strikingly, signaling by the master metabolic regulator AMP-activated protein kinase (AMPK) represents a functionally critical node of the CD2 network which regulates granule polarization towards the MTOC in CTLs. Granule trafficking is driven by active AMPK enriched on adjacent lysosomes, illustrating a novel signaling cross-talk between vesicular compartments in CTLs. Our results thus establish CD2 signaling as key for regulating cytotoxic killing and granule polarization in freshly isolated CTLs and strengthens the rationale to choose CD2 and AMPK as therapeutic targets to boost CTL activity.