key: cord-0708345-5k0ukosd authors: Duckworth, Brigette C.; Groom, Joanna R. title: Conversations that count: Cellular interactions that drive T cell fate date: 2021-02-14 journal: Immunol Rev DOI: 10.1111/imr.12945 sha: d5f73013900c50a5aadd6278c06cf6a22abd86e6 doc_id: 708345 cord_uid: 5k0ukosd The relationship between the extrinsic environment and the internal transcriptional network is circular. Naive T cells first engage with antigen‐presenting cells to set transcriptional differentiation networks in motion. In turn, this regulates specific chemokine receptors that direct migration into distinct lymph node niches. Movement into these regions brings newly activated T cells into contact with accessory cells and cytokines that reinforce the differentiation programming to specify T cell function. We and others have observed similarities in the transcriptional networks that specify both CD4+ T follicular helper (T(FH)) cells and CD8+ central memory stem‐like (T(SCM)) cells. Here, we compare and contrast the current knowledge for these shared differentiation programs, compared to their effector counterparts, CD4+ T‐helper 1 (T(H1)) and CD8+ short‐lived effector (T(SLEC)) cells. Understanding the interplay between cellular interactions and transcriptional programming is essential to harness T cell differentiation that is fit for purpose; to stimulate potent T cell effector function for the elimination of chronic infection and cancer; or to amplify the formation of humoral immunity and longevity of cellular memory to prevent infectious diseases. interactions occur, and the dendritic cell subsets involved, plays a critical role in establishing the gene networks that determine effector and memory differentiation for both CD4+ and CD8+ T cells. 1, [7] [8] [9] [10] [11] [12] [13] [14] [15] Following activation, CD4+ T cells can differentiate into a diverse set of effector helper cells. Each helper lineage is specified by distinct transcriptional regulators and cytokines that orchestrate a coordinated attack for the eradication of a specific type of pathogen. 16, 17 T follicular helper (T FH ) cells are a distinct subset that promotes B cell maturation into high-affinity plasma and memory cells. [18] [19] [20] Unlike the other CD4+ T cell effectors, T FH differentiation occurs irrespective of the type of pathogen to support germinal center formation, and thus, these cells are an essential link between the cellular and humoral responses. In contrast to CD4+ T cell differentiation, the diversity in CD8+ T cells has only been more recently appreciated. In addition to the varied effector responses, CD8+ T cell differentiation leads to a heterogeneous pool of memory subsets. CD8+ T cell memory can be broadly grouped into either central memory or effector memory populations, which were originally defined by their distinct homing potential. 21, 22 Central memory cells continually recirculate in blood and lymph and are found in secondary lymphoid organs, while effector memory cells can recirculate through, or reside within peripheral tissues. Each of these pools contains specialized memory populations. Recently, a subpopulation of central memory cells, termed stem-like memory cells (T SCM ), has been described in humans and mice. 23 T SCM are programmed to promote self-renewal and repress terminal differentiation. [24] [25] [26] By seeding the effector cell pool, T SCM mediate long-term immunity to chronic infection, provide superior recall responses to secondary infection, and control tumor growth following immunotherapy. 23 From the initial description of CD8+ T SCM cells, comparisons have been made concerning their relatedness to CD4+ T FH cells. 24, 27 These similarities are not only reflected in the cell-surface markers that define these subsets, but also the transcriptional regulators that determine their specific differentiation. Understanding these similarities may highlight the cellular interactions, cytokines, and lymphoid niches that promote these differentiation pathways. Investigating this potential could reveal new avenues to harness CD4+ T FH and CD8+ T SCM cells and steer vaccine design to focus on the T cell responses that would provide the greatest humoral response combined with long-lived, robust cellular memory. In this review, we compare and contrast our current knowledge for the differentiation of CD4+ T FH and CD8+ T SCM cells and their effector counterparts T H1 and T SLEC cells. We discuss how the details of one system may inform the other, and the emerging technologies that may help address remaining questions. Combined with recent discoveries, future work which aims to understand the environmental and cellular interactions that support one or both of these T cell differentiation fates will drive the formulation of target vaccines that establish reliable, protective, and long-lived immunological memory. In addition, as both T FH and T SCM responses have been shown to promote the clearance of tumors following checkpoint blockade, this knowledge has the potential to inform and optimize the revival of T cell responses during chronic infection or for cancer immunotherapy. 25, 28, 29 2 | D IVER S IT Y OF CD 4+ AND CD8+ T CELL D IFFERENTIATI ON Following infection, CD4 + T cells differentiate into distinct T-helper subsets that mediate clearance of and protection against diverse pathogens. 17, 30 This dynamic T cell differentiation permits an exceptional flexibility to tailor responses for the clearance of distinct pathogens and protection from reinfection. T H1 cells secrete interferonγ (IFNγ) and tumor necrosis factorα (TNFα) and promote cellular immune responses mainly to intracellular pathogens, such as viruses and tumors. T H2 cells are formed during helminth and allergy challenges and secrete interleukin (IL)-4, IL-5, and IL-13. T H17 cells are identified through their production of IL-17A and IL-17F and are increased in the context of extracellular bacteria or fungi challenges. 16, 17 It is important to note that not every immune response contains a single lineage, but rather there is a rich and diverse continuum of CD4+ T cell effectors. In addition, considerable plasticity exists between T H subsets to fine-tune responses. However, as the differentiation of these lineage pathways is determined by the cytokine milieu present during infection, CD4+ T cell responses are skewed toward a T H population that is tuned to respond to a distinct class of pathogen. Thus, flexibility within T H functions insures immune responses are context-appropriate to facilitate host protection against a wide range of pathogens. Another essential role for CD4+ cells is to support and instruct the germinal center response, leading to the production of high-affinity antibody-producing cells and memory B cells. This task is performed by a distinct population known as T FH cells. The differentiation of T FH is distinct from the other T H lineages, as this population is required to differentiate alongside each of the effector T H subsets irrespective of the class of pathogen challenge. Still, there exists critical cytokine and transcriptional tipping points that can lead to a preference for differentiation toward either T FH and CD4+ T cell effectors. 9 Functional heterogeneity within T FH populations is a newly established concept. During B cell interactions within the germinal center, T FH cells can secrete multiple cytokines, IL-21, IL-4, IL-2, IL-9, IL-10, IL-13, and IFNγ. 19, [31] [32] [33] [34] The potential to secrete a specific cytokine combination represents functionally distinct T FH subpopulations not only between distinct infectious and pathogenic settings but also over the course of an infection. 32, 35, 36 In particular, T FH subpopulations defined by their distinct cytokine expression have been shown to influence the production of pathogenic antibodies in allergy and asthma in both humans and mice. 32, 36 Heterogeneity within T FH populations was first appreciated in human studies, through the investigation of circulating peripheral blood T FH subsets. 37, 38 While these cells exhibit lower expression of T FH identifying proteins CXCR5 and PD-1, they still resemble those found within the tonsil and lymph nodes. [39] [40] [41] Interestingly, circulating T FH cells are defined by their expression of the chemokine receptors CCR6 and CXCR3. Studies assessing circulating T FH in patients with inborn errors of immunity have shown that these populations are independently transcriptionally regulated. 42 In these patients, STAT1 deficiency leads to a loss in the CCR6+ population and a reciprocal accumulation of the CXCR3+ circulating T FH cells. Several studies have investigated the relationship between T FH subpopulations and the development of vaccine response or immune protection following infection. While some indicate that CXCR3+ circulating T FH cells can be used as a biomarker of immune protection, 43, 44 others demonstrate that protection of humoral responses is negatively correlated with CXCR3+ subpopulations. 38, 42, 45, 46 Still in other settings, the relationship between T FH heterogeneity and humoral response is less clear. This is the case in convalescent COVID-19 patients, where CCR6+ T FH cells were negatively correlated with SARS-CoV-2 neutralizing antibodies, while the remaining CCR6-CXCR3−/+ population correlated with antibody levels. 47 It is currently unknown if the heterogeneity of human T FH changes over time; however, it is more likely that this is imprinted by their developmental path and determined by the infection or vaccination environment. In animal models, we have demonstrated that diversity exists in the formation of T FH cells between different viral infections. 8 This diversity is transcriptionally regulated. Specifically, we have identified a T-betdependent and T-bet-independent process for T FH differentiation using the experimental systems of acute lymphocytic choriomeningitis virus (LCMV) and influenza. 8 Until recently, the diversity within CD8+ T cell differentiation was restricted to a bifurcation between effector and memory. However, it is now appreciated that substantial heterogeneity exits in each of these broad paths. 22, 48 The most common definition of CD8+ T cell effectors is found following viral infections and resemble CD4+ T H1 cells, both in terms of their transcriptional requirements and effector molecule production. These cells are identified via their surface expression of killer cell lectin-like receptor subfamily G, member 1 (KLRG1). CD8+ T cell effector cells are critical for the clearance of viral-infected cells due to their high production of effector molecules (such as IFNγ and granzyme B). 22, 49 This population of effector cells undergoes considerable contraction following pathogen clearance and are therefore referred to as short-lived effector T cells (T SLEC ). As T SLEC represent the CD8+ T cell equivalent of T H1 CD4+ T cells, so too do the less common T C2 and T C17 cells relate to T H2 and T H17 cells, respectively. However, both T C2 differentiation and T C17 differentiation are more strongly associated with aberrant and pathogenic immune responses than targeting responses toward diverse pathogenic infections. 50 Indeed, differentiation toward IL-5-producing-T C2 cells is associated with the development of severe asthma following infection. 50 In contrast, the population of IL-17-producing T C17 cells were originally suspected to be an abnormality of genetic modifications in mice. T C17 cells are enriched with the compound deletion of the T-box transcription factors T-bet and EOMES or T-bet and Blimp, or the deletion of TCF-1. [51] [52] [53] However, in recent years, knowledge of this subset has increased to define its role in protection against fungal infection and microbial colonization of the skin. [51] [52] [53] In addition, T C17 cells have also been found associated with gastrointestinal cancers and responses during influenza viral infection, making them novel targets for immunotherapy. 54 In recent years, a population of CXCR5+ follicular cytotoxic T cells was identified during chronic infection. 37, 55 Many of the key cell-surface markers and characteristic properties of these cells resemble that of a newly described T SCM memory population. Follicular cytotoxic T cells were observed within B cell follicles, while T SCM accumulate in the T cell zone. The literature has coalesced behind the T SCM nomenclature and this population will be discussed in greater detail in the following sections. 24 25, 27, 57 While the mechanisms that underlie the enhanced multipotency of T SCM are undefined, this population is of exceptional interest for its therapeutic potential. 23, 58 The effector memory population contains both CD127hi and CD127lo populations with low CD62L expression. These markers, along with the absence of CCR7, allow these cells to enter and circulate through peripheral tissues. 22 Previously, this confounded the identification of resident memory T cells, which are permanently retained in the periphery. 59 Furthermore, a recent report has transcriptionally dissected the CD8+ T effector memory population to identify distinct, terminally differentiated effector memory cells, termed terminal-T EM in both humans and mice. 60 Despite their persistence for over 70 days postinfection, these cells display some hallmarks of T SLEC cells, including KLRG1 expression. Importantly, this study showed T EM exhibit potent cytotoxicity, but have limited recall potential. This population was depleted after 3-5 months postinfection. As the T EM population has been previously conflated with the effector memory, this has redefined the classical characteristic attributed to this memory population. In addition to effector memory cells that are found recirculating in both lymphoid and non-lymphoid tissue, a tissue resident memory (T RM ) population also exists that is restrained within peripheral tissue, local to the site of initial infection. 59 In these sites, T RM cells dominate the local response upon re-challenge infection. 61, 62 In addition, the scanning behavior of T RM cells in the skin can prevent the outgrowth of melanoma and is associated with a better prognosis in breast cancer patients. 63, 64 The diversity in cell-surface markers and transcriptional signatures observed in CD8+ T cell T RM populations, located in distinct anatomical sites, is imprinted by the pathogen and the tissue environment. 65 Unlike the pathogen-driven differentiation of CD4+ T cells, for In addition to the correlation between cell-surface markers, there exist common attributes consistent within the transcriptional networks that are established between various subsets of CD4+ and CD8+ T cells. These networks act in concert with the extrinsic environment; first to move cells into specific niches, then to re-enforce differentiation based on the cytokines present in these regions ( Figure 1 ). Here, we explore some of the central nodes of the transcriptional programs that are shared between CD4+ T H1 and CD8+ T SLEC effector cells and alternatively, those that specify differentiation for CD4+ T FH and CD8+ central memory cells and T SCM cells. Within each of these CD4+ and CD8+ T cell branches, these regulators act to establish cell ontogeny and define functional outcomes. Understanding these shared transcriptional pathways may reveal shared environmental cues, such as cytokines and cellular interactions, which promote T cell differentiation. Here, we focus on central transcriptional nodes with implications for cell location and position. Bcl6 (BTB-POZ; bric-a-bric, tramtrack, broad complex-poxvirus zinc finger) and Blimp1 (B lymphocyte-induced maturation protein 1; encoded by Prdm1) are a pair of reciprocal and antagonistic transcription factors which play a key role in determining memory and effector fate decisions in T cells. 66 Specifically, the transcriptional repressor Blimp1 drives terminal effector differentiation in both CD8+ and CD4+ T cells. 66, 67 Conversely, Bcl6 is vital for the generation and maintenance of CD8+ T cell memory and also promotes T FH development in CD4+ T cells. 27, [68] [69] [70] [71] Bcl6 is the master regulator of T FH cell fate. It is vital for humoral immunity, regulating B cell help and germinal center formation. 69, 70 Early Bcl6 expression is induced by CD28 activation and cytokines such as IL-6, which activates STAT3 signaling to enhance Bcl6 expression and drive T FH development. Bcl6 acts primarily to downregulate Blimp1 and to prevent effector T H fates. 9,69,72 Bcl6 also represses Gata3, the T H2 master regulator, RORγt, required for F I G U R E 1 Circular relationship between transcriptional and environmental regulators of T cell differentiation. Following initial contact with dendritic cells, T cells begin to upregulate the transcription factors that regulate diverging fates. This leads to the upregulation or maintenance of chemokine regulators such as CXCR3, CXCR5, and CCR7. According to this expression, newly activated cells move into new regions and are exposed to a cytokine milieu that further feeds forward differentiation to re-enforce the transcriptional program and function of each cell subset T H17 differentiation, and T-bet, the T H1 master transcription factor, to further limit alternate T H fates. 19, 69, 71, 73 As discussed in detail below, Bcl6 controls cell migration by silencing promoter regions of genes such as Ccr7, Ccr6, S1pr1, and Psgl1 to promote retention within the lymph node and migration toward the T:B border. 74 Bcl6 further delays T cell egress by repressing KLF2, a transcription factor which initiates expression of S1PR1 and CD62L, revealing a similar mechanism observed in tissue resident CD8+ memory T cells to prevent lymphoid tissue homing. 75, 76 Furthermore, overexpression of Bcl6 led to increased PD-1, CXCR5, CXCR4, and SAP which are critical for T FH migration to the B cell follicles and interaction with B cells. [77] [78] [79] These interactions in turn reinforce Bcl6 expression as they progress into the B cell follicle and participate in germinal center reactions. The role of Bcl6 in T FH cells has informed studies investigating its role in the development and maintenance of CD8+ T cell memory. Here, as for CD4+ T cells, Bcl6 is inversely expressed to Blimp1, with higher levels of Bcl6 found in memory cells, particularly in T SCM cells compared to effectors. As memory precursors mature into central memory cells, Bcl6 expression progressively accumulates while Blimp1 expression decreases. 80 Interestingly, in CD8+ T cells, this expression is dependent on IL-21, while in T FH differentiation, the requirement for IL-21 remains controversial. 9 In memory precursors, Bcl6 binds directly to the Tcf7 locus, driving TCF-1 expression, which is critical for memory differentiation. 27 83 However, this function may be in concert with expression of T-bet, which directly regulates CXCR3. 52, 88 Unlike CD8+ effector T cells, early development of effector CD4+ T cells does not depend on Blimp1 expression. 89, 90 Rather, Blimp1 is expressed later in CD4+ T cell differentiation and as such is associated with highly committed, cytokine-producing CD4+ T H cell subsets. 87, 90 This profile is similar to its expression profile in differentiated CD8+ effector cells and in plasma cells, both which have an increased secretion capacity. 66 T-box transcription factors, T-bet, and eomesodermin (Eomes) are essential in the differentiation and function of effector and memory T cells. Similar to Blimp1 and Bcl6, the relative expression of T-bet and Eomes acts to tip the balance between terminally differentiated effector and memory fates. In CD8+ T cells, increasing T-bet expression promotes effector lineages early in T cell development, while Eomes is required to promote and sustain memory fate. 49, 91, 92 Similarly, in the CD4+ lineage, T-bet is considered a master regulator of driving formation of T H1 cells, while restricting non-T H1 effector fates. 93 In both precursor and committed cell subsets, T-bet plays a role repressing alternative T fates. In addition to direct repression, this is achieved by forming complexes with other lineage-defining transcription factors, such as RORgt, GATA3, and Bcl6, to block T H17 , T H2 , and T FH differentiation, respectively. 9,94,95 Formation of these complexes sequesters these other lineage-defining factors away from their DNA binding sequences and thus blocks their action. 91, 96 Additionally, T-bet cooperates with RUNX3 to antagonize GATA3 activity and also silences the Il4 locus to repress IL-4 expression. 97, 98 In CD8+ T cells, T-bet expression is graded according to the level of inflammation. 49 High levels of T-bet expression instruct a transcriptional program that is distinct from cells with moderate T-bet expression. In this setting, T-bet collaborates with the transcription factor ZEB2 to turn on the cytotoxic, terminally differentiated T SLEC differentiation program. 99 Combined, these studies show that in both CD4+ and CD8+ T cells, T-bet is a master at driving effector differentiation through the co-operation and coercion of other transcriptional regulators. A central role of T-bet is the direct activation of genes that are essential for effector T cell function. As such, T-bet is specifically binding directly to the Ifng locus to drive expression of the canonical T H1 cytokine IFNγ. 93, 100, 101 This source of IFNγ is an essential primer for the immune system and can subsequently induce T-bet expression in naive CD4 T cells, which do not express T-bet, in a STAT1-dependent manner. 102 Thus, T H1 -produced IFNγ production functions as a positive feed-forward loop to drive further T H1 differentiation and induce IFN-inducible chemokines to form peripheral and lymph node inflammatory niches. 7, 103 Further to this, T-bet directly binds and regulates the expression of the chemokine receptor, CXCR3. This expression further re-enforces the inflammatory loop generated by T-bet, as it moves cells into regions of inflammation via CXCR3. In these regions, T-bet via IFNγ production induces local expression of the CXCR3 ligands, CXCL9, and CXCL10, leading to further recruitment to the site. 7, 88 Expression of T-bet is induced by multiple inflammatory cytokines. The most characterized of these is IL-12 through the activation of STAT4. 30, 49, 94, 95, 101 For CD4+ T cells, multiple cytokines can tip the balance toward T-bet expression and T H1 differentiation including IL-2, type I IFN, and IL-12. 8, 11, 104 Alternatively, others such as IL-6 reduce T-bet expression and increase Bcl6 to promote T FH development. 9 In CD8+ T cells, T-bet and Eomes are expressed by distinct populations, representing reciprocal differentiation paths. 105 T-bet is highest early in CD8+ effectors but is downregulated in memory cells, while Eomes expression is low in early effectors but upregulated later as memory cells emerge. 106 As such, the phenotype, function, and persistence of memory and effector CD8+ T cells are sensitive to the relative expression of Eomes and T-bet. 49, [105] [106] [107] T-bet and Eomes share significant homology and perform partially redundant roles in CD8+ T cells, including regulation of IFNγ and granzyme B. 51, 108 Initial T-bet expression in naive CD8+ T cells is induced by T cell receptor (TCR) and IFN signaling. As mentioned, this is further amplified in effector T cells by IL-12-mediated signals in addition to mTOR activity. 49, 109, 110 In contrast, Eomes is induced in early effectors in a Runx-dependent manner, enhanced by IL-2 signaling, but is limited by IL-12 and mTOR. [109] [110] [111] Eomes expression increases further in memory cells in response to WNT signaling and TCF-1. 26, 85, 106, 112 In memory CD8+ T cells, Eomes plays a critical role in cell survival and homeostatic regulation by increasing expression of IL2RB (CD122) and IL-15 signaling to promote homeostatic proliferation. 49, 85, 105 Importantly, during secondary challenge, Eomes-deficient memory precursors elicit an impaired recall response, generate less central memory, and demonstrate reduced secondary expansion. 112 and IL2Ra expression. The blocking of IL-2 signaling promotes T FH over T H1 cell differentiation. 69, 119, 120 In addition, TCF-1 is enriched at the IL-6 receptor gene locus, which suggests a role for TCF-1 in increasing IL-6 responsiveness, a vital cytokine for T FH differentiation. 121 Indeed, loss of TCF-1 leads to a decrease in IL-6Ra and IL-6. 118 While the mechanism is unclear, TCF-1 deficiency also reduces the expression of Achaete-scute homologue-2 (ASCL2). ASCL2 is required to downregulate CCR7, allowing migration to the T:B border. 122 Additionally, TCF-1 directly targets costimulatory marker inducible T cell co-stimulator (ICOS), another signaling molecule important for T FH cell commitment and growth. 118 Thus, TCF-1 acts in multiple ways to both initiate and reinforce T FH differentiation. Distinct from other T H subsets, T FH cells form a critical component of the memory pool, that repress exhaustion in chronic infection and maintain stem-like potential. 27, 123, 124 Upon re-exposure, this population re-initiated B cell helper function. 125 The generation of these memory cells was impaired by the loss of TCF-1, suggesting that TCF-1 plays an essential role for CD4+ T cell memory stemness, particularly in T FH cells. 126 In the last decade, many studies have revealed that TCF-1 plays a key role in the formation and persistence of memory CD8+ T cells. 26, 127 The Tcf7 gene is expressed highly in naive and memory CD8+ T cells, but is decreased in T SLEC cells. 54 The rapid downregulation of TCF-1 during effector differentiation is mediated, in part, by IL-12. 128 Early studies proposed that memory CD8+ T cell differentiation required Wnt signaling and β-catenin binding to TCF-1 to establish cell fate; however, the role of Wnt ligands in vivo remains disputed. 26, 127, 129 In the absence of TCF-1 during infection, memory formation is restricted while effectorassociated genes, including Blimp1, T-bet, ID2, are reciprocally upregulated. 26, 127, 128 In keeping with this, several studies have found that TCF-1 and the effector molecule granzyme B are reciprocally expressed. 56 Loss of TCF-1 further impairs the expansion of memory CD8+ T cells upon secondary challenge, indicating that TCF-1 is essential for both memory formation and secondary expansion. 26 professional antigen-presenting cells and have been classified into two major branches. 133 Type 1 cDCs (cDC1) are considered efficient in presenting cell-associated antigens through cross-presentation, while type 2 (cDC2) dendritic cells can perform some presentation but are traditionally thought to be focused on activation of CD4+ T cells. 133 These distinct subsets are strategically located within the different lymphoid regions. 13, 134 Thus, within these distinct niches or "rooms" T cells engage with dendritic cells and other accessory cells of influence to co-ordinate the efficient eradication of pathogens or cancer and to establish a pool of memory cells for long-lived protection. The effector differentiation of both CD4+ and CD8+ cells occurs in parallel, with strong correlations in the transcription factors, cellular partners, and migration cues that co-ordinate this differentiation path. 1 CXCR3 is a chemokine receptor that is highly expressed on both CD4+ T H1 and CD8+ T SLEC cells. Previously, it was thought that this chemokine moves fully differentiated cells out of secondary lymphoid organs and into peripheral sites of inflammation. 103 Following the observation that CXCR3 is upregulated well before CD4+ T cells leave the lymph node, we identified a counter intuitive role for this receptor in T H1 differentiation. 7 In this setting, newly activated cells moved from the T cell paracortex located in the center of lymph nodes, into the interfollicular regions (IFRs), between B cell follicles at the lymph node periphery ( Figure 2 ). The IFR provides a unique lymph node niche that has a rich stromal cell network. 135 Here, antigen can be deposited, either during particulate The lymph node compartments where T cell differentiation occurs. The specific transcriptional program of T H1 or T SLEC effectors, T FH, and T SCM cells moves cells into spatially distinct regions of draining lymph nodes viral infection or by using specific vaccination strategies that target antigen to this region. 1, 136, 137 Using a dual CXCR3 ligand reporter mouse, we demonstrated that CXCR3+ CD4+ T cell movement is directed by the expression of CXCL9 and CXCL10 in this region. 7 While this study did not reveal the precise dendritic cell or stromal cell population that move T cells into this area, we have now demonstrated that type 2 conventional dendritic cells (cDC2) express high levels CXCL10 in the IFR. 138 Similar to CD4+ T H1 differentiation, CD8+ effectors are also found in IFRs and this migration correlates with robust formation of effector function. 139 CD8+ T cells in IFRs form clusters around cDC2 cells consistent with their expression of CXCL10. 134 A potential splenic correlate of this niche is the marginal zone or red pulp, where CD8+ T SLEC cells have also been shown to position in a CXCR3-dependent manner. 140, 141 Multiple studies have demonstrated that expression of CXCR3 is essential for CD8+ effector differentiation. [141] [142] [143] As outlined above, CXCR3 expression is principally regulated by the transcription factor T-bet. 103 Consistent with this relationship, T-bet deficiency resembles the loss of effector differentiation observed with loss of CXCR3. 49, 143 We have shown the first observable expression of T-bet in CD8+ T cells is found within pockets of IFRs; however, it is likely that moderate T-bet induction is sufficient for CXCR3 expression and migration into this region. 138 While CD8+ T cells and cDC2s cluster in IFRs, it is unclear if this interaction is mediated through cDC2-derived CXCL10, or if stromal cell expression moves T cells into this region. We have shown that CD4+ T cells preferentially interact with CXCL10+ expressing DCs, but the specific location of these interactions and the DC subset was not defined in this study. 7 In addition, there are other accessory cells located in this region that may drive the differentiation toward T H1 and T SLEC formation. While relatively scarce in secondary lymphoid organs, monocyte-derived dendritic cells (moDCs) can induce both T H1 and T H17 responses. 133, 145, 146 Further, moDC promotion of T H1 differentiation is associated with their location in IFRs following vaccination. 137 Recently, Bosteel et al provided an important advance to the dendritic cell literature, revealing that these cells are not capable of antigen processing; however, they can express high levels of IL-12 to polarize effector T cells. [147] [148] [149] In addition to moDCs, an inflammatory subpopulation of cDC2 cells, named infDCs, has recently been characterized. 145 Given the inflammatory milieu in IFRs following viral infection, this type I IFN-dependent population may be a critical regulator of T cell effector fate. 145 The identity of the key cytokine or cytokines produced by dendritic cells or accessory cells in this region that promotes effector differentiation remains to be fully elucidated. IL-12 is a good candidate for this as it has been demonstrated to steer T cell differentiation toward both CD4+ T FH and CD8+ T SLEC cell formation and away from other fates, in a T-bet dependent manner. 128, 146, 150 Despite not being located within draining lymph node IFRs, cDC1 cells are commonly associated with T H1 responses due to their high production of IL-12. 16 Indeed, in ex vivo cultures, cDC1s promote greater formation of T H1 cells. 151 This suggests that within IFRs in vivo, it is other accessory cells, either infDCs or moDCs that are present that can produce the high levels of IL-12 needed to skew CD4+ and CD8+ T cell effector differentiation. In addition to IL-12, other cytokines such as IL-2, type I, and II IFNs have been demonstrated to promote T H1 and/or T SLEC differentiation. 9 The neighbor cell producing these additional cytokines is not clear, but may include other T cells with opposing fates as well as specific dendritic cell populations. 11,104 The differentiation of T FH cells is dynamically regulated through time and in spatially distinct regions of secondary lymphoid organs. 74 This process takes place in three distinct steps and each involves a unique set of interacting partners that imprint and reinforce differentiation into functional, germinal center resident T FH cells. 16, 152, 153 Interacting dendritic cells are generally considered essential for directing the initial phases of T FH differentiation. 154 Several studies have shown that depletion of cDC2s reduced the amount of T FH differentiation, suggesting they are the prominent dendritic cell regulator of this lineage. Early contact between CD4+ T cells and dendritic cells leads to the upregulation of Bcl6; however, during early stages of differentiation, Bcl6 can be co-expressed with T-bet, the T H1 defining factor. 95, 101 Thus, this initial activating event is not sufficient to define early fate bifurcation. These contacts do, however, result in the directed movement from the paracortex to the T:B cell border. 153 In addition to Bcl6, this migration is instructed by the ASCL2 helixloop-helix transcription factor that acts in multiple ways to drive T FH ontogeny. 122 Expression of ASCL2 leads to the upregulation of CXCR5 and downregulation of CCR7. It is worth noting that the initial location of pre-T FH cells at the T:B border occurs independently of Bcl6 and CXCR5, suggesting the downregulation of CCR7 and loss of retention in the paracortex is critical for migration. 155, 156 Instead early T FH migration to the T:B border requires the G protein-coupled receptor, EBI2 (GPR183). 157 In these regards, the initial stage of pre-T FH migration resembles that of T H1 cells, although it is clear that T FH cells do not reach the outer limits of the IFRs, as shown for T H1 cells. 7, 101, 158 The T:B border is a critical site where decisions between T H1 and T FH differentiation are made. 11, 74, 152, 153 Distinct infections can elicit a unique set of inflammatory signals and recruitment of interacting partners that can shift the balance of differentiation either toward or away from T FH differentiation. 11 Viral infections that induce a high expression of type I IFNs lead to increased dendritic cell production of IL-6 and skew differentiation toward T H1 cells. Further at the T:B border, the upregulation of EBI2 promotes CD4+ T cell interactions with IL-2 quenching cDC2s, which favors T FH differentiation. 157 Again, this work suggests that cDC2s are more permissive for T FH differentiation than for T H1 , and that for the latter additional accessory cells are required to enter this site and direct effector polarization. Critically, in this location, pre-T FH cells form long-lived contacts with B cells through ICOS:ICOS-ligand interactions, which leads to further upregulation of the canonical T FH genes, Bcl6, Cxcr5, and Il21. 77, 78, 153, 156 Interestingly, these ICOS:ICOSL interactions are formed with bystander and not antigen-specific B cells at the T:B border. 156 Although the importance of engagements with both cDC2s and B cells is well established, it is unclear if the three cells are required to interact at the same time at the T:B border. Further, one study has demonstrated that, during Plasmodium infection, dendritic cell interactions were dispensable, and B cells were solely responsible for the generation of T FH responses. 159 In contrast, augmenting antigen presentation can make T FH cell generation independent of B cells, highlighting the context-dependent nature of this process. 160 Increasing CXCR5 expression directs T FH cells into the CXCL13-expressing B cell follicles, to take part in germinal center reactions. 78, 155 Interestingly, once CXCR5 permits entry into the germinal center, it is no longer required for the maintenance of cells in this location. 161 This is the final step in the differentiation process and allows T FH cells to accomplish their role to select high-affinity B cells. 74 Despite the shared transcriptional regulation, there are significant distinctions in location of CD4+ T FH cells and CD8+ T SCM cells. The first description of T SCM (formerly known as follicular cytotoxic) cells was found due to their expression of CXCR5. Some researchers found these cells positioned according to CXCR5 expression in the B cell follicles, in both models of chronic infection and in the spleens of HIV patients. 37, 55 In contrast to this, others showed that they instead remained in the T cell paracortex of secondary lymphoid organs. 24 To clarify this discrepancy, we have shown that the T SCM precursors are retained in the T cell zone following viral infection, and their differentiation is enhanced in the absence of CXCR3. 138 While the transcriptional regulation of T cell effector molecules is well established, less attention has been placed on the transcriptional regulators of T cell location. 17, 30, 53 Delving into this further, we showed that TCF-1, the key transcriptional regulator of T SCM cells, binds to the Ccr7 locus. 138 Supporting this, multiple studies have demonstrated that CCR7 is downregulated in the absence of TCF-1 or in T cell populations that lack TCF-1. 27, 53, 165 The retained expression of CCR7 on T SCM is in contrast to T FH cells, where this receptor is downregulated rapidly following the upregulation of Bcl6. 155 For T FH cells, the expression of CCR7 acts as a counterbalance retention signal, such that when CCR7 expression is maintained, cells are not capable of entering the B cell follicle, even when CXCR5 is overexpressed. 155 Therefore, it is likely that as T SCM cells co-express both CCR7 and CXCR5, the retention within the paracortex is maintained to block migration out of this region. As discussed above, the key transcriptional difference between these populations could be the expression of ASCL2 in T FH cells, which orchestrates the upregulation of CXCR5 and downregulation of CCR7. 122 The role of ASCL2 in T SCM has so far not been investigated, but, unlike CCR7 it has not been reported to be expressed in transcriptional characterization studies. 24, 25, 56, 165, 166 Despite T SCM being positioned in the lymph node paracortex or splenic T cell zone, 24 it remains unclear if these precursors require contact with B cells at the T:B border in a similar manner to that shown for T FH cells. Initial characterization of these cells demonstrated they co-expressed T FH cell-surface markers ICOS and Slamf6, both of which facilitate T FH cell engagement with B cells. 55 A recent report showed transcriptional expression of EBI2, a molecule that is similarly expressed by pre-T FH cells and directs them to the T:B border. 157, 166 Again, it is unclear if in this region T SCM cells contact B cells or the IL-2 quenching dendritic cells producing the EBI2 ligand. 157 Further, when T SCM cells were transferred into B celldeficient μMT recipients they were less capable of inhibiting viral replication than those transferred into wild-type mice. 37 These data suggest that while T SCM cells may not need B cells to form, they potentially provide essential signals to function. The specific dendritic cell subset that promotes differentiation toward T SCM remains unknown. Conventional DC1 cells reside in the paracortex of lymph nodes, positioning themselves as interacting partners during T SCM differentiation. 134 Dynamic imaging studies have shown that CD8+ T cells interact with cDC1 cells. 10, 12 In these interactions, cDC1 cells act as conduits to facilitate CD8+ and CD4+ T cell interactions. 10, 12 This three-cell interaction facilitates the formation of memory, although T SCM differentiation was not assessed in either of these studies. While these cells highly express CXCL9, loss of CXCL9 does not appear to alter T SCM differentiation in vivo. 138 Previous work has shown that these interactions are alternatively instructed by CCR5. 167 Unlike cDC2s, cDC1s are thought to be a more homogeneous population 133 ; however, it could be that subsets of dendritic cells with a defined imprinting potential reside in the paracortex. In addition, T SCM may be maintained in the T cell zone by directly contacting the gp38+ CCL21expressing fibroblastic reticular cells (FRCs) located here. 140 is one cytokine that has been described to specifically promote T SCM expansion and coincidentally also promotes the expansion of T FH cells. 168 Although there is more work to be done to identify other factors that specifically direct differentiation toward T SCM , an alternative hypothesis is that positioning in the T cell paracortex instead protects these precursors away from other regions in lymph nodes where they may come into contact with inflammatory accessory cells or cytokines that promote effector and terminal differentiation. Supporting this hypothesis, it is the absence of inflammatory singles such as IL-12 and type I IFN that steer differentiation away from T SCM . 27, 128 We have recently shown that altering the location of CD8+ T cells by blocking CXCR3 responsiveness restricts cells to the paracortex. This change in T cell position, from the IFRs to the center of the lymph node, coincides with a decrease in effector differentiation and increased T SCM precursor formation. 138 Combined, it is clear that the migration of CD4+ and CD8+ T cells is driven by the dynamic regulation of chemokine receptors. This is controlled at the transcriptional level where early T-bet expression leads to the upregulation of CXCR3 in T H1 and T SLEC cells; TCF-1 insures the retention of T SCM in the T cell paracortex via CCR7 expression; and T FH cells localize in the B cell follicle due to expression of Bcl6 which instructs CXCR5 upregulation. Curiously, we have shown that early precursors of both T FH cells and T SCM cells can also upregulate CXCR3. 8, 138 In these instances, it appears that the CCR7 and CXCL13 migration cues counterbalance CXCR3 ligands to override the movement of non-effector cells into the locations (paracortex and B cell follicles) where these cells further differentiate. As discussed, the location where CD4+ and CD8+ T cell differentiation occurs and been elucidated. Within these regions, several accessory cells are associated with skewing toward a particular cell type; however, the precise interactions remain poorly defined. Interactions that lead to T FH differentiation are the most clearly defined to require staged interactions with cDC2 cells and B cells. 16, 74, 152 Effector T cell differentiation can also be associated with cDC2 engagement and in some conditions with the presence of inflammatory moDCs. In contrast, T SCM cells likely require contact with cDC1 cells within the T cell paracortex. Additionally, simultaneous contact with CD4+ T cells and cDC1 cells is required for promotion of CD8+ T memory formation. The serial engagement of T cells with distinct immune populations, which has been dynamically studied by multi-photon microscopy, suggests distinct roles of dendritic cell subsets during T cell differentiation. Instead, it is likely that these events work together in an additive manner to guide the transcriptional programming of T cells and determine the balance between effector and memory subsets. In this regard, movement of T cells into specific regions facilitates additive interactions that promote a single fate. In addition to DC, moDC and B cell interactions, non-immune cells are emerging as important modulators of T cell fate. Lymphoid stromal cells represent important cell organizers that enable T cell and B cell recruitment to secondary lymphoid tissues. 169 FRCs are found in close contact with T cells and act as guiding paths for dynamic scanning of naive T cells in search of their cognate antigen. 169, 170 Previously, we have described the upregulation of CXCR3 ligands in the IFRs following vaccination. 7 FRCs express CXCL9, CXCL10, and CXCL11 within the paracortex and IFRs of resting mice and following infection. 171, 172 These studies demonstrate that FRCs not only guide the location of cells in steady state, but can dynamically redistribute cells during an immune response. This role for stromal cells in facilitating interactions between immune cells is well established. As the stromal cell compartment becomes increasingly disorganized with age, dysfunction in both the dendritic cell and stromal cell compartment may both contribute to the dampened efficacy of vaccination with age. 173, 174 Emerging studies suggest that in addition to this structural role, stromal cells, in particular FRCs, may directly influence T cell differentiation. When added to human T cell activation cultures, FRCs led to decreased proliferation and memory cell formation of both CD4+ and CD8+ T cells. 175 Interestingly, this inhibition was relieved in combination with IL-6 and TGFβ and PD-L2 blockade, factors known to regulate either T FH or T SCM cell differentiation. Another study demonstrated that FRC co-culture limits the production of CD8+ T cell IFNγ, but increased T cell production of IL-2. 176 Importantly, this was independent of the block in T cell proliferation that is mediated through FRC iNOS production. Consistent with the IL-2 production, FRCs altered the epigenome of T cells leading to heightened differentiation to memory and persistence in vivo. 176 179, 180 More work needs to be done to validate that the interactions defined with these genetic models are indeed critical during a normal immune response. To help overcome this issue, a novel human tissue in situ assay has been developed to dissect the regulators of T cell differentiation in live tissue explants. 175 Vaccines are one of public health's greatest achievements, preventing many millions of illnesses and deaths every year. 181 Traditional vaccination strategies have focused on the overall magnitude of the immune response produced. However, we can now appreciate how to manipulate immune responses to promote the most valuable T cell protection for future infectious challenge. Indeed, a preference toward CD4+ T FH cells to boost humoral responses and long-lived CD8+ T SCM cell differentiation is preferred rather than exuberant effector response. While all current vaccine strategies provide protection via humoral immunity, many of the recalcitrant pathogens for which no vaccine is currently available [including human immunodeficiency virus (HIV), Mycobacterium tuberculosis, and Plasmodium species] are resistant to humoral immunity. 182 Strategies that specifically direct the generation of memory CD8 + T cells could aid in eliminating these intracellular pathogens. [181] [182] [183] Furthermore, CD8 + T cells have the potential to be utilized in the development of therapeutic vaccines to establish potent effector function against chronic infection and cancer. 181 In contrast, rapid proliferative responses with robust cytokine and effector molecule production of CD4+ T H1 and CD8+ T SLEC populations may be preferred where strategies are focused on overcoming an immediate challenge such as these settings. Therefore, the type of T cells required in each of these vaccination contexts is distinct. Thus, there is potential to exploit the shared transcriptional and cellular interactions that determine CD4+ and CD8+ effector T cells, or those that determine T FH and T SCM . Some new vaccination methods may influence the location of T cells within secondary lymphoid organs, which could, in turn, generate T cells that are fit for purpose depending on the response required. The structure and anatomy of lymph nodes, along with the distinct positioning of T cells within them, allow for the delivery of drugs or antigens directly into specific sites. 184 The ability of these platforms to target distinct lymph node niches relies on specific design factors. Several vaccine vehicles can be used to target delivery to lymph nodes; in doing so, this improves the efficacy of T cell responses. 185 A key approach for this is via the use of nanoparticles, such as silica and protein nanoparticles, for targeted lymph node delivery of subunit vaccines. [184] [185] [186] [187] [188] These platforms increase the size of antigen which assists in the uptake and deposition within lymph nodes. We and others have demonstrated that these systems favorably affect lymphatic uptake and preferentially lodge in the lymph node IFRs. 136, 189, 190 While this strategy enhanced germinal center formation, this was done in the absence of increased T FH differentiation in both animal and non-human primates. 191 Consistent with the increased recruitment of T cells to the IFR being associated with effector differentiation, this system enhanced the efficacy of anti-cancer vaccines, compared to soluble vaccination. 186 A recent study has demonstrated that the route used to deliver nanoparticle vaccines dramatically alters T cell differentiation. 166 Specifically, intravenous delivery heightened differentiation toward T SCM , while subcutaneous delivery resulted in more terminally differentiated effector cells. Therefore, it appears that the route of nanoparticle entry into secondary lymphoid organs may alter where antigens are deposited. This demonstrates that it is not the magnitude of response postvaccination that matters, but the specific path of T cell differentiation promoted. In this system, despite the lower amplification of antigen-specific CD8+ T cells following intravenous administration, the differentiation toward T SCM differentiation was essential to clear tumors cells more rapidly following checkpoint blockade. 166 Compared to subcutaneous delivery where antigen landed in IFRs with extended up take of MoDCs, intravenous delivery in the spleen was very transiently presented by cDC1s and MoDCs and the expression of IL-12 and IFNα again was transient compared to settings where T SLEC cells were preferentially made. 166 The increased efficacy and safety of these nanoparticle vaccines lie in the ability to recapitulate the deposition of viral particles in the IFR. In addition, the structure-based design of self-assembling protein nanoparticles allows multiple epitopes to be complexed in a highly immunogenic manner, allowing multivalent presentation of antigen. This system has been used to elicit potent neutralizing immunity using a dual-antigen self-assembling SARS-CoV-2 nanoparticle in mice and non-human primates. 192 Excitingly, these vaccines also provide a structure-based design for the generation of broadly reactive universal vaccine platforms that target complete classes of viral strains, such as coronavirus or influenza. 188, 192 An alternative strategy is the investigation of how different adjuvants deposit antigen into spatially distinct regions. This has recently been investigated for the promotion of T H1 cell differentiation in vivo. 137 This study compared soluble Toll-like receptor and antigen delivered in the mineral oil emulsion, incomplete Freund's adjuvant (IFA). The use of the oil emulsion formulation altered the antigen deposition, moving it to the IFRs, rather than the medulla of draining lymph nodes. In addition, this approach promoted the recruitment of inflammatory monocytes which expressed CXCL10 to drive antigenspecific CD4+ T cells into this region. 137 Although not tested, presumably this would have also been associated with increased CD8+ T cell effector differentiation, but come at a loss of T FH and T SCM . Other newer adjuvant formulations, such as GLA-SE, have been shown to heighten T FH and memory T cell formation; however, as yet how these alter the deposition of antigen or inflammatory signals has not been demonstrated. 193 A final approach that aims to exploit specific T cell:DC interactions is via the targeting of specific dendritic cell populations. 194 This strategy exploits our understanding of T:DC interactions to target antigen to restrict presentation to a particular dendritic subtype. The optimal way to do this is to generate a genetic fusion of the antigen to the Fc portion of the antibody heavy chain to target a dendritic cell-specific surface molecule. The two molecules that have shown the most potential for this strategy are using Fc fusions proteins for targeting CLEC-9A, which is specifically expressed on cDC1s or targeting to DEC-205 which is more broadly expressed dendritic cells and langerin cells. 194 Targeting DEC-205 with adjuvants results in potent effector responses for both CD4+ and CD8+ T cells with strong humoral responses. The addition of adjuvant in this system is critical as without it, there are minimal effector responses and tolerance mechanisms related to regulatory T cells are induced. 195 Similarly, without adjuvant, targeting CLEC-9A also results in the induction of CD8+ T cell tolerance. As discussed above, the choice of adjuvant can steer and tailor immune responses and this is true also, in combination with CLEC-9A targeting. 195 However, in the absence of adjuvant, CLEC-9A drives potent CD4+ T FH differentiation, germinal formation, and humoral responses, that exceed that seen with DEC-205 targeting. 197, 198 Interestingly, targeting cDC1s via CLEC-9A positions these cells at the T:B border of lymph nodes and allows more contact with antigen-specific B cells and T FH progenitors in this region. 199 Combined, these two targeting approaches hold promise to promote specific T cell engagement with both dendritic cells and/ or antigen-specific B cells. While the focus of studies to date has investigated CD4+ and CD8+ effector T cell differentiation and function, it remains to be seen how these individual targeting strategies promote the generation of long-lived memory. Again, it is likely that adjuvant selection with each of these approaches will determine outcomes, highlighting the flexibility of these strategies. Given the potential of harnessing both T FH and T SCM cells, the next questions in the field will be to more completely understand and identify the cellular contacts that promote formation of these subsets. In recent years, a host of new technologies and analysis pipelines have been developed to overcome some of the challenges for understanding the cellular interactions that govern transcriptional signatures of T cell fates. The most powerful of these incorporate advancements in imaging modalities combined with innovative transcriptional analysis. One technology that is particularly suitable to determine cell interactions in a particular region is NICHE-seq. 200 This combines a photoactivatable fluorescent marker to color-in regions of interest using two-photon laser scanning microscopy. Following dissociation of tissues, cells are sorted to identify niche components which are then analyzed by single cell RNA-seq (scRNA-seq). This method has been used to exceptional effect to characterize the T:B border CD4+ T cell priming niche during viral infection. 11 This revealed increased proportions of monocytes and dendritic cells following LCMV infection, when T FH differentiation is inhibited, compared to recombinant VSV where T FH differentiation is promoted. As discussed above, gene signatures within the niche also demonstrated increased type I IFN, which was subsequently shown to control T FH differentiation in this setting. 11 A further application of this technology would be to combine NICHE-seq with a comprehensive ligand:receptor bioinformatic analysis to broadly identify the cellular interactome within a specific region. 201 The current tool kit available to bioinformatically resolve cellular interactions has recently been reviewed. 202 These analyses could also be complemented assessing the physically interacting cells (PIC-seq) approach. 203 This method calibrates tissue dissociation protocols to insure interacting cell conjugates are preserved prior to scRNA-seq analysis. Following conjugate RNA-seq, the interacting partners are bioinformatically separated. Of interest, some of the receptor:ligand and downstream transcriptional events can be associated with specific interacting cells. 203 Combining these approaches within a specific lymphoid niche offers new opportunities to understand how inflammatory cues and directly interacting cells facilitate T cell differentiation. More advanced methods have been developed to analyze specific T cell interactions. A proximity-dependent labeling system that crosses cell:cell interfaces known as LIPSTIC (Labelling Immune Cell Partnerships by SorTagging Intercellular Contacts) has been developed to investigate CD40:CD40L interactions. 204 In this system, ligands and receptors are genetically fused to the Staphylococcus aureus transpeptidase sortase A (SrtA), or a tag residue. When the ligand and receptors interact, Srt A catalyzes the transfer of substrate onto the tagged receptor, recording the history of interaction that can be detected by flow cytometry. This study revealed that initial vaccine-induced interactions occur between 24 and 72 hours following T cell transfer and these interactions were primarily with cDC2 as opposed to cDC1s. 204 This approach is highly adaptable to other receptor:ligand interactions and cell types of interest. An extension of this approach, termed FucolD, provides another option for interaction-dependent labeling of T cells using cell-surface enzymatic fucosyl-biotinylation. 205 In this system, antigen-specific T cells were labeled by their interacting dendritic cell partners within the tumor environment. Importantly, this approach is a proximity-based labeling system, meaning that, unlike LIPSTIC, the receptor mediating the interaction is not required to be known prior to experimentation. Given the transcriptional similarities between CD4+ and CD8+ effectors, as well as the similarities between T FH and T SCM , we have discussed in this review what is currently known for these subsets and contrasted the spatial location and environmental cues that promote specific differentiation toward each of these paths. While the secondary lymphoid niches that imprint specific T cell differentiation have been elucidated, there remains much left to understand regarding the specific interactions within these sites that direct cell fate. This information is key to understanding the ways to harness T cell differentiation for therapeutic and vaccine interventions and will allow the generation of T cells that are fit for purpose; either to stimulate potent T cell effector function for the elimination of chronic infection and cancer, or to amplify the formation of humoral immunity and longevity of cellular memory to prevent current and emerging infectious diseases. We thank Stephen L Nutt for critical reading of the manuscript. 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Efficient and versatile manipulation of the peripheral CD4+ T-cell compartment by antigen targeting to DNGR-1/CLEC9A Targeting antigen to Clec9A primes follicular Th cell memory responses capable of robust recall Targeting antigen to mouse dendritic cells via Clec9A induces potent CD4 T cell responses biased toward a follicular helper phenotype Display of native antigen on cDC1 that have spatial access to both T and B cells underlies efficient humoral vaccination Spatial reconstruction of immune niches by combining photoactivatable reporters and scRNA-seq Lung single-cell signaling interaction map reveals basophil role in macrophage imprinting Deciphering cellcell interactions and communication from gene expression Dissecting cellular crosstalk by sequencing physically interacting cells Monitoring T celldendritic cell interactions in vivo by intercellular enzymatic labelling Detecting tumor antigen-specific T cells via interaction-dependent fucosyl-biotinylation The author has no conflict of interest to declare.