CD4+ T cells support cytotoxic T lymphocyte priming by controlling lymph node input

Yosuke Kumamoto; Lisa M. Mattei; Stephanie Sellers; Geoffrey W. Payne; Akiko Iwasaki

doi:10.1073/pnas.1100567108

New Research In

Edited by Philippa Marrack, Howard Hughes Medical Institute, National Jewish Health, Denver, CO, and approved April 15, 2011 (received for review January 11, 2011)

Abstract

Rapid induction of CD8⁺ cytotoxic T lymphocyte (CTL) responses is critical to combat acute infection with intracellular pathogens. CD4⁺ T cells help prime antigen-specific CTLs in secondary lymphoid organs after infection in the periphery. Although the frequency of naïve precursors is very low, the immune system is able to efficiently screen for cognate CTLs through mechanisms that are not well understood. Here we examine the role of CD4⁺ T cells in early phases of the immune response. We show that CD4⁺ T cells help optimal CTL expansion by facilitating entry of naïve polyclonal CD8⁺ T cells into the draining lymph node (dLN) early after infection or immunization. CD4⁺ T cells also facilitate input of naïve B cells into reactive LNs. Such “help” involves expansion of the arteriole feeding the dLN and enlargement of the dLN through activation of dendritic cells. In an antigen- and CD40-dependent manner, CD4⁺ T cells activate dendritic cells to support naïve lymphocyte recruitment to the dLN. Our results reveal a previously unappreciated mode of CD4⁺ T-cell help, whereby they increase the input of naïve lymphocytes to the relevant LN for efficient screening of cognate CD8⁺ T cells.

Antigen-specific cytotoxic T lymphocytes (CTLs) provide crucial protection against infection by viruses and intracellular bacteria. Previous studies have indicated that CD4⁺ T-cell help is important for the development and maintenance of CTLs. CD4⁺ T cells help the CD8⁺ T-cell response by at least two mechanisms (1 –4): first, through direct contact between CD4⁺ and CD8⁺ T cells and, second, through licensing of dendritic cells (DCs) for antigen presentation to CD8⁺ T cells (5). DCs that have been activated by CD4⁺ T cells enable efficient primary and secondary CD8⁺ T-cell responses by inducing expression of CD25 and CD127 on antigen-specific CD8⁺ T cells. In addition, when DCs encounter activated antigen-specific CD4⁺ T cells, they attract polyclonal CD8⁺ T-cell precursors through secretion of CCL3 and CCL4, resulting in more efficient memory CTL generation (3).

Lymphocyte priming takes place within secondary lymphoid tissues, such as the lymph node (LN) and spleen. Individual naïve C57BL/6 mice (B6) contain a total of only ∼20–200 peptide-specific CD4⁺ T cells (6) and ∼20–1,200 peptide-specific CD8⁺ T cells (7) in their spleen and LNs. Despite this rare frequency, the process of T-cell priming is remarkably efficient because of both macroscopic and microscopic mechanisms (8). At the level of the whole organism, inflammation induces a dramatic increase in the number of naïve cells recruited to the reactive LN early after infection or immunization, resulting in LN swelling, a long-recognized sign of infection. LN hyperplasia is preceded by an increase in LN vasculature (9). The expansion of the arteriole feeding the LN enables more efficient screening for cognate lymphocytes, which promotes an effective adaptive immune response (10). However, the role of specific cell types involved in this process remains unclear.

In the present study, we use a variety of approaches to address the role of CD4⁺ T cells in the induction of primary CD8⁺ T-cell responses. We use a herpes simplex virus (HSV) infection model because immunity to HSV is characterized by a strong CTL response (11), and, in contrast to most infectious agents in which CD4⁺ T-cell help is only needed to generate memory CTLs (12 –15), CD4⁺ T cells are needed to generate the primary CTL response to HSV (5, 16–21). Here, we demonstrate that CD4⁺ T cells are required for increased cellular input to the draining LN (dLN) after HSV2 infection. CD4⁺ T cells were required for remodeling of the LN feed arteriole to a larger diameter. Similar requirements for CD4⁺ T cells in LN hyperplasia were found after immunization with DCs. Using these systems, we demonstrate a critical role of DC–CD4⁺ T-cell interaction through major histocompatibility complex class II (MHCII)– T-cell receptor (TCR) and CD40–CD40 ligand (CD40L) in mediating naïve lymphocyte input to the reactive LN. Ultimately, enhanced recruitment of naïve lymphocytes results in the rapid onset of CTL responses caused by the increased screening capacity of the reactive LN. These results reveal a CD8⁺ T-cell–extrinsic role of CD4⁺ T-cell help in the primary immune response through increasing naïve lymphocyte input to the sites of lymphocyte priming.

Results

LN Expansion Is Impaired After Infection and Immunization in CD4⁺ T-Cell–Deficient Mice.

To examine the role of CD4⁺ T-cell help during an immune response, WT, MHCII-deficient, or CD4-deficient mice transplanted with naïve gBT-I cells were infected vaginally with thymidine kinase–defective HSV2 (TK⁻HSV2). TK⁻HSV2 infects and triggers robust immune responses without causing neuropathogenesis (22). The gBT-I cells, which express a transgenic TCR specific for HSV glycoprotein B, were used to track antigen-specific CTLs (5, 23). Although draining iliac LNs underwent robust expansion in WT recipients, the degree of expansion was significantly reduced in MHCII-deficient and CD4-deficient hosts, both of which lack a normal CD4⁺ T-cell compartment (ΔCD4T) (Fig. 1A). A similar observation was reported by a previous study (21). The absence of maximal LN hyperplasia in ΔCD4T mice was not explained solely by the absence of CD4⁺ T cells in ΔCD4T mice because the non-CD4⁺ T-cell compartment, including CD8⁺ T cells and B cells, was also significantly reduced in these hosts (Fig. S1). Although LN size in ΔCD4T mice was slightly reduced compared with WT mice at steady state, this reduction was accounted for by the lack of CD4 T cells in ΔCD4T mice (Fig. S1). Thus, these data suggest that CD4⁺ T cells contribute to inducible LN enlargement but not to the steady-state function of a LN. In addition, dLN cellularity was significantly reduced in ΔCD4T mice after footpad injection of TK⁻HSV2 or bone marrow–derived DCs (BMDCs) (Fig. S2 A and B), indicating that the requirement of CD4⁺ T cells for LN expansion is not restricted to the vaginal HSV infection model.

Fig. 1.

CD4⁺ T cells are required for optimal dLN enlargement and CTL development after vaginal HSV2 infection. WT, MHCIIKO, or CD4KO mice were injected with 1 × 10⁶ naïve gBT-I cells and vaginally infected with 1 × 10⁶ pfu of TK⁻HSV2. Numbers of total cells (A), gBT-I CD8⁺ T cells (B), and percentages of gBT-I cells (C) in the dLN are shown. Each symbol represents one mouse. Data are pooled from two or more independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001 by one-way ANOVA (naïve, 2 and 4 dpi) and by two-tailed Student's t test (7 dpi).

Of note, LNs in ΔCD4T mice showed a significant increase in size and cellularity compared with the uninfected controls (Fig. 1A), albeit to a lesser extent than WT mice, suggesting the presence of CD4⁺T-cell–dependent and –independent mechanisms of LN enlargement in WT mice. Interestingly, injection of ΔCD4T mice with CpG in the absence of antigen led to similar increase in cellularity of the non-CD4 compartment in the dLNs (Fig. S2A), suggesting that LN hyperplasia induced by innate stimuli alone mainly invokes a CD4⁺T-cell–independent mechanism. Here, we focus on the role and mechanism of the CD4-dependent LN enlargement in CD8⁺ T-cell immunity.

CD4⁺ T Cells Help Primary CD8⁺ T-Cell Responses Through Increasing Cellular Input.

After intravaginal infection with TK⁻HSV2, the number of gBT-I cells was significantly reduced in ΔCD4T recipient mice compared with WT hosts (Fig. 1B). Surprisingly, however, the percentage of gBT-I cells within the dLN was comparable among the hosts at all time points tested (Fig. 1C). DCs are unable to induce CD25 expression on CD8 T cells without receiving help from antigen-specific CD4⁺ T cells (“helpless”) (5). CD25 controls late-phase expansion and differentiation of antigen-specific effector CD8⁺ T cells (6). However, this mode of DC activation cannot account for the reduced gBT-I cell number in HSV2-infected ΔCD4T hosts at 2 d postinfection (dpi) (Fig. 1) because the up-regulation of CD25 occurred only after multiple cell divisions (Fig. S3) and no detectable division was observed at this time (Fig. S4). These data suggest that the reduced magnitude of primary CD8⁺ T-cell accumulation in the dLN of ΔCD4T hosts is largely dictated by the minimal increase in dLN cellularity in ΔCD4T hosts, and not by a cell-intrinsic defect of gBT-I cells.

To address this possibility, we assessed the activation and differentiation status of gBT-I cells. At 2 dpi, the numbers of gBT-I cells expressing high levels of an activation marker, CD69, were reduced in ΔCD4T hosts (Fig. 2A). This finding was not attributable to reduced efficiency of antigen presentation to gBT-I cells because the proportion of CD69⁺ cells within the transferred gBT-I population was comparable between WT and ΔCD4T hosts (Fig. 2B). Expression levels of CD69 in gBT-I cells and endogenous polyclonal CD8T cells were also intact in the absence of CD4⁺ T cells (Fig. 2C). Because the majority of the transferred gBT-I cells remained undivided at this time point (Fig. S4), any putative differences in cell division could not account for the difference in gBT-I cell numbers. In addition, effector CTL differentiation, as measured by granzyme B levels (Fig. 2D), was comparable at later time points in the LN and the spleen of ΔCD4T mice. Examination of the cell division rate of transferred gBT-I cells revealed no differences at 4 d after TK⁻HSV2 infection (Fig. 2E). Together, these data indicate that, although late-stage CTL effector functions, including IFNγ and TNFα secretion (21) and CD25 up-regulation (5, 6, 21), depend on CD4 help, the reduced ability of helpless gBT-I cells to accumulate in ΔCD4T hosts at early stages is not attributable to a cell-intrinsic deficiency. Rather, it appears to be linked to the defect in LN expansion.

Fig. 2.

Primary CTL responses in ΔCD4T hosts are reduced in magnitude but are phenotypically intact. WT, MHCIIKO, or CD4KO mice were injected with carboxyfluorescein diacetate succinimidyl ester (CFSE)–labeled naïve gBT-I cells and infected as in Fig. 1. (A–C) Expression of CD69 2 d after infection. Numbers of CD69⁺ gBT-I cells per dLN (A) and percentages of CD69⁺ cells among total gBT-I cells in the dLN (B) are shown. Data are pooled from three independent experiments. *P < 0.05, **P < 0.01 by one-way ANOVA. In C, CD69 expression on gBT-I cells (thick line), endogenous CD8⁺ T cells (dashed line), and CD8⁺ T cells in naïve WT mouse (gray histogram) are shown. (D and E) Intracellular granzyme B expression on 4 and 7 dpi (D) and CFSE dilution on 4 dpi (E) in the donor gBT-I cells in indicated organs. Gray histograms indicate isotype staining (D) or uninfected controls (E).

CD4⁺ T Cells Support Primary CD8⁺ T-Cell Responses Through Interaction with DCs.

To understand the mechanism by which CD4⁺ T cells support LN expansion, we asked whether this process requires the interaction of CD4⁺ T cells with MHCII⁺ antigen-presenting cells (APCs). First, we examined whether the APCs in ΔCD4T hosts are intrinsically defective in inducing accumulation of gBT-I cells within the dLN upon HSV infection. To this end, we partially reconstituted ΔCD4T hosts with WT polyclonal CD4⁺ T cells before gBT-I transfer. Consistent with previous reports (5, 6, 21), CD25 expression was impaired in gBT-I cells in ΔCD4T hosts (Fig. S3). However, ∼10% reconstitution of the CD4⁺ T-cell compartment in CD4-deficient, but not MHCII-deficient, hosts partially restored the expression of CD25 on activated gBT-I cells (Fig. S3) and dLN enlargement after footpad infection with HSV2 (Figs. S5 A and B and S6C). In addition, DCs from Cd4^−/− mice were able to induce LN expansion in WT, but not in Cd4^−/− hosts (Fig. S2C). These data indicate that help is mediated via the ability of CD4⁺ T cells to interact with APCs through MHCII and that such help can be provided transiently in situ.

Next, we examined the type of MHCII⁺ APC responsible for supporting CD4⁺ T-cell–dependent LN enlargement. We found that injection of DCs, but not B cells, induced LN enlargement (Fig. S5C). In addition, depletion of DCs before HSV2 infection abolished LN enlargement (Fig. S5D), indicating that DCs are both required and sufficient to induce dLN hyperplasia, consistent with previous studies (24, 25). Although B-cell injection was not sufficient to induce LN enlargement in helpless mice, B cells are known to mediate inflammation-induced lymphangiogenesis and LN remodeling (26, 27). To examine the requirement of B cells in HSV-induced LN hyperplasia, we infected WT or μMT mice, which lack B cells, in the footpad with TK⁻HSV2. Although total cellularity of the dLN was smaller in μMT mice compared with WT mice (Fig. S5E), the size of the non–B-cell compartment was comparable (Fig. S5F), indicating that B cells are not required to increase cell input into the dLN. Thus, DCs, but not B cells, are causally involved in CD4⁺ T-cell–dependent LN enlargement.

CD4⁺ T-Cell–Mediated LN Expansion Requires MHCII and CD40 Expression by DCs.

Next, we examined the molecules involved in LN expansion after CD4⁺ T–DC interaction. Injection of BMDCs alone is known to induce LN enlargement without the need for antigen pulsing (Fig. S2 B and C) (24, 25). Such LN hyperplasia is unlikely to be caused by LPS contamination of the BMDCs because we did not observe any signs of activation in the WT BMDC preparation (Fig. S2D). Furthermore, BMDC-intrinsic TLR signaling is not responsible for DC-induced LN hyperplasia because cellularity was comparable in the dLNs of mice injected with WT or MyD88/Toll/interleukin-1 receptor-domain-containing adaptor inducing interferon β (TRIF) double-deficient BMDCs (Fig. S2E). In contrast, we observed a significant defect in the ability of MHCIIKO BMDCs to induce expansion of the dLNs (Fig. 3A). These data indicate that BMDCs must interact with CD4⁺ T cells through MHCII to mediate LN expansion. Activation of DCs by CD4⁺ T cells through CD40 and CD40L interaction represents one important mechanism of CD4⁺ T-cell help (17 –19). Thus, we assessed the role of CD40–CD40L interaction in LN enlargement. A significant reduction in the degree of LN expansion upon injection of CD40-deficient BMDCs was observed compared with WT BMDCs (Fig. 3B), indicating that CD40 signaling in DCs is required to mediate LN enlargement. The incomplete LN enlargement induced by MHCII- and CD40-deficient BMDCs is not explained by impaired migration of these DCs because they reached the dLN at levels comparable to WT BMDCs (Fig. 3C). Together, these data indicate that CD4⁺ T cells provide help through the engagement of both MHCII and CD40 on DCs.

Fig. 3.

DCs induce dLN enlargement in an MHCII- and CD40-dependent manner. (A and B) BMDCs (1 × 10⁶ cells) prepared from indicated mice were injected into WT mice in the footpad. Total LN cellularity in the draining popliteal LNs at day 4 are shown. Data are representative of two or more independent experiments. (C) BMDCs (1 × 10⁶ cells) prepared from indicated mice were labeled with CFSE and injected into the footpad of WT mice. The number of migrated donor BMDCs was calculated from dLNs harvested 2 dpi. **P < 0.01 by one-way ANOVA.

CD4⁺ T Cells Promote Increased Recruitment of Naïve Lymphocytes to the Draining LN.

We hypothesized that the CD4⁺ T-cell–dependent increase in dLN cellularity results in optimal CD8⁺ T-cell expansion by increasing naïve polyclonal CD8⁺ T-cell input into the reactive LN. To avoid complications from putative developmental bias in CD8⁺ T-cell population in ΔCD4T mice, first we monitored accumulation of adoptively transferred WT naïve polyclonal CD8⁺ T cells in the dLN after footpad infection of TK⁻HSV2. To this end, polyclonal CD8⁺ T cells were purified from the spleen and LNs of naïve WT mice and transferred into WT or ΔCD4T recipient mice that had been infected in the footpad for 12 h with TK⁻HSV2. At 12 h after the transfer of the CD8⁺ T cells, the draining ipsilateral and nondraining contralateral popliteal LNs were removed, and the donor CD8⁺ T cells were counted. At this time point, the total cellularity in the dLN was not significantly different between WT and ΔCD4T mice, although the dLN enlargement was evident in all mice compared with the nondraining LN counterpart (Fig. 4A), suggesting that the CD4⁺T-cell–independent mode of LN enlargement precedes the CD4⁺T-cell–dependent mode of hyperplasia. In contrast, the number of donor CD8⁺ T cells in the dLN was significantly reduced in the ΔCD4T hosts, suggesting that CD4⁺ T “help” accelerates the accumulation of polyclonal naïve CD8⁺ T cells within the dLN starting from very early time points (Fig. 4A).

Fig. 4.

CD4⁺ T cells increase inflammation-induced naïve CD8⁺ T-cell influx in the dLN through vascular remodeling. (A) Naïve polyclonal CD8⁺ T cells (1.5 × 10⁶ cells per recipient) were retro-orbitally transferred into WT, MCHIIKO, or CD4KO mice that had been infected for 12 h with 1 × 10⁶ pfu TK⁻HSV2 in the footpad. Numbers of total cells (Left and Center) and donor CD8⁺ T cells (Right) in the contralateral [nondraining LN (ndLN)] or ipsilateral (dLN) popliteal LN 12 h after the transfer of CD8⁺ T cells are shown. Data are representative of two independent experiments. **P < 0.01, ***P < 0.001 by one-way ANOVA. (B) Naïve splenocytes and LN cells were isolated from CD45.1⁺ congenic WT mice, and 5 × 10⁷ cells were retro-orbitally transferred into WT or CD4KO mice that had been infected for 43 h with 1 × 10⁶ pfu TK⁻HSV2 in the vagina. At 2 h after the donor cell transfer, the draining iliac LNs and nondraining brachial LNs were removed, and the donor cells were counted. Relative cellularity normalized to WT average (Left) or absolute cell numbers in the iliac LNs (Center and Right) are shown. Similar results were observed in an experiment conducted at 24 h postinfection. *P < 0.05 by two-tailed t test. (C) Internal vessel diameter of the arteriole feeding the inguinal LN after vaginal infection with TK⁻HSV2 in WT or CD4-deficient mice. Bars indicate mean ± SD. **P < 0.01, ***P < 0.001 compared with 0 dpi by two-tailed t test (n = 4–11 mice per group).

Next, we asked whether such CD4⁺ T-cell–dependent accumulation of CD8⁺ T cells in the dLN is (i) explained by enhanced entry of lymphocytes and (ii) applicable to other routes of infection because different sets of DCs are involved in T-cell priming after footpad vs. vaginal HSV infection (28). WT and CD4-deficient mice were infected with TK⁻HSV2 intravaginally and then injected with spleen and LN cells from naïve congenic CD45.1⁺ WT mice. Two hours after the transfer, the draining iliac LNs and the nondraining brachial LNs were harvested, and donor cell numbers were compared. The number of donor cells was significantly reduced in the draining iliac LNs, but not the nondraining brachial LNs, in ΔCD4T hosts (Fig. 4B Left). These results suggest that the impaired accumulation of naïve CD8⁺ T cells in ΔCD4T mice is accounted for, at least in part, by the reduced lymphocyte entry into the dLNs in these hosts. Of note, we observed a similar requirement for CD4⁺ T cells for the recruitment CD8⁺ T cells and B cells (Fig. 4B Center and Right), suggesting that this mode of CD4⁺T-cell help is not specific to CD8⁺ T cells and that the CD4⁺ T cells may also help B-cell responses through increased input.

Cognate CD4⁺ T Cells Enable LN Expansion.

Thus far, our data do not distinguish whether CD4⁺ T cells exert help in an antigen-specific manner. Although injection of antigen-unpulsed DCs or CpG was sufficient to drive LN hyperplasia (Fig. S2), it is impossible to rule out the contributions from unintended antigens. For instance, unpulsed BMDC injection alone is known to induce an antigen-specific antibody response against FBS contained in the culture media (29). To determine whether antigen specificity of CD4⁺ T cells is required to increase dLN cellularity, we assessed naïve CD8⁺ T-cell accumulation in OT-II TCR transgenic mice on recombination activating gene (RAG)-deficient background. In these mice, the only CD4⁺ T cells available for help are specific to ovalbumin and not to HSV-2. Footpad infection of OT-II/Rag-1^−/− mice with TK⁻HSV2 resulted in a significant increase in LN cellularity compared with the Rag-1^−/− counterpart, mainly accounted for by the influx of OT-II TCR transgenic T cells (Fig. S6A). However, accumulation of naïve polyclonal CD8⁺ T cells in these mice was comparable to the Rag-1^−/− mice (helpless) (Fig. S6B). One caveat to this experimental approach is that Rag^−/− mice lack all lymphocytes, making it difficult to compare cellular influx in the context of normal LN composition. To compare influx of endogenous lymphocyte populations in a more physiological setting in the presence or absence of cognate CD4⁺ T cells, ΔCD4 mice reconstituted with either WT polyclonal CD4⁺ T cells (containing a rare population of HSV-specific T cells) or with OT-II/Rag1^−/− CD4⁺ T cells (nonspecific to HSV-2) were infected with TK⁻HSV2 in the footpad. Four days later, LN cellularity and numbers of endogenous B and CD8⁺ T cells were measured (Fig. S6C). These data clearly indicated that (i) antigen-nonspecific CD4⁺ T cells are not sufficient to support infection-induced cellular influx to the dLN and (ii) CD4⁺ T cells must interact with MHCII⁺ APCs to bring about this change because MHCII^−/− recipients of polyclonal CD4⁺ T cells failed to undergo LN expansion. Together, these data indicate that CD4⁺ T cells promote accumulation of naïve lymphocytes to the dLN after antigen-specific interaction with MHCII⁺ APCs.

CD4⁺ T Cells Support Expansion of the Arteriole Feeding the Reactive LN.

The increase in naïve lymphocyte entry into the reactive LN may be attributable to an increase in blood flow. We previously demonstrated that the dLN feed arteriole remodels to a larger diameter upon infection or immunization to increase supply of naïve lymphocytes into the dLN (10). To examine whether CD4⁺ T cells might mediate larger lymphocyte input by inducing arteriole remodeling, the vessel diameter of the primary arteriole feeding the inguinal dLN was measured in WT and CD4-deficient mice after vaginal infection with TK⁻HSV2. Consistent with our previous report (10), both resting and maximal vessel diameter expanded after infection in WT mice (Fig. 4C), indicating that the arteriole had undergone remodeling. The increase in arteriolar diameter was observed as early as 1 d after infection. In contrast, the arteriole failed to expand in CD4-deficient mice at all time points tested (Fig. 4C), indicating that CD4⁺ T cells are required for infection-induced arteriole remodeling. Collectively, these data indicate that CD4⁺ T-cell help is required for immunization-induced increase in lymphocyte entry into reactive LN through arteriolar remodeling and expansion.

Discussion

The immune system faces the daunting task of screening for cognate lymphocytes for a given antigen. Because the precursor frequencies of naïve T and B lymphocytes are extremely low, its capacity to rapidly find and induce expansion of cognate lymphocytes are critical in combating an acute infection. Our data establish a previously undescribed role of CD4⁺ T-cell help in the initiation of an immune response, namely increasing naïve lymphocyte input into the reactive LN. Our data are consistent with a model by which DCs migrating into the LN interact with CD4⁺ T cells through MHCII and receive help through CD40. This process is accompanied by an expansion of the LN feed arteriole, promoting the enhanced recruitment of polyclonal naïve CD8 T cells into the LN and resulting in more efficient screening for cognate CD8⁺ T cells (10). This mode of CD4⁺ T-cell help likely occurs before and/or in parallel to the other help pathways known to be mediated by CD4⁺ T cells, such as licensing of DCs for efficient formation of effector and memory cells (5) and guiding of CD8⁺ T cells to antigen-presenting DCs within the LNs (3). Although this mode of help depends on TCR engagement by MHCII plus peptide complex, it occurs very rapidly (24 h) and is likely mediated by a small number of cognate CD4⁺ T cells engaging antigen-presenting DCs. In fact, naïve CD4⁺ T cells are known to constitutively express abundant CD40L and engage CD40 on APCs (30).

Our data indicate that there are two separate pathways that mediate LN enlargement. The first pathway is innate, in that it does not require CD4⁺ T cells or specific antigen. This type of enlargement is observed at very early time points after immunization (12 h) (Fig. 4A), in the absence of adaptive immunity (Fig. S6A) (25), or after inoculation of CpG without antigen (Fig. S2A). We do not yet understand the mechanism that governs the innate stage of LN expansion. However, we start to see signs of CD4⁺ T-cell–dependent LN input even at this stage because donor CD8⁺ T-cell entry into dLN is enhanced in the presence of CD4⁺ T cells (Fig. 4A Right). The second pathway is adaptive, in that it requires antigen-specific CD4⁺ T cells and DC interaction. This latter pathway is evident after 24 h of infection (Fig. 4 B and C) and is mediated by remodeling of feed arteriole to a larger diameter. During infection or immunization, LN enlargement likely reflects a combination of these two pathways because varying degrees of CD4-independent enlargement is observed under most conditions tested.

We found that CD4⁺ T cells are necessary to remodel the LN feed arteriole to a larger diameter. We observed a striking impairment of the primary feed arteriole to enlarge in the absence of CD4⁺ T cells after HSV-2 infection. Of note, the average diameter of the LN feed arteriole in naïve Cd4^−/− mice was consistently larger than in the WT counterpart. These data suggest that CD4⁺ T cells also regulate vascular function at the steady state. The precise mechanism by which CD4⁺ T cells support arteriole remodeling remains unclear. It is known that antigen-bearing DCs migrating from inflamed tissues preferentially wrap around high endothelial venules, enabling the DCs to scan the arriving lymphocytes at their site of entry into the LN (31). It is possible that “helped” DCs transmit signals to the high endothelial venules, inducing remodeling of the arteriole upstream. Although the requirement of CD40 in BMDCs to induce full enlargement of the dLN (Fig. 3A) suggests that licensing of DC by CD4⁺ T cells is involved in this process, it is also possible that antigen-specific CD4⁺ Th1 cells themselves, after interacting with DCs, directly enhance naïve lymphocyte entry. Indeed, IFN-γ, a major effector cytokine produced by antigen-specific Th1 cells, is known to activate high endothelial venules and enhance attachment and transendothelial migration of lymphocytes (32 –35). However, this latter possibility is likely only relevant at later time points and would not explain LN enlargement at 24 h. Future studies are needed to determine the mechanism by which arteriole remodeling is induced by CD4⁺ T–DC interaction.

Our results demonstrated that naïve lymphocyte input is significantly impaired in ΔCD4T mice. However, we have not ruled out the possibility that CD4⁺ T cells also induce LN hyperplasia by increasing retention of lymphocytes within the reactive LNs. It is known that LN shutdown occurs rapidly after infection or immunization (36), and recent studies illustrate the underlying molecular mechanisms (37). It is possible that CD4⁺ T–DC interaction leads to a more efficient shutdown of lymphocyte egress, which would contribute to LN enlargement and more efficient activation of cognate CD8⁺ T cells and B cells.

We believe that there are multiple pathways that link CD4⁺ T cells and enhanced LN input, some of which might use molecules that are redundant with CD40. For instance, TLR stimulation or overt infection could induce signals that either override the requirement for CD4⁺ T-cell help altogether or induce CD40-independent signals in DCs and other cells to achieve LN enlargement. The immune system is set up to maximize the recruitment of naïve antigen-specific CD8⁺ T cells in response to infection in WT animals (38). Our data suggest that CD4⁺ T-cell help in maximizing naïve lymphocyte input to the reactive LN is critical in achieving such efficient screening for cognate antigen by naïve CD8⁺ T cells. In addition, this mode of CD4⁺ T-cell help is likely not restricted to CD8⁺ T cells because polyclonal B-cell entry into reactive LNs was also severely reduced in helpless mice. Finally, our current findings may in part explain the severe immune deficiencies in CD8⁺ T and B-cell compartments observed after depletion of CD4⁺ T cells during the AIDS stage of HIV-1 infection.

Methods

Mice.

B6, B6.SJL-Ptprc^aPep3^b/BoyJ (congenic CD45.1 mice on B6 background), B6.129S-H2^dlAb1-Ea, Cd40^−/−, and Cd4^−/− mice were purchased from the National Cancer Institute and Jackson Laboratories. The gBT-I TCR transgenic mice (5, 23), which are specific for the immunodominant HSV glycoprotein B peptide gB_498–505, and CD11c-DTR mice (39), which express diphtheria toxin receptor under the CD11c promoter, were previously described. All procedures used in this study complied with federal guidelines and institutional policies of the Yale Animal Care and Use Committee and University of Northern British Columbia Animal Use Committee.

Mouse Treatment.

For vaginal HSV infection, mice were treated with Depo-Provera at 4 d before infection and then inoculated with 10⁶ pfu TK⁻HSV2 in the vagina (40). The draining iliac LNs were harvested at indicated time points. For footpad injection, TK⁻HSV2, CpG2216, splenic DCs, splenic B cells, or BMDCs were injected into the hind footpad in a 20-50 μl volume. The ipsilateral draining or contralateral nondraining popliteal LNs were harvested at indicated time points. For DC depletion, WT or CD11c-DTR mice were injected i.p. with diphtheria toxin (300 ng per mouse; Sigma) 1 d before infection with TK⁻HSV2 (10⁶ pfu) in the footpad. Popliteal LNs were harvested at 2 dpi. DC depletion was confirmed by flow cytometry.

Intravital Microscopy.

LN feed arteriole diameter was measured as previously described (10). Briefly, a midline incision was made along the ventral surface of the abdominal cavity of anesthetized mice. The skin was retracted and pinned onto a pedestal while continuously superfused with a bicarbonate-buffered physiological salt solution equilibrated with 5% CO₂/95% N₂ (pH 7.4, 34 °C). Surrounding adipose tissue was cleared, and the main arterial segment lying adjacent to the LN was visualized and evaluated. The preparation was secured on an intravital microscope, and the feed arteriole was observed at a total magnification of ∼950 Å using video microscopy. Internal vessel diameter was determined from the width of the red blood cell column by using a video caliper. The same vessel segment was studied in each mouse per group. The resting internal diameter was measured after 60-min equilibration with physiological salt solution. The preparation was then equilibrated with sodium nitroprusside (100 mM; Sigma-Aldrich) to obtain maximal diameter in vivo.

Statistical Analysis.

Statistical significance was tested either by two-tailed Student's t test or by one-way ANOVA followed by Tukey's post test using GraphPad PRISM software (Version 4.0b; GraphPad Software). Data are presented as mean ± SEM.

Details on adoptive transfer of lymphocytes and DCs, preparation of BMDCs, and flow cytometric analysis can be found in SI Methods.

Acknowledgments

We thank R. Medzhitov for critical reading of the manuscript. This work is supported by National Institutes of Health Grants AI054359 and AI062428 (to A.I.) and an award from the American Heart Association (to L.M.M.). Y.K. was an Astellas Foundation for Research on Metabolic Disorders fellow. A.I. is a recipient of the Burroughs Wellcome Investigators in Pathogenesis of Infectious Disease Award.

Footnotes

↵¹Y.K. and L.M.M. contributed equally to this work.
↵²To whom correspondence should be addressed. E-mail: akiko.iwasaki{at}yale.edu.

Author contributions: Y.K., L.M.M., S.S., G.W.P., and A.I. designed research; Y.K., L.M.M., and S.S. performed research; Y.K., L.M.M., S.S., G.W.P., and A.I. analyzed data; and Y.K., L.M.M., and A.I. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1100567108/-/DCSupplemental.

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1. Williams MA,
2. Bevan MJ
(2007) Effector and memory CTL differentiation. Annu Rev Immunol 25:171–192.
OpenUrl CrossRef PubMed
↵
1. Castellino F,
2. Germain RN
(2006) Cooperation between CD4⁺ and CD8⁺ T cells: When, where, and how. Annu Rev Immunol 24:519–540.
OpenUrl CrossRef PubMed
↵
1. Behrens G,
2. et al.
(2004) Helper T cells, dendritic cells and CTL immunity. Immunol Cell Biol 82:84–90.
OpenUrl CrossRef PubMed
↵
1. Smith CM,
2. et al.
(2004) Cognate CD4(+) T cell licensing of dendritic cells in CD8(+) T cell immunity. Nat Immunol 5:1143–1148.
OpenUrl CrossRef PubMed
↵
1. Obar JJ,
2. et al.
(2010) CD4⁺ T cell regulation of CD25 expression controls development of short-lived effector CD8⁺ T cells in primary and secondary responses. Proc Natl Acad Sci USA 107:193–198.
OpenUrl Abstract/FREE Full Text
↵
1. Obar JJ,
2. Khanna KM,
3. Lefrançois L
(2008) Endogenous naive CD8⁺ T cell precursor frequency regulates primary and memory responses to infection. Immunity 28:859–869.
OpenUrl CrossRef PubMed
↵
1. Bajénoff M,
2. et al.
(2007) Highways, byways and breadcrumbs: Directing lymphocyte traffic in the lymph node. Trends Immunol 28:346–352.
OpenUrl CrossRef PubMed
↵
1. Herman PG,
2. Yamamoto I,
3. Mellins HZ
(1972) Blood microcirculation in the lymph node during the primary immune response. J Exp Med 136:697–714.
OpenUrl Abstract
↵
1. Soderberg KA,
2. et al.
(2005) Innate control of adaptive immunity via remodeling of lymph node feed arteriole. Proc Natl Acad Sci USA 102:16315–16320.
OpenUrl Abstract/FREE Full Text
↵
1. Koelle DM,
2. et al.
(1998) Clearance of HSV-2 from recurrent genital lesions correlates with infiltration of HSV-specific cytotoxic T lymphocytes. J Clin Invest 101:1500–1508.
OpenUrl PubMed
↵
1. Janssen EM,
2. et al.
(2003) CD4⁺ T cells are required for secondary expansion and memory in CD8⁺ T lymphocytes. Nature 421:852–856.
OpenUrl CrossRef PubMed
↵
1. Shedlock DJ,
2. Shen H
(2003) Requirement for CD4 T cell help in generating functional CD8 T cell memory. Science 300:337–339.
OpenUrl Abstract/FREE Full Text
↵
1. Sun JC,
2. Bevan MJ
(2003) Defective CD8 T cell memory following acute infection without CD4 T cell help. Science 300:339–342.
OpenUrl Abstract/FREE Full Text
↵
1. Sun JC,
2. Williams MA,
3. Bevan MJ
(2004) CD4⁺ T cells are required for the maintenance, not programming, of memory CD8⁺ T cells after acute infection. Nat Immunol 5:927–933.
OpenUrl CrossRef PubMed
↵
1. Bennett SR,
2. Carbone FR,
3. Karamalis F,
4. Miller JF,
5. Heath WR
(1997) Induction of a CD8⁺ cytotoxic T lymphocyte response by cross-priming requires cognate CD4⁺ T cell help. J Exp Med 186:65–70.
OpenUrl Abstract/FREE Full Text
↵
1. Bennett SR,
2. et al.
(1998) Help for cytotoxic-T-cell responses is mediated by CD40 signalling. Nature 393:478–480.
OpenUrl CrossRef PubMed
↵
1. Ridge JP,
2. Di Rosa F,
3. Matzinger P
(1998) A conditioned dendritic cell can be a temporal bridge between a CD4⁺ T-helper and a T-killer cell. Nature 393:474–478.
OpenUrl CrossRef PubMed
↵
1. Schoenberger SP,
2. Toes RE,
3. van der Voort EI,
4. Offringa R,
5. Melief CJ
(1998) T-cell help for cytotoxic T lymphocytes is mediated by CD40-CD40L interactions. Nature 393:480–483.
OpenUrl CrossRef PubMed
1. Jennings SR,
2. Bonneau RH,
3. Smith PM,
4. Wolcott RM,
5. Chervenak R
(1991) CD4-positive T lymphocytes are required for the generation of the primary but not the secondary CD8-positive cytolytic T lymphocyte response to herpes simplex virus in C57BL/6 mice. Cell Immunol 133:234–252.
OpenUrl CrossRef PubMed
↵
1. Rajasagi NK,
2. et al.
(2009) CD4⁺ T cells are required for the priming of CD8⁺ T cells following infection with herpes simplex virus type 1. J Virol 83:5256–5268.
OpenUrl Abstract/FREE Full Text
↵
1. Jones CA,
2. Taylor TJ,
3. Knipe DM
(2000) Biological properties of herpes simplex virus 2 replication-defective mutant strains in a murine nasal infection model. Virology 278:137–150.
OpenUrl CrossRef PubMed
↵
1. Mueller SN,
2. Heath W,
3. McLain JD,
4. Carbone FR,
5. Jones CM
(2002) Characterization of two TCR transgenic mouse lines specific for herpes simplex virus. Immunol Cell Biol 80:156–163.
OpenUrl CrossRef PubMed
↵
1. Martín-Fontecha A,
2. et al.
(2003) Regulation of dendritic cell migration to the draining lymph node: Impact on T lymphocyte traffic and priming. J Exp Med 198:615–621.
OpenUrl Abstract/FREE Full Text
↵
1. Webster B,
2. et al.
(2006) Regulation of lymph node vascular growth by dendritic cells. J Exp Med 203:1903–1913.
OpenUrl Abstract/FREE Full Text
↵
1. Angeli V,
2. et al.
(2006) B cell-driven lymphangiogenesis in inflamed lymph nodes enhances dendritic cell mobilization. Immunity 24:203–215.
OpenUrl CrossRef PubMed
↵
1. Kumar V,
2. et al.
(2010) Global lymphoid tissue remodeling during a viral infection is orchestrated by a B cell-lymphotoxin-dependent pathway. Blood 115:4725–4733.
OpenUrl Abstract/FREE Full Text
↵
1. Lee HK,
2. et al.
(2009) Differential roles of migratory and resident DCs in T cell priming after mucosal or skin HSV-1 infection. J Exp Med 206:359–370.
OpenUrl Abstract/FREE Full Text
↵
1. Tzeng TC,
2. et al.
(2010) CD11c(hi) dendritic cells regulate the re-establishment of vascular quiescence and stabilization after immune stimulation of lymph nodes. J Immunol 184:4247–4257.
OpenUrl Abstract/FREE Full Text
↵
1. Lesley R,
2. Kelly LM,
3. Xu Y,
4. Cyster JG
(2006) Naive CD4 T cells constitutively express CD40L and augment autoreactive B cell survival. Proc Natl Acad Sci USA 103:10717–10722.
OpenUrl Abstract/FREE Full Text
↵
1. Bajénoff M,
2. Granjeaud S,
3. Guerder S
(2003) The strategy of T cell antigen-presenting cell encounter in antigen-draining lymph nodes revealed by imaging of initial T cell activation. J Exp Med 198:715–724.
OpenUrl Abstract/FREE Full Text
↵
1. Kraal G,
2. Schornagel K,
3. Savelkoul H,
4. Maruyama T
(1994) Activation of high endothelial venules in peripheral lymph nodes. The involvement of interferon-gamma. Int Immunol 6:1195–1201.
OpenUrl Abstract/FREE Full Text
↵
1. Manolios N,
2. Geczy CL,
3. Schrieber L
(1988) High endothelial venule morphology and function are inducible in germ-free mice: A possible role for interferon-gamma. Cell Immunol 117:136–151.
OpenUrl CrossRef PubMed
↵
1. Hendriks HR,
2. Korn C,
3. Mebius RE,
4. Kraal G
(1989) Interferon-gamma-increased adherence of lymphocytes to high endothelial venules. Immunology 68:221–226.
OpenUrl PubMed
↵
1. May MJ,
2. Ager A
(1992) ICAM-1-independent lymphocyte transmigration across high endothelium: Differential up-regulation by interferon gamma, tumor necrosis factor-α and interleukin 1β. Eur J Immunol 22:219–226.
OpenUrl PubMed
↵
1. Hall JG,
2. Morris B
(1965) The immediate effect of antigens on the cell output of a lymph node. Br J Exp Pathol 46:450–454.
OpenUrl PubMed
↵
1. Cyster JG
(2007) Specifying the patterns of immune cell migration. Novartis Found Symp 281:54–61, discussion 61–54, 208–209.
OpenUrl PubMed
↵
1. van Heijst JW,
2. et al.
(2009) Recruitment of antigen-specific CD8⁺ T cells in response to infection is markedly efficient. Science 325:1265–1269.
OpenUrl Abstract/FREE Full Text
↵
1. Jung S,
2. et al.
(2002) In vivo depletion of CD11c⁺ dendritic cells abrogates priming of CD8⁺ T cells by exogenous cell-associated antigens. Immunity 17:211–220.
OpenUrl CrossRef PubMed
↵
1. Zhao X,
2. et al.
(2003) Vaginal submucosal dendritic cells, but not Langerhans cells, induce protective Th1 responses to herpes simplex virus-2. J Exp Med 197:153–162.
OpenUrl Abstract/FREE Full Text

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Proceedings of the National Academy of Sciences: 108 (21)

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Article Classifications

Biological Sciences
Immunology

[1] ↵
Arens R,
Schoenberger SP
(2010) Plasticity in programming of effector and memory CD8 T-cell formation. Immunol Rev 235:190–205.
OpenUrl PubMed

[2] Arens R,

[3] Schoenberger SP

[4] ↵
Williams MA,
Bevan MJ
(2007) Effector and memory CTL differentiation. Annu Rev Immunol 25:171–192.
OpenUrl CrossRef PubMed

[5] Williams MA,

[6] Bevan MJ

[7] ↵
Castellino F,
Germain RN
(2006) Cooperation between CD4⁺ and CD8⁺ T cells: When, where, and how. Annu Rev Immunol 24:519–540.
OpenUrl CrossRef PubMed

[8] Castellino F,

[9] Germain RN

[10] ↵
Behrens G,
et al.
(2004) Helper T cells, dendritic cells and CTL immunity. Immunol Cell Biol 82:84–90.
OpenUrl CrossRef PubMed

[11] Behrens G,

[12] et al.

[13] ↵
Smith CM,
et al.
(2004) Cognate CD4(+) T cell licensing of dendritic cells in CD8(+) T cell immunity. Nat Immunol 5:1143–1148.
OpenUrl CrossRef PubMed

[14] Smith CM,

[15] et al.

[16] ↵
Obar JJ,
et al.
(2010) CD4⁺ T cell regulation of CD25 expression controls development of short-lived effector CD8⁺ T cells in primary and secondary responses. Proc Natl Acad Sci USA 107:193–198.
OpenUrl Abstract/FREE Full Text

[17] Obar JJ,

[18] et al.

[19] ↵
Obar JJ,
Khanna KM,
Lefrançois L
(2008) Endogenous naive CD8⁺ T cell precursor frequency regulates primary and memory responses to infection. Immunity 28:859–869.
OpenUrl CrossRef PubMed

[20] Obar JJ,

[21] Khanna KM,

[22] Lefrançois L

[23] ↵
Bajénoff M,
et al.
(2007) Highways, byways and breadcrumbs: Directing lymphocyte traffic in the lymph node. Trends Immunol 28:346–352.
OpenUrl CrossRef PubMed

[24] Bajénoff M,

[25] et al.

[26] ↵
Herman PG,
Yamamoto I,
Mellins HZ
(1972) Blood microcirculation in the lymph node during the primary immune response. J Exp Med 136:697–714.
OpenUrl Abstract

[27] Herman PG,

[28] Yamamoto I,

[29] Mellins HZ

[30] ↵
Soderberg KA,
et al.
(2005) Innate control of adaptive immunity via remodeling of lymph node feed arteriole. Proc Natl Acad Sci USA 102:16315–16320.
OpenUrl Abstract/FREE Full Text

[31] Soderberg KA,

[32] et al.

[33] ↵
Koelle DM,
et al.
(1998) Clearance of HSV-2 from recurrent genital lesions correlates with infiltration of HSV-specific cytotoxic T lymphocytes. J Clin Invest 101:1500–1508.
OpenUrl PubMed

[34] Koelle DM,

[35] et al.

[36] ↵
Janssen EM,
et al.
(2003) CD4⁺ T cells are required for secondary expansion and memory in CD8⁺ T lymphocytes. Nature 421:852–856.
OpenUrl CrossRef PubMed

[37] Janssen EM,

[38] et al.

[39] ↵
Shedlock DJ,
Shen H
(2003) Requirement for CD4 T cell help in generating functional CD8 T cell memory. Science 300:337–339.
OpenUrl Abstract/FREE Full Text

[40] Shedlock DJ,

[41] Shen H

[42] ↵
Sun JC,
Bevan MJ
(2003) Defective CD8 T cell memory following acute infection without CD4 T cell help. Science 300:339–342.
OpenUrl Abstract/FREE Full Text

[43] Sun JC,

[44] Bevan MJ

[45] ↵
Sun JC,
Williams MA,
Bevan MJ
(2004) CD4⁺ T cells are required for the maintenance, not programming, of memory CD8⁺ T cells after acute infection. Nat Immunol 5:927–933.
OpenUrl CrossRef PubMed

[46] Sun JC,

[47] Williams MA,

[48] Bevan MJ

[49] ↵
Bennett SR,
Carbone FR,
Karamalis F,
Miller JF,
Heath WR
(1997) Induction of a CD8⁺ cytotoxic T lymphocyte response by cross-priming requires cognate CD4⁺ T cell help. J Exp Med 186:65–70.
OpenUrl Abstract/FREE Full Text

[50] Bennett SR,

[51] Carbone FR,

[52] Karamalis F,

[53] Miller JF,

[54] Heath WR

[55] ↵
Bennett SR,
et al.
(1998) Help for cytotoxic-T-cell responses is mediated by CD40 signalling. Nature 393:478–480.
OpenUrl CrossRef PubMed

[56] Bennett SR,

[57] et al.

[58] ↵
Ridge JP,
Di Rosa F,
Matzinger P
(1998) A conditioned dendritic cell can be a temporal bridge between a CD4⁺ T-helper and a T-killer cell. Nature 393:474–478.
OpenUrl CrossRef PubMed

[59] Ridge JP,

[60] Di Rosa F,

[61] Matzinger P

[62] ↵
Schoenberger SP,
Toes RE,
van der Voort EI,
Offringa R,
Melief CJ
(1998) T-cell help for cytotoxic T lymphocytes is mediated by CD40-CD40L interactions. Nature 393:480–483.
OpenUrl CrossRef PubMed

[63] Schoenberger SP,

[64] Toes RE,

[65] van der Voort EI,

[66] Offringa R,

[67] Melief CJ

[68] Jennings SR,
Bonneau RH,
Smith PM,
Wolcott RM,
Chervenak R
(1991) CD4-positive T lymphocytes are required for the generation of the primary but not the secondary CD8-positive cytolytic T lymphocyte response to herpes simplex virus in C57BL/6 mice. Cell Immunol 133:234–252.
OpenUrl CrossRef PubMed

[69] Jennings SR,

[70] Bonneau RH,

[71] Smith PM,

[72] Wolcott RM,

[73] Chervenak R

[74] ↵
Rajasagi NK,
et al.
(2009) CD4⁺ T cells are required for the priming of CD8⁺ T cells following infection with herpes simplex virus type 1. J Virol 83:5256–5268.
OpenUrl Abstract/FREE Full Text

[75] Rajasagi NK,

[76] et al.

[77] ↵
Jones CA,
Taylor TJ,
Knipe DM
(2000) Biological properties of herpes simplex virus 2 replication-defective mutant strains in a murine nasal infection model. Virology 278:137–150.
OpenUrl CrossRef PubMed

[78] Jones CA,

[79] Taylor TJ,

[80] Knipe DM

[81] ↵
Mueller SN,
Heath W,
McLain JD,
Carbone FR,
Jones CM
(2002) Characterization of two TCR transgenic mouse lines specific for herpes simplex virus. Immunol Cell Biol 80:156–163.
OpenUrl CrossRef PubMed

[82] Mueller SN,

[83] Heath W,

[84] McLain JD,

[85] Carbone FR,

[86] Jones CM

[87] ↵
Martín-Fontecha A,
et al.
(2003) Regulation of dendritic cell migration to the draining lymph node: Impact on T lymphocyte traffic and priming. J Exp Med 198:615–621.
OpenUrl Abstract/FREE Full Text

[88] Martín-Fontecha A,

[89] et al.

[90] ↵
Webster B,
et al.
(2006) Regulation of lymph node vascular growth by dendritic cells. J Exp Med 203:1903–1913.
OpenUrl Abstract/FREE Full Text

[91] Webster B,

[92] et al.

[93] ↵
Angeli V,
et al.
(2006) B cell-driven lymphangiogenesis in inflamed lymph nodes enhances dendritic cell mobilization. Immunity 24:203–215.
OpenUrl CrossRef PubMed

[94] Angeli V,

[95] et al.

[96] ↵
Kumar V,
et al.
(2010) Global lymphoid tissue remodeling during a viral infection is orchestrated by a B cell-lymphotoxin-dependent pathway. Blood 115:4725–4733.
OpenUrl Abstract/FREE Full Text

[97] Kumar V,

[98] et al.

[99] ↵
Lee HK,
et al.
(2009) Differential roles of migratory and resident DCs in T cell priming after mucosal or skin HSV-1 infection. J Exp Med 206:359–370.
OpenUrl Abstract/FREE Full Text

[100] Lee HK,

[101] et al.

[102] ↵
Tzeng TC,
et al.
(2010) CD11c(hi) dendritic cells regulate the re-establishment of vascular quiescence and stabilization after immune stimulation of lymph nodes. J Immunol 184:4247–4257.
OpenUrl Abstract/FREE Full Text

[103] Tzeng TC,

[104] et al.

[105] ↵
Lesley R,
Kelly LM,
Xu Y,
Cyster JG
(2006) Naive CD4 T cells constitutively express CD40L and augment autoreactive B cell survival. Proc Natl Acad Sci USA 103:10717–10722.
OpenUrl Abstract/FREE Full Text

[106] Lesley R,

[107] Kelly LM,

[108] Xu Y,

[109] Cyster JG

[110] ↵
Bajénoff M,
Granjeaud S,
Guerder S
(2003) The strategy of T cell antigen-presenting cell encounter in antigen-draining lymph nodes revealed by imaging of initial T cell activation. J Exp Med 198:715–724.
OpenUrl Abstract/FREE Full Text

[111] Bajénoff M,

[112] Granjeaud S,

[113] Guerder S

[114] ↵
Kraal G,
Schornagel K,
Savelkoul H,
Maruyama T
(1994) Activation of high endothelial venules in peripheral lymph nodes. The involvement of interferon-gamma. Int Immunol 6:1195–1201.
OpenUrl Abstract/FREE Full Text

[115] Kraal G,

[116] Schornagel K,

[117] Savelkoul H,

[118] Maruyama T

[119] ↵
Manolios N,
Geczy CL,
Schrieber L
(1988) High endothelial venule morphology and function are inducible in germ-free mice: A possible role for interferon-gamma. Cell Immunol 117:136–151.
OpenUrl CrossRef PubMed

[120] Manolios N,

[121] Geczy CL,

[122] Schrieber L

[123] ↵
Hendriks HR,
Korn C,
Mebius RE,
Kraal G
(1989) Interferon-gamma-increased adherence of lymphocytes to high endothelial venules. Immunology 68:221–226.
OpenUrl PubMed

[124] Hendriks HR,

[125] Korn C,

[126] Mebius RE,

[127] Kraal G

[128] ↵
May MJ,
Ager A
(1992) ICAM-1-independent lymphocyte transmigration across high endothelium: Differential up-regulation by interferon gamma, tumor necrosis factor-α and interleukin 1β. Eur J Immunol 22:219–226.
OpenUrl PubMed

[129] May MJ,

[130] Ager A

[131] ↵
Hall JG,
Morris B
(1965) The immediate effect of antigens on the cell output of a lymph node. Br J Exp Pathol 46:450–454.
OpenUrl PubMed

[132] Hall JG,

[133] Morris B

[134] ↵
Cyster JG
(2007) Specifying the patterns of immune cell migration. Novartis Found Symp 281:54–61, discussion 61–54, 208–209.
OpenUrl PubMed

[135] Cyster JG

[136] ↵
van Heijst JW,
et al.
(2009) Recruitment of antigen-specific CD8⁺ T cells in response to infection is markedly efficient. Science 325:1265–1269.
OpenUrl Abstract/FREE Full Text

[137] van Heijst JW,

[138] et al.

[139] ↵
Jung S,
et al.
(2002) In vivo depletion of CD11c⁺ dendritic cells abrogates priming of CD8⁺ T cells by exogenous cell-associated antigens. Immunity 17:211–220.
OpenUrl CrossRef PubMed

[140] Jung S,

[141] et al.

[142] ↵
Zhao X,
et al.
(2003) Vaginal submucosal dendritic cells, but not Langerhans cells, induce protective Th1 responses to herpes simplex virus-2. J Exp Med 197:153–162.
OpenUrl Abstract/FREE Full Text

[143] Zhao X,

[144] et al.

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