Graphical AbstractHighlights d Mouse and human tumors harbor relatively undifferentiated Tcf1 + PD-1 + CD8 + T cells d These intratumoral cells have expansion, regeneration, and differentiation capacity d They produce differentiated Tcf1 À PD-1 + CD8 + T cells in response to immunotherapy d These stem-like cells are critical for tumor control in response to immunotherapy In BriefSince chronic activation promotes terminal T cell differentiation (exhaustion), it has remained unclear how checkpoint blockade mediates a proliferative response of tumorinfiltrating T cells. Siddiqui et al. identify intratumoral, tumor-reactive Tcf1 + PD-1 + CD8 + T cells that display stem-like properties and that promote tumor control in response to vaccination and checkpoint blockade immunotherapy. SUMMARYCheckpoint blockade mediates a proliferative response of tumor-infiltrating CD8 + T lymphocytes (TILs). The origin of this response has remained elusive because chronic activation promotes terminal differentiation or exhaustion of tumor-specific T cells. Here we identified a subset of tumor-reactive TILs bearing hallmarks of exhausted cells and central memory cells, including expression of the checkpoint protein PD-1 and the transcription factor Tcf1. Tcf1 + PD-1 + TILs mediated the proliferative response to immunotherapy, generating both Tcf1 + PD-1 + and differentiated Tcf1 À PD-1 + cells. Ablation of Tcf1 + PD-1 + TILs restricted responses to immunotherapy. Tcf1 was not required for the generation of Tcf1 + PD-1 + TILs but was essential for the stem-like functions of these cells. Human TCF1 + PD-1 + cells were detected among tumor-reactive CD8 + T cells in the blood of melanoma patients and among TILs of primary melanomas. Thus, immune checkpoint blockade relies not on reversal of T cell exhaustion programs, but on the proliferation of a stem-like TIL subset.

Mucocutaneous leishmaniasis is caused by infections with intracellular parasites of the Leishmania Viannia subgenus, including Leishmania guyanensis. The pathology develops after parasite dissemination to nasopharyngeal tissues, where destructive metastatic lesions form with chronic inflammation. Currently, the mechanisms involved in lesion development are poorly understood. Here we show that metastasizing parasites have a high Leishmania RNA virus–1 (LRV1) burden that is recognized by the host Toll-like receptor 3 (TLR3) to induce proinflammatory cytokines and chemokines. Paradoxically, these TLR3-mediated immune responses rendered mice more susceptible to infection, and the animals developed an increased footpad swelling and parasitemia. Thus, LRV1 in the metastasizing parasites subverted the host immune response to Leishmania and promoted parasite persistence.

Under conditions of chronic antigen stimulation, such as persistent viral infection and cancer, CD8 T cells may diminish effector function, which has been termed “exhaustion.” Expression of inhibitory Receptors (iRs) is often regarded as a hallmark of “exhaustion.” Here we studied the expression of eight different iRs by CD8 T cells of healthy humans, including CTLA-4, PD1, TIM3, LAG3, 2B4, BTLA, CD160, and KLRG1. We show that many iRs are expressed upon activation, and with progressive differentiation to effector cells, even in absence of long-term (“chronic”) antigenic stimulation. In particular, we evaluated the direct relationship between iR expression and functionality in CD8 T cells by using anti-CD3 and anti-CD28 stimulation to stimulate all cells and differentiation subsets. We observed a striking up-regulation of certain iRs following the cytokine production wave, in agreement with the notion that iRs function as a negative feedback mechanism. Intriguingly, we found no major impairment of cytokine production in cells positive for a broad array of iRs, as previously shown for PD1 in healthy donors. Rather, the expression of the various iRs strongly correlated with T cell differentiation or activation states, or both. Furthermore, we analyzed CD8 T cells from lymph nodes (LNs) of melanoma patients. Interestingly, we found altered iR expression and lower cytokine production by T cells from metastatic LNs, but also from non-metastatic LNs, likely due to mechanisms which are not related to exhaustion. Together, our data shows that expression of iRs per se does not mark dysfunctional cells, but is rather tightly linked to activation and differentiation. This study highlights the importance of considering the status of activation and differentiation for the study and the clinical monitoring of CD8 T cells.

During chronic infection, pathogen-specific CD8(+) T cells upregulate expression of molecules such as the inhibitory surface receptor PD-1, have diminished cytokine production and are thought to undergo terminal differentiation into exhausted cells. Here we found that T cells with memory-like properties were generated during chronic infection. After transfer into naive mice, these cells robustly proliferated and controlled a viral infection. The reexpanded T cell populations continued to have the exhausted phenotype they acquired during the chronic infection. Thus, the cells underwent a form of differentiation that was stably transmitted to daughter cells. We therefore propose that during persistent infection, effector T cells stably differentiate into a state that is optimized to limit viral replication without causing overwhelming immunological pathology.