Although recent evidence demonstrates heterogeneity among CD8+ T cells during chronic infection, developmental relationships and mechanisms underlying their fate decisions remain incompletely understood. Using single-cell RNA and TCR sequencing, we traced the clonal expansion and differentiation of CD8+ T cells during chronic LCMV infection. We identified immense clonal and phenotypic diversity, including a subset termed intermediate cells. Trajectory analyses and infection models showed intermediate cells arise from progenitor cells before bifurcating into terminal effector and exhausted subsets. Genetic ablation experiments identified that type I IFN drives exhaustion through an IRF7-dependent mechanism, possibly through an IFN-stimulated subset bridging progenitor and exhausted cells. Conversely, Zeb2 was critical for generating effector cells. Intriguingly, some T cell clones exhibited lineage bias. Mechanistically, we identified that TCR avidity correlates with an exhausted fate, whereas SHP-1 selectively restricts low-avidity effector cell accumulation. Thus, our work elucidates novel mechanisms underlying CD8+ T cell fate determination during persistent infection and suggests two potential pathways leading to exhaustion.

Our previous reports demonstrated that the magnitude of NK cell mediated cytotoxicity correlated directly with the stage and levels of differentiation of tumor cells. In addition, we have shown previously that activated NK cells induce growth inhibition of cancer cells through induction of differentiation resulting in the resistance of tumor cells to NK cell mediated cytotoxicity through secreted cytokines, as well as direct NK-tumor cell contact. In this report we show that in comparison to IL-2+anti-CD16mAb treated NK cells, activation of NK cells by probiotic bacteria sAJ2 in combination with IL-2 and anti-CD16mAb substantially decreases tumor growth and induces maturation, differentiation, and resistance of Oral Squamous Cancer Stem Cells (OSCSCs), Mia-Paca (MP2) stem-like/poorly differentiated pancreatic tumors, and healthy Stem Cells of Apical Papillae (SCAPs) through increased secretion of IFN-γ and TNF-α, as well as direct NK-tumor cell contact. Tumor resistance to NK cell mediated killing induced by IL-2+anti-CD16mAb+sAJ2 treated NK cells is mediated by both IFN-γ and TNF-α since antibodies to both, and not each cytokine alone, was able to restore tumor sensitivity to NK cells. Increased surface expression of CD54, B7H1, and MHC class I on NK differentiated tumors was mediated by IFN-γ since the addition of anti-IFN-γ abolished their increase and restored the ability of NK cells to trigger cytokine and chemokine release; whereas, differentiated tumors inhibited cytokine release by the NK cells. Monocytes synergized with NK cells in the presence of bacteria to induce regulated differentiation of stem cells through secretion of IL-10 resulting in resistance to NK cell mediated cytotoxicity and inhibition of cytokine release. Therefore, probiotic bacteria condition activated NK cells to provide augmented differentiation of cancer stem cells resulting in inhibition of tumor growth, increased tumor differentiation and decreased inflammatory cytokine release.

Natural killer (NK) cells are known to target cancer stem cells and undifferentiated tumors. In this paper, we provide a novel strategy for expanding large numbers of super-charged NK cells with significant potential to lyse and differentiate cancer stem cells, and demonstrate the differences in the dynamics of NK cell expansion between healthy and cancer patients. Decline in cytotoxicity and lower IFN-γ secretion by osteoclast-expanded NK cells from cancer patients correlates with faster expansion of residual contaminating T cells within purified NK cells, whereas healthy donors’ osteoclasts continue expanding super-charged NK cells while limiting T cell expansion for up to 60 days. Similar to patient NK cells, NK cells from tumor-bearing BLT humanized mice promote faster expansion of residual T cells resulting in decreased numbers and function of NK cells, while NK cells from mice with no tumor continue expanding NK cells and retain their cytotoxicity. In addition, dendritic cells in contrast to osteoclasts are found to promote faster expansion of residual T cells within purified NK cells resulting in the decline in NK cell numbers from healthy individuals. Addition of anti-CD3 mAb inhibits T cell proliferation while enhancing NK cell expansion, however, expanding NK cells have lower cytotoxicity but higher secretion of IFN-. Expansion and functional activation of super-charged NK cells by osteoclasts is dependent on IL-12 and IL-15. Thus, in this report we not only provide a novel strategy to expand super-charged NK cells, but also demonstrate that rapid and sustained expansion of residual T cells within the purified NK cells during expansion with dendritic cells or osteoclasts could be a potential mechanism by which the numbers and function of NK cells decline in cancer patients and in BLT humanized mice.

Therapeutic role of NK cells in solid tumors was challenged previously even though their role in hematological malignancies has clearly been established. Furthermore, functions and numbers of NK cells are greatly suppressed in oral cancer patients necessitating effective future NK immunotherapeutic strategies to aid in the control of disease. The humanized-BLT (hu-BLT) mice were used to implant stem-like/undifferentiated oral tumors to study the role of super-charged NK cells with and without feeding with AJ2 probiotic bacteria. Implanted CSC/undifferentiated tumors resected from NK-injected mice exhibited differentiated phenotype, grew slowly, and did not cause weight loss, whereas those from tumor-bearing mice without NK-injection remained relatively more stem-like/poorly-differentiated, grew faster, and caused significant weight loss. Moreover, in vitro NK-differentiated tumors were sensitive to chemotherapeutic drugs, and when implanted in the oral-cavity grew no or very small tumors in mice. When NK-mediated differentiation of tumors was blocked by IFN-γ and TNF-α antibodies before implantation, tumors grew rapidly, remained stem-like/poorly-differentiated and became resistant to chemotherapeutic drugs. Loss of NK cytotoxicity and decreased IFN-γ secretion in tumor-bearing mice in PBMCs, splenocytes, bone marrow derived immune cells and enriched NK cells was restored by the injection of super-charged NK cells with or without feeding with AJ2. Much greater infiltration of CD45⁺ and T cells were observed in tumors resected from the mice, along with the restored secretion of IFN-γ from purified T cells from splenocytes in NK-injected tumor-bearing mice fed with AJ2 probiotic bacteria. Thus, super-charged NK cells prevent tumor growth by restoring effector function resulting in differentiation of CSCs/undifferentiated-tumors in hu-BLT mice.

Topchyan P, et al. Immune Netw. 2023 Oct;23(5):e41. https://doi.org/10.4110/in.2023.23.e41