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

Joyce K. Hua,b, Takashi Kagaria,1, Jonathan M. Clingana,b, and Mehrdad Matloubiana,2 aDivision of Rheumatology, Department of Medicine, and Rosalind Russell Medical Research Center for Arthritis, and bGraduate Program in Biomedical Sciences, University of California, San Francisco, CA 94143 AUTHOR SUMMARY

Killer T cells, also known as CD8 T cells, Localization terminally differentiated effector cells are essential for protection against viru- WT CD8 T cells CXCR3 KO CD8 T cells and long-lived memory cells. ses and many other intracellular patho- Naive CD8 T cells, mature but inactive

gens. In response to an infection, Red pulp killer T cells that have not yet encoun- pathogen-specific CD8 T cells rapidly tered their respective , continu- zone proliferate and differentiate into a het- Marginal ously circulate through peripheral erogeneous population of effector cells, lymphoid organs, such as the spleen and the majority of which undergo lymph nodes, in search of an after the infection is controlled. How- capable of activating their pathogen- ever, a small fraction of the effector cells fighting functions. Within these lymphoid

survive, becoming self-renewing and cell zone T tissues, naive CD8 T cells are mostly lo- long-lived memory T cells that provide calized in so-called “T zones” through AntigenAntigen WT effector CCD8D8 T celcelll B cecellll

rapid protection against subsequent the binding of homeostatic chemokines MMUNOLOGY CXCL9CXCL9 CXCR3CXCR3 KOKO effector CCD8D8 challenges from the same pathogen. Be- Differentiation CCL19 and CCL21 to the cell-surface cause a naive CD8 T cell can differenti- WT CD8CD8 T cells CXCR3CXCR3 KOKO CD8CD8 T cellscells receptor CCR7 (4). Activation of T cells Contraction/MemoryContraction/Memory CContraction/Memoryontraction/Memory ate into both short-lived effector cells ExpansionExpansion GGenerationeneration ExpansionExpansion Generation through T-cell receptor engagement sets and long-lived memory cells, identifying into motion a series of programmatic the signals and processes that affect this NaiveNaive EffectorEffector Memory changes that includes down-regulation of WTWT Short-liveShort-livedd cell fate is of great interest. Chemokines, CXCR3CXCR3 KOKO MemorMemoryy precursoprecursorr QualityQuality CCR7 and up-regulation of a number a family of small proteins that act as of inflammatory chemokine receptors, chemoattractants to mobilize lympho- Fig. 1. Contribution of the CXCR3 receptor to which then allow effector CD8 T cells to cytes, regulate the localization of CD8 T CD8 T-cell differentiation. In the spleen, the traffic to sites of inflammation, where (B and T cells) are separated from the cells within lymphoid organs and their ligands for these receptors are expressed. red pulp areas by a cellular layer, termed the fl subsequent recruitment to sites of in- marginal zone, consisting mostly of macrophages The ligands for CXCR3, the in amma- fection and inflammation. In this study, that act as a first line of defense against blood- tory chemokine receptor on which this we examine the role of one chemokine borne pathogens. In our study, marginal zone study focuses, are CXCL9 and CXCL10. receptor, CXCR3, on the fate of CD8 macrophages are infected with LCMV and express Using mice infected with lymphocytic T cells. We show that CXCR3 is up- the CXCR3 receptor ligand CXCL9. Activated choriomeningitis virus (LCMV) as regulated on the surface of activated effector CD8 T cells express CXCR3 and move a model, we find that the expression of CD8 T cells and guides these cells toward toward CXCL9 in marginal zone areas, where they both ligands is up-regulated soon after areas of the spleen that are rich in anti- are exposed to antigen. Consequently, WT CD8 T the infection of cells in vivo. Further- cells encounter more antigen and inflammation gens, thereby reducing the quantity and than CXCR3-deficient cells. As a result, CXCR3- more, the CXCR3 receptor itself is up- quality of the long-lived memory T-cell expressing cells tend to become short-lived regulated as soon as the third day after population. These findings provide evi- effector cells instead of long-lived memory infection on LCMV-specific CD8 T cells dence that positioning of T cells within precursors. In contrast, CD8 T cells that lack CXCR3 and is uniformly expressed by day 5 on specific microenvironments of lymphoid are localized away from marginal zone areas and, both terminal effector and memory tissues can affect their differentiation, as a result, are less exposed to antigen and precursor CD8 T cells. Using both loss- fl and hence influence the overall quality in ammatory stimuli. This leads to the generation of-function and gain-of-function of the immune response. of more long-lived memory CD8 T cells that are approaches, we asked whether CXCR3 qualitatively better than WT cells. In the past decade, researchers have plays a role in effector CD8 T-cell dif- significantly advanced our understanding ferentiation and fate determination. We 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 Author contributions: J.K.H., T.K., and M.M. designed research; J.K.H., T.K., J.M.C., and memory cells (2). The proportion of T cells that enter each M.M. performed research; J.K.H. and J.M.C. analyzed data; and J.K.H. and M.M. wrote the differentiation pathway is influenced by several factors (3). For paper. example, increased exposure to antigen and inflammatory stimuli The authors declare no conflict of interest. encourages CD8 T cells to differentiate into short-lived effector *This Direct Submission article had a prearranged editor. cells, whereas decreased exposure to these stimuli enhances 1Present address: Frontier Research Laboratories, Daiichi Sankyo Co., Ltd., Tokyo 134- the production of long-lived memory cells. However, very little is 8630, Japan. known about how chemokines and other cues that guide lym- 2To whom correspondence should be addressed. E-mail: [email protected]. phocyte trafficking and positioning regulate access to antigen See full research article on page E118 of www.pnas.org. and inflammatory stimuli, and hence affect the balance between Cite this Author Summary as: PNAS 10.1073/pnas.1101881108.

www.pnas.org/cgi/doi/10.1073/pnas.1101881108 PNAS | May 24, 2011 | vol. 108 | no. 21 | 8535–8536 Downloaded by guest on October 2, 2021 adoptively cotransferred equal numbers of differentially marked memory T cells. To assess whether forced retention of effector WT and CXCR3 KO virus-specific T cells into naive hosts and CD8 T cells within the splenic T zone areas and away from in- followed the differentiation of these cells at various times after flammation would have the same effect, we studied virus-specific infection. We found that on the eighth day after infection, at T cells that constitutively expressed the chemokine receptor the peak of CD8 T-cell expansion, terminally differentiated WT CCR7 [CCR7 transgenic (Tg)]. In contrast to normal T cells, cells consistently outnumbered CXCR3 KO cells. In contrast, on which down-regulate CCR7 expression on activation, the CCR7 the 15th day after infection, once the majority of terminally Tg T cells maintain surface expression of a functional receptor. differentiated effector CD8 T cells had died, more CXCR3 KO We found that during the early course of infection, CCR7 Tg memory precursor cells remained than WT cells. In comple- virus-specific CD8 T cells remained within the splenic T zone mentary overexpression studies, retroviral-mediated restoration and away from the infected cells. Furthermore, a greater pro- of CXCR3 expression in CXCR3 KO CD8 T cells led to the portion of CCR7 Tg T cells compared with WT cells developed development of more terminal effector cells at the expense of into memory precursor cells. These results provide strong evi- memory precursor cells. These results suggest that CXCR3 ex- dence that localization of T cells within the microenvironment of pression on CD8 T cells affects the balance between terminal lymphoid organs during the course of an infection can affect effector and memory precursor cells. In a long-term follow-up CD8 T-cell differentiation as well as the magnitude and quality study of coadoptively transferred WT and CXCR3 KO virus- of the long-lived memory T-cell population. specific T cells, we consistently found more CXCR3-deficient In this study, we showed that alterations in chemokine re- memory CD8 T cells than WT cells. In addition, CXCR3 KO ceptor expression redistribute effector CD8 T cells within lym- CD8 T cells transitioned more rapidly into a more phenotypically phoid tissues and have an impact on the differentiation and mature memory T-cell population and had a larger recall re- generation of memory CD8 T cells (Fig. 1). Further studies on sponse than WT cells, suggesting that in addition to having an the role of various chemokine receptors and other guidance cues early impact on the number of memory precursor cells, CXCR3 will advance our understanding of the interplay between these expression affects the quantity and quality of long-lived memory signals, the localization within tissue microenvironments, and the fl ’ CD8 T cells, which could potentially in uence the body s abi- shaping of the effector and memory T-cell responses. In addi- lity to ward off a similar infection years down the road. This tion, it will be interesting to determine what role chemokine fi nding has important implications for vaccine design, where the receptors play in the repeated exposure of CD8 T cells to antigen ultimate goal is to produce a large number of high-quality during a chronic viral infection and whether these receptors have fi memory CD8 T cells that can ght off the invading organism an impact on T-cell exhaustion. Our findings suggest that che- fl before too much damage is in icted on the infected person. mokine receptors like CXCR3 may provide a target for vacci- Our investigation into the mechanism of this process revealed nation methods aimed at generating a more robust memory CD8 that during the course of infection, effector CD8 T cells colo- T-cell response as protection against future infections or cancer. calized with antigen within lymphoid organs in a CXCR3- fi dependent manner. On the third to fth days after infection, the 1. Kaech SM, Wherry EJ (2007) Heterogeneity and cell-fate decisions in effector and majority of WT virus-specific T cells were found in areas of the memory CD8+ T cell differentiation during viral infection. Immunity 27:393–405. spleen where both viral antigen and CXCL9 are abundant. In 2. Gerlach C, et al. (2010) One , multiple fates in CD8+ T cell differentiation. contrast, most of the CXCR3 KO effector CD8 T cells were J Exp Med 207:1235–1246. fl located within the T-zone areas of the spleen, away from anti- 3. Haring JS, Badovinac VP, Harty JT (2006) In aming the CD8+ T cell response. Immunity 25:19–29. gens. These results suggest that the expression of CXCR3 in- 4. Cyster JG (2005) Chemokines, sphingosine-1-phosphate, and cell migration in secondary creased exposure to antigen and inflammation, resulting in fewer lymphoid organs. Annu Rev Immunol 23:127–159.

8536 | www.pnas.org/cgi/doi/10.1073/pnas.1101881108 Hu et al. Downloaded by guest on October 2, 2021