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. Author manuscript; available in PMC: 2013 Dec 14.
Published in final edited form as: Immunity. 2012 Nov 1;37(6):1091–1103. doi: 10.1016/j.immuni.2012.08.016

CXCR3 chemokine receptor-ligand interactions in the lymph node optimize CD4+ T helper 1 cell differentiation

Joanna R Groom 1,3, Jillian Richmond 1, Thomas T Murooka 1, Elizabeth W Sorensen 1, Jung Hwan Sung 2, Katherine Bankert 1, Ulrich H von Andrian 2, James J Moon 1, Thorsten R Mempel 1, Andrew D Luster 1,*
PMCID: PMC3525757  NIHMSID: NIHMS414353  PMID: 23123063

SUMMARY

Differentiation of naïve CD4+ T cells into T helper (Th) cells is a defining event in adaptive immunity. The cytokines and transcription factors that control Th cell differentiation are understood, however it is not known how this process is orchestrated within lymph nodes (LNs). Here we have shown that the CXCR3 chemokine receptor was required for optimal generation of interferon (IFN)-γ secreting Th1 cells in vivo. Using a CXCR3 ligand reporter mouse, we found that stromal cells predominately expressed the chemokine ligand CXCL9 while hematopoietic cells expressed CXCL10 in LNs. Dendritic cell (DC)-derived CXCL10 facilitated T cell-DC interactions in LNs during T cell priming while both chemokines guided intranodal positioning of CD4+ T cells to interfollicular and medullary zones. Thus, different chemokines acting on the same receptor can function locally to facilitate DC-T cell interactions and globally to influence intranodal positioning, and that both functions contribute to Th1 cell differentiation.

INTRODUCTION

CD4+ T cells play a central role in orchestrating adaptive immune responses. Naïve CD4+ T cells are activated in draining lymph nodes (dLNs) by cognate antigen loaded dendritic cells (DCs) where they differentiate into one of several lineages of helper T cell subsets, such as T helper type 1 (Th1), Th2, Th17, T follicular helper (Tfh) and induced T regulatory (iTreg) cells (Zhu et al., 2010). For example, Th1 cells secrete interleukin-2 (IL-2), interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α), and promote cellular immune responses mainly to intracellular pathogens and tumors. Differentiation into a particular helper subset is guided by extrinsic cytokine cues that induce lineage-specifying transcription factors. While the cytokines and transcription factors that control this process are relatively well understood, far less is known about how this process is temporally and spatially orchestrated within dLN in vivo, where it initially takes shape.

CD4+ T cells require persistent antigen throughout their expansion to differentiate (Obst et al., 2005). Multiple successive phases of T cell priming have been described based on dynamic interactions between T cells and DCs in dLNs (Mempel et al., 2004; Miller et al., 2004). First, T cells sporadically interact with DCs, leading to an initial increase in T cell activation. Subsequently, T cells undergo sustained interactions with DCs, which are likely required to induce T helper cell differentiation. Diminished contact stability at this time, leads to reduction in IFN-γ production in settings where Th1 cells are induced (Hugues, 2010). Finally, repeated engagements of DC by the daughters of newly activated CD4+ T cells may also be required to optimally differentiate into IFN-γ producing Th1 cells (Celli et al., 2005; Itano et al., 2003).

The migration of priming T cells to different LN compartments during antigenic priming may expose T cells to different types of antigen presenting cells (APCs) and provide them with unique differentiation cues. For instance, activated migratory and LN resident DCs localize to different dLN regions, including the deep and the superficial paracortex, as well as the interfollicular T cell zones and medullary regions (Hickman et al., 2008; Itano et al., 2003; Tang and Cyster, 1999). In the course of this antigenic priming, T cells may redistribute to these peripheral regions of the LN, and once there, may be exposed to other APC types or cytokine cues than those present in the deep paracortex (Hickman et al., 2008).

The induction of chemokine receptors is intertwined with CD4+ T cell differentiation. The differentiation of a particular helper subset instructs the upregulation of a specific set of homing receptors, which guide effector cells out of the lymphoid compartment and into otherwise restricted peripheral sites of inflammation (Bromley et al., 2008). How chemokines participate in the process of Th cell differentiation is not known but there are two likely possibilities. First, DCs may use chemokines to promote encounters with antigen-specific T cells (Friedman et al., 2006; Molon et al., 2005). Second, chemokines expressed in specific LN regions may influence more globally the intranodal positioning of priming T cells to particular microenvironments, to bring these cells in contact with the appropriate APCs or accessory cells important for their differentiation (Tang and Cyster, 1999).

CXCR3 is the receptor for the interferon-inducible chemokines CXCL9 (MIG), CXCL10 (IP-10), and CXCL11 (I-TAC). CXCR3 expression on activated T cells is important for the amplification of IFN-γ-dependent recruitment into peripheral sites of infection and autoimmune responses (Groom and Luster, 2011a). However, CXCR3 ligands are also expressed in dLNs during to Th1 cell differentiation (Martin-Fontecha et al., 2004; Yoneyama et al., 2002).

We sought to determine the role of CXCR3 receptor-ligand interactions in CD4+ T cell differentiation in dLNs using two models of CD4+ T cell priming to address the potential roles for this chemokine system described above: promoting DC-T interactions and intranodal positioning of T cells. The first model examined interactions of DCs and CD4+ T cells in the LN that induce Th1 cell polarization. In this model, antigen-pulsed DCs were injected subcutaneously into the footpad of mice from where they migrate into dLNs and interact with cognate antigen-specific T cells (Ingulli et al., 1997; Miller et al., 2004). The second model examined the global positioning of CD4+ T cell in the reactive LN required for optimal Th1 cell generation. Here, soluble antigen was introduced into peripheral tissue to mimic the physiological delivery of antigen to LNs via the lymph, where both migratory and resident DCs may influence T cell priming. To characterize chemokine expression in the dLNs, we developed a transgenic (Tg) mouse that reports the expression of both CXCL9 and CXCL10, called REX3 (Reporting the Expression of CXCR3 ligands). Using these in vivo models of immunization and REX3 Tg mice, we found that the CXCR3 system regulated the local interactions of antigen-specific CD4+ T cell with cognate antigen-loaded DCs in the LN, as well as the global intranodal positioning of CD4+ T cells following antigen-induced activation, both of which contribute to Th1 cell differentiation.

RESULTS

Rapid upregulation of CXCR3 in CD4+ T cells correlates with IFN-γ production

To study the in vivo development of Th1 cells, we first outlined the kinetics of CXCR3 upregulation by antigen-specific CD4+ T cells in LNs using a controlled activated LN reaction (Figure 1A). Expanded DCs were pulsed with ovalbumin (OVA) protein activated with lipopolysaccharide (LPS) and PolyI:C (Figure S1B), and subcutaneously transferred into the footpads of naïve mice. Purified CD11c+MHC-IIhi DCs contained both CD8+ and CD11b+ subpopulations, however, only CD11b+ cells successfully migrated to the LN (Figure S1A,C). Twenty-four hours (hr) following DC injections, mice were given naïve OVA-specific CD4+ T cells isolated from OTII Tg mice. Two hr following T cell transfer, CD62L blocking antibody was given to synchronize T cell activation by inhibiting further entry into dLNs (Mempel et al., 2004). Following T cell transfer, the dLNs were harvested and OTII cells were assessed for accumulation, proliferation and expression of CD44 and CXCR3. Although slower than CD44 upregulation, the initially low CXCR3 expression was upregulated within 24 hr after T cell transfer, prior to T cell proliferation (Figure 1B–E). Following the first cycles of proliferation, CXCR3+ cells peaked with the majority of OTII cells expressing CXCR3 (Figure 1D,E). The frequency of CXCR3+ cells then decreased slightly, but remained above 50% throughout the activated LN reaction prior to cells leaving the LN (Figure 1B,D). IFN-γ production peaked in this model at 60 hours (Figure S1D,E). At this time, cells producing IFN-γ had a higher mean fluorescence intensity (MFI) of CXCR3 expression than cells that failed to produce cytokine, and cells expressing CXCR3 were more likely to be IFN-γ producers (Figure 1F,G). Thus, CXCR3 is upregulated and remains high on antigen-specific T cells in dLNs and correlates with their production of IFN-γ.

Figure 1. CXCR3 expression is upregulated rapidly in draining LNs (dLNs) and correlates with IFN-γ expression.

Figure 1

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(A) Experimental protocol. Antigen-pulsed DCs were injected 24 hr before i.v. OT II cells. (Phenotype of transferred DCs Figure S1). CD62L blocking antibody was given 2 hr following OTII cell transfer and every 24 hr. At times indicated, dLNs (popliteal) and non-dLNs (bracial) were harvested to assess T cell activation. Time course of (B) cell numbers (C) CD44 and (D,E) CXCR3 cell surface induction on transferred OTII cells. (F,G) Correlation between IFN-γ+ cells and CXCR3 mean fluorescence intensity (MFI). Data are representative of 3 independent experiments (n=4–6).

CXCR3 is required for optimal Th1 cell-associated cytokine production and activation of OTII cells

CXCR3 and its ligands play an important role in the trafficking of effector Th1 CD4+ T cells into inflamed peripheral tissues. However, CXCR3 is upregulated in dLNs well before T cell egress (Figure 1). Whether CXCR3 also influences the generation of Th1 cells is unknown. To investigate this, we co-transferred WT and Cxcr3−/− OTII cells following DC injection. At 60 hr post T cell transfer, the frequency of Cxcr3−/− OTII cells IFN-γ-producing cells was reduced by ~50% compared to WT OTII cells (Figure 2A,B). A less pronounced, but significant reduction was observed for IL-2 and TNF-α. As no difference was seen in the overall numbers of WT and Cxcr3−/− cells in the dLN, this decrease in cytokine-producing cells translated into a decrease in the total number of polyfunctional (cells producing IL-2, TNF-α, and IFN-γ) OTII cells (Figure 2C). Cxcr3−/− OTII cells also displayed markedly lower surface activation markers CD25, CD40L (CD152) and CD69 than co-transferred WT OTII cells (Figure 2D).

Figure 2. CXCR3 is required for optimal Th1 differentiation following transfer of OVA-pulsed DCs.

Figure 2

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WT and Cxcr3−/− OTII cells were co-transferred into WT hosts that received OVA-pulsed DCs. 60 hr post T cell transfer dLNs were harvested. (A) Plots of IFN-γ and TNF-α production by WT and Cxcr3−/− OTII cells. (B) Frequency of IFN-γ+, IL-2+, TNF-α+ WT (square) and Cxcr3−/− (triangle) OTII cells. (C) Total numbers of polyfunctional (producing IL-2, TNF-α and IFN-γ) WT and Cxcr3−/− OTII cells in dLNs. (D) MFI of CD40L, CD69, CD25 at 24–36 hr post T cell transfer of WT and Cxcr3−/− OTII cells. In (B–D) the line connects paired WT and Cxcr3−/− OTII cells transferred into the same WT host. (E) CMFDA labeled cell proliferation following WT (grey) and Cxcr3−/− (dashed) OTII cell co-transfer. No treat mice (without CD62L blocking were included to show peaks with continual T cell entry). (F) Frequency of TNF-α+IFN-γ+ WT (grey) and Cxcr3−/− (open) OTII cells in mice without, or with CD62L blocking alone or with FTY720. (G) Kinetics of WT and Cxcr3−/− OTII cell induction of IFN-γ+TNF-α+. Data are representative of 3 independent experiments (n=6–8). (See Figure S2A,B for transfer of WT and Cxcr3−/− cells into separate hosts and Figure S2C for in vitro polarization of WT and Cxcr3−/− OTII cells).

To address if the decrease in IFN-γ+ cells seen in the Cxcr3−/− population was due to competition for interactions with DCs, naïve WT or Cxcr3−/− OTII cells were transferred into separate DC-injected hosts and assessed for effector cytokine production. In this non-competitive setting, Cxcr3−/− OTII cells displayed the same reduced frequency of IFN-γ+TNF-α+ cells as seen in the co-transfer experiments (Figure S2A,B).

To eliminate the requirement for cells to migrate towards OVA-pulsed DCs, WT and Cxcr3−/− OTII cells were co-cultured and differentiated in vitro. In these conditions, the cells producing IFN-γ were evenly split between the T cells of each genotype (Figure S2C), indicating that there was a unique requirement for Cxcr3 expression in vivo. Although the numbers of WT and Cxcr3−/− OTII cells present in the dLNs was comparable, a possible defect in proliferation could account for decreased production of polyfunctional Th1 cells in the Cxcr3−/− OTII cells. However, CMFDA dilution profiles of WT and Cxcr3−/− OTII cells were overlapping 60 hr following T cell transfer, when cell entry into the LN was or was not blocked with CD62L antibody treatment (Figure 2E). To ensure that differences in IFN-γ+ production by Cxcr3−/− OTII cells were not due to differences in their egress from dLN into peripheral tissues (Yoneyama et al., 2002), mice were either left untreated, treated with CD62L blocking antibody as above, or treated with both CD62L antibody along with FTY720, which induces the sequestration of T cells in lymphoid organs by modulating S1P1 and inhibiting LN T cell egress (Matloubian et al., 2004). Neither blocking T cell egress, nor allowing continued T cell entry into dLNs altered the decrease in effector cytokine production seen in Cxcr3−/− cells compared to WT OTII cells (Figure 2F). Finally, we performed a kinetic experiment of IFN-γ production. While, the maximum frequency of IFN-γ producing cells varied throughout analysis (Figure S1D,E), at each time point co-transferred Cxcr3−/− cells demonstrated a similar decrease in IFN-γ production, compared to WT OTII cells (Figure 2G). Collectively, these data show that CXCR3 expression is important for optimizing Th1 CD4+ T cell responses. This effect was not observed in vitro and was not related to proliferation, different kinetics of IFN-γ expression, or early egress of Cxcr3−/− T cells from LNs.

Both CXCR3 ligands are induced in dLN during Th1 cell differentiation

Given the role for CXCR3 on CD4+ T cells during their differentiation to Th1 effector cells, we investigated the expression of CXCR3 ligands during inflammatory LN reactions. We focused on the CXCR3 ligands CXCL9 and CXCL10, as the third CXCR3 ligand, CXCL11 is not expressed in C57BL/6 mice at the protein level as demonstrated by immunoblot (Figure S3A,B) due to a frame shift mutation (Sierro et al., 2007). To examine the expression of CXCR3 ligands at times relevant to Th1 cell differentiation, we first examined the upregulation of Cxcl9 and Cxcl10 by RNA analysis of whole draining and non-draining LNs during our activated DC transfer LN model. Both ligands were highly upregulated in dLNs, while they remained minimally expressed in non-dLNs (Figure S3C,D).

CXCL10 expression by antigen-presenting DCs is important for Th1 cells differentiation

CXCL9 and CXCL10 have been observed in various models to display overlapping as well as unique functions (Groom and Luster, 2011b). In this regard, it has been unclear which, if any, of the CXCR3 ligands is the major contributor towards promotion of Th1 cell differentiation. Expanded WT, Cxcl9−/− and Cxcl10−/− DCs were pulsed and activated as described above, and injected into naïve mice. At 60 hr post OTII T cell transfer, DCs expanded from Cxcl10−/− cells were not capable of supporting the induction of IFN-γ+TNF-α+ producing OTII cells. In contrast, Cxcl9−/− DCs were as capable as WT DCs in inducing IFN-γ+TNF-α+ producing OTII cells (Figure 3A,B). Of note, OTII T cell IFN-γ production in mice receiving Cxcl10−/− DCs was reduced to a greater degree than Cxcr3−/− T cells co-transferred with WT T cells (Figure 2). This may be due to an additional influence of DC-derived CXCL10 on host accessory cells such as natural killer (NK) cells and plasmacytoid dendritic cells (pDCs), which express CXCR3, and are known to influence Th1 cell differentiation (Martin-Fontecha et al., 2004; Yoneyama et al., 2004). To eliminate other intrinsic defects in Cxcl10−/− DCs, we determined that these cells were capable of migrating to dLNs and facilitating the early upregulation of CD25 on OTII cells in vivo, and inducing IFN-γ production in vitro, to the same degree as WT and Cxcl9−/− DCs (Figure S3E,F,G).

Figure 3. DC-derived CXCL10 optimizes Th1 responses and identification of chemokine-expressing DCs during T cell priming.

Figure 3

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(A,B) Pulsed WT, Cxcl9−/− and Cxcl10−/− DCs were transferred to hosts prior to OTII T cells. 60 hr post T cell transfer, dLN T cells were harvested. (A) Plots of IFN-γ and TNF-α production of WT OTII cells activated by DCs of indicated genotype. (B) Frequency of TNF-α+IFN-γ+ WT OTII cells activated by WT (black), Cxcl9−/− (open), or Cxcl10−/− (grey) DCs. (C) Schematic of REX3 Tg construct indicating insertion of RFP into the Cxcl9 locus and BFP into the Cxcl10 locus of the RP-24-164O11 BAC. Open box non-coding exons and black box coding exons of Cxcl9 and Cxcl10 genes; red box RFP ORF, blue box BFP ORF, and stripped box SV40 poly A site; FRT Flippase Recognition Target and loxP Cre recombinase site. (D) CMFDA labeled DCs expanded from REX3 Tg were pulsed, stimulated and injected into WT mice. Plot and bar graph indicating frequency of REX3 negative (open), CXCL10-BFP only (grey), and CXCL10-BFP and CXCL9-RFP double positive (stripe) DCs. (E) Tracked REX3++ (expressing CXCL10-BFP and CXCL9-RFP) and REX3− (REX3 negative) DCs MFI of CD40, CD86 at 24–36 hr post OTII cell transfer. Histogram and MFI plots are shown. (F–I) DCs expanded from REX3 Tg mice injected in WT mice. 24 hr later, WT and Cxcr3−/− OTII cells were transferred i.v. MP-IVM of exposed dLN was performed at 6–8 hr post T cell transfer. (F) Intravital multiphoton micrograph of representative REX3+ DC interactions with WT and Cxcr3−/− OTII cells. Numbered arrowheads indicate long-lived interactions between REX3+ DCs and WT (green) or Cxcr3−/− (red) OTII cells. White lines indicate tracks of WT and Cxcr3−/− cell centroids engaging in short-lived interactions with REX3+ DCs. Elapsed time in minutes:seconds. Bar indicates 30 µm. (G) Mean 3D track velocity, confinement ratio and arrest coefficient of WT and Cxcr3−/− OTII cells. Dashed boxes around WT (green) Cxcr3−/− (red) OTII cells indicate the frequency of cells in the specified region; 3D track velocity >10 µm/min; confinement ration >0.4; arrest coefficient <0.2 (not arrested). (H) Frequency of WT and Cxcr3−/− OTII cells in long term (open) short-lived (grey) or without (stripe) DC interactions. (I) Short-lived interactions between REX3+ DCs and either WT or Cxcr3−/− OTII cells. Duration of contacts from movies imaged 6–8 hr post T cell transfer. Only interactions where the initiation and termination of the interactions were observed were analyzed. Data are representative of 3 independent experiments (n=3–6). (See Figure S3A,B for confirmation that C57BL/6 mice do not produce CXCL11 protein; Figure S3C,D for RNA expression of Cxcl9 and Cxcl10 in whole dLN or non-dLNs in mice receiving OTII cells or not; Figure S3E–G for confirmation of Cxcl10−/− DC tracking and function; Figure S3H,I for BAC Tg construction and PCR genotyping of REX3 Tg mice; Figure S3J–M; Movie S1 for representative imaging performed 6–8 hr post T cell transfer and Figure S4 for quantification of imaging performed at 24–27 hr post T cell transfer).

Generation of REX3 Transgenic mice

While RNA analysis of whole dLNs (Figure S3C,D) established that CXCL9 and CXCL10 are expressed at times relevant to Th1 cell differentiation in vivo, it offered little information on the precise timing, location, and cell types expressing these ligands. Therefore, we generated a reporter mouse in which spectrally distinct fluorescent reporter proteins report the RNA expression of the CXCR3 ligands, Cxcl9 and Cxcl10. To create Reporting the Expression of CXCR3 ligands (REX3) Tg mice, we inserted red fluorescent protein (RFP) at the start codon of Cxcl9 and inserted blue fluorescent protein (BFP) at the start codon of Cxcl10 in a CXCR3-ligand containing bacterial artificial chromosome (BAC) (Figure 3C and Figure S3H). Accurate reporting of Cxcl9 and Cxcl10 expression was confirmed by correlating the induction of RNA transcripts and protein with induction of FPs in stimulated REX3 Tg DCs (Figure S3J–L). CXCL9 immunostaining on REX3 Tg dLNs co-stained with the expression of the CXCL9-RFP reporter, indicating most of the chemokine protein detected is presented by cells producing it and not dispersed throughout the LN (Figure S3M).

Chemokine-expressing DCs display increased activation

To visualize the expression of CXCL9 and CXCL10 by transferred, antigen-presenting DCs throughout the inflamed LN model, REX3 Tg DCs were pulsed with antigen, activated, and Carboxymethyl fluorescein diacetate (CMFDA) labeled prior to subcutaneous footpad injections into WT mice. CMFDA+ DCs were then tracked to assess reporter expression (Figure 3D). The majority of transferred DCs expressed both CXCL9-RFP and CXCL10-BFP within 12 hr of T cell transfer. In addition, smaller populations of CXCL10-BFP single expressers and double negative DCs were detected, however, single positive CXCL9-RFP DCs were never observed. The frequency of cells in each of these populations remained stable throughout the time course (Figure 3D). We assessed if there were any activation differences between DCs expressing REX3+ FPs and those without expression. While no difference was seen in expression of major histocompatibility complex (MHC) class II between these populations, REX3++ DCs (dually expressing CXCL9-RFP and CXCL10-BFP) had increased expression of the activation markers CD40 and CD86 compared to DCs without reporter expression (Figure 3E). Thus, identifying chemokine reporter cells allowed us to observe that both CXCL9 and CXCL10 are expressed by DCs at times relevant to Th1 cell differentiation, and that this expression correlates with increased activation of APCs.

Cxcr3−/− OTII cells display altered behavior in dLNs during CD4+ T cell priming

In vitro, production of CXCR3 ligands by DCs enhances their ability to attract T cells (Padovan et al., 2002). We therefore tested if this might be relevant in vivo. The use of multiphoton microscopy (MP-IVM) has revealed that the duration of T cell-DC interactions correlates with efficient T cell priming and IFN-γ production (Hugues, 2010). To gain insight into why Cxcr3−/− OTII CD4+ T cells do not optimally differentiate into Th1 cells, we characterized their in vivo movements using MP-IVM. When draining popliteal LNs were surgically exposed, transferred REX3 Tg DCs could be visualized. However, CXCL9-reporting RFP photo-bleached rapidly, leading to visualization of transferred DC solely through BFP-expression in time-lapse recording. Six to eight hr after i.v. co-transfer of labeled WT and Cxcr3−/− OTII T cells, both populations were capable of forming stable contacts with DCs (Figure 3F,H). However, while WT cells were almost uniformly engaged in stable contacts, as reflected by low migratory velocity, low confinement ratio, and high arrest coefficients (Figure 3G), a 3–4 fold larger fraction (~25%) of Cxcr3−/− cells compared to WT cells failed to form long-lasting interactions (Figure 3G). Even 24 h after transfer, when both T cell populations had resumed motile behavior, Cxcr3−/− OTII cells were less confined in their migration (Figure S4). Analysis of the contacts of T cells with CXCL10-BFP-expressing DC showed that the majority of Cxcr3−/− OTII cells did not visibly engage with these cells (Figure 3H, I). When the length of short-lived DC-T cell interactions was assessed, contact times of Cxcr3−/− OTII cells with REX3+ DCs were indeed shorter than those of WT OTII cells (Figure 3F,H,I). Combined, data generated by transfer of antigen-pulsed DCs established that Cxcr3−/− CD4+ cells cannot maximally differentiate into IFN-γ+ Th1 cells, which correlated with their reduced ability to interact with CXCL10-expressing antigen-presenting DCs.

Immunization requires CXCR3 for optimal Th1 cell differentiation

Thus far, our results addressed the requirement for CXCR3 ligands produced by CD11b+ antigen-presenting DCs in the dLN; however, CXCL9 and CXCL10 are expressed by multiple hematopoietic and stromal cells residing within dLNs (Gattass et al., 1994; Martin-Fontecha et al., 2004). We therefore investigated if chemokines produced by these cells were also important for optimal Th1 cell responses, reasoning that immunization with soluble antigen may correlate more with vaccination strategies than the transfer of antigen-pulsed DCs. For these studies, naïve host mice were immunized with OVA protein with LPS and PolyI:C subcutaneously. Twenty-four hr later, mice were co-transferred with naïve WT and Cxcr3−/− OTII cells, which were assessed for the production of IFN-γ and TNF-α 60 hr post-T cell transfer (Figure 4A). As seen when using transferred antigen-pulsed DCs (Figure 2), following immunization with soluble antigen, CXCR3 ligand-receptor interactions were required for optimal differentiation of OTII cells towards a Th1 cell phenotype (Figure 4B,C,D).

Figure 4. CXCR3 is required for optimal Th1 differentiation following OVA and TLR-ligand immunization.

Figure 4

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(A) Experimental protocol. Host mice were immunized with OVA and LPS and PolyI:C prior to i.v. co-transfer of WT and Cxcr3−/− OTII cells. CD62L blocking antibody 2 hr following OTII cell. 60 hr post T cell transfer, dLNs T cells were harvested to assess cytokine production. (B) Plots and (C) fold change of IFN-γ and TNF-α production by WT and Cxcr3−/− OTII cells. (D) Total numbers of polyfunctional (producing IL-2, TNF-α and IFN-γ) WT and Cxcr3−/− OTII cells in dLNs. Line connects paired WT and Cxcr3−/− OTII cells transferred into the same WT host. (F–G) LN sections from REX3 Tg mice. CXCL9-RFP, red; CXCL10-BFP, blue; B220 and CD19 immunostaining white. (E) Unimmunized REX3 Tg LN. (F) REX3 Tg dLNs post immunization. Right panels shows grey scale of B220 and CD19 immunostaining (top), CXCL9-RFP (middle) and CXCL10-BFP (lower) of LN harvested at 36 hr post T cell transfer. Bar indicates 500 µm. Data are representative of 3 independent experiments (n=3–6).

We next used REX3 Tg mice as hosts in this model to identify the pattern of expression of CXCL9 and CXCL10 in dLNs. In unimmunized REX3 Tg mice, popliteal LNs showed little expression of either CXCL9-RFP or CXCL10-BFP (Figure 4E). However, during the inflamed LN reaction, expression was greatly increased, peaking at 24–36 hr following T cell transfer, before declining again at 60 hr (Figure 4F). Surprisingly, while some double positive staining was observed, it appeared that CXCL9 and CXCL10 had strikingly different expression patterns. CXCL10 was primarily expressed in the LN medulla, the site into which the most of the lymph and lymph-borne antigens drain. Meanwhile, the strongest CXCL9-RFP expression was seen in interfollicular areas. These areas serve as transit corridors for lymphocytes migrating into and out of T cell areas, and are also areas where T cells may survey DCs. In contrast, only scattered single- and dual-expressing cells for each chemokine were seen in the T cell zone where T cell-DC interactions are traditionally thought to occur (Figure 4F) (Ingulli et al., 1997; Tang and Cyster, 1999). During the time course undertaken, there was minimal expression of either CXCR3 ligand inside B cell follicles (Figure 4F). These expression data using the REX3 Tg mice suggest the development of dramatic chemokine gradients in different regions of dLNs following immunization.

Non-redundant roles for CXCL9 and CXCL10 in Th1 cell differentiation following immunization

To investigate the patterns of chemokine expression further, we examined BM chimeras with REX3 Tg BM being transferred into irradiated WT hosts or vice versa. Strikingly, the majority of CXCL10-BFP expression was in the BM-derived hematopoietic compartment (Figure 5A). In contrast, CXCL9-RFP was predominantly expressed by the radio-resistant stromal cells located in the interfollicular and medulla areas of the dLN (Figure 5B).

Figure 5. CXCL9 and CXCL10 have non-redundant roles in promoting OTII cell IFN-γ responses following host immunization.

Figure 5

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(A–B) BM chimeras created with (A) REX3 Tg BM into WT hosts and (B) WT BM into REX3 Tg hosts. Reconstituted mice were immunized and transferred with OTII cells. 24–36 hr post T cell transfer, dLNs reporter protein expression (CXCL9-RFP, red; CXCL10-BFP, blue; B220 and CD19 immunostaining white). Bar indicates 500 µm. Higher magnification images are from regions indicated (I, 2, 3). (C) WT, Cxcl9−/− and Cxcl10−/− host mice were immunized into the footpad 24 hr prior to adoptive transfer of WT OTII cells. At 60 hr post T cell transfer dLNs were harvested and restimulated to assess cytokine production. Plots of IFN-γ and TNF-α production by WT OTII cells transferred into WT, Cxcl9−/− or Cxcl10−/− hosts. (D) Frequency of TNF-α+IFN-γ+ transferred OTII cells in WT (black), Cxcl9−/− (open) and Cxcl10−/− (grey) hosts 60 hr following immunization. (E) BM chimeras of WT hosts reconstituted with WT (black), Cxcl9−/− (open), Cxcl10−/− (grey) BM. Chimeras were immunized and transferred with WT OTII cells. At 60 hr post T cell transfer, dLNs were harvested to assess cytokine production. Fold change of frequency of TNF-α+IFN-γ+ transferred cells in indicated BM chimeras is shown. (F) BM chimeras of WT (black), Cxcl9−/− (open), Cxcl10−/− (grey) hosts reconstituted with WT BM. As in E, dLN cytokine production. Fold change of frequency of TNF-α+IFN-γ+ transferred cells in indicated BM chimeras is shown. Data are representative of 2–3 independent experiments (n=4–8).

We next assessed the role of each chemokine in CD4+ T cell responses using ligand-deficient mice following immunization. Surprisingly, both CXCL9 and CXCL10 were non-redundantly required for optimal Th1 cell differentiation following immunization (Figure 5C, D). This was in contrast to the DC transfer model, where only CXCL10-expressing antigen-pulsed DCs were required for maximal Th1 cell differentiation (Figure 3A,B). To determine which compartment was primarily required for maximum induction of Th1 cell differentiation, given the difference in expression of CXCL9 and CXCL10 seen in REX3 Tg BM chimeras, we immunized BM chimeric mice generated with WT, Cxcl9−/− or Cxcl10−/− BM transplanted into irradiated WT mice. WT host mice reconstituted with Cxcl10−/− BM showed a defect in maximal Th1 cell differentiation, while mice containing WT stromal cells with Cxcl9−/− hematopoietic cells were capable of maximal induction of IFN-γ+TNF-α+ cells (Figure 5E). Finally, we performed BM chimera experiments where WT BM was used to reconstitute irradiated WT, Cxcl9−/− or Cxcl10−/− mice. In this setting, mice lacking either stromal-derived CXCL9 or CXCL10 displayed reduced numbers of IFN-γ+TNF-α+ T cells (Figure 5F). These data correlate with the expression data (Figure 5A,B), and demonstrate that CXCL10 produced by the hematopoietic compartment is critical, while both CXCL9 and CXCL10 produced by stromal cells are important.

CXCL9 and CXCL10 gradients promote peripheralization of T cells in the LN

As the majority of chemokine expressing cells were located peripheral to the T cell zone, we asked if, following immunization, antigen-specific CD4+ T cells migrate out of the T cell zone towards these areas in a CXCR3-dependent manner. We therefore investigated the intranodal location of WT and Cxcr3−/− OTII cells in the T cell zone, interfollicular zone surrounding B cell follicles, or in the LN medulla (as described in Figure S5) before and after immunization. In unimmunized mice receiving either WT or Cxcr3−/− OTII cells, transferred cells were predominantly located in the T cell zone (Figure 6A). In immunized hosts, 24–36 hr following T cell transfer, WT cells were located in the interfollicular and medulla regions, while the majority of Cxcr3−/− OTII cells remained in the T cell zone (Figure 6A). Again, in unimmunized mice, co-transferred WT and Cxcr3−/− OTII cells were similarly located in the T cell zone. Following immunization, compared to Cxcr3−/− OTII cells, WT OTII cells had a greater propensity to move into the periphery of the dLN, where CXCR3 ligands are highly expressed (Figure 6B,C and Figure 4F). We next investigated the localization of WT OTII cells in immunized WT, Cxcl9−/− or Cxcl10−/− hosts. WT OTII cells were similarly located in the T cell zone in unimmunized WT, Cxcl9−/− and Cxcl10−/− hosts (Figure 6D,E). 24–36 hr post T cell transfer into immunized hosts, WT OTII cells in WT hosts migrated from the T cell zone into either the peripheral regions of the dLN (Figure 6D,E). However, WT OTII cells transferred into Cxcl9−/− hosts remained in the T cell zone, but showed some migration toward the medullary region, where predominantly CXCL10 was induced (Figure 6D,E and Figure 4F). WT OTII cells transferred into Cxcl10−/− hosts also remained in the T cell zone, but conversely, showed some migration toward the interfollicular areas of the dLN, where predominantly CXCL9 was induced (Figure 6D,E and Figure 4F). Combined, these data highlight the importance of intranodal migration during Th1 cell responses and the requirement for CXCR3 ligands, expressed by hematopoietic and stromal cells, in directing movement of T cells out of the T cell zone of dLNs for maximal Th1 cell differentiation.

Figure 6. CXCR3 ligands determine intranodal location of newly activated OTII cells following immunization.

Figure 6

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(A) Immunized and unimmunized WT hosts received Actin-GFP WT or Cxcr3−/− OTII cells. T cell location 24–36 hr post T cell transfer. (B–C) Unimmunized (top panels) and immunized (lower panels; 2 representative LNs shown) WT hosts received co-transferred labeled WT (CMTMR, orange) and Cxcr3−/− (CMFDA, green) OTII cells. T cell location 24–36 hr post T cell transfer. Regions of LN were determined as indicated in Figure S5. (B) Representative snapshots of WT and Cxcr3−/− location in dLNs. (C) Quantification of WT and Cxcr3−/− OTII cell location in dLN 24–36 hr post T cell transfer. † indicates p<0.05; * indicates p<0.001 between WT and Cxcr3−/− OTII cells. (D–E) Immunized and unimmunized WT, Cxcl9−/−, and Cxcl10−/− hosts received transferred WT Actin-GFP OTII cells. T cell location 24–36 hr post T cell transfer. (D) Representative snapshots of WT and Cxcr3−/− location in dLNs. (E) Quantification of WT OTII cell locations in indicated hosts with and without immunization. * indicates p<0.05 between WT and Cxcl9−/− hosts; † indicates p<0.05 between WT and Cxcl10−/− hosts. Bar indicates 200 µm.

CXCR3 receptor-ligand interactions promote Th1 cell differentiation in response to viral infection

While the DC transfer and immunization protocols outlined above allow for the discrimination of factors important for the differentiation of Th1 cells during a synchronized T cell response, it remained important to determine if these mechanisms are relevant during a response to an intracellular pathogen, such as lymphocytic choriomeningitis virus (LCMV), that induces a strong Th1-type immune response (Varga and Welsh, 2000). To address this, we evaluated the endogenous antigen-specific CD4+ T cell response in WT, Cxcl9−/−, Cxcl10−/−, or Cxcr3−/− mice at the peak of acute LCMV infection. T cells responding to the dominant CD4+ T cell epitope for LCMV were detected using MHC II restricted gp66 tetramers (Moon et al., 2011; Oxenius et al., 1995) (Figure 7A). Within the gp66tet+CD44+ population, the frequency of IFN-γ+TNF-α+ cells was determined. Endogenous LCMV-specific CD4+ T cells in Cxcl9−/− and Cxcl10−/− mice displayed a deficiency in maximal Th1 cell differentiation (Figure 7B,C). IFN-γ production by gp66tet+CD44+ cells in Cxcr3−/− mice was even further reduced, suggesting CXCR3 was required on cells other than CD4+ for Th1 cell responses. To determine the importance of CXCR3 exclusively on CD4+ cells, the frequency of IFN-γ+TNF-α+ cells following LCMV infection was examined in mixed BM chimeras where WT and Cxcr3−/− BM was used to reconstitute irradiated Rag1−/− mice. Again, Cxcr3−/− antigen-specific CD4+ T cells displayed a reduced frequency of IFN-γ production, compared to WT cells in the same hosts (Figure 7D,E). Together, these data validate our immunization models, indicating that CXCR3 receptor-ligand interactions optimizes Th1 differentiation during a response to a natural infectious pathogen.

Figure 7. CXCR3 is required for maximal endogenous antigen-specific Th1 cell differentiation during infection.

Figure 7

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8 days following i.v. LCMV infection, splenocytes from mice were harvested, restimulated, and tetramer enriched for detection of (A) LCMV gp66 tetramer-specific cells and (B,C) IFN-γ and TNF-α production. (D,E) BM chimeras of mixed WT and Cxcr3−/− BM in Rag1−/− hosts were infected with LCMV and harvested for detection of IFN-γ and TNF-α production from LCMV-tetramer-positive cells. Data are representative of 2 independent experiments (n=4).

DISCUSSION

The differentiation of naïve CD4+ T cells into Th cell subsets in LNs draining sites of infection and inflammation determines the type (e.g., Th1, Th2, Th17) of immune response that a pathogen or foreign antigen will elicit. We chose to study the CXCR3 chemokine system in Th1 cell development because of all the chemokine receptors, CXCR3 is most associated with Th1 cells (Groom and Luster, 2011a). Expression of CXCR3 by newly activated CD4+ T cells correlated with their ability to produce IFN-γ and was required for optimal effector cytokine responses. Our findings suggest that a chemokine-dependent loop exists between priming CD4+ T cells and antigen-presenting DCs, as expression of CXCL9 and CXCL10 was increased in LNs that received antigen-specific T cells. By tracking REX3++ chemokine-expressing DCs, we have shown that they have higher expression of the activation markers CD86 and CD40. Signaling through CD40 has been shown to increase the expression of CXCL10 by DCs, and CD40L is increased on CXCR3-expressing T cells, indicating that this chemokine pathway may be important for the licensing of DCs during CD4+ T cell priming (Quezada et al., 2004). Alternatively, the upregulation of CXCL10 may be through the production of Type I and/or Type II IFNs produced by antigen-presenting DCs themselves, transferred T cells, or by accessory cells, such as NK cells or pDCs, required for CD4+ Th1 cell effector differentiation (Cervantes-Barragan et al., 2012; Martin-Fontecha et al., 2004).

We have shown that this chemokine-dependent loop between priming CD4+ T cells and antigen-presenting DCs leads to differences in the intranodal behaviour of CXCR3-competent and Cxcr3-deficient T cells during the second phase of T cell priming, which is important for T cell IFN-γ production (Hugues, 2010). Cxcr3−/− OTII formed fewer and shorter interactions with CXCL10+ (REX3+) DCs, indicating CXCL10-dependent tethering is important for T cell outcome. Interestingly, this difference appeared to occur prior to the massive upregulation of CXCR3 surface on WT OTII cells, suggesting either initial upregulation of CXCR3 was not detected by flow cytometry due to receptor internalization or that that the low basal expression of CXCR3 expression by naïve WT cells was important for this phenotype (Rabin et al., 1999). Along with increasing the tethering of DCs to T cells, CXCL10-CXCR3 interactions may also increase synapse formation, facilitating productive communication between these cells (Friedman et al., 2006; Molon et al., 2005). CXCR3 has been shown to be upregulated in vitro during the differentiation of multiple CD4+ T cell lineages (Rabin et al., 2003; Sallusto et al., 1998), therefore, it may be that CXCR3 is required for all CD4+ effector T cells, or that, generation of other Th cell subsets have unique spatial requirements in the reactive LN that are controlled by other chemokine systems.

Our study has also established the concept that the location of T cells within the LN is important for specific Th polarization and that intranodal positioning of T cells is controlled, at least in part, by intranodal chemokine gradients. We found that CD4+ T cells move out of the T cell zone, to the outer LN following initial activation and upregulation of CXCR3 and that this process is controlled by CXCR3 ligands, which are induced predominantly in these peripheral regions of the LN. Recent studies have demonstrated that CD169+ macrophages and other APCs present in the subcapsular sinus (SCS), interfollicular and medulla regions of dLNs efficiently trap antigen following lymph-borne viral challenge (Hickman et al., 2008; Iannacone et al., 2010). Importantly, loss of these macrophages results in reduced Type I IFN production, suggesting a potential stimulus for CXCL9 and CXCL10 production following infection (Iannacone et al., 2010). Therefore, CXCR3 upregulation by recently activated T cells allows them to move to areas where they are poised to interact with antigen-presenting SCS macrophages or DC subsets, which have received cross-presented antigen, potentially providing T cells increased antigen stimulation and/or unique cytokine signals (Hickman et al., 2008).

Several studies have indicated the importance for CD8+ T cells to leave the T cell zone and migrate into peripheral LN areas to interact with pathogen-loaded APCs (Hickman et al., 2008). Our study has expanded on these findings indicating that this process of intranodal relocation is also important for the development of Th1 CD4+ cells. In addition, we now have shown that this movement is regulated by the ligands for CXCR3, and using the REX3 Tg mouse, we have identified the location and cell types expressing the chemokines responsible for this redistribution. Thus, our data suggests that the CXCR3 chemokine system is a key mediator of T cell peripheralization in the reactive LN following infection and immunization. Since CXCL10 is induced by TLRs and Type I interferon, our data offer an explanation for why pathogen-activated macrophages and DCs in the LN periphery produce CXCL10 leading to the subsequent peripheralization of T cells. Further, as has been demonstrated in peripheral tissue (Groom and Luster, 2011a; Nakanishi et al., 2009), IFN-γ brought to these regions by early T cell emigrants likely amplifies the recruitment signal through the induction of CXCL9 and more CXCL10.

As Cxcr3 is a direct transcriptional target of Tbet (Tbx21), it is likely that our findings extend to cell fate decisions between effector and memory differentiation. Recently, CD4+ T cells lacking Tbet have been described to preferentially differentiate into memory cells (Marshall et al., 2011; Pepper et al., 2011). Our study offers an explanation for these finding, suggesting that Tbet-dependent CXCR3 expression predisposes cells to become effector CD4+ Th1 cells, as opposed to memory cells. Similar observations have recently been made for CD8+ T cells where Cxcr3-deficient CD8+ Tcells locate to different areas of the spleen and preferentially become memory cells over effectors (Hu et al., 2011; Kohlmeier et al., 2011; Kurachi et al., 2011).

Our findings thus demonstrate unique spatial requirements for CD4+ T cells during differentiation, which could have important implications in the design of potent Th1 cell-inducing vaccines. Our results, as well as the tools used to obtain them, lay the foundation for future studies aimed at identifying other factors that regulate Th1 cell responses in dLNs and peripheral inflamed tissues, and assessing the importance of the CXCR3 chemokine system in T cell fate decisions. Indeed, the development of the REX3 Tg mouse should be a valuable tool for the analysis of productive immune responses against infectious pathogens and for the rational design and analysis vaccines.

EXPERIMENTAL PROCEDURES

Mice

C57BL/6 (BL/6), CD90.1 and OTII mice were obtained from Jackson Laboratory. REX3 Tg mice in the C57BL/6 background were generated in our laboratory. All mice, including Cxcr3−/− (Hancock et al., 2000), Cxcl9−/− (Park et al., 2002) and Cxcl10−/− (Dufour et al., 2002) mice in the C57BL/6 background were housed under specific pathogen-free conditions. All infectious work was performed in designated BL2+ workspaces. All procedures were approved by the Massachusetts General Hospital Subcommittee on Research and Animal Care or by Harvard Committee on Microbiological Safety. See Supplemental Methods for details of other mouse strains used.

Cell preparation and Immunizations

DCs were CD11c+ purified (Miltenyi) from mice implanted with Flt-3L B16 cells and pulsed with 10 µM OVA protein (Worthington) for 1 hr prior to 1 µg/ml LPS and PolyI:C (InvivoGen) for another 1 hr. Tracked cells were labeled with for 15 min at 37°C with 2 µM chloromethylfluorescein diacetate (CMFDA; Molecular Probes) prior to injection of 5×105 DCs into the footpad. Mice were immunized with 20 µg/ml OVA with 1 ng/ml LPS and PolyI:C. 24 hr post DC transfer or immunization, mice were given immunomagnetic selected 5×106 CD4+CD62L+ (Miltenyi) T cells prepared from OTII mice. To synchronize T cell responses, animals received 100 µg CD62L monoclonal antibody Mel-14 (100 µg per mouse; BioXcell). To block T cell egress 1 mg/kg FTY720 (Cayman Chemicals) was given i.p 12 hr following T cell transfer. For imaging and localization experiments transferred cells were either labeled with CMFDA or CMTRA as above, alternatively Actin-GFP or Actin-RFP crossed to OTII Tg mice were used.

Generation of REX3 Tg mice

Targeting constructs for Cxcl9-RFP and Cxcl10-BFP were inserted into the RP24-164O11 BAC (CHORI), which contained the Cxcl9 and Cxcl10 genes. See Supplemental Methods for details.

Cell Isolation and Flow Cytometry

Popliteal (draining) and brachial (non-draining) LNs were harvested, pooled and massaged using tweezers to single cell suspensions. For staining antibodies see Supplemental Methods. For analysis of polyfunctional T cell responses, cells were incubated with 20 µg/ml OVA (323–339) peptide (Peptides International) and 2 µg/ml αCD28 (Biolegend). Following 1 hr, 10 µg/ml Brefeldin A (GolgiPlug; BD Biosciences) was added for an additional 3 hr. Following surface staining, cells were fixed and permeablized using Fix&Perm kit (Invitrogen), and stained for intracellular cytokines. Cells were resuspended in FACS buffer (2% FCS in PBS) and acquired on an LSR Fortessa flow cytometer (BD Biosciences) and analyzed using FlowJo software (Tree Star).

Immunofluorescence Staining

LNs were harvested into PLP buffer (0.05 M phosphate buffer containing 0.2 M L-lysine [pH 7.4], 2 mg/ml NaIO4, 10 mg/ml paraformaldehyde) fixed for 5–12 hr, and dehydrated in 30% sucrose prior to embedding in OCT freezing media (Sakura Fineteck). 16 µm frozen sections were cut on a CM3050S cryostat (Leica). Sections were blocked in PBS containing 0.1 % Triton X-100 (Sigma) and 10 % goat serum (Jackson Immunoresearch) and stained in PBS (0.01 % Triton X-100 and 5 % goat serum). Images were acquired on a LSM510 confocal microscope (Carl Zeiss Mircoimaging). T cell regions were identified and labeled using immunostaining of B cell follicles and LN architecture, as described in Figure S5. Regions and cells were defined using IMARIS image analysis software (Bitplane), and center point spots were included in snapshot images.

LCMV infection and detection of gp66+ tetramer positive cells

Mice were given 104 focus forming units of (Armstrong) LCMV i.v. 8 days prior to harvest. Splenocytes from LCMV infected mice were restimulated with gp61-80 (AnaSpec) for 4 hr in the presence of Brefeldin A. Tetramer+ cells were labeled with PE-gp66 tetramer for 1 hr and enriched using anti-PE magnetic microbeads (Moon et al., 2007).

Multiphoton Intravital Microscopy and Image Analysis

Performed as previously described (Mempel et al., 2004) and in Supplemental Methods.

Statistical Analysis

Paired two tailed Student’s t tests were used for data analysis and generation of p values, for experiments when T cells from WT and Cxcr3−/− OTII T cells were co-transferred (GraphPad Software). ANOVA using post-tukey test for multiple comparisons was used for experiments comparing more than 2 samples. P is shown for all significant (p<0.05) analyses. All data are represented as mean with individual data points representing individual samples while time course data show mean with SEM error bars. Bar graph data show standard deviation error bars.

Supplementary Material

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HIGHLIGHTS.

  • CXCR3 is required on CD4+ T cells for optimal Th1 cell differentiation in vivo.

  • CXCL9 and CXCL10 are both required for optimal Th1 cell IFN-γ production in vivo.

  • CXCR3 enhances T cell-DC interactions in LNs for optimal Th1 cell differentiation.

  • CXCR3 positions priming T cells in LNs for optimal Th1 cell differentiation.

ACKNOWLEDGMENTS

Work was supported by the NIH grants CA069212 (to A.D.L.) and AI078897 (to U.H.v.A). J.R.G is supported by the NH&MRC, Australia (Fellowship 516791). J.H.S. is supported by a Samsung Scholarship. We thank Dr. David Alvarez for assistance with LCMV infections, Dr. Joshua Farber for the Cxcl9−/− breeding pair, and Dr. Craig Gerard for the Cxcr3−/− breeding pair.

Footnotes

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