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.

Lymphoid follicles are B-cell-rich compartments of lymphoid organs that function as sites of B-cell antigen encounter and differentiation. CXC chemokine receptor-5 (CXCR5) is required for B-cell migration to splenic follicles, but the requirements for homing to B-cell areas in lymph nodes remain to be defined. Here we show that lymph nodes contain two types of B-cell-rich compartment: follicles containing follicular dendritic cells, and areas lacking such cells. Using gene-targeted mice, we establish that B-lymphocyte chemoattractant (BLC/BCA1) and its receptor, CXCR5, are needed for B-cell homing to follicles in lymph nodes as well as in spleen. We also find that BLC is required for the development of most lymph nodes and Peyer's patches. In addition to mediating chemoattraction, BLC induces B cells to up-regulate membrane lymphotoxin alpha1beta2, a cytokine that promotes follicular dendritic cell development and BLC expression, establishing a positive feedback loop that is likely to be important in follicle development and homeostasis. In germinal centres the feedback loop is overridden, with B-cell lymphotoxin alpha1beta2 expression being induced by a mechanism independent of BLC.

Interleukin 7 is essential for the survival of naive T lymphocytes. Despite its importance, its cellular source in the periphery remains poorly defined. Here we report a critical function for lymph node access in T cell homeostasis and identify T zone fibroblastic reticular cells in these organs as the main source of interleukin 7. In vitro, T zone fibroblastic reticular cells were able to prevent the death of naive T lymphocytes but not of B lymphocytes by secreting interleukin 7 and the CCR7 ligand CCL19. Using gene-targeted mice, we demonstrate a nonredundant function for CCL19 in T cell homeostasis. Our data suggest that lymph nodes and T zone fibroblastic reticular cells have a key function in naive CD4(+) and CD8(+) T cell homeostasis by providing a limited reservoir of survival factors.

Directional guidance of cells via gradients of chemokines is considered crucial for embryonic development, cancer dissemination, and immune responses. Nevertheless, the concept still lacks direct experimental confirmation in vivo. Here, we identify endogenous gradients of the chemokine CCL21 within mouse skin and show that they guide dendritic cells toward lymphatic vessels. Quantitative imaging reveals depots of CCL21 within lymphatic endothelial cells and steeply decaying gradients within the perilymphatic interstitium. These gradients match the migratory patterns of the dendritic cells, which directionally approach vessels from a distance of up to 90-micrometers. Interstitial CCL21 is immobilized to heparan sulfates, and its experimental delocalization or swamping the endogenous gradients abolishes directed migration. These findings functionally establish the concept of haptotaxis, directed migration along immobilized gradients, in tissues.

Stromal cells (SCs) establish the compartmentalization of lymphoid tissues critical to the immune response. However, the full diversity of lymph node (LN) SCs remains undefined. Using droplet-based single-cell RNA sequencing, we identified nine peripheral LN non-endothelial SC clusters. Included are the established subsets, Ccl19 T-zone reticular cells (TRCs), marginal reticular cells, follicular dendritic cells (FDCs), and perivascular cells. We also identified Ccl19 TRCs, likely including cholesterol-25-hydroxylase cells located at the T-zone perimeter, Cxcl9 TRCs in the T-zone and interfollicular region, CD34 SCs in the capsule and medullary vessel adventitia, indolethylamine N-methyltransferase SCs in the medullary cords, and Nr4a1 SCs in several niches. These data help define how transcriptionally distinct LN SCs support niche-restricted immune functions and provide evidence that many SCs are in an activated state.