Expression of chemokine receptor CXCR3 on T cells affects the balance between effector and memory CD8 T-cell generation

Author Summary

Killer T cells, also known as CD8 T cells, are essential for protection against viruses and many other intracellular pathogens. In response to an infection, pathogen-specific CD8 T cells rapidly proliferate and differentiate into a heterogeneous population of effector cells, the majority of which undergo apoptosis after the infection is controlled. However, a small fraction of the effector cells survive, becoming self-renewing and long-lived memory T cells that provide rapid protection against subsequent challenges from the same pathogen. Because a naive CD8 T cell can differentiate into both short-lived effector cells and long-lived memory cells, identifying the signals and processes that affect this cell fate is of great interest. Chemokines, a family of small proteins that act as chemoattractants to mobilize lymphocytes, regulate the localization of CD8 T cells within lymphoid organs and their subsequent recruitment to sites of infection and inflammation. In this study, we examine the role of one chemokine receptor, CXCR3, on the fate of CD8 T cells. We show that CXCR3 is up-regulated on the surface of activated CD8 T cells and guides these cells toward areas of the spleen that are rich in antigens, thereby reducing the quantity and quality of the long-lived memory T-cell population. These findings provide evidence that positioning of T cells within specific microenvironments of lymphoid tissues can affect their differentiation, and hence influence the overall quality of the immune response.

In the past decade, researchers have significantly advanced our understanding of CD8 T-cell differentiation (1). Elegant lineage tracing studies have established that the progeny of a single CD8 T cell can differentiate into both short-lived effector cells and long-lived memory cells (2). The proportion of T cells that enter each differentiation pathway is influenced by several factors (3). For example, increased exposure to antigen and inflammatory stimuli encourages CD8 T cells to differentiate into short-lived effector cells, whereas decreased exposure to these stimuli enhances the production of long-lived memory cells. However, very little is known about how chemokines and other cues that guide lymphocyte trafficking and positioning regulate access to antigen and inflammatory stimuli, and hence affect the balance between terminally differentiated effector cells and long-lived memory cells.

Naive CD8 T cells, mature but inactive killer T cells that have not yet encountered their respective antigens, continuously circulate through peripheral lymphoid organs, such as the spleen and lymph nodes, in search of an antigen capable of activating their pathogen-fighting functions. Within these lymphoid tissues, naive CD8 T cells are mostly localized in so-called “T zones” through the binding of homeostatic chemokines CCL19 and CCL21 to the cell-surface receptor CCR7 (4). Activation of T cells through T-cell receptor engagement sets into motion a series of programmatic changes that includes down-regulation of CCR7 and up-regulation of a number of inflammatory chemokine receptors, which then allow effector CD8 T cells to traffic to sites of inflammation, where ligands for these receptors are expressed. The ligands for CXCR3, the inflammatory chemokine receptor on which this study focuses, are CXCL9 and CXCL10. Using mice infected with lymphocytic choriomeningitis virus (LCMV) as a model, we find that the expression of both ligands is up-regulated soon after the infection of cells in vivo. Furthermore, the CXCR3 receptor itself is up-regulated as soon as the third day after infection on LCMV-specific CD8 T cells and is uniformly expressed by day 5 on both terminal effector and memory precursor CD8 T cells. Using both loss-of-function and gain-of-function approaches, we asked whether CXCR3 plays a role in effector CD8 T-cell differentiation and fate determination. We adoptively cotransferred equal numbers of differentially marked WT and CXCR3 KO virus-specific T cells into naive hosts and followed the differentiation of these cells at various times after infection. We found that on the eighth day after infection, at the peak of CD8 T-cell expansion, terminally differentiated WT cells consistently outnumbered CXCR3 KO cells. In contrast, on the 15th day after infection, once the majority of terminally differentiated effector CD8 T cells had died, more CXCR3 KO memory precursor cells remained than WT cells. In complementary overexpression studies, retroviral-mediated restoration of CXCR3 expression in CXCR3 KO CD8 T cells led to the development of more terminal effector cells at the expense of memory precursor cells. These results suggest that CXCR3 expression on CD8 T cells affects the balance between terminal effector and memory precursor cells. In a long-term follow-up study of coadoptively transferred WT and CXCR3 KO virus-specific T cells, we consistently found more CXCR3-deficient memory CD8 T cells than WT cells. In addition, CXCR3 KO CD8 T cells transitioned more rapidly into a more phenotypically mature memory T-cell population and had a larger recall response than WT cells, suggesting that in addition to having an early impact on the number of memory precursor cells, CXCR3 expression affects the quantity and quality of long-lived memory CD8 T cells, which could potentially influence the body's ability to ward off a similar infection years down the road. This finding has important implications for vaccine design, where the ultimate goal is to produce a large number of high-quality memory CD8 T cells that can fight off the invading organism before too much damage is inflicted on the infected person.

Our investigation into the mechanism of this process revealed that during the course of infection, effector CD8 T cells colocalized with antigen within lymphoid organs in a CXCR3-dependent manner. On the third to fifth days after infection, the majority of WT virus-specific T cells were found in areas of the spleen where both viral antigen and CXCL9 are abundant. In contrast, most of the CXCR3 KO effector CD8 T cells were located within the T-zone areas of the spleen, away from antigens. These results suggest that the expression of CXCR3 increased exposure to antigen and inflammation, resulting in fewer memory T cells. To assess whether forced retention of effector CD8 T cells within the splenic T zone areas and away from inflammation would have the same effect, we studied virus-specific T cells that constitutively expressed the chemokine receptor CCR7 [CCR7 transgenic (Tg)]. In contrast to normal T cells, which down-regulate CCR7 expression on activation, the CCR7 Tg T cells maintain surface expression of a functional receptor. We found that during the early course of infection, CCR7 Tg virus-specific CD8 T cells remained within the splenic T zone and away from the infected cells. Furthermore, a greater proportion of CCR7 Tg T cells compared with WT cells developed into memory precursor cells. These results provide strong evidence that localization of T cells within the microenvironment of lymphoid organs during the course of an infection can affect CD8 T-cell differentiation as well as the magnitude and quality of the long-lived memory T-cell population.

In this study, we showed that alterations in chemokine receptor expression redistribute effector CD8 T cells within lymphoid tissues and have an impact on the differentiation and generation of memory CD8 T cells (Fig. 1). Further studies on the role of various chemokine receptors and other guidance cues will advance our understanding of the interplay between these signals, the localization within tissue microenvironments, and the shaping of the effector and memory T-cell responses. In addition, it will be interesting to determine what role chemokine receptors play in the repeated exposure of CD8 T cells to antigen during a chronic viral infection and whether these receptors have an impact on T-cell exhaustion. Our findings suggest that chemokine receptors like CXCR3 may provide a target for vaccination methods aimed at generating a more robust memory CD8 T-cell response as protection against future infections or cancer.

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Fig. 1.

Contribution of the CXCR3 receptor to CD8 T-cell differentiation. In the spleen, the lymphocytes (B and T cells) are separated from the red pulp areas by a cellular layer, termed the marginal zone, consisting mostly of macrophages that act as a first line of defense against blood-borne pathogens. In our study, marginal zone macrophages are infected with LCMV and express the CXCR3 receptor ligand CXCL9. Activated effector CD8 T cells express CXCR3 and move toward CXCL9 in marginal zone areas, where they are exposed to antigen. Consequently, WT CD8 T cells encounter more antigen and inflammation than CXCR3-deficient cells. As a result, CXCR3-expressing cells tend to become short-lived effector cells instead of long-lived memory precursors. In contrast, CD8 T cells that lack CXCR3 are localized away from marginal zone areas and, as a result, are less exposed to antigen and inflammatory stimuli. This leads to the generation of more long-lived memory CD8 T cells that are qualitatively better than WT cells.