Antigen-Induced but Not Innate Memory CD8 T Cells Express

Antigen-Induced but Not Innate Memory CD8 T Cells Express NKG2D and Are Recruited to the Lung Parenchyma upon Viral Infection This information is current as of September 27, 2021. Morgan Grau, Séverine Valsesia, Julien Mafille, Sophia Djebali, Martine Tomkowiak, Anne-Laure Mathieu, Daphné Laubreton, Simon de Bernard, Pierre-Emmanuel Jouve, Erwan Ventre, Laurent Buffat, Thierry Walzer, Yann Leverrier and Jacqueline Marvel Downloaded from J Immunol published online 9 April 2018 http://www.jimmunol.org/content/early/2018/04/06/jimmun ol.1701698 http://www.jimmunol.org/

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2018 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published April 9, 2018, doi:10.4049/jimmunol.1701698 The Journal of Immunology

Antigen-Induced but Not Innate Memory CD8 T Cells Express NKG2D and Are Recruited to the Lung Parenchyma upon Viral Infection

Morgan Grau,* Se´verine Valsesia,* Julien Maﬁlle,* Sophia Djebali,* Martine Tomkowiak,* Anne-Laure Mathieu,* Daphne´ Laubreton,* Simon de Bernard,† Pierre-Emmanuel Jouve,† Erwan Ventre,* Laurent Buffat,† Thierry Walzer,* Yann Leverrier,* and Jacqueline Marvel*

The pool of memory-phenotype CD8 T cells is composed of Ag-induced (AI) and cytokine-induced innate (IN) cells. IN cells have been described as having properties similar to those of AI memory cells. However, we found that pathogen-induced AI memory cells Downloaded from can be distinguished in mice from naturally generated IN memory cells by surface expression of NKG2D. Using this marker, we described the increased functionalities of AI and IN memory CD8 T cells compared with naive cells, as shown by comprehensive analysis of cytokine secretion and gene expression. However, AI differed from IN memory CD8 T cells by their capacity to migrate to the lung parenchyma upon inflammation or infection, a process dependent on their expression of ITGA1/CD49a and ITGA4/ CD49d integrins. The Journal of Immunology, 2018, 200: 000–000. http://www.jimmunol.org/ ne hallmark of the adaptive immune system is its ability lymphoid organs, but a subset of them, the tissue-resident memory to respond more quickly and more strongly to previously cells (TRM), settles within nonlymphoid tissues where they pro- O encountered Ags. This immunological memory relies on vide increased protection against secondary pathogen infections the generation of cells that display increased reactivity toward the (3, 4). TRM are long-lived sessile cells in most tissues except the previously encountered Ag. Protection against intracellular path- lung, where they need to be replenished from the circulating pool ogens or tumor-derived Ag is conferred in part by Ag-induced of memory cells (5, 6). (AI) memory CD8 T cells. Indeed, AI memory CD8 T cells Memory-phenotype CD8 T cells or innate (IN) memory cells can have improved functional properties compared with naive cells, also be generated through several alternative pathways that are making them more potent to rapidly eliminate infected cells upon independent of foreign Ag exposure (7). Memory CD8 T cells by guest on September 27, 2021 reinfection (1, 2). These AI memory cells are found in secondary generated through lymphopenia-induced proliferation (LIP) were the first IN memory cells to be described (8–12). This pathway *Centre International de Recherche en Infectiologie, INSERM, U1111, Universite´ depends on strong IL-7 stimulation of naive CD8 T cells (because Claude Bernard Lyon 1, CNRS, UMR5308, E´ cole Normale Supe´rieure de Lyon, of the increased availability of this gc cytokine in the lymphopenic † Universite´ de Lyon, F-69007 Lyon, France; and Altrabio, F-69007 Lyon, France host) combined with weak TCR stimulation through self-peptide/ ORCIDs: 0000-0002-4503-5202 (S.V.); 0000-0002-0567-5387 (S.D.); 0000-0003- MHC complexes (13–15). Other gc cytokines also support the 3161-8295 (M.T.); 0000-0002-2893-7001 (A.-L.M.); 0000-0001-9506-6755 (D.L.); 0000-0002-3390-0924 (S.d.B.); 0000-0002-3076-8841 (P.-E.J.); 0000-0002-0857- generation of IN memory cells. In vivo, strong IL-2 stimulation 8179 (T.W.); 0000-0002-4227-5446 (Y.L.); 0000-0001-6241-459X (J. Marvel). through injection of IL-2/anti–IL-2 Ab complexes was shown to Received for publication December 11, 2017. Accepted for publication March 12, drive the generation of IN memory CD8 T cells from naive TCR 2018. transgenic cells (16). Similarly, the characterization of several This work was supported by INSERM, CNRS, Universite´ de Lyon, E´ cole Normale mutant mouse strains revealed that strong IL-4 stimulation of CD8 Supe´rieure de Lyon, Re´gion Rhoˆne-Alpes, and Agence Nationale de la Recherche (Grant 12-RPIB-0011). M.G. was a Re´gion Rhoˆne-Alpes Ph.D. fellow. single-positive thymocytes or naive CD8 T cells leads to IN memory cell generation (17–19). Moreover, naive BALB/c mice M.G. and J. Marvel conceived and designed experiments; M.G., S.V., J. Mafille, S.D., M.T., A.-L.M., D.L., and E.V. performed and analyzed experiments; T.W., Y.L., and have an increased proportion of memory-phenotype CD8 T cells J. Marvel analyzed experiments; S.d.B., P.-E.J., and L.B. provided bioinformatics because of higher levels of circulating IL-4 compared with naive support; J. Marvel, M.G., and Y.L. wrote the manuscript. C57BL/6 mice (20). Conversely, naive mice deficient for IL-4 The microarray data presented in this article have been submitted to the Gene production or signaling have a reduced frequency of IN memory Expression Omnibus (http://www.ncbi.nlm.nih.gov/geo/) under accession number GSE111137. CD8 T cells (21, 22). In physiological conditions, LIP memory Address correspondence and reprint requests to Dr. Jacqueline Marvel, Team Immu- cells generation occurs during the neonatal period in naive mice nity and Cytotoxic Lymphocytes, Centre International de Recherche en Infectiologie, (21, 23, 24) and Th2 immune responses might also favor the ´ INSERM, U1111, Universite´ Claude Bernard Lyon 1, CNRS, UMR5308, Ecole generation of IN memory CD8 T cells (25). Normale Supe´rieure de Lyon, Universite´ de Lyon, 21 Avenue Tony Garnier, 69365 Lyon Cedex 07, France. E-mail address: [email protected] Hence, among CD8 T cells specific for foreign Ag never en- The online version of this article contains supplemental material. countered by specific pathogen-free (SPF) naive mice, 10–20% Abbreviations used in this article: AI, Ag-induced; Flu, recombinant influenza; i.n., of cells display a memory phenotype. These cells are also re- intranasal; IN, innate; KO, knockout; LIP, lymphopenia-induced proliferation; PC, ferred to as virtual memory (VM) cells (26). Importantly, equal principal component; SPF, specific pathogen-free; TRM, tissue-resident memory cell; numbers of these V cells were found in naive germ-free mice, V , virtual memory; VV, vaccinia virus. M M indicating that their generation is independent of microbiota- Copyright Ó 2018 by The American Association of Immunologists, Inc. 0022-1767/18/$35.00 derived Ag (26). Therefore, in physiological conditions, the pool

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1701698 2 INNATE VERSUS Ag-INDUCED MEMORY CD8 T CELLS of memory-phenotype CD8 T cells is composed of two classes of C57BL/6 mice by i.v. injection. OT-I or F5 naive cells were also trans- cells: AI and IN. ferred to immunocompetent mice that further received i.p. injections of AI and IN CD8 memory cells are generated through distinct 1.5 mg of IL-2 or IL-4 (PeproTech) immunocomplexed to anti–IL-2 (S4B6; Bio X Cell) or anti-IL-4 (11B11; Bio X Cell) Ab, during seven consecutive pathways but express a similar array of surface markers, which has days. hampered their demarcation. Interestingly, experimentally generated TCR transgenic OT-I memory CD8 T cells do not express Cell preparation and flow cytometry CD49d compared with AI OT-I memory CD8 T cells. This lack of To discriminate between TRM and circulating memory CD8 T cells, in vivo CD49d expression has been used to identify and characterize OVA intravascular staining was performed as previously described (31). Briefly, and vaccinia virus (VV)–specific clones of IN memory cells mice were injected i.v. with 3 mg of CD45-BV421 Ab (BioLegend) diluted m generated in physiological conditions (26). In parallel, it has been in 200 l of sterile PBS (Life Technologies, Saint Aubin, France) and were sacrificed 2 min after injection by overdose of pentobarbital. Blood sam- shown that compared with naive cells, these unconventional Ag- ples (100 ml) were collected on EDTA by retro-orbital bleeding. Spleen specific memory cells (IN or VM cells) are able to mount an ef- and lymph nodes were harvested, mechanically disrupted, and filtered ficient response against pathogen infection with increased func- through a sterile 100-mm nylon mesh filter (BD Biosciences). To collect tional properties including augmented IFN-g production and bronchoalveolar lavages, the trachea was exposed and cannulated with a 24-gauge plastic catheter (BD Biosciences) and lungs were lavaged twice proliferative response (18, 26, 27). However, the comparison be- with 1 ml of cold sterile PBS. Lungs were enzymatically digested using a tween IN and AI memory CD8 T cells in terms of phenotype, specific dissociation kit and following manufacturer’s instructions (Mil- function, and gene expression profile has not been performed. tenyi Biotec). Upon strong TCR triggering, CD8 T cells express high levels of Surface staining was performed on single-cell suspensions from each organ for 30 min at 4˚C with the appropriate mixture of mAbs diluted in the NK cell receptor NKG2D (28), and AI memory cells express Downloaded from staining buffer (PBS supplemented with 1% FCS [Life Technologies] and NKG2D (29). In contrast, IL-4–induced IN cells do not express 0.09% NaN3 [Sigma-Aldrich, Saint Quentin-Fallavier, France]). To iden- NKG2D (29). Therefore, we hypothesized that NKG2D could be tify B8R-specific memory CD8 T cells, dextramer staining was performed differently expressed between AI and IN memory CD8 T cell for 20 min at room temperature using B8R dextramer (Immudex) before populations. surface staining. The following Abs (clones) were used for surface staining: NKG2D (CX5), CD45.1 (A20), CD45.2 (104), CD122 (TM-b1), In this study, we demonstrate that the expression of NKG2D is CD62L (MEL-14), CD8 (53-6.7), CD44 (IM7.8.1), CXCR3 (Cxcr3-173), restricted to AI memory CD8 T cell populations. Using NKG2D as CD49a (HA 31-8), CD49d (R1-2), CD29 (eBioHMb1-1), CD11c (N418). http://www.jimmunol.org/ a marker of AI cells, we performed an extensive comparison of AI VM CD8 T cells were from nonimmunized naive C57BL/6 mice. Spleen + and IN cells within the natural pool of memory CD8 T cells. Our CD8 T cells were enriched using a MACS CD8a T Cell Isolation Kit II for mouse (Miltenyi Biotec), and VM were identified as B8R-specific results indicate that although IN CD8 T cells share many features hi 2 CD44 CD49 CD8 T cells (using CD11b, CD11c, CD19 and NK1.1 as with AI memory cells, only AI cells are recruited toward the lung a dump gate). To perform intracellular cytokine staining, cells were fixed parenchyma upon inflammation or infection. and permeabilized using CytoFix/CytoPerm (BD Pharmingen). To detect transcription factors, a Foxp3 Kit (eBioscience) was used to fix and permeabilize cells. The following Abs (clones) were used for intra- Materials and Methods cellular staining: IFN-g (XMG1.2), CCL5 (2E9), Tbet (4B10), and Mice Eomes (Dan11mag). All analyses were performed on a BD Biosciences FACS LSR II or Fortessa and analyzed with FlowJo software (Tree Star, by guest on September 27, 2021 F5 TCR [B6/J-Tg(CD2-TcraF5,CD2-TcrbF5)1Kio/Jmar] transgenic Ashland, OR). mice were provided by Prof. D. Kioussis (National Institute of Medical Research, London, U.K.) and backcrossed on CD45.1 C57BL/6 back- In vitro stimulation and cytokines production measurements ground (30). The F5 TCR recognizes the NP68 peptide from influenza A virus (ASNENMDAM) in the context of H2-Db.OT-ITCRtrans- For measurements of cytokine production, 5 3 104 naive and memory genic [B6/J-Tg(Tcra,Tcrb)1100Mjb/Crl], CD45.2 (C57BL/6J) and (NKG2D2 and NKG2D+) CD8 T cells sorted from VV-infected mice were CD45.1 (B6.SJL-PtprcaPepcb/BoyCrl), C57BL/6J, BALB/c, and OF1 cultured for 12 h with plate-bound anti-CD3 Ab (145-2C11, 10 mg/ml; BD mice were purchased from Charles River Laboratories (L’Arbresle, Biosciences), soluble anti-CD28 Ab (37.51, 1 mg/ml; BD Biosciences), France). The OT-I TCR recognizes the OVA257–264 peptide from and IL-2 (2%). Supernatants were collected and cytokine production was chicken OVA (SIINFEKL) in the context of H2-Kb.Micewerebredor measured by bead-based multiplexing technology for IL-1a, IL-1b, IL-3, housed under SPF conditions in our animal facility (AniRA-PBES, IL-4, IL-9, IL-10, IL-13, IL-17, IFN-g, TNF-a, and CCL2/3/4/5 (Bio-Plex Lyon, France). All experiments were approved by our local ethics Pro; Bio-Rad) or by ELISA for CCL1, CCL5, and IFN-g (Mouse DuoSet; committee (CECCAPP, Lyon, France) and accreditations have been R&D Systems). For flow cytometry measurements of cytokine production obtained from governmental agencies. at the single-cell level, 1 3 105 NKG2D2 and NKG2D+ memory CD8 T cells, sorted from VV-infected mice, were cultured for 6 h with VV- Pathogens and mouse immunization infected (multiplicity of infection = 10) DC2.4 cells or with PMA (20 ng/ml) and ionomycin (1 mg/ml) in the presence of GolgiStop (BD Biosciences). The recombinant influenza (Flu) virus strain WSN encoding the NP68 Alternatively, 1 3 106 total splenocytes from VV-infected mice were epitope (Flu-NP68) was produced by reverse genetics from the A/WSN/33 cultured for 5 h with plate bound anti-CD3 Ab and soluble anti-CD28 Ab H1N1 strain. The recombinant VV, expressing the NP68 epitope (VV- or with IL-12 (10 ng/ml; R&D Systems), IL-18 (10 ng/ml; MBL), and IL-2 NP68), was engineered from the Western Reserve strain by Dr. D.Y.-L. (10 ng/ml; PeproTech). Teoh, in Prof. Sir Andrew McMichael’s laboratory at the Medical Re- search Council (Human Immunology Unit, Institute of Molecular Medi- Protection assay cine, Oxford, U.K.). The Listeria monocytogenes strain 10403s was produced from clones grown from organs of infected mice. For immuni- To evaluate the degree of protection associated with each CD8 T cells zation, anesthetized mice received intranasal (i.n.) administration of Flu population, mice were transferred with 1 3 105 naive, NKG2D2 or 5 5 + (2 3 10 tissue culture ID50), VV (2 3 10 PFU) or poly(I:C) (30 mg) in NKG2D memory CD8 T cells from Flu-infected mice. The next day, host 3 6 20 ml of PBS or i.v. L. monocytogenes (2 3 10 ) administration in 200 ml mice were infected with a lethal dose (1 3 10 tissue culture ID50) of Flu. PBS. For some indicated experiments, mice received i.p. administration of Mice weight loss was measured each day, for up to 12 d, postinfection. 1 3 106 PFU VV in 200 ml of PBS. Mice that lost more than 20% of initial body weight were euthanized. TCR transgenic memory CD8 T cells TCR repertoire analysis To generate AI TCR transgenic memory CD8 T cells, 2 3 105 naive Naive, NKG2D2 and NKG2D+ memory CD8 T cells were sorted from CD45.1 F5 CD8 T cells were transferred in C57BL/6 mice by i.v. injec- VV-infected mice (50 d postinfection). Cells were lysed and multiplex tion. The next day, mice were infected with VV-NP68 as described above. PCR were performed by ImmunID (Grenoble, France) on genomic DNA to To generate IN TCR transgenic memory CD8 T cells, 1 3 106 naive OT-I detect V(D)J rearrangements at the TCR b-chain locus. For each cell CD8 T cells were transferred in sublethally irradiated (600 rad) CD45.1 population, the percentage of TCR repertoire diversity was calculated as The Journal of Immunology 3 the ratio between the number of observed V(D)J recombinations and the Results theoretical number of V(D)J recombinations (i.e., 209). NKG2D expression identifies polyclonal AI memory CD8 T cell Transcriptome analysis populations F5 TCR transgenic memory CD8 T cells were generated as described We tested the hypothesis that NKG2D expression could dis- above. Eighty days after VV-NP68 infection, CD45.1 F5 memory CD8 criminate AI from IN memory cells. Indeed, IN cells gener- 2 + T cells as well as host’s NKG2D and NKG2D memory CD8 T cells ated following injection of IL-2 or IL-4 Ab complexes were sorted from five pools of spleens, each from eight mice. CD8 (Fig. 1A, Supplemental Fig. 1) or after lymphopenic proliferation T cells were enriched by negative selection (MACS CD8a+ TCellIso- lation Kit II for mouse); then, memory CD8 T cell (CD8+CD44+)were (Supplemental Fig. 2) do not express NKG2D at their cell surface. sorted by FACS based on the expression of NKG2D (purity .98%). In agreement with this hypothesis, in nonimmunized SPF mice, Naive F5 and polyclonal CD8 T cells were sorted from pools of three the majority (more than 90%) of splenic memory-phenotype CD8 spleens from naive F5 and C57BL/6 mice respectively. Total RNA was T cells (i.e., CD44hi), which are mainly IN cells (7), do not ex- extracted from dry cell pellets according to the “Purification of total 2 RNA from animal and human cells” protocol of the RNeasy Micro Kit press NKG2D (Fig. 1B). Similarly, VM cells are mainly NKG2D (Qiagen, Hilden, Germany). Purity and integrity of the RNA was (Fig. 1C). In contrast, almost all B8R-specific memory CD8 assessed on the Agilent 2100 Bioanalyzer (Agilent, Palo Alto, CA). Total T cells (CD44hi B8R+) from C57BL/6 mice infected with VV RNA from each sample was amplified, labeled, and hybridized to mouse express NKG2D (Fig. 1D). Following infection with VV, the GeneChip HT MG-430 PM Plates as described in the Affymetrix Gen- hi eChip 39 IVT PLUS Reagent Kit User Manual (Affymetrix, Santa Clara, frequency of CD44 effector or memory CD8 T cells expressing CA). Affymetrix CEL files were analyzed in R using the appropriate NKG2D was increased by .100-fold in the effector phase leading packages from the Bioconductor suite (https://www.bioconductor.org/). + to a 10-fold expansion of the NKG2D subset in the memory Downloaded from Raw probe signals were background corrected using the maximum phase (Fig. 1E, Supplemental Fig. 3). This was not specific to VV likelihood estimation of the normal–exponential mixture model (32), because at 55 d postinfection by Flu or bacteria L. monocytogenes, normalized using the variance stabilization normalization (33) followed + hi by a quantile normalization (34). Summarization was performed using there was also a strong amplification of the NKG2D CD44 CD8 the median (35) and using a modified version 17.1 of the Entrez-Gene– T population in the blood (Fig. 1E). In contrast, the number of based reannotated chip description file. Noninformative genes were fil- memory-phenotype NKG2D2 cells remained stable when com- tered using the I/NI algorithm (36). Linear models were applied using the paring the naive and the memory phase. Importantly, NKG2D2 limma package to compute the average expression level for each cell ∼ http://www.jimmunol.org/ type. A random effect was introduced to account for the paired design. cells contained a fraction ( 25%) of T effector memory CD8 + Statistical contrasts were then applied to compute differential expression T cells. The fraction of T effector memory cells among NKG2D between the different cell types. The empirical Bayes method was used memory cells decreased with time following activation to reach on to compute moderated p values that were then corrected for multiple average 40% of memory cells 6 wk postinfection (data not comparisons using the Benjamini and Hochberg’s false discovery rate controlling procedure. shown). These results could be extended to other mouse strains: in BALB/c mice and in the outbred mouse strain OF1 VV infection Measurement of transcription factors expression by qPCR induced the generation of NKG2D+ memory CD8 T cells Naive, NKG2D2, and NKG2D+ memory CD8 T cells were sorted from (Supplemental Fig. 4).

VV-infected mice (50 d postinfection). Total RNA was extracted using To further validate NKG2D as a marker of AI memory T cells, by guest on September 27, 2021 Trizol reagent according to manufacturer’s instructions (Life Technolo- we next analyzed NKG2D expression by VV-specific memory gies). Total RNA was digested using turbo DNA-free DNase (Life Tech- T cells recognizing other epitopes than B8R. Indeed, VV harbors at nologies) to avoid genomic contamination. Quality and absence of genomic DNA contamination were assessed with a Bioanalyzer (Agilent, least 40 epitopes recognized by CD8 T cells (37). To extend this Massy, France). We used a High-Capacity RNA-to-cDNA Kit (Life analysis to the whole viral epitope repertoire, memory CD8 T cells Technologies) to generate cDNA for PCR amplification. PCR was carried from VV-immunized mice were restimulated with VV-infected out with a SYBR Green–based kit (FastStart Universal SYBR Green dendritic cells. More than half of NKG2D+ memory-phenotype Master; Roche, Basel, Switzerland) on a StepOnePlus instrument (Applied CD8 T cells produced IFN-g following VV restimulation, Biosystems, Calrlsbad, CA). Primers were designed using the Roche Web 2 site (Universal Probe Library Assay Design Center). whereas only ∼5% of NKG2D memory-phenotype CD8 T cells did (Fig. 1F). This lack of IFN-g production was not due to a Transwell assay functional defect of NKG2D2 memory-phenotype CD8 T cells, as 1 3 106 CD8 T cells purified from VV-infected mice (50 d postinfection) restimulation with PMA and ionomycin led to IFN-g production. were added to the upper chamber of polycarbonate transwell inserts (5 mm As some epitopes of VV might be expressed in a delayed fashion pore size; Corning). The lower chamber was filled with complete DMEM and might not be presented in the time frame used for in vitro medium (6% FCS, 10 mM HEPES, 50 mM22MΕ,50mg/ml gentamicin, L restimulation, we also performed in vivo rechallenge of memory 2mM -glutamine, all from Life Technologies) alone or supplemented 2 with CXCL10 (100 ng; PeproTech). After 2 h of incubation at 37˚C, 7% CD8 T cells (Fig. 1G). Although NKG2D and NKG2D+ memory CO2, transmigrated cells were collected in the lower chamber and were CD8 T cells from VV-infected mice equally grafted in the spleen washed and stained for CD8, CD44, and NKG2D. The absolute number of of host mice, only NKG2D+ memory-phenotype CD8 T cells had transmigrated cells was determined by flow cytometry by adding a known number of fluorescent beads (Flow-Count Fluorosphere; Beckman Coul- strongly proliferated 7 d after rechallenge with VV, as revealed by ter). Results are expressed as the migration index, which represents the the number of donor cells recovered in the spleen and lung of host fold increase in the number of transmigrated cells in response to chemo- mice (Fig. 1G). In contrast, the number of donor NKG2D2 attractant over the nonspecific cell migration (medium alone). memory-phenotype CD8 T cells remained close to the number CD3 Ab-directed cytotoxicity assay observed in unimmunized host mice, revealing marginal expansion following VV rechallenge. NKG2D+ memory CD8 T cell P815 target cells were labeled with Cell Trace Violet (Thermo Fisher) and incubated with anti-CD3 Ab (2C11, 10 mg/ml) for 30 min at 37˚C. Target proliferation was strictly dependent on Ag recognition, as infec- cells were cultured for 12 h with naive, NKG2D2, or NKG2D+ memory tion with the heterologous pathogen L. monocytogenes did not CD8 T cells sorted from VV-infected C57BL/6 mice (80 d postinfection). lead to their expansion (Fig. 1G). Finally, we compared the ca- P815 viability was assessed using LIVE/DEAD fixable dye (Life Tech- pacity of NKG2D+ and NKG2D2 memory-phenotype CD8 T cells nologies) followed by fixation with 1% paraformaldehyde (Sigma-Aldrich) to protect naive mice against a lethal dose of virus. To do so, naive followed by flow cytometry analysis. The percentage of specific lysis is the 2 + percentage of P815 cell death in samples containing CD8 T cells after CD8 T cells as well as NKG2D and NKG2D memory CD8 subtracting the percentage of spontaneous P815 cell death. T cells from Flu-immune mice were transferred in naive hosts that 4 INNATE VERSUS Ag-INDUCED MEMORY CD8 T CELLS Downloaded from http://www.jimmunol.org/ by guest on September 27, 2021

FIGURE 1. NKG2D expression identifies polyclonal AI memory CD8 T cell populations. (A) CD45.2 OT-I or F5 TCR transgenic naive CD8 T cells were adoptively transferred in immunocompetent congenic mice. The next day, hosts received i.p. injections of the indicated gc cytokine Ab complex, as described in the Materials and Methods. Thirty days after transfer, the expression of NKG2D by OT-I CD8 T cells was assessed by flow cytometry. Black histogram: OT-I CD8 T cells, gray histogram: host’s CD8 T cells. The percentage of transferred CD45.2 cells expressing NKG2D is indicated (IL-2: 1 experiment, n = 3 mice; IL-4: 3 experiments, n = 10 mice). (B) NKG2D expression by memory-phenotype (CD44hi) spleen CD8 T cells from 6-wk naive C57BL/6 mice. Black histogram: CD44hi CD8 T cells, gray histogram: naive CD8 T cells. The percentage of NKG2D+ cells among CD44hi CD8 T cells is hi 2 shown (two experiments, n = 12 mice). (C) NKG2D expression by VM CD8 T cells (B8R-specific CD44 CD49 from spleen of nonimmunized naive C57BL/6 mice). Black histogram: VM CD8 T cells, gray histogram: naive CD8 T cells. The percentage of VM cells expressing NKG2D is indicated (two experiments, n = 10 mice). (D) B8R-specific memory CD8 T cells (B8R+) from VV-immunized C57BL/6 mice (.100 d postinfection) were assessed for their expression of NKG2D (black histogram). Naive CD8 T cells from same mice were used as control. The percentage of NKG2D+ cells among B8R- specific cells is shown (five experiments, n = 5–7 mice per experiment). (E) C57BL/6 mice were infected with VV (i.n.), Flu (i.n.) or L. monocytogenes (i.v.), and the number of total, NKG2D2, and NKG2D+, CD44hi CD8 T cells was measured in the blood 55 d following pathogen infection. Graph shows mean expansion index (6 SD) of the indicated CD44hi CD8 T cell populations. The dotted line represents an absence of expansion (index = 1). One representative experiment out of two (n = at least 6 mice per group and per experiment). *p , 0.05, **p , 0.01, Wilcoxon matched-pairs signed rank test. (F) NKG2D+ and NKG2D2 memory CD8 T cells were sorted from VV-infected C57BL/6 mice (55 d postinfection). Cells were stimulated for 6 h with VV- infected DC2.4 cells (DC + VV) or with PMA/ionomycin (PMA/iono) in the presence of GolgiStop. The percentage of (Figure legend continues) The Journal of Immunology 5 were infected with a lethal dose of Flu. NKG2D+ memory CD8 cytokine/chemokines CCL5, CCL1, and IFN-g than IN cells T cells induced a significant protection of host mice as more than (Fig. 2D). AI memory cells can also produce IFN-g upon stimu- 40% of them survived the infection (Fig. 1H). This is in contrast to lation by innate signals such as IL-12 and IL-18, a property known naive cells and NKG2D2 memory CD8 T cells that conferred no as the IN function of memory CD8 T cells (38). A fraction of IN protection. Altogether, these results indicate that NKG2D ex- memory cells produced IFN-g upon IL-12/IL-18 stimulation, al- pression identifies AI memory CD8 T cells in polyclonal settings, beit at a lower frequency than AI memory CD8 T cells (Fig. 2E). allowing them to be distinguished from IN memory CD8 T cells Taken together, these results indicate that although AI and IN that do not express this marker. memory CD8 T cells produced a similar pattern of cytokines following stimulation, AI produce higher amounts of cytokines AI and IN memory CD8 T cells are distinguished by their TCR than IN memory T cells. repertoire and cytokine secretion capacity Next, taking advantage of NKG2D as a marker discriminating AI Transcriptome analysis of AI and IN memory CD8 T cells and IN memory CD8 T cells, we compared these two populations in To further characterize the two subsets defined by NKG2D ex- terms of TCR repertoire and effector functions. A multiplex PCR pression, we compared their transcriptome using microarrays. F5 identifying b-chain locus V(D)J rearrangements showed that, as TCR transgenic CD8 T cells were transferred in naive mice before expected, AI memory CD8 T cells have a less diverse TCR rep- infection with VV-NP68 to establish an internal control of AI ertoire than naive CD8 T cells, reflecting Ag selection (Fig. 2A). memory CD8 T cells. Eighty days postinfection, F5 memory cells In contrast, IN memory CD8 T cells have a TCR repertoire that is as well as host’s IN and AI memory CD8 T cell populations were as diverse as that of naive CD8 T cells. A principal component sorted, and their transcriptome was analyzed. Naive CD8 T cells Downloaded from (PC) analysis indicated that the TCR repertoire was more similar were sorted from naive F5 and C57BL/6 mice. We first performed within the NKG2D+ subset of different mice than within the CD8 a PC analysis that shows that 70% of the variability of the samples cells (i.e., the naive, NKG2D+, and NKG2D2 cells of a given is explained by the first two PC. Samples were aligned along PC1 mouse) (Fig. 2A). As IN cells have a diverse repertoire that seems according to their differentiation stage, whereas PC2 highlighted a to differ in its composition from naive CD8 T cells, we tested if IN difference between monoclonal F5 TCR transgenic T cells and

cells could participate in a primary immune response against a polyclonal CD8 T cells (Fig. 3A). F5 and polyclonal CD8 T cells http://www.jimmunol.org/ pathogen (i.e., whether they can mount responses against un- differed by the expression of few genes, among which those known foreign epitopes) and compared their responsiveness to coding for the TCR, reﬂecting the monoclonality of the F5 CD8 naive cells. Equal numbers of CD45.1 naive and CD45.2 IN population (Supplemental Fig. 5A), and one set of genes encoding memory CD8 T cells were sorted from naive mice and cotrans- the inhibitory NK cell receptors (Klra) were expressed by poly- ferred to congenic CD45.1/CD45.2 hosts that were infected the clonal AI memory cells but not F5 memory cells. Accordingly, next day with VV or L. monocytogenes. Seven days postinfection, these Ly49 receptors are exclusively expressed by a small fraction the contribution of transferred cell populations to the primary of polyclonal memory CD8 T cells in contrast to TCR transgenic response was determined in the spleen (Fig. 2B). Both naive and F5 memory cells (Supplemental Fig. 5B). This property is shared

IN memory cells expanded during the primary immune response by both IN and AI polyclonal memory cells (Supplemental Fig. by guest on September 27, 2021 and became NKG2D+. We also evaluated the relative contribution 5C). Importantly, all genes differentially expressed by AI memory of the two grafted cell populations to the NKG2D+ CD44hi ef- CD8 T cells compared with naive cells were also differentially fector cells response. During VV infection, naive and IN memory expressed by F5 CD8 T cells when comparing memory to naive CD8 T cells generated almost equal numbers of NKG2D+ CD44hi cells, confirming their AI nature (data not shown). We then effector cells. In contrast, during L. monocytogenes infection, IN compared AI and IN polyclonal memory populations. IN memory memory CD8 T cells generated more NKG2D+ CD44hi effector cells were positioned closer to AI memory cells than to naive CD8 cells compared with naive cells (Fig. 2B). In conclusion, IN cells T cells on the PC1 axis, confirming their memory differentiation have a more diversified repertoire than AI cells and can contribute (Fig. 3A). Indeed, AI and IN memory CD8 T cell transcriptomes to a primary T cell response. differ in the expression levels of several genes encoding tran- One key property of memory CD8 T cells is the rapid and in- scription factors, effector molecules, NK cell receptors, chemo- creased production of cytokines and chemokines upon TCR kine receptors, and integrins (Fig. 3B). Compared with IN cells, stimulation. We therefore compared the capacity of polyclonal IN AI memory CD8 T cells express higher levels of genes encoding and AI memory and naive CD8 T cells to produce cytokines and effector molecules involved in the killing of target cells through chemokines in response to TCR stimulation. Early after TCR cytotoxicity. Accordingly, this memory cell population displays triggering, both IN and AI cells produced a broader array of a higher capacity to mediate killing, in anti-CD3 redirected cy- cytokines/chemokines than naive cells. These factors were also totoxicity assay (Supplemental Fig. 6). Importantly, AI memory secreted in larger amounts by memory T cells of both subsets CD8 T cells express higher levels than IN cells of transcrip- (Fig. 2C). Importantly, the same pattern of cytokines/chemokines tion factors that promote the full differentiation of memory CD8 was produced by IN and AI memory cells, although AI cells T cells, such as Tbet, ID2, Zeb2, and Blimp-1 (Fig. 3C, 3D). produced at least a 10-fold higher quantity of the poised memory In contrast, IN memory CD8 T cells show increased levels of

IFN-g+ cells was measured by intracellular cytokine staining. Graph shows the mean percentage (6 SD) of IFN-g+ cells among each cell population. One representative experiment out of three (n = 5 mice per experiment). **p , 0.01, Mann–Whitney U test. (G) NKG2D+ and NKG2D2 memory CD8 T cells were sorted from VV-infected CD45.1 C57BL/6 mice (50 d postinfection) and 105 cells were transferred in separate CD45.2 C57BL/6 congenic mice. The next day, hosts were infected with VVor L. monocytogenes or left uninfected. Graphs show the mean numbers (6 SD) of CD45.1 donor cells recovered 7 d postinfection in the indicated organs. One representative experiment out of four (n = 6 mice per group and per experiment). **p , 0.01, Mann–Whitney U test. (H)Splenic naive, NKG2D+ and NKG2D2 memory CD8 T cells were cell-sorted from Flu-infected C57BL/6 mice (45 d postinfection) and 105 cells were transferred in separate host mice. The next day, hosts were infected with a lethal dose of Flu. Graph shows the percentage of survival observed among each group of mice. One representative experiment out of two (n = 10 mice per group and per experiment). *p , 0.05, log-rank (Mantel–Cox) test. ns, not signiﬁcant. 6 INNATE VERSUS Ag-INDUCED MEMORY CD8 T CELLS Downloaded from http://www.jimmunol.org/ by guest on September 27, 2021

FIGURE 2. Characterization of IN memory CD8 T cells. (A) Naive, IN and AI memory CD8 T cells were sorted from VV-infected C57BL/6 mice (50 d postinfection), and multiplex PCR was performed on genomic DNA to detect VbJb rearrangements among each cell population. Left graph shows the mean percentage (6 SD) of TCR repertoire diversity, calculated as described in Materials and Methods.*p , 0.05, **p , 0.01, Mann–Whitney U test (n = 5 mice). Right: PC analysis based on the presence or absence of each possible VbJb rearrangement among each cell population. This representation shows the distribution of CD8 T cell populations regarding to the composition of their VbJb TCR repertoire. Each form of symbol represents a mouse and lines connect cell populations sorted from the same mouse. (B) Naive and IN memory CD8 T cells were sorted from naive CD45.1/CD45.2 and CD45.2 C57BL/6 mice respectively and were cotransferred at a 1:1 ratio in congenic CD45.1 C57BL/6 mice. Host mice were infected with VV or L. monocytogenes or left uninfected (Ø). Left graph shows the mean number (6 SD) of CD44hi NKG2D+ CD8 T cells in the spleen generated from transferred cell populations 7 d postinfection. Right graph shows the contribution of each transferred CD8 T cell population to the total CD44hi NKG2D+ CD8 T cells generated from transferred cells (mean percentage 6 SD). One representative experiment out of two (n = 5 mice per group and per experiment). *p , 0.05, **p , 0.01, Mann–Whitney U test. (C) Equal number of naive, IN, and AI memory CD8 T cells were sorted from VV-infected mice (Figure legend continues) The Journal of Immunology 7 transcription factors that promote a less differentiated state of if Abs directed against CD49a and CD49d could alter the re- memory CD8 T cells, such as Eomes and ID3. The pattern of cruitment of AI cells to the infected lung. Ab treatment did not transcription factors expressed by AI memory cells is thus in ac- affect AI cell numbers in spleen and blood (Supplemental Fig. 8). cordance with their more differentiated state compared with IN. In contrast, it significantly inhibited AI cell recruitment to the lung parenchyma (Fig. 5D). Altogether, these results indicate that fol- AI memory CD8 T cells have an increased capacity to enter lowing lung infection, AI memory cells are the main memory inflamed peripheral tissues compared with IN memory cells subset recruited to the lung parenchyma, a process that is de- One important characteristic of memory cells is their capacity to pendent on CXCR3 and CD49a/CD49d integrin expression. circulate and migrate to inflamed tissues (39–41). As IN cells express fewer memory-specific chemokine receptors and integrins than AI cells, we compared their ability to traffic to the lung upon Discussion inflammation or infection. Inflammation was first induced in mouse In this study, we demonstrated that NKG2D surface expression is lungs by the TLR3 agonist poly(I:C) that induces the production of restricted to AI memory CD8 T cells, allowing their discrimination type-I IFN and its downstream chemokines (CXCL9, 10, and 11) from IN memory CD8 T cells generated under physiological (42). Memory CD8 T cells containing similar numbers of IN and AI conditions. We showed that this dichotomy is conserved in different populations were purified from VV-infected mice and transferred mouse strains and in response to infection by different pathogens. into host mice that then received i.n. injection of poly(I:C). The Expression of NKG2D by VM CD8 T cells has recently been 2 recruitment of AI and IN memory cells in different organs was reported at the mRNA level (46). We found that sorted NKG2D assessed 2 d after poly(I:C) injection. The same numbers of donor memory-phenotype IN cells containing the VM cells expressed Downloaded from AI and IN memory CD8 T cells were found in the spleen and in higher levels of mRNA encoding NKG2D compared with naive the blood of recipient mice injected with PBS or poly(I:C) cells (fold change = 2, Supplemental Fig. 9). However, we found + hi (Supplemental Fig. 7), as reflected by a cell number ratio close to 1 that the small fraction of B8R VM cells (0.02%) within CD44 2 2 (Fig. 4A). As expected the recruitment of donor memory CD8 CD49d CD8 T cells found in naive mice are mainly NKG2D at + T cells within the lung parenchyma and airways was induced fol- the protein level (Fig. 1C). Importantly, NKG2D AI memory CD8 T cells expressed much higher levels of NKG2D mRNA than lowing poly(I:C) injection (Supplemental Fig. 7). However, AI http://www.jimmunol.org/ memory CD8 T cells were preferentially recruited compared with IN memory cells in quiescent cells, which could explain the dif- IN cells (Fig. 4A). Following lung infection by a pathogen, spleen ference observed in NKG2D protein levels. Thus, the surface memory cells are recruited to the lung in an Ag-independent fashion expression of NKG2D protein is restricted to AI memory CD8 (43, 44). To analyze the capacity of AI and IN memory CD8 T cells T cells. to be attracted to the infected lung, VV-specific memory CD8 Taking advantage of NKG2D expression, we compared IN T cells containing NKG2D2 and NKG2D+ populations were iso- memory CD8 T cells to pathogen-induced memory CD8 T cells. lated from VV-immune mice and transferred into host mice that had We performed a transcriptome comparison of IN and AI memory been immunized 2 d before with Flu virus, and the recruitment was CD8 T cell populations. Our results clearly demonstrated that VV- + assessed 2 d later. Again, AI memory CD8 T cells were preferen- induced memory cells, whether polyclonal (NKG2D CD8 T cells) by guest on September 27, 2021 tially recruited to the lung during Flu infection, accumulating within or monoclonal (F5 TCR transgenic CD8 T cells), share the same the parenchyma (Fig. 4B). In conclusion, our results show that upon transcriptome. IN cells have also acquired a genetic program inflammation or infection, AI memory CD8 T cells enter the lung typical of memory; nevertheless, these cells are less differentiated parenchyma more efficiently than IN memory CD8 T cells. compared with AI memory CD8 T cells. In agreement with a previous study (47), IN cells did not express a specific gene ex- Recruitment of AI memory CD8 T cells into the inflamed lung pression pattern that could indicate an independent differentiation is CXCR3- and CD49a/CD49d-dependent pathway. This suggests that IN memory CD8 T cells represent an Memory CD8 T cell trafficking to the lung parenchyma and airways intermediate stage of differentiation between naive and AI mem- has been reported to be dependent on ITGA1-4 integrins and ory CD8 T cells rather than a distinct CD8 T cell lineage. This is chemokine receptors CXCR3 (39, 41, 45). Using CXCR3 also supported by their cytokine secretion pattern in response to knockout (KO) mice, we confirmed that CXCR3 expression is TCR stimulation, which is similar to that of AI memory CD8 required for AI memory cell recruitment to the lung parenchyma T cells. Differentiation of memory cells is regulated by different (Fig. 5A). However, the differential recruitment of AI and IN pairs of transcription factors, such as Tbet and Eomes, Blimp1/ memory subsets did not involve CXCR3, as both memory cell Bcl6, or ID2/ID3 (48). Interestingly, genes encoding for these types expressed a similar level of CXCR3 and migrated strongly transcription factors are differentially expressed between IN and toward CXCL10 in a transwell assay (Fig. 5B). By contrast, AI AI memory CD8 T cells. Indeed, AI memory CD8 T cells express cells differ from IN cells by the expression level of several integrin higher levels of genes encoding for transcription factors that mRNA; namely, Itga1, Itga4, and Itgb1 encoding for the a- and promote memory CD8 T cell full differentiation (Tbx21, Id2, b-chains of VLA1 (CD49a, CD29) and VLA4 (CD49d, CD29), Prdm1, Zeb2). In contrast, IN memory CD8 T cells express higher respectively (Fig. 3B) (26, 46), a difference confirmed at the levels of genes encoding transcription factors that favor a less protein level by flow cytometry analysis (Fig. 5C). We thus tested differentiated state (Id3, Eomes). Thus, the expression pattern of

(.100 d postinfection) and were restimulated for 12 h with anti-CD3 plus anti-CD28 Abs in the presence of IL-2. Radar plots show the mean amount of each cytokine produced, measured by multiplex. One experiment (n = 5 mice). (D) Graph shows the mean amount (6 SD) of memory-associated poised cytokines produced by CD8 T cell populations, measured by ELISA. One representative experiment out of three (n = 5 mice per experiment). **p , 0.01, Mann–Whitney U test. (E) Production of IFN-g by naive, IN, and AI memory CD8 T cells from VV-infected mice (50 d postinfection) was measured by intracellular cytokine staining following 5 h of restimulation with anti-CD3 plus anti-CD28 Abs or with a mixture of IL-12/IL-18/IL-2. Graph shows the mean percentage (6 SD) of IFN-g+ cells among each CD8 T cell population. A pool of two representative experiments is shown (n = 4 mice in total). *p , 0.05, Mann–Whitney U test. ND, not detected; ns, not signiﬁcant. 8 INNATE VERSUS Ag-INDUCED MEMORY CD8 T CELLS Downloaded from http://www.jimmunol.org/ by guest on September 27, 2021

FIGURE 3. Transcriptome analysis of AI and IN memory CD8 T cells. Naive CD45.1 F5 TCR transgenic CD8 T cells were adoptively transferred in congenic host mice that were subsequently immunized with VV-NP68. Eighty days postinfection, F5 (TCR transgenic) memory cells and polyclonal NKG2D2 and NKG2D+ memory CD8 T cells from the host were sorted from five independent groups of eight mice. As a control, naive F5 and polyclonal CD8 T cells were sorted from five independent groups of three naive F5 and C57BL/6 mice, respectively. The transcriptome of these cell populations was compared by microarrays. (A) PC analysis was performed on whole microarray data. Left graph shows the distribution of samples according to PC1 and PC2. Right graph shows the percentage of variance explained by successive PC. (B) The main genes differently expressed between polyclonal IN and AI memory CD8 T cells are listed. Fold changes and p values are indicated for each gene. The family to which each group of genes belongs is also indicated. (C) Naive, IN, and AI memory CD8 T cells were sorted from VV-infected mice (50 d postinfection). The expression level of several transcription factors by each cell population was assessed by quantitative PCR. Graph shows the mean fold increase (6 SD) compared with naive CD8 T cells. One representative experiment out of two (n = 5 mice per experiment). (D) The expression of Tbet and Eomes by naive, IN, and AI memory CD8 T cells from VV-infected mice (50 d postinfection) was assessed by flow cytometry. Graphs show mean of mean fluorescence intensity (6 SD) of each transcription factor. One representative experiment out of two (n = 4 mice per experiment). *p , 0.05, Mann–Whitney U test. The Journal of Immunology 9

FIGURE 4. IN cells have a reduced capacity to access inﬂamed peripheral tissues compared with AI memory CD8 T cells. (A) CD45.2 memory CD8 T cells were puriﬁed from VV-infected mice (i.p. immunization) and transferred into congenic CD45.1 mice. The next day, host mice received i.n. administration of poly

(I:C) or PBS. Two days later, the numbers of donor IN Downloaded from and AI memory CD8 T cells were measured in various organs. Graph shows the mean ratios (6 SD) between donor IN and AI memory CD8 T cells in spleen, blood, and lung. Lung intravascular staining was performed to differentiate cells in the vasculature and in the parenchyma. One representative experiment out of two (n = 5 mice per group per experiment). (B) CD45.2 memory http://www.jimmunol.org/ CD8 T cells were puriﬁed from VV-infected mice (i.p. immunization) and transferred into congenic CD45.1 mice immunized with Flu virus (i.n. immunization) 2 d earlier. Graphs show mean numbers (6 SD) of donor IN and AI memory CD8 T cells in the spleen, the blood, and the lung (total or in the parenchyma) 2 d later. **p , 0.01, Mann–Whitney U test. by guest on September 27, 2021

transcription factors observed in IN and AI memory CD8 T cell specificity (43, 44, 49), although pathogen-specific spleen memory populations fits with the observed differentiation state. In agree- T cells also rapidly gain access to the tissue (2). ment with their transcription factors expression pattern, AI Previous studies have demonstrated that the CD49d integrin is memory CD8 T cells express higher levels of genes encoding for differentially expressed between B8R-specific IN and AI memory effector molecules, such as granzymes, perforin, and Fas ligand, CD8 T cells (22, 26). Transcriptome comparison of IN and AI cells confirming that these cells are more differentiated and are able to revealed here that other integrin chains are also differentially kill a potential target more rapidly. expressed by AI and IN memory cells. This was confirmed at the One major difference between naive and memory cells is the protein level: AI memory CD8 T cells expressed higher levels of capacity of memory cells to access and reside within tissues pa- several integrins (CD29, CD49a, CD49d) compared with naive or renchyma. Following a pulmonary infection, pathogen-specific IN memory CD8 T cells. These integrins play a key role in im- CD8 effector cells enter the lung parenchyma, and under spe- mune cell migration, allowing them to exit blood vessels and cific signaling, some of them differentiate in TRM that ensure a access peripheral tissues (45). Indeed, ITGA1/B2 (CD29, CD49a) robust response if a secondary infection occurs (3, 4). In the lung, and ITGA4/B2 (CD29, CD49d) play an essential role in the ex- in contrast to other tissues, the population of TRM wanes over travasation of CD8 T cells in the lung or the brain, respectively time (5, 6). Thus, long-term protection of the lung relies on the (50, 51). Accordingly, we found that upon lung inflammation, AI recruitment of secondary memory cells stored in lymphoid tissues. memory CD8 T cells were preferentially recruited within the lung Indeed, following infection of the lung, inflammation rapidly in- parenchyma, and we demonstrated that this process was depen- duces the recruitment of memory cells independently of their Ag dent on integrins. In contrast, IN memory CD8 T cells remained 10 INNATE VERSUS Ag-INDUCED MEMORY CD8 T CELLS

FIGURE 5. Role of CXCR3 and CD49a/ CD49d for entry of AI memory CD8 T cells into inflamed lung. (A) Memory CD8 T cells purified from VV-infected mice, C57BL/6 mice, or CXCR3 KO mice (i.p. immunization) were transferred into congenic hosts. Thirty-five days later, host mice received i.n. administration of Flu virus. Two days later, the number of donor IN and AI memory CD8 T cells was measured in lung parenchyma. One representative experiment out of two. *p , 0.05, Mann–Whitney U test. (B) Histogram shows the expression of CXCR3 by naive, IN, and AI memory CD8 T cells from VV- infected mice (50 d postinfection). Gray histogram: control isotype. Graph shows the migration of naive, IN, and AI memory CD8 T cells from VV-infected mice toward CXCL10 in a transwell assay (mean 6 SD). One representative experi- Downloaded from ment out of three (n = 5 mice per experiment). *p , 0.05, **p , 0.01, Mann–Whitney U test. (C) The expression of CD29, CD49a, and CD49d by naive, IN, and AI memory CD8 T cells from VV-infected mice (50 d postinfection) was assessed by flow cytometry. Gray histogram: control isotype. One representative experiment http://www.jimmunol.org/ out of three (n = 2 mice per experiment). (D) Memory CD8 T cells were purified from VV-infected mice (i.n. immunization), incubated or not with 1 mg/ml anti-CD49a and anti-CD49d Abs, and then transferred into congenic mice that had been immunized i.n. 2 d before with Flu virus. The next day, host mice received i.p. administration of 250 mg anti-CD49a plus anti-CD49d

Abs or PBS. Two days later, the number of donor by guest on September 27, 2021 IN and AI memory CD8 T cells was measured in lung parenchyma. Graph shows mean numbers (6 SD) of donor IN and AI memory CD8 T cells. A pool of two representative experiments is shown (n =13miceintotal).**p , 0.01, Mann–Whitney U test.

within the lung vasculature. Memory CD8 T cell trafficking to the infection more efficiently than naive CD8 T cell do. Similarly, lung parenchyma and airways has been reported to be dependent results obtained in mice that are deficient in IL-4–induced IN cells of the chemokine receptor CXCR3 (41). In agreement, CXCR3 indicate a decreased capacity to control a primary lymphocytic KO AI memory cells were not recruited to the lung. Importantly, choriomeningitis virus infection in the absence of these cells (52). we did not observe any differential CXCR3 expression or The protection conferred by IN cells could be direct, as a result of CXCL10-induced migration between IN and AI memory CD8 their enhanced effector functions, or indirect, through the help to T cells. This indicates that CXCR3 expression is necessary but not naive CD8 T cells (53). However, our results show that they are sufficient to access the inflamed lung parenchyma. not recruited to the inflamed lung parenchyma because of the lack IN memory CD8 T cells have a TCR repertoire as diverse as that of ITGA1/4 integrins expression. This is in line with the dem- of naive CD8 T cells, although their repertoire does not completely onstration that VM CD8 T cells do not confer protection against overlap. This diversified TCR repertoire could allow IN memory L. monocytogenes upon gut infection (46). CD8 T cells to contribute to multiple immune responses. In line The decreased capacity of IN memory cells to access non- with this, we showed that IN memory CD8 T cells participated in lymphoid tissues in response to inflammatory chemokines could be primary immune responses against two pathogens, namely VV and essential for the prevention of autoimmunity. Because of its gen- L. monocytogenes. Participation of IN memory CD8 T cells in eration process, the population of IN memory CD8 T cells might be primary immune responses against infectious pathogens could preferentially generated from naive cells with increased sensitivity significantly increase the efficiency of these responses. Indeed, we for self-antigens. Indeed, CD5hi naive CD8 T cells, which have an showed that physiologically generated IN cells produced a lot increased sensitivity to self-antigens, are more prone to undergo more cytokines than naive cells when stimulated through their LIP compared with CD5lo naive cells (54, 55). Moreover, CD5hi TCR. In line with this, Lee et al. (27) demonstrated that OVA- naive cells are more predisposed to become VM CD8 T cells specific IN memory CD8 T cells cleared L. monocytogenes–OVA compared with CD5lo naive cells. Of note, increased lymphopenia The Journal of Immunology 11 drives the development of autoaggressive T cells in NOD mice. 20. Lee, Y. J., K. L. Holzapfel, J. Zhu, S. C. Jameson, and K. A. Hogquist. 2013. Steady-state production of IL-4 modulates immunity in mouse strains and is This mechanism accounts partly for the development of dia- determined by lineage diversity of iNKT cells. Nat. Immunol. 14: 1146–1154. betes in this mouse model (56). The exclusion from peripheral 21. Akue, A. D., J.-Y. Lee, and S. C. Jameson. 2012. Derivation and maintenance of tissues of IN memory CD8 T cells that show increased reac- virtual memory CD8 T cells. J. Immunol. 188: 2516–2523. 22. Sosinowski, T., J. T. White, E. W. Cross, C. Haluszczak, P. Marrack, L. Gapin, tivity compared with naive cells might thus be important to and R. M. Kedl. 2013. CD8a+ dendritic cell trans presentation of IL-15 to naive avoid autoimmunity. CD8+ T cells produces antigen-inexperienced T cells in the periphery with In conclusion, NKG2D is a novel marker of AI memory cells. memory phenotype and function. J. Immunol. 190: 1936–1947. 23. Le Campion, A., C. Bourgeois, F. 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Immunol. 182: 1429– acknowledge the contribution of SFR BioSciences (UMS3444-CNRS/US8- 1438. INSERM, Ećole Normale Supe´rieure de Lyon, Universite´ de Lyon) facilities 26. Haluszczak, C., A. D. Akue, S. E. Hamilton, L. D. S. Johnson, L. Pujanauski, L. Teodorovic, S. C. Jameson, and R. M. Kedl. 2009. The antigen-specific CD8+ and their staff, especially T. Andrieu and S. Dussurgey (AniRA-Cytome´trie) T cell repertoire in unimmunized mice includes memory phenotype cells bearing and J.L. Thoumas, C. Angleraux and J.F. Henry (AniRA-PBES). markers of homeostatic expansion. J. Exp. Med. 206: 435–448. 27. Lee, J.-Y., S. E. Hamilton, A. D. Akue, K. A. Hogquist, and S. C. Jameson. 2013. Virtual memory CD8 T cells display unique functional properties. Proc. Natl. Downloaded from Disclosures Acad. Sci. USA 110: 13498–13503. The authors have no financial conflicts of interest. 28. Rabinovich, B. A., J. Li, J. Shannon, R. Hurren, J. Chalupny, D. Cosman, and R. G. Miller. 2003. 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Antigen-induced but not innate memory CD8 T cells express NKG2D and are recruited to the lung parenchyma upon viral infection

Morgan Grau1, Séverine Valsesia1, Julien Mafille1, Sophia Djebali1, Martine Tomkowiak1, Anne-Laure Mathieu1, Daphné Laubreton1, Simon de Bernard2, Pierre-Emmanuel Jouve2, Erwan Ventre1, Laurent Buffat2, Thierry Walzer1, Yann Leverrier1, Jacqueline Marvel1. IL-2 IL-4

99.5 99.7 CD44

99.9 100 CXCR3

Figure S1: Cytokine-induced memory CD8 T cells. Purified naive CD45.2 OTI CD8 T cells were adoptively transferred in immunocompetent congenic CD45.1 C57BL/6 mice. The next day, hosts received intra-peritoneal injections of the indicated γc cytokine antibody-complex as described in the methods. 30 days after transfer, the expression of memory markers CD44 and CXCR3 by CD8 T cells was assessed by flow cytometry. Black histogram: OTI CD8 T cells, grey histogram: host’s CD8 T cells. The percentage of OTI cells expressing each protein is indicated on each graph. One representative experiment out of four is shown (n = 3 mice per experiment). 12 88 1.5 98.5 96.5 3.5 CD45.2 CD44 CXCR3 NKG2D

Figure S2: Lymphopenia-induced memory cells do not express NKG2D. Purified naive CD45.2 OT-I CD8 T cells were adoptively transferred into irradiated CD45.1 C57BL/6 mice. 30 days later, the expression of CD44, CXCR3 and NKG2D by OT-I CD8 T cells and endogeneous CD8 T cells was assessed by flow cytometry. Grey plots: endogenous CD8 T cells, red plots: OT-I CD8 T cells. One representative experiment out of three (n = 4 mice per experiment). Numbers indicate the percentages among CD45.2+ OTI CD8 T cells. Total CD44 hi NKG2D- NKG2D+ 107 107 107 blood 106 ** 106 106 / ml of ns ** 5 5 5 cells 10 10 10 Cells / ml of blood Cells / ml of blood Cells / ml of blood

CD8 T T CD8 104 104 104 0 20 40 60 0 20 40 60 0 20 40 60

DaysDays post-infectionpost infection Days post-infection Days post-infection

Figure S3: Kinetic of NKG2D expression by CD44hi CD8 T cell populations in the blood of VV-infected mice. C57BL/6 mice were infected with VV and the number of total, NKG2D- and NKG2D+ CD44hi CD8 T cells was measured in the blood at various time points post-infection. Graphs show for each time point the mean number (+/- SD) of each CD44hi CD8 T cell population. Dotted lines mark the mean number of cells observed before infection. One representative experiment out of two (n = 10 mice per experiment). Wilcoxon matched-pairs signed rank test, ns: not significant, **: pvalue < 0.01. Naive A D0 D55 B CD44hi

15 37.4 BALB/c 4.7 *** Total CD44hi 10 * NKG2D-

5 NKG2D+

10.2 40.2 OF1 Expansion index 0 BALB/c OF1 NKG2D

Figure S4: NKG2D identifies AI memory cells in BALB/c and OF1 mouse strains. BALB/c and OF1 mice were infected with VV. (A) NKG2D expression by blood-derived memory phenotype (CD44hi CD122+, black histogram) and naive (grey histogram) CD8 T cells was measured by flow cytometry before (D0) and 55 days after infection (D55). The percentage of NKG2D+ cells are indicated. (B) Graph shows mean expansion index (+/- SD) of each memory phenotype CD8 T cell populations following infection by VV. For each cell population, the expansion index was calculated as the ratio between the number of cells observed in the blood of each individual mouse 55 days post infection and the number of cells observed before infection. The dotted line represents an absence of cell population expansion (Index = 1). One representative experiment out of two (n = 13 mice per group and per experiment). Wilcoxon matched-pairs signed rank test, *: pvalue < 0.05, **: pvalue < 0.01, ***: pvalue < 0.001. A

Symbol Name fold-change p-value CTSE cathepsin E x24.7 2.749e-33 KLRA7 killer cell lectin-like receptor, subfamily A, member 7 /11.9 1.636e-23 TRDC T cell receptor delta, constant region x9.4 1.063e-35 TRBV14 T cell receptor beta, variable 14 /7.2 3.724e-18 IFITM3 interferon induced transmembrane protein 3 x7.2 1.428e-27 TRBV5 T cell receptor beta, variable 5 /5.9 1.785e-18 PDCD1 programmed cell death 1 /5.5 2.65e-19 KLRA1 killer cell lectin-like receptor, subfamily A, member 1 /5.1 2.042e-17 KLRE1 killer cell lectin-like receptor family E member 1 /5.1 4.219e-23 CD5 CD5 antigen /5.0 2.029e-20 IFITM1 interferon induced transmembrane protein 1 x4.6 5.165e-17 CD6 CD6 antigen /4.3 6.466e-20 KLRA7 killer cell lectin-like receptor, subfamily A, member 7 /3.3 1.655e-15 GZMA granzyme A /3.2 4.426e-11 IFITM2 interferon induced transmembrane protein 2 x3.0 6.04e-12 CD163L1 CD163 molecule-like 1 /2.9 1.325e-23 ST8SIA1 ST8 alpha-N-acetyl-neuraminide alpha-2,8-sialyltransferase 1 x2.7 4.464e-14 B4GALNT4 beta-1,4-N-acetyl-galactosaminyl transferase 4 x2.6 2.399e-9 RAMP3 receptor (calcitonin) activity modifying protein 3 /2.5 4.658e-6 TRBJ2-2 T cell receptor beta joining 2-2 /2.5 1.802e-11 DAPL1 death associated protein-like 1 /2.5 3.725e-10 IKZF2 IKAROS family zinc finger 2 /2.5 4.274e-12 GPR34 G protein-coupled receptor 34 x2.4 1.56e-16 ITGA6 integrin alpha 6 /2.4 6.563e-9 AA467197 expressed sequence AA467197 x2.3 3.391e-12 EZH2 enhancer of zeste homolog 2 (Drosophila) /2.2 2.223e-12 CDH1 cadherin 1 x2.2 2.067e-8 CAR2 carbonic anhydrase 2 /2.2 3.405e-10 KLRG1 killer cell lectin-like receptor subfamily G, member 1 /2.2 2.29e-8 UNC5CL unc-5 homolog C (C. elegans)-like /2.1 1.456e-15 ACPP acid phosphatase, prostate /2.1 5.527e-9 2810417H1 3RIK RIKEN cDNA 2810417H13 gene /2.1 1.078e-10 RRM2 ribonucleotide reductase M2 /2.1 5.469e-9 EMP1 epithelial membrane protein 1 x2.1 2.52e-6 TTC39C tetratricopeptide repeat domain 39C x2.1 4.228e-11 ITM2A integral membrane protein 2A /2.0 3.024e-12 PSEN2 presenilin 2 /2.0 4.365e-13 FNDC3B fibronectin type III domain containing 3B /2.0 9.262e-15

B C

F5 memory cells 36.9 8.6 Host’s memory CD8 T cells NKG2D % of max 42.6 11.9

Ly49 A/E/F/G2 Ly49 A/E/F/G2 Figure S5: Transcriptome of polyclonal AI memory CD8 T cells and TCR transgenic memory CD8 T cells differ by the expression of Ly49A, E, F and G2 receptors. (A) Genes differentially expressed with a FC higher >2 and a p value < 0.001, when comparing F5-TCR transgenic and polyclonal-AI memory CD8 T cells are shown. Genes encoding NK receptors are highlighted in orange. Genes that are differentially expressed between F5 (naive or memory) and polyclonal CD8 (naive or memory) are highlighted in green. Of note, increased expression of the TCR delta chain transcript in F5 TCR transgenic CD8 T cells is a well characterized phenomenon, that results from the absence of alpha chain rearrangement that is associated with TCR transgene expression (Wilson, A., de Villartay, J. P. & MacDonald, H. R. T cell receptor delta gene rearrangement and T early alpha (TEA) expression in immature alpha beta lineage thymocytes: implications for alpha beta/gamma delta lineage commitment. Immunity 4, 37–45 (1996).). (B) Expression of Ly49A, E, F and G2 receptors by F5 and polyclonal memory CD8 T cells from VV-NP68–infected mice (50 days post infection) was assessed by flow cytometry. (C) Expression of Ly49A, E, F and G2 receptors and NKG2D by memory CD8 T cells from VV-infected mice (50 days post infection). Percentages in the corresponding quadrants are indicated. One representative experiment out of two (n = 2 mice per experiment). 80

Naives IN AI

40 % of speciﬁc lysis % of speciﬁc

0 0.01 0.1 0.3 1 3 CD8 T cells : Target

Figure S6: IN memory CD8 T cells have reduced cytotoxic capacity compared to AI memory cells. Anti-CD3 coated P815 cells were incubated at various ratio with naive, IN or AI memory CD8 T cells sorted from VV-infected mice (80 days post-infection) for 16h. The percentage of specific lysis was determined as described in the methods. One representative experiment out of two (a pool of 5 mice was used for each experiment). Lung parenchyme

3Spleen×1003 Blood* 05 04 4 10 ns 1.5 10 ns IN ns NKG2D- NKG2D- 3 1005 NKG2D+ns NKG2D+AI CD8 T cells CD8 T cells 03 1.0 1004 hi hi 2×10 CD8 T Cells CD8 2 1005 hi 5.0 1003 1 1005 03 0 1×10 0.0 PBS Poly I/C PBS Poly I/C # of donor CD44 donor of # # of donor CD44 donor of # # of donors CD44

0 Lung WT CXCR3 KO Blood capilaries Parenchyma Airways 5.0 1004 * 04 * 04 03 5.0 10 ** 3.0 10 NKG2D- +* Virus3.0 10 NKG2D-NKG2D+ NKG2D- NKG2D-

CD8 T cells ** *

hi NKG2D+ NKG2D+ NKG2D+ CD8 T cells CD8 T cells CD8 T cells

hi 2.5 1004 hi hi 2.5 1004 1.5 1004 1.5 1003

ns 0.0 * 0.0 PBS Poly I/C 0.0 0.0 # of donor CD44 donor of # PBS Poly I/C PBS Poly I/C PBS Poly I/C # of donor CD44 donor of # CD44 donor of # CD44 donor of #

Figure S7: IN have a reduced capacity to access peripheral tissues compared to AI memory CD8 T cells. CD8 T cells were purified from VV-infected mice (intraperitoneal immunization) and transferred into congenic mice. The next day, host mice received intranasal administration of poly(I:C) or PBS. Two days later, the number of donor IN and AI memory CD8 T cells was measured in various organs. Graphs show mean number (+/- SD) of donor NKG2D- and NKG2D+ memory CD8 T cells in different organs or compartment. One representative experiment out of two (n = 5 mice per group per experiment). CD44+ NKG2D+ parmi CD8+ transférés

3×1004

2×1004 CD8 T Cells CD8 T hi

** 1×1004

ns # of donors CD44

0 Flu + + + + + + CD49a/CD49d - + - + - +

Lung Spleen Blood

Figure S8: Distribution of AI memory cells. Memory CD8 T cells were purified from VV-infected mice (intranasal immunization), incubated or not with 1 µg/ml anti-CD49a and anti-CD49d antibodies, and then transferred into congenic mice that had been immunized intra-nasaly 2 days before with Flu virus. The next day, host mice received intraperitoneal administration of 250 µg/ml anti-CD49a plus anti- CD49d antibodies or PBS. Two days later, the number of donor AI memory CD8 T cells was measured in lung, spleen and blood. Graph shows mean numbers (+/- SD) of donor AI memory CD8 T cells. A pool of two representative experiments is shown (n = 13 mice in total). Mann-Whitney test, *: pvalue < 0.05, **: pvalue < 0.01. 4000 3000 2000 1000 150

expression (a.u.) expression 100 50 Klrk1 0 Naive IN AI

Figure S9: Expression of NKG2D mRNA by naive, IN and AI memory CD8 T cells. Naive, IN and AI memory CD8 T cells were purified as described in Figure 3. Analysis of Klrk1 expression was performed by microarray analysis. Graph shows mean expression from 4 to 5 replicates (+/- SD).