Cannulation studies have revealed that thoracic duct lymph (46–48) as well as efferent lymph collected after passage through one or more LNs is mainly constituted by T lymphocytes (6, 43, 44). More than 90% of lymphocytes in efferent lymph were shown to have initially entered the LN through high endothelial venules (HEVs) (39, 43). CD4⁺ T cells enter and recirculate through LNs more rapidly than CD8⁺ T cells (27). Accordingly, CD4⁺ T cells constitute the major cellular fraction in efferent lymph and outnumber CD8⁺ T cells at a ratio higher than that in blood (49, 50). Most T cells in efferent lymph collected from sheep exhibit a naïve phenotype, with a reported increase in the proportion of memory T cells in older animals (44, 51, 52).

Antigenic stimulation of LNs often leads to distinct phases in the efferent lymph response: an initial “LN shutdown” where lymphocyte output is decreased; a “recruitment phase” where lymphocyte output rises above resting levels; and a “resolution phase” where lymphocyte output and cellular composition return to resting levels (53–55). While in most cases a sequential egress of CD4⁺ and then CD8⁺ T cells has been reported (56–58), the dominance of a particular lymphocyte subset in efferent lymph appears to be dependent on the antigenic stimulus (45, 59–61).

Compared to efferent lymph, the cellularity of afferent lymph is much lower (5–10%) under homeostatic conditions (6, 43, 44). While αβ T lymphocytes represent the most abundant cell type of afferent lymph (80-90%), DCs (5–15%), monocytes, B cells, and few granulocytes are also routinely found in steady-state afferent lymph (39, 43). CD4⁺ T cells in afferent lymph collected from sheep outnumber CD8⁺ T cells by approximately fourfold to fivefold (6, 43, 44). As reported in sheep, CD4⁺ T cells are the dominant cell type in afferent lymph collected from superficial dermal LVs of healthy humans (62–64). T cells in afferent lymph of both humans and sheep exhibit an effector-memory (T_EM) phenotype, characterized by elevated expression of common T cell activation markers, adhesion molecules, and effector cytokines (44, 45, 63, 64). Although γδ T cells are present in large numbers in afferent lymph from sheep (65), they are almost non-existent in lymph or blood in humans (63, 64) and so are not further discussed here.

As cannulation of LVs is difficult in mice, a lot of our current knowledge of the T cell populations migrating through afferent LVs in mice has come from other experimental techniques used to investigate leukocyte trafficking (see Box 1). Specifically, these include adoptive transfer experiments or experiments performed in transgenic mice in which migrating leukocytes can be tracked by photoconversion of endogenously expressed fluorescent proteins [e.g., Kaede mice (28)—see Box 1]. Conclusions drawn from these approaches in mice are in accordance with earlier cannulation studies in larger animals. Moreover, they have revealed that the CD4⁺ T cell dominance in afferent lymph results from more efficient CD4⁺ T cell migration from the skin to the dLN (7, 31). In Kaede mice, the majority of CD4⁺ T cells that migrated from the skin to the dLN expressed the common T cell activation marker CD44 as well as the skin-homing molecules C–C chemokine receptor type 4 (CCR4) and E-selectin ligands (30, 31). Approximately 25% of CD4⁺ T cells that migrated from the skin to the dLN were also found to express the T_reg transcription factor FOXP3⁺ (30). Similarly, others have reported that adoptively transferred T_regs enter afferent LVs and migrate from the skin to dLN in mice (66–68). Notably, T_regs are phenotypically similar to T_EM and are only distinguishable when specific T_reg markers are used. The fact that FOXP3, the most widely used T_reg marker, was only described approximately 13 years ago might explain why T_regs have thus far not been reported from cannulation studies performed in sheep and humans (which frequently date back to earlier times).

In contrast to the conventional viewpoint that naïve T cells exclusively recirculate between blood and SLOs, low numbers of naïve T cells have also been found in both homeostatic and inflamed non-lymphoid tissues and have been suggested to circulate through afferent LVs (20, 69, 70). Indeed, in adoptive transfer experiments in mice, naïve T cells were shown to avidly migrate from the skin to dLN (7, 10). However, it is important to consider that the majority of endogenous CD4⁺ T cells in the skin have an effector/memory-like phenotype (10, 71). Correspondingly, cannulation studies in humans and sheep, and studies in Kaede mice, suggest that naïve T cells constitute only a minor subset of T cells in afferent lymph under both steady-state (30, 44, 64) and inflammatory conditions (12, 30, 72).

Cannulation studies in sheep have revealed that acute skin inflammation, e.g., elicited by injection of complete Freund’s adjuvant (CFA), induced a dramatic increase in granulocyte numbers in skin-draining afferent lymph, whereas CD4⁺ and CD8⁺ T cells initially remained fairly stable (12, 53, 72, 73). By contrast, chronic inflammation, resulting from CFA-induced granuloma formation, was shown to lead to a substantial increase in CD4⁺ and CD8⁺ T cell output in skin-draining afferent lymph (12, 72). Contrastingly, in Kaede mice, an acute contact hypersensitivity response elicited a striking increase in the number of T cells that migrated from the skin to the dLN (30). However, it needs to be considered that numbers of T cells in steady-state lymph of laboratory mice might be unnaturally low, because of the sterile housing conditions that lead to the formation of a reduced pool of effector-memory T cells populating peripheral tissues (74).

Early findings that pertussis toxin (a natural inhibitor of Gα_i-protein-coupled receptors, such as chemokine receptors) inhibited the export of mature T cells from the thymus (84), suggested that egress of T cells from the LN could also be an active process. Studies on the immunosuppressive activity of Fingolimod (FTY720), a now approved treatment for multiple sclerosis (85), incited further research on the molecular mechanism of T cell exit from LNs. FTY720 induces sequestration of lymphocytes in SLOs through retention and “log jamming” of lymphocytes on the abluminal side of the lymphatic sinuses, thereby inhibiting lymphocyte egress into circulation and migration to sites of disease (86–88). Besides histologic analysis of lymphatic sinuses, efferent lymph cannulation studies and LN egress experiments, in which T cell homing into LNs is first blocked and T cell numbers subsequently quantified over time, have been instrumental for studying T cell exit into efferent LVs (see Box 1).

Several studies have shown that the egress-blocking activity of FTY720 can mainly be attributed to the action of FTY720 on sphingosine-1-phosphate (S1P) receptors, in particular, S1P receptor 1 (S1P₁) expressed on T cells (8, 9, 89, 90). The natural ligand of S1P₁ is S1P, an endogenous sphingolipid that mediates diverse cellular processes, including cell survival, cytoskeletal rearrangements, and cellular chemotaxis (91, 92). S1P levels in tissues are tightly controlled by sphingosine kinase 1 and 2 (Sphk1/2)-mediated production and S1P degradation, which depends on S1P lyase and other enzymes (77, 93). While erythrocytes, red blood cells, and the blood endothelium constitute major cellular sources of plasma S1P, lymph S1P is derived independently from the blood (91, 94). In fact, LECs were identified as the major source of S1P in lymph (41).

S1P levels in the blood and in lymph are much higher than in lymphoid organs (77, 95). Low concentrations of S1P in lymphoid tissues and S1P abundance in lymph was shown to create a gradient across LECs, which induces transmigration of S1P₁-expressing T cells into the lymphatic sinuses and egress into efferent lymph (93, 96): acting as a functional antagonist, FTY720 induces downregulation and degradation of S1P₁ in T cells, thereby inhibiting S1P-mediated chemotaxis across the lymphatic sinuses (8). Similar to FTY720 treatment, adoptively transferred S1P₁-deficient T cells were found to “log jam” around medullary and cortical sinuses and failed to egress into efferent lymph (8, 9, 23, 41). An analogous egress defect could also be evoked when the S1P gradient in LNs was experimentally destroyed, by inhibiting S1P lyase (77), or upon genetic deletion of Sphk1 and Sphk2 in LECs (41).

Similar to FTY720, high concentrations of S1P are capable of inducing S1P₁ internalization in T cells (92, 97). Consequently, T cells in blood express low levels of S1P₁ (95). Following entry into LNs via HEVs, T cells begin to upregulate S1P₁ (95). Given that entry into LN sinuses, and subsequent egress from the LN, is S1P₁ dependent, T cell transit time through the LN is in some manner dependent on S1P₁-mediated resensitization to S1P in lymph. In addition to S1P-induced receptor internalization, the C-type lectin CD69 has also been reported to regulate S1P₁ surface expression in T cells. CD69 is an early T cell activation marker and is upregulated in T cells by various inflammatory mediators, such as type I interferons (78, 93). CD69 has been shown to interact with S1P₁, thereby inducing a receptor conformation similar to the ligand bound state, leading to S1P₁ internalization and degradation (78, 79). CD69 expression by recently activated T cells therefore serves to inhibit the egress promoting function of S1P₁ (24, 78, 79). However, activated T cells only transiently express CD69 (98). Accordingly, once activated T cells have undergone several rounds of division and have downregulated CD69, they start to re-express S1P₁ and appear in circulation (8, 22). Akin to CD69 regulated surface expression of S1P₁ on recently activated T cells, T cell receptor signaling (the first signal of T cell activation) has been reported to induce transcriptional downregulation of S1P₁ (8). Transcriptional restoration of S1P₁ is also likely to regulate T cell egress during an immune response.

In addition to S1P₁, CCR7 expression levels in T cells also impact the time T cells spend in LNs. Upon antigen recognition, activated T cells downregulate CCR7 (22). Fibroblastic reticular cells within the LN produce CCL21 and help generate a gradient where CCL21 levels are highest toward the LN center and decrease toward the peripheral medullary areas (25, 99). In addition to mediating intranodal positioning, migration, and motility (75), CCR7 also confers T cell retention within LNs (22). T cells devoid of CCR7 (CCR7^−/−) egressed more rapidly than their wild-type (WT) counterparts, whereas transgenic T cells overexpressing CCR7 were retained in the LN for longer periods of time (22). Treatment with pertussis toxin restored egress competence of S1P₁-deficient lymphocytes and in mixed bone marrow chimeras FTY720 treatment increased the number of CCR7^−/− T cells found in efferent lymph relative to their WT counterparts (22). Collectively, these findings suggest that CCR7 on T cells promotes their retention in LNs and that egress signals through S1P₁ in part overcome CCR7-mediated retention (22). Interestingly, more CCR7^+/− than WT T cells entered sinuses, suggesting that the interplay between CCR7-mediated retention and S1P₁-mediated egress occurs at the level of entry into sinuses (22). More recently, it has also been reported that C–X–C chemokine receptor 4 (CXCR4) on T cells synergizes with CCR7 to retain both naïve and activated T cells in LNs (80).

While it is well established that adhesion molecules and their integrin ligands play an important role in T cell entry into LNs through HEVs (100), not much is known about their role in T cell egress across lymphatic sinuses. A role for leukocyte function-associated antigen 1 (LFA-1) in delaying egress of T cells across lymphatic sinuses has recently been suggested. Following the probing of the surface of LN sinuses, CD4⁺ T cells devoid of LFA-1 had a greater tendency to egress across sinuses and spent less time in the LN than their WT counterparts (26). This distinction was lost in mice lacking the major LFA-1 ligand intercellular adhesion molecule 1 (ICAM-1) (26).

In addition to LFA-1, the common lymphatic endothelial and vascular endothelial receptor-1 (CLEVER-1), as well as the macrophage mannose receptor (MR) or its ligand L-selectin have been implicated in T cell migration across lymphatic sinuses: when performing adhesion assays on LN sections, antibody-mediated blockade of CLEVER-1 or MR reduced binding of lymphocytes to sinus endothelium (81, 82). However, the in vivo involvement of these receptors in LN egress has not been demonstrated thus far. On the other hand, a possible role for the integrin α9 subunit in lymphocyte egress from inflamed LNs has recently been reported (83). Integrin α9β1 is a well-described binding partner of the extracellular matrix component tenascin-C, and both α9 and tenascin-C reportedly are upregulated in medullary and cortical LN sinuses during inflammation. The study revealed that tenascin-C binding to LEC-expressed α9β1 induced S1P production in LECs, establishing a mechanistic link between α9 integrin expression and S1P₁-mediated T cell egress. In fact, antibody-based blockade of α9 or tenascin-C deficiency resulted in impairment of T cell egress from inflamed LNs, reminiscent of treatment with FTY720 (83).

T cell egress from LNs has not only been studied at the population level but also at the single-cell level using IVM (see Box 1). Such studies have confirmed previous histology-based studies showing that T cell migration and egress occurs both at the level of the cortical and medullary sinuses (23, 101). T cells were observed entering sinuses at multiple locations, however, occasionally two or more T cells entered at specific entry “hot spots” (23, 101). In cortical sinuses without flow, T cells migrated at the same speed as those in the parenchyma and occasionally exited sinuses back into the LN parenchyma (23, 24). In larger cortical sinuses with flow, T cells were more rounded, shared fairly uniform velocities, and had a lower frequency of exit back into the parenchyma (23, 24). T cells in the macrophage-rich medullary sinuses appeared to become poorly mobile and occasionally exited the sinuses and returned to the T cell zone (23). Following migration of T cells through cortical and medullary sinuses, T cells were released into the subcapsular region near the efferent vessel and moved off rapidly with lymph flow (23). Overall, T cell transit time through the LN appears to be determined by random walk encounters with lymphatic sinuses (24). Only at the level of the sinus do S1P₁-expressing T cells start to sense S1P in lymph, which triggers their exit into the lymphatic compartment (22–24).

Classical definitions outline that non-lymphoid tissue homing T_EM are devoid of CCR7 (119). However, in humans, CCR7 is expressed on the majority of T cells in blood, including those that express adhesion molecules required for homing to non-lymphoid tissue (120). Consistent with these findings, 40–50% of all skin-associated CD4⁺ T cells in humans (121) and mice (31) express CCR7. Several studies have identified CCR7 and its ligand CCL21, which is constitutive expressed by LVs (107, 122), as one of the most important drivers of T cell migration to dLNs: adoptive transfer experiments (7, 10) and experiments performed in Kaede mice (31) have shown that compared to WT T cells, significantly fewer (in the order of 10–20%) CCR7^−/− CD4⁺ or CD8⁺ T cells migrated from the skin to the dLN. Moreover, in a model of allergic airway inflammation, CCR7^−/− CD4⁺ T_EM cells accumulated in the lung and airways (114). Similarly, CD4⁺ T_EM have been shown to accumulate within the epithelial tissues of CCR7^−/− mice (123), and CCR7^−/− T_regs accumulated in inflamed skin (124). Although CCR7 appears to be crucial for T cell exit from homeostatic and acutely inflamed skin, its contribution to T cell exit from chronically inflamed skin appears to be more limited (11, 12). In the case of DCs, IVM studies have recently revealed that the CCR7/CCL21 axis mediates DC migration toward and into LVs (106, 122) and also impacts the directionality of DC crawling within lymphatic capillaries (107). By contrast, the exact contribution of CCR7 to T cell migration through afferent lymphatics has not been addressed so far.

Besides CCR7/CCL21, the second best described chemotactic pathway involved in T cell exit from skin is S1P₁/S1P. As mentioned, LECs are considered the major contributor to S1P levels in lymph (41). Overexpression of S1P₁ in CD8⁺ T cells prevented “settling” of T_RM in the intestine, kidney, salivary gland, and skin, suggesting S1P₁ enhanced exit via afferent LVs (125). Similar to S1P₁-overexpressing CD8⁺ T cells, CD69-deficient CD8⁺ T cells failed to persist in skin after HSV infection, and treatment with an S1P₁ agonist restored their retention within the skin (126). Correspondingly, surface expression of CD69 and transcriptional loss of S1P₁ is a hallmark for CD8⁺ T_RM (127–130).

In contrast to CD8⁺ T_RM, tissue-resident CD4⁺ T cells have been less well characterized and studied. In a study using Kaede mice (see Box 1), Bromley and colleagues identified one population of CD4 memory T cells that remained in the skin and a second population, termed recirculating memory CD4⁺ T cells (T_RCM), that migrated from the skin to the dLN (31). T_RCM expressed a novel cell surface phenotype (CCR7^int/+, CD62L^int, CD69⁻, CD103^+/−, CCR4^+/−, and E-selectin ligands⁺) and migrated in a CCR7-dependent manner (31). These cells displayed a trafficking behavior distinct from classical T_EM or T_CM cells in such that T_RCM migrated from skin to dLNs, and from circulation back into sites of unspecific cutaneous inflammation (31). The role of S1P in CD4⁺ T cell egress from skin has been addressed by two other recent studies (10, 12). Treatment of adoptively transferred T cells or of recipient mice with FTY720 or S1P significantly reduced T cell migration to the dLN (10, 12). Interestingly, acute inflammation was shown to increase S1P levels in the skin and also resulted in reduced migration of CD4⁺ T cells to the dLN (10). This suggests that acute inflammation might induce T cell retention in the tissue.

T cells that have migrated from the skin to the dLN display high expression of CCR7, CXCR4, and S1P₁ (7, 10). In contrast to the involvement of CCR7 and S1P₁, CXCR4 was reported to have no role in T cell migration from homeostatic (10) or inflamed skin to the dLN (11). By contrast, in a pancreatic islet transplantation model, CCR2, CCR5, and CXCR3 reportedly contributed to the migration of natural T_regs (nT_regs) from the allograft to the dLN (66, 68). While LECs constitutively produce CXCL12, CCL21, and S1P (41, 131), they are also able to upregulate inflammatory chemokines under conditions of tissue inflammation (131, 132). This upregulation occurs in a stimulus-specific manner (131) and may serve to fine-tune leukocyte recruitment into LVs. Although not specifically studied so far, changes in the chemokine expression profile of LECs might also explain the reduced CCR7 and S1P dependence of T cell tissue exit observed from chronically but not from acutely inflamed skin (12). On the other hand, it has to be considered that most studies investigating T cell tissue exit have been performed using adoptively transferred T cells, which might not completely reflect the chemokine (or adhesion molecule) requirements of endogenous T cells.

T cell recirculation through afferent LVs is thought to contribute to immune surveillance by constantly replenishing the T cell pool in peripheral tissues with new antigenic specificities. However, increasing evidence suggests that recirculating T cells do not provide complete protection of peripheral tissues, and that T_RM play a more important role in this process (104, 138). Although mainly studied for CD8⁺ T cells and in a limited number of infection models, T_RM (typically CD69^hi, CD103^hi, E-cadherin^hi, S1PR1lo, and CCR7^lo) have been shown to provide immediate protection against reinfection (104, 139). Current evidence suggests that T_RM differentiate from T_eff, remain resident within the tissue for long periods of time (>1 year in mice) and predominate at sites of infection or inflammation (104, 140, 141). Although there is some evidence that T_RM proliferate locally, it is unknown whether T_RM are ever replaced by circulating T cells (139, 142, 143). The protective mechanisms of T_RM are not yet fully known, but evidence suggests that T_RM functionally delay pathogen spread and further act as an antigen-specific sensor that “sounds the alarm” for the recruitment of circulating T cells (104). The relative contribution of resident and circulating T cells in pathogen clearance remains unknown and might be highly context dependent, e.g., dependent on the type of infection and the specific requirement for CD4⁺ or CD8⁺ T cells for immune control (104, 139).

Previous studies have shown that the local ratio of T_regs to T_eff at inflamed sites is a critical determinant for the outcome of inflammation (144–146). In support of this notion, adoptively transferred CCR7^−/− T_regs that accumulated in the skin of mice controlled Th1-mediated inflammation more efficiently than WT T_regs (124). While these findings suggest that retention of T_regs within peripheral tissue promotes resolution of inflammation, large numbers of T_regs reportedly exit the skin during a cutaneous immune response in mice (30).

CD4⁺ T_regs control both priming and expansion of T_eff in SLOs and the activation of T_eff in the skin (147–150). Several islet allograft survival studies highlight T_reg migration to dLNs as a prerequisite for efficient downregulation of the ongoing allograft response (66–68, 151). Only T_regs within the skin, or having previously exited the skin via afferent LVs, reportedly displayed an activated phenotype (66). Upon adoptive transfer of egress-incompetent T_reg into the graft, graft survival was shorter than that for WT T_regs (66–68). Similarly, in a study using Kaede mice, T_regs that migrated from inflamed skin had an activated phenotype, inhibited immune responses more robustly than LN-resident T_regs, and were able to recirculate back to the skin (30). These findings suggest that T_regs that have exited the skin via afferent LVs restrict LN immune responses (and consequently tissue inflammation) and recirculate back to inflamed tissue to help further control local immune responses.

The extent of tissue inflammation often correlates with the number and composition of infiltrating T cells, which itself is dependent on T cell recruitment from blood, survival in the tissue, and, last but not least, on T cell exit through afferent LVs. Interestingly, two recent studies have shown that the ability of T cells to exit inflamed tissues has an impact on the degree of tissue inflammation. In mouse models of delayed-type hypersensitivity and TNF-driven Crohn’s-like ileitis, reduced exit of CCR7^−/− T cells from the site of inflammation translated into enhanced and prolonged inflammation (152, 153). Similarly, T cells overexpressing CCR7 had an enhanced capacity to exit from inflamed skin and accelerated resolution of inflammation (152). However, depending on the experimental setup, these experiments might have to be interpreted with caution because of the confounding influence of autoimmunity observed in CCR7^−/− mice, which might be due to other factors in addition to limited exit from peripheral tissues (76).

While recruitment into tissue is independent of the antigen specificity of T cells (154, 155), exit of T cells from inflamed tissues appears to be at least in part antigen dependent (152, 156). In a mouse model of delayed-type hypersensitivity, transgenic CD4⁺ Th1 cells, co-injected with DCs that were pulsed with cognate antigen, displayed reduced migration from inflamed skin to the dLN relative to polyclonal CD4⁺ Th1 cells (152). Similarly, a significantly reduced number of antigen-specific cytotoxic CD8⁺ T cells (Tc1), in comparison to antigen-unspecific Tc1 cells, migrated from the lung to the dLN in influenza-infected animals (156). These findings suggest that upon recognition of antigen, T cells have an impaired “tissue exit program” and are retained at the effector site, while antigen non-specific bystander T cells continue to exit via the afferent LVs in a CCR7-dependent manner (156). This mechanism is likely in place to reduce unnecessary tissue damage through bystander T cells.