NKG2A is a late immune checkpoint on CD8 T cells and marks repeated stimulation and cell division

The surface inhibitory receptor NKG2A forms heterodimers with the invariant CD94 chain and is expressed on a subset of activated CD8 T cells. As antibodies to block NKG2A are currently tested in several efficacy trials for different tumor indications, it is important to characterize the NKG2A(+) CD8 T cell population in the context of other inhibitory receptors. Here we used a well-controlled culture system to study the kinetics of inhibitory receptor expression. Naive mouse CD8 T cells were synchronously and repeatedly activated by artificial antigen presenting cells in the presence of the homeostatic cytokine IL-7. The results revealed NKG2A as a late inhibitory receptor, expressed after repeated cognate antigen stimulations. In contrast, the expression of PD-1, TIGIT and LAG-3 was rapidly induced, hours after first contact and subsequently down regulated during each resting phase. This late, but stable expression kinetics of NKG2A was most similar to that of TIM-3 and CD39. Importantly, single-cell transcriptomics of human tumor-infiltrating lymphocytes (TILs) showed indeed that these receptors were often coexpressed by the same CD8 T cell cluster. Furthermore, NKG2A expression was associated with cell division and was promoted by TGF-beta in vitro, although TGF-beta signaling was not necessary in a mouse tumor model in vivo. In summary, our data show that PD-1 reflects recent TCR triggering, but that NKG2A is induced after repeated antigen stimulations and represents a late inhibitory receptor. Together with TIM-3 and CD39, NKG2A might thus mark actively dividing tumor-specific TILs.Experimental cancer immunology and therap

Host genetics and tumor environment determine the functional impact of neutrophils in mouse tumor models

Background Neutrophils have been reported to have protumor, antitumor or neutral effects in cancer progression. The underlying causes for this functional variability are not clear. Methods We studied the role of neutrophils in six different mouse tumor models by intratumoral injection of antimicrobial peptides or vaccination. Changes in systemic and intratumoral immune cells were analyzed by flow-cytometry and mass-cytometry. The role of neutrophils was studied by antibody-mediated neutrophil depletion. Neutrophils from different mouse strains were compared by RNA sequencing. Results The antimicrobial peptide Omiganan reduced the growth of TC-1 tumors in BL/6 mice and CT26 tumors in BALB/c mice. No significant effects were observed in B16F10, MC38 and 4T1 tumors. Growth delay was associated with increased abundance of neutrophils in TC-1 but not CT26 tumors. Systemic neutrophil depletion abrogated Omiganan efficacy in TC-1 but further reduced growth of CT26, indicating that neutrophils were required for the antitumor effect in TC-1 but suppressed tumor control in CT26. Neutrophils were also required for a therapeutic vaccine-induced T-cell mediated control of RMA tumors in BL/6 mice. Clearly, the circulating and intratumoral neutrophils differed in the expression of Ly6G and CD62L, between TC-1 and CT26 and between blood neutrophils of tumor-naive BL/6 and BALB/c mice. RNA-sequencing revealed that neutrophils from BL/6 mice but not BALB/c mice displayed a robust profile of immune activation, matching their opposing roles in TC-1 and RMA versus CT26. Conclusions Neutrophil functionality differs strongly between mouse strains and tumor types, with consequences for tumor progression and therapy.Molecular Epidemiolog

The clonal origin and clonal evolution of epithelial tumours

While the origin of tumours, whether from one cell or many, has been a source of fascination for experimental oncologists for some time, in recent years there has been a veritable explosion of information about the clonal architecture of tumours and their antecedents, stimulated, in the main, by the ready accessibility of new molecular techniques. While most of these new results have apparently confirmed the monoclonal origin of human epithelial (and other) tumours, there are a significant number of studies in which this conclusion just cannot be made. Moreover, analysis of many articles show that the potential impact of such considerations as patch size and clonal evolution on determinations of clonality have largely been ignored, with the result that a number of these studies are confounded. However, the clonal architecture of preneoplastic lesions provide some interesting insights — many lesions which might have been hitherto regarded as hyperplasias are apparently clonal in derivation. If this is indeed true, it calls into some question our hopeful corollary that a monoclonal origin presages a neoplastic habitus. Finally, it is clear, for many reasons, that methods of analysis which involve the disaggregation of tissues, albeit microdissected, are far from ideal and we should be putting more effort into techniques where the clonal architecture of normal tissues, preneoplastic and preinvasive lesions and their derivative tumours can be directly visualized in situ