Although FLT3 ligand (FLT3L, CD135) is a primary factor driving the differentiation of conventional dendritic cells (cDCs), whether it contributes to determining the ratio of type 1 (cDC1) versus type 2 (cDC2) cells in both lymphoid and nonlymphoid tissues is unclear. In this Top Read, Audiger and Lesage (p. 2117) sought to characterize cDC differentiation in five common inbred mouse strains, exploring factors that contribute to cDC distribution in the spleen. Quantification of bone marrow pre-cDC1 and pre-cDC2 precursors committed to the cDC1 and cDC2 lineages revealed that mice with fewer pre-cDC1 had more cDC1. This disparity was associated with variations in FLT3L expression in cDC subsets. Consistent with this observation, the authors demonstrated that increased systemic FLT3L concentration biased cDC differentiation toward the cDC1 phenotype, to the detriment of the cDC2 phenotype, suggesting that cDC1 precursors are more sensitive to FLT3L than cDC2 precursors. Furthermore, whereas FLT3L is required throughout cDC2 differentiation, it was dispensable during late-stage cDC1 differentiation. Thus, this study demonstrates that tight regulation of FLT3L levels throughout cDC differentiation dictates the cDC1 to cDC2 ratio in lymphoid organs.

In this Top Read, Aagaard et al. (p. 2146) demonstrate that a heterologous prime-boost strategy against Mycobacterium tuberculosis improved CD4 T cell responses. The authors primed mice using Bacillus Calmette–Guérin (BCG) and followed with a subunit booster containing Ags from either BCG (H65) or an Ag absent from the BCG vaccine (H74). Both subunit vaccines were equally protective in naive mice following challenge. However, when used as a booster, H74 significantly reduced bacterial load in BCG-primed mice, whereas H65 failed to enhance BCG-elicited protection. Treatment with either BCG alone or BCG in combination with the H65 boost resulted in highly differentiated CD4 T cells, wherein the majority produced IFN-γ. Animals boosted with H74 had significantly more M. tuberculosis–specific CD4 T cells producing TNF-α alone or in combination with IL-2, suggesting these cells were less differentiated. Additionally, BCG priming followed by H65 boost, but not by H74 boost, inhibited CD4 T cell migration to the lung parenchyma. Together, these data provide important insights into how boosts with complementing M. tuberculosis Ag can overcome pre-existing BCG immunity and enhance anti–M. tuberculosis responses.

To date, CRISPR/Cas9 genetic alteration of Ag-experienced memory CD8 T cells has not been successful. In this Top Read, Kurup et al. (p. 2222) demonstrated that CRISPR/Cas9-mediated gene disruption prevented cytokine- or Ag-induced proliferation of memory CD8 T cells in vivo. Interestingly, targeting of genes not known to directly influence the proliferative abilities of memory CD8 T cells also precluded cellular proliferation, indicating that genomic damage may hinder memory T cell proliferation. Consistent with these observations, targeted disruption of the proapoptotic transcription factor p53, which is an integral part of the DNA damage response pathway, prevented CD8 T cells from undergoing apoptotic death in response to CRISPR/Cas9-induced DNA damage. Importantly, temporary inhibition of p53 function via small interfering RNA facilitated targeted gene disruption by CRISPR/Cas9 and functional alteration of Ag-experienced CD8 T cells in vivo. Thus, the authors demonstrated that temporary inhibition of p53 function facilitates reliable gene disruption and functional transformation of memory CD8 T cells in vivo.

T cell immunoreceptor with Ig and ITIM domains (TIGIT) is an immunoreceptor protein expressed on T and NK cells that functions as an inhibitory receptor. In this Top Read, Yang et al. (p. 2156) describe the antitumor activity of an anti-TIGIT Ab called T4. This Ab was identified from a phage display library and was selected both for its optimal binding kinetics to both murine and human TIGIT and for its ability to inhibit the interaction between TIGIT and its cognate ligand, CD155. Despite its specificity, the antitumor effects of T4 did not depend on CD155 expression on the tumor. Instead, FcγR engagement was essential for antitumor activity, and modifications that enhanced FcγR binding improved antitumor efficacy. Treatment with T4 decreased the proportion of regulatory T cells within tumors in an Fc receptor– and NK cell-dependent manner, resulting in an increased CD8 T antitumor activity. T4-treated mice also had effective antitumor CD8 T cell memory and were able to clear tumors upon rechallenge. These data demonstrate the potential of TIGIT as an immunotherapy target in cancer.

As a key mediator of leukocyte trafficking during inflammation, CCL22 is constitutively expressed at high levels in lymphoid organs during homeostasis, where it recruits regulatory T cells (Tregs) to dendritic cells (DCs). In this Top Read, Piseddu et al. (p. 2056) aimed to identify the mechanisms regulating constitutive CCL22 expression. Using both human PBMCs and murine splenocytes, the authors demonstrated that CD11c+ cDCs are exclusive producers of CCL22 in secondary lymphatic organs during homeostasis. However, DCs required T cell help to produce CCL22 in vitro, and this was dependent on T cell–derived GM-CSF. Consistent with these observations, adoptive transfer of wild-type T cells or administration of GM-CSF restored low levels of CCL22 in lymphoid organs of Rag−/− mice. Thus, this study demonstrates that T cell–derived GM-CSF is a key inducer of CCL22 and a central regulator of immunity in lymphoid organs.

Although the priming, differentiation, and modulation of Ag-specific CD8+ and CD4+ effector T cells (TE) by chemokines has been well documented, the actual range, regulation, and role of T cell–derived chemokines remains incompletely understood. In this Top Read, Eberlein et al. (p. 2169) sought to define the entire spectrum of chemokines produced by Ag-specific T cells and to delineate properties pertaining to temporospatial organization of chemokine expression patterns, synthesis and secretion kinetics, and cooperative regulation. Using flow cytometry to survey production of 37 individual chemokines, the authors demonstrated that TE are a significant source of chemokines in wild-type mice. Robust chemokine expression was observed for CCL3, CCL4, CCL5, and XCL1. One to two percent of TE also synthesized CXCL2, and a very small subset produced CCL1 or CCL9/10. Thus, the authors concluded that these six chemokines are indicative of highly activated T cells. Additional studies demonstrated that these chemokines are uniquely regulated and are a consistent component of the TE response across different acute and chronic infectious disease models or protective vaccination. Furthermore, T cells were found to be the major hematopoietic source of CCL1/3/4/5 production, and CCL5 was observed to have a unique temporospatial organization of synthesis, storage, and secretion associated with its targeted release at the forefront of the mature CD8+ TE response.

In a second study by Davenport et al. (p. 2188), the authors extended their analysis of chemokine kinetics to pathogen-specific memory T cells (TM). Similar to TE, TM were a robust source of six select chemokines (CCL1/3/4/5, CCL9/10, and XCL1) and displayed an invariant signature across acute and chronic infections. The authors also investigated kinetics of these chemokines to identify discrete traits subject to temporal modulation and to define acute and chronic infection. Chemokine production dominated the earliest stages of CD8+ TM recall responses due to rapid synthesis/secretion kinetics of CCL3/4/5 and low activation thresholds of CCL1/3/4/5 and XCL1. Constitutive CCL5 expression by CD8+ TM was a unique functional signature of prior Ag experience. CCL1 induction identified highly functional CD8+ and CD4+ TM subsets. Long-term maintenance of CD8+ TM was associated with a significant increase in XCL1 production capacity. Whereas TM chemokine profiles induced by chronic viral pathogens exhibited discrete functional defects, recall responses and partial virus control in chronic infection were not impacted by the absence of major TM chemokines. Collectively, these characteristics establish TM-derived chemokines as prominent, robust, and rapidly mobilized components of the recall response. In addition, the unique TM chemokine signatures in acute/chronic infections may be of diagnostic value and correlate with protective capacities or potential outcomes.