CXCR4 is critical for CD8⁺ memory T cell homeostatic self-renewal but not rechallenge self-renewal

Abstract

Central memory (CM) CD8⁺ T cells “remember” prior encounters because they maintain themselves through cell division in the absence of ongoing challenge (homeostatic self-renewal) as well as reproduce the central memory fate while manufacturing effector cells during secondary antigen encounters (rechallenge self-renewal). We tested the consequence of conditional deletion of the bone marrow (BM) homing receptor CXCR4 on antiviral T cell responses. CXCR4-deficient CD8⁺ T cells have impaired memory cell maintenance due to defective homeostatic proliferation. Upon rechallenge, however, CXCR4-deficient T cells can re-expand and renew the central memory pool while producing secondary effector cells. The critical BM-derived signals essential for CD8⁺ T cell homeostatic self-renewal appear to be dispensable to yield self-renewing, functionally asymmetric cell fates during rechallenge.

Introduction

A key feature of adaptive immunity is the capacity to develop long-lived memory T cells that control recurrent or persistent infection. Memory CD8⁺ T cells are heterogeneous and can be divided into two main subsets, central memory (CM) and effector memory (EM). CM (CD44^hi CD62L^hi) cells, which preferentially home to secondary lymphoid organs, have longer life spans and greater capacity for homeostatic proliferation than EM (CD44^hi CD62L^lo) cells (1). In the absence of rechallenge, CM CD8⁺ T cells slowly replenish themselves to maintain stable size of the cell population. Upon rechallenge, CM T cells produce differentiated effector cells while renewing the CM cell fate through asymmetric cell division (2), thereby avoiding depletion of cells needed to respond to subsequent or persistent challenge (3).

CM T cells preferentially accumulate and undergo homeostatic proliferation in the BM (4-6). The functional consequences of BM homing on homeostatic self-renewal or rechallenge self-renewal, however, have not been directly evaluated. CXCR4 binds to CXCL12 and has an essential role in homing of HSCs to the BM (7). Here we analyzed the impact of the lack of CXCR4 on CD8⁺ T cell responses to LCMV infection. CM CXCR4-deficient T cells exhibit defective homeostatic self-renewal, which correlates with impaired homing to the BM. Upon rechallenge, however, CM CXCR4-deficient T cells can proliferate efficiently and differentiate while self-renewing. Thus, homeostatic self-renewal and re-challenge self-renewal are mechanistically separable regenerative properties of memory T cells.

Materials and Methods

Mice and infections

All animal work was performed in accordance with Columbia University Institutional Animal Care and Use Committee guidelines. CXCR4^F/F mice (8) expressing or not expressing Granzyme B-Cre (9) were infected as intact animals. For adoptive transfer experiments, naive CD8⁺ P14 T cells were sorted from WT Thy1.1/1.1 and CXCR4^F/F-CD4-Cre⁺ Thy1.1/1.2 mice. From each genotype, 10³ cells were transferred i.v. into WT Thy1.2/1.2 recipients one day prior to infection. Mice were infected with 2 × 10⁵ PFU of LCMV Armstrong strain by i.p. injection. For rechallenges, 5 × 10⁵ CFU Listeria monocytogenes expressing GP33–41 (gp33) were injected i.v. Results depict the percentage of CXCR4-deficient P14 T cells among transferred cells at the indicated time post infection when normalized to the proportion of CXCR4-deficient P14 T cells among transferred cells at the time of transfer. To label of sinusoidal lymphocytes, mice were injected intravenously with 1μg of anti-CD45.2 mAb coupled to PE (BD Biosciences) and sacrificed 2 min after mAb injection as previously described (10). To asses proliferation, mice were treated with 2mg of BrdU (Sigma-Aldrich) i.p. every 2 d for 15 d prior to tissue harvest and analysis.

Flow cytometry

Single cell suspensions of spleen, BM and lymph nodes (LN, pool of mesenteric and subcutaneous) were stained with a LIVE/DEAD Fixable Dead Cell Stain Kit (Invitrogen) prior to staining with indicated antibodies. H-2D^b GP33-41 tetramer was used to identify LCMV-specific CD8⁺ T cells. The following mAb from BD Biosciences, BioLegend or eBioscience were used: anti-CD4 (RMA4-5), anti-CD8a (53-6.7), anti-CD19 (1D3), anti-CD44 (IM7), anti-CD62L (MEL14), anti-CD127 (A7R34), anti-KLRG1 (2F1), anti-Thy1.1 (Ox-7), and anti-Thy1.2 (53-21). BrdU incorporation was assessed using a BrdU Flow Kit (BD Biosciences) according to manufacturer’s instructions. Cells were analyzed on an LSR II (BD Biosciences) and data were analyzed with FlowJo software (Treestar).

Statistical analyses

Statistical analyses were performed using 2-tailed t-tests run on Prism Version 5 (GraphPad) software. Levels of significance are expressed as follows: *p < .05, **p < .01, and ***p < .001.

Results and Discussion

CXCR4 promotes homing of naive and CM CD8⁺ T cells to the BM

Both HSCs and CM T cells face similar demands of long-term homeostatic renewal and the capacity to produce differentiated progeny while self-renewing the less differentiated fate. We, therefore, tested whether CM T cells, like HSCs (11), require BM homing to ensure durability during homeostasis and differentiation. We used mice with a conditional allele of CXCR4 (8) to assess the role of BM homing in CD8⁺ T cell responses to LCMV infection. CXCR4^F/F mice were bred to CD4-Cre mice to avoid defects in double negative thymocytes that were observed in CXCR4^F/F-Lck-Cre⁺ mice (12). Compared to CXCR4-proficient animals, the BM of CXCR4^F/F-CD4-Cre⁺ mice contained approximately 8-fold fewer naive and 14-fold fewer CM CD8⁺ T cells relative to wild-type (WT) controls (Fig. 1A). The abundance of CXCR4-deficient EM CD8⁺ T cells in the BM was normal, consistent with prior studies showing impaired migration of CM but not EM phenotype cells toward a CXCL12 gradient (4).

FIGURE 1.

CXCR4 controls homing of naive and CM CD8⁺ T cells to the BM. Flow cytometric analysis of CD8⁺ T cells populations in WT and CXCR4^F/F-CD4-Cre⁺ mice. (A) Absolute numbers of Naive, CM, and EM CD8⁺ T cells in indicated organs. Data are mean ± SEM of 7 mice in 3 independent experiments. (B) Sinusoidal staining of BM cells. The frequencies of total sinusoidal CD8⁺ T cells (“CD8”) and the indicated CD8⁺ T cell subsets (Naive = CD44^loCD62L^hi; CM = CD44^hiCD62L^hi; EM = CD44^hiCD62L^lo) for WT and CXCR4 KO are depicted. Data are mean ± SEM of 5 mice in 3 independent experiments.

CXCL12-abundant reticular (CAR) cells are an important BM stromal source of homeostatic cytokines and a putative niche that could trans-present IL-15 to T and NK cells in a cognate fashion (5, 6, 13-19). We therefore asked whether CXCR4-deficient T cells are even capable of accessing BM parenchyma or if instead are simply circulating in BM capillary sinusoids. Intrasinusoidal cells were identified by injection of CD45.2 antibody (10). Most CXCR4-proficient cells were in the BM parenchyma proper, whereas the majority of naive CXCR4-deficient CD8⁺ T cells and a substantial fraction of CXCR4-deficient memory CD8⁺ T cells were intrasinusoidal (Fig. 1B). CXCR4 may thus play a critical role in homing of naive and memory CD8⁺ T cells into the BM parenchyma.

CXCR4 is dispensable for CD8⁺ T cell effector expansion

To assess the antiviral response, we generated peripheral chimeras by transferring WT Thy1.1/1.1 and CXCR4^F/F-CD4-Cre⁺ (KO) Thy1.1/1.2 P14 TCR-transgenic T cells into WT Thy1.2/1.2 recipients, prior to infection of recipients with LCMV. After infection, we evaluated recovery of KO P14 T cells normalized to the proportion of KO P14 T cells present on the day of adoptive transfer. At d 8 after infection, there was a specific defect in CXCR4-deficient P14 T cells populating the BM, but no significant defect in blood, spleen or LN (Fig. 2A, 2B).

FIGURE 2.

CXCR4 is dispensable for CD8⁺ T cell effector functions but required for memory cell maintenance. (A and B) Frequencies of KO P14 T cells at indicated times post LCMV infection in blood (A) and organs (B). P14 donor T cells were identified using Thy1 congenic disparity. KO P14 T cell recovery is normalized to the frequency of KO P14 T cells on the day of reconstitution. (C and D) Number of gp33 tetramer⁺ CD8⁺ T cells in blood (C) and organs (D) of CXCR4^F/F-GzmB-Cre⁻ (WT) and CXCR4^F/F-GzmB-Cre⁺ (KO) mice at indicated times post LCMV infection. Data are mean ± SEM of 25 mice in 3 independent experiments (A), are mean ± SEM of at least 6 mice per time point in 3 independent experiments (B), are mean ± SEM of at least 5 mice per group and are representative of 4 independent experiments (C), are mean ± SEM of at least 6 mice in 2 independent experiments (D).

CD4-Cre-expressing P14 TCR-transgenic T cells have largely completed gene deletion prior to transfer (20), while Granzyme B-Cre-expressing T cells will undergo most deletion rapidly following antigen activation (21). We therefore analyzed endogenous T cell responses to virus in mice with deletion of CXCR4 following infection. Tetramer staining of gp33-specific T cells in CXCR4^F/F-Granzyme (Gzm) B-Cre⁺ versus CXCR4^F/F-GzmB-Cre⁻ mice at d 8 revealed little difference in relative numbers of tetramer⁺ CD8⁺ T cells in the blood (Fig. 2C) or in the absolute numbers of gp33 tetramer⁺ CD8⁺ T cells in other organs (Fig. 2D). Together these results suggest that CXCR4-deficient T cells can undergo normal effector cell clonal expansion whether CXCR4 is deleted prior to or following antigen challenge. It is possible that the pre-challenge loss of CXCR4 in CD4-Cre-expressing cells compared to acute loss of CXCR4 in Granzyme B-Cre-expressing cells may explain the difference in onset of the BM homing phenotype.

CXCR4 maintains the CD8⁺ T cell memory pool

Serial analysis of mice that had received WT P14 T cells and CXCR4^F/F-CD4-Cre⁺ P14 T cells revealed that the ratio of WT:KO P14 T cells was stable in the blood until d 30, after which CXCR4-deficient cellularity began to decline. By d 140, KO P14 T cells dropped to 40% of their original representation (Fig. 2A). To rule out the possibility that recipient mice preferentially rejected CXCR4-deficient P14 cells, we followed the LCMV response of CXCR4^F/F-GzmB-Cre⁺ versus CXCR4^F/F-GzmB-Cre⁻ littermate controls using gp33 tetramers. Similar to the analysis of P14 transgenic T cells, we found that CXCR4-deficient tetramer⁺ cells were present in the blood stably until d 30 (Fig. 2C). By d 140, however, CXCR4^F/F-GzmB-Cre⁺ mice had 3-fold fewer gp33 tetramer⁺ CD8⁺ T cells in the blood relative to CXCR4^F/F-GzmB-Cre⁻ mice.

In both the P14 chimeras and the CXCR4^F/F-GzmB-Cre⁺ mice, we observed a pronounced impairment of memory CD8⁺ T cells in the BM after resolution of infection. In addition to the BM defect, there was also a defect in representation of CXCR4-deficient memory CD8⁺ T cells in the spleen and lymph nodes (LN) (Fig. 2B, 2D). The results so far suggest a critical role for CXCR4 and BM homing in the long-term maintenance of the memory CD8⁺ T cell population, but a limited role for homeostatic BM conditioning on the effector expansion of a naive T cell upon antigen challenge.

CXCR4 promotes homeostatic self-renewal of CM CD8⁺ T cells

After viral clearance, CM CD8⁺ T cells gradually and steadily express increasing abundance of the transcription factor Eomesodermin, and its target gene CD122, a key receptor of IL-15 responsiveness (22-24). The phenotype of Eomesodermin-deficient memory CD8+ T cells, which also have reduced expression of CXCR4, is marked by progressively poorer occupancy of BM and a selective loss of CM CD8⁺ T cells (13). At d 140 following LCMV infection, we found that the proportion of CXCR4-deficient CM cells was most obviously deficient in the BM (Fig. 3A), although the total number of CXCR4-deficient CM was reduced in both BM and in LN, another characteristic location of CM cells (Fig. 3B). As might be predicted from their similarity to cells deficient in Eomesodermin or IL-15 signaling (13, 14, 16, 19), we found that CXCR4-deficient CM T cells undergo reduced homeostatic proliferation as assessed by BrdU incorporation, but without obvious evidence of increased apoptosis (Fig. 3C). The defect in homeostatic proliferation was more pronounced for CXCR4-deficient CM than EM CD8⁺ T cells (Fig. 3C, 3D). Although impaired homeostasis of CXCR4-deficient memory cells may be due to defective BM homing and reduced homeostatic division of CM cells, it ultimately seems to impact the entire CD8⁺ memory T cell pool since total EM cell numbers are also significantly reduced in BM and spleen (Fig. 3B).

FIGURE 3.

CXCR4 promotes CM CD8⁺ T cell homeostatic proliferation and maintenance. (A) Frequency of CM (CD44^hiCD62L^hi) cells among gp33 tetramer⁺ CD8⁺ T cells in CXCR4^F/F-GzmB-Cre⁻ (WT) and CXCR4^F/F-GzmB-Cre⁺ (KO) mice at indicated time and organ post LCMV infection. Virtually all non-CM gp33 tetramer⁺ CD8⁺ T cells were EM cells (CD44^hiCD62L^lo). (B) Number of gp33 tetramer⁺ CD8⁺ T cells (Total, CM, and EM) in the indicated organs of CXCR4^F/F-GzmB-Cre^- (WT, filled bars) and CXCR4^F/F-GzmB-Cre⁺ (KO, open bars) 140 d post LCMV infection. Data are mean ± SEM of at least 5 mice per group in 2 independent experiments at d 8 and 6 mice in 2 independent experiments for d 140. (C and D) Frequency of annexin-V⁺ and BrdU⁺ cells among splenic CD8⁺ gp33 tetramer⁺ CM (C) or EM (D) T cells 140 d post LCMV infection. Data are mean ± SEM of 6 mice in 2 independent experiments.

CXCR4 is not required for rechallenge self-renewal of CM CD8⁺ T cells

CM CD8⁺ T cells function much like adult stem cells, homeostatically maintaining themselves in the absence of recurrent infection and asymmetrically producing differentiated progeny while renewing themselves during rechallenge (2). Despite the critical role of CXCR4 and BM homing in maintaining homeostatic proliferation of CM CD8⁺ T cells, we found that CXCR4-deficient memory CD8⁺ T cells underwent normal and possibly heightened re-expansion upon rechallenge with Listeria monocytogenes expressing gp33 (Fig. 4A, 4B). Importantly, CXCR4-deficient memory CD8⁺ T cells retained the capacity to maintain a population of CD44^hiCD62L^hi CM cells while generating CD44^hiCD62L^lo secondary effector cells (Fig. 4C).

FIGURE 4.

Normal rechallenge self-renewal of CXCR4-deficient memory CD8⁺ T cells. (A) At d 140 post LCMV infection, WT and KO mice were rechallenged with Listeria monocytogenes expressing gp33. Numbers of gp33 tetramer⁺ CD8⁺ T cells per million blood cells are shown for indicated times post LCMV infection and at 5 d following rechallenge. Data are mean ± SEM of at least 5 mice per group and are representative of 2 independent experiments. (B) Fold expansion of gp33 tetramer⁺ CD8⁺ T cells during the 5 d following rechallenge. Data are mean ± SEM of at least 7 mice in 2 independent experiments. (C) Percentage of splenic CD62L^hi gp33 tetramer⁺ CD8⁺ T cells at 5 d after rechallenge. Data are mean ± SEM of at least 7 mice in 2 independent experiments.

The results presented here suggest that homeostatic and rechallenge self-renewal are differentially dependent on CXCR4 and BM homing, probably owing to differences in the signaling requirements for each type of self-renewal. Homeostatic proliferation, which seems to be CXCR4-dependent, is thought to be primarily cytokine-driven and antigen-independent (25) whereas rechallenge differentiation, which seems to be CXCR4-independent, is induced by antigen (26), occurs mainly in the spleen and LNs, and achieves functional self-renewal by asymmetric production of CM and secondary effector cells (2). In chronic infection, exhausted T cells no longer undergo homeostatic self-renewal (27) but progenitors stimulated by antigen can divide and reproduce themselves while simultaneously producing more differentiated cells that suppress viral burdens (3). The importance of rechallenge self-renewal in maintaining equilibrium with the virus is finally unveiled when continual antigen-induced division ultimately results in the collapse of both forms of self-renewal (3, 27). Recently, there has been interest in identifying stem cell-like CD8⁺ T cells with durability and protective capacity (28, 29). Our results suggest that the long-term maintenance and protective capacity of stem cell-like T cells might not be regulated by the same signals, which will be an important consideration in the design of vaccines.