Differential Kinetics of Antigen Dependency of CD4 + and CD8+ T Cells Hannah Rabenstein, Anne C. Behrendt, Joachim W. Ellwart, Ronald Naumann, Marion Horsch, Johannes Beckers and This information is current as Reinhard Obst of October 7, 2021. J Immunol published online 17 March 2014 http://www.jimmunol.org/content/early/2014/03/17/jimmun ol.1302725 Downloaded from

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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 © 2014 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published March 17, 2014, doi:10.4049/jimmunol.1302725 The Journal of Immunology

Differential Kinetics of Antigen Dependency of CD4+ and CD8+ T Cells

Hannah Rabenstein,*,1,2 Anne C. Behrendt,*,2 Joachim W. Ellwart,† Ronald Naumann,‡ Marion Horsch,x Johannes Beckers,x,{ and Reinhard Obst*

Ag recognition via the TCR is necessary for the expansion of specific T cells that then contribute to adaptive immunity as effector and memory cells. Because CD4+ and CD8+ T cells differ in terms of their priming APCs and MHC ligands we compared their requirements of Ag persistence during their expansion phase side by side. Proliferation and effector differentiation of TCR transgenic and polyclonal mouse T cells were thus analyzed after transient and continuous TCR signals. Following equally strong stimulation, CD4+ T cell proliferation depended on prolonged Ag presence, whereas CD8+ T cells were able to divide and differentiate into effector cells despite discontinued Ag presentation. CD4+ T cell proliferation was neither affected by Th lineage or memory differentiation nor blocked by coinhibitory signals or missing inflammatory stimuli. Continued CD8+ T cell prolif- Downloaded from eration was truly independent of self-peptide/MHC-derived signals. The subset divergence was also illustrated by surprisingly broad transcriptional differences supporting a stronger propensity of CD8+ T cells to programmed expansion. These T cell data indicate an intrinsic difference between CD4+ and CD8+ T cells regarding the processing of TCR signals for proliferation. We also found that the presentation of a MHC class II–restricted peptide is more efficiently prolonged by dendritic cell activation in vivo than a class I bound one. In summary, our data demonstrate that CD4+ T cells require continuous stimulation for clonal expansion, whereas CD8+ T cells can divide following a much shorter TCR signal. The Journal of Immunology, 2014, 192: http://www.jimmunol.org/ 000–000.

cell receptor–mediated recognition of antigenic peptides the cells regain motility and divide every 4–6 h, if not faster (1), presented by MHC class I and II molecules is necessary for several d to expand the 15–1500 clonal precursors per mouse for clonal expansion and effector cell differentiation of up to 100,000-fold. This expansion and differentiation of rare T+ + CD8 and CD4 T cells, respectively. Initial TCR signals, inte- pathogen-specific T cell precursors to effector clones is crucial for grated by microclusters and synaptic structures, arrest the T cells adaptive immunity and survival (2, 3). On the relevance of Ag in contact with the APC allowing for sustained signals initiating recognition following the initial divisions, however, a consensus blasting and massive de novo gene transcription. One to 2 d later, has not been reached. by guest on October 7, 2021 For CD8+ T cells, it has been shown that antigenic signals beyond an initial period are not required for proliferation (4–10), + *Institute for Immunology, Ludwig-Maximilians-University Munich, 80336 Munich, thus supporting the notion of early programming of CD8 T cell Germany; †Institute for Molecular Immunology, Helmholtz Zentrum Munich, 81377 expansion (11, 12). However, there is also data that CD8+ Tcell Munich, Germany; ‡Transgenic Core Facility, Max Planck Institute of Molecular Cell x expansion is correlated with the continued duration of TCR Biology and Genetics, 01307 Dresden, Germany; Institute of Experimental Genetics, Helmholtz Zentrum Munich, 85764 Neuherberg, Germany; and {Department of Ex- stimulation in the context of both infections and sterile vacci- perimental Genetics, Center of Life and Food Sciences Weihenstephan, Technical nations (13–21). The reason for this discrepancy is currently un- University Munich, 85350 Freising-Weihenstephan, Germany clear and cannot be explained by differences of protocol, exper- 1 Current address: Immunogenetics, Center for Human Genetics and Laboratory Med- imental systems or availability of help or inflammation. icine, Martinsried, Germany. The use of an Ag-independent proliferative phase of T cells has 2H.R. and A.C.B. contributed equally to this work. been instrumental for the expansion of lines and clones in vitro, Received for publication October 18, 2013. Accepted for publication February 13, 2014. especially by including growth factors and cytokines in the cul- tures. Two studies supplied evidence for the notion that TCR- This work was supported by German Research Council Grants SFB571-B8 and + SFB1054-B7 (to R.O.), Bundesministerium fur€ Bildung und Forschung Grant triggered CD4 T cells keep proliferating in the absence of Ag NGFNplus 01GS0850 (to J. B.), and the Munchner€ Medizinische Wochenschrift and cytokines (22, 23), whereas others did not support such an (2010) (to H.R.). early-programming scenario but rather supplied evidence for The microarray data presented in this article have been deposited in the Gene Ex- the importance of maintained TCR signals (24–27). Most of the pression Omnibus database (http://www.ncbi.nlm.nih.gov/gds) under accession num- + ber GSE49063. experiments done in vivo supported the Ag dependency of CD4 Address correspondence and reprint requests to Dr. Reinhard Obst, Institute for T cells throughout the expansion phase (8, 13, 28–33). Immunology, Ludwig-Maximilians-University Munich, Goethestrasse 31, 80336 Mu- Although clonal expansion is at the center of adaptive immu- nich, Germany. E-mail address: [email protected] nity, very little is known about cell cycle regulation in normal The online version of this article contains supplemental material. lymphocytes (34). It has been noted in many infections of mice Abbreviations used in this article: B6, C57BL/6; BM, bone marrow; DC, dendritic and humans that the magnitude of the CD8+ T cell response is larger cell; dox, doxycycline; dtg, double-transgenic; FC, fold change; Ii, invariant chain; + LN, lymph node; MCC, moth cytochrome c; MCMV, murine CMV; pMHC, peptide- than that of CD4 T cells and an intrinsic difference between MHC; rTA, reverse transactivator; tet, tetracycline; TIM, tetracycline-inducible in- the subsets has been invoked (35, 36). However, whether Ag variant chain with MCC; TSO, tetracycline-inducible signal sequence with OVA; wt, requirements in the expansion phase differ between CD4+ and wild-type. CD8+ T cells has been asked in few studies that came to different Copyright Ó 2014 by The American Association of Immunologists, Inc. 0022-1767/14/$16.00 conclusions (8, 13, 32). Thus, we examined the Ag dependency of

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1302725 2 DIFFERENTIAL Ag DEPENDENCY OF T CELL SUBSETS

CD4+ and CD8+ T cell proliferation side by side in a way that Cells were fixed and permeabilized with buffers from BioLegend (fix/perm excludes cross-talk between the subsets. Because pathogens have buffer) or eBioscience (FoxP3 staining buffer), according to the manu- developed numerous ways to undermine T cell priming and affect facturers’ instructions. Flow cytometric analyses were performed on FACSCanto II flow cytometers using the FACSDiva software (BD). Data the immune response by inflammation, we used noninfectious were analyzed with the FloJo 8.8.7 software (Tree Star), the cells shown are methods where priming conditions and Ag removal are clearly DAPI or Fixable Viable Dye (eBioscience)–negative singlet lymphocytes, defined, are the same for both subsets, and are not affected by according to forward and side scatter properties. The proliferative index N APC type and MHC class differences. We found that following based on CFSE dilution profiles was calculated as the average number of divisions compared with unstimulated control cells. strong priming, CD8+ T cells are able to divide in an Ag-independent way whereas CD4+ T cells are not. These findings correlated with Cell preparation and stimulation in vitro the more efficiently extended presentation of a MHC class II–re- Lymph node (LN) and splenocyte suspensions were prepared in DMEM/5 stricted epitope following dendritic cell (DC) activation compared mM HEPES/1% BSA subsets purified magnetically (Miltenyi Biotec) using with a class I–restricted peptide. This suggests, we speculate, a positive selection with biotinylated CD4 or CD8 mAbs or negative selec- coevolution of MHC class and the proliferative responses of the tion with mAbs against CD11c, CD11b, GR1, CD49b, CD45R, CD4, or CD8 and anti-biotin beads to .90% purity. TCR transgenic or polyclonal CD4+ or corresponding T cell subset. + CD8 T cells were cultured for 2 d in 96-well round-bottom plates (Sar- stedt) precoated with 10 mg/ml anti-CD3 (145-2C11) and anti-CD28 mAbs Materials and Methods (37.51; both from BioXCell) or untreated plates at 1–2 3 105 cells/well in Mice RPMI 1640 medium (PAA) supplemented with 10% FCS (Life Technol- m AND (Tg(TcrAND)53Hed) (37), OT-1 (Tg(TcraTcrb)1100Mjb) (38), in- ogies), 2 mM glutamine, 50 M 2-ME, antibiotics (complete medium), variant chain-moth cytochrome c (Ii-MCC) (Tg(H2-Ea-Cd74/MCC)37GNnak) and 5 ng/ml IL-7 (Immunotools). For Th1 and Th2 differentiation of AND (39), Ii-reverse transactivator (rTA) (Tg(Cd74-rtTA)#Doi), tet-inducible T cells in vitro, cells were stimulated with anti-CD3 and anti-CD28 in Downloaded from complete medium containing 5 ng/ml IL-12 and 20 mg/ml anti–IL-4 mAb modified Ii MCC (Tg(tetO-Cd74/MCC)#Doi) (28), CD11c-b2m and tm1Jae tmiLpc (11B11) for Th1 conditioning or 50 ng/ml IL-4 and 50 mg/ml anti–IFN-g K14-b2m transgenics, and B2m (40), and Cd274 mice (41) have been described previously. Animals were kept on the C57BL/6 (B6) and mAb (XMG1.2; both BioXCell) for Th2 conditioning. Following stim- the B10.BR/SgSnJ (The Jackson Laboratory) backgrounds. The CD45.1 or ulation, cells were CFDA-SE labeled and transferred as described pre- CD90.1 congenic markers for AND and OT-1 TCR transgenic T cells were viously (42). originally derived from B6.SJL-Ptprca Pepcb/BoyJ and B6.PL-Thy1a/CyJ Generation of rested effector CD4+ T cells animals (The Jackson Laboratory), respectively. All animals were housed http://www.jimmunol.org/ and bred, and experiments were conducted, at the Institute for Immunology Rested effector cells were generated according to Ref. 43. Briefly, 0.5 3 in compliance with German federal guidelines and approved by the gov- 105 naive LN AND T cells were cultured with 105 irradiated splenocytes ernment of Upper Bavaria (Az. 55.2-1-54-2531-106-08). from Ii-MCC transgenic mice that constitutively express Ek/MCC com- plexes on DCs and B cells (39) in 96-well plates in complete medium Transgene construct containing 80 U/ml IL-2 for 4 d. APCs were removed by centrifugation The tetracycline (tet)-inducible signal sequence with OVA (TSO) trans- over a Ficoll cushion (LSM1077; PAA), and cells were cultured for an gene consists of the Kb signal sequence (from pUC19-H-2Kb, provided by additional 3 d without IL-2. B. Arnold, German Cancer Research Center, Heidelberg, Germany), fol- In vivo killing assay lowed by the sequence encoding OVA257–264, two stop codons, and the human growth hormone splice substrate under the control of the tet re- Congenically marked B6 splenocytes were cultured for 4 h in complete sponsive elements of pTRE-tight (BD Clontech). Restriction digested medium with or without 1 mg/ml SIINFEKL peptide (Peptides and Ele- by guest on October 7, 2021 transgene DNA was separated from plasmid sequences by agarose/crystal phants). Cells were then labeled with 5 nM (pulsed) or 5 pM CFDA-SE violet electrophoresis and purified for injection into C57BL/6 zygotes (unpulsed) and 2.5 3 106 of each transferred into recipients of OT1 cells by Nucleobond column purification (Macherey-Nagel). Seven founders 3 d before. Splenocytes were analyzed 1 d later. were identified by PCR with primers RO235 (59-CTCATCTCAAACAA- GAGCCA-39) and RO236 (59-CACTGCTTACTTCCTGTACC-39) and Animal treatments crossed to Ii-rTA transgenics (28). DCs from double transgenic offspring For blocking coinhibitory interactions, animals were injected i. p. with 200 were tested for proliferation of OT-1 T cells in the presence of titrated amounts mg blocking mAbs to PD-1 (J43), PD-L1 (10F.9G2), CTLA-4 (UC10- of doxycycline (dox). One line (number 7) exhibited dox-dependent Ag pre- 4F10-11), LFA-1 (M17.4), or control Ab (polyclonal Armenian hamster sentation of the OVA peptide by DCs and all experiments described were IgG; rat IgG2a; all from BioXCell). For DC activation, 20–50 mg anti- done with this line. CD40 (FGK45.5; Miltenyi Biotec or BioXCell) were given i.p. Where 6 Bone marrow chimeras indicated mice were injected i. p. with 2 3 10 PFU murine CMV (MCMV) (provided by T. Brocker, Munich, Germany). For gene induction, B6 and B10.BR animals were irradiated (5 Gy) with a [137Cs] source twice mice were fed with 100 mg/ml dox (Sigma-Aldrich) diluted in water low in (6–48 h apart), transferred with 5 3 106 bone marrow (BM) cells from divalent cations (Volvic, Danone Waters) supplied ad libitum. The use of double-transgenic (dtg)-O or -M donors and supplied with 2 g/l neomycin dox-treated nontransgenic and untreated dtg recipients did not make sulfate and 0.1 g/l polymyxin b sulfate (Applichem) in the drinking water. a difference so that antibiotic side effects have been excluded. The animals were used 4–6 wk later for adoptive transfers. Cell sorting and RNA preparation Abs and flow cytometry For 2-d transcriptome analysis, lymphocytes were sorted and cultured as Single-cell suspensions were stained in DMEM/5 mM HEPES/1% BSA on described and sorted to .99% purity on a MoFlo cell sorter (Cytomation, ice. Fluorochrome-conjugated Abs were CD4 (clone RM4-5), CD5 (53- Fort Collins, CO). Transferred CD4+ or CD8+ T cells were stained and 7.3), CD8 (53-6.7), CD11b (M1/70), CD11c (N418), CD44 (IM7), CD45R CD4+/CD8+CD45.1+DAPI2 cells were sorted twice, the second time with (RA3-6B2), CD45.1 (A20), CD45.2 (104), CD49b (DX5), CD62L (MEL- a purity of 99–100% directly into TRIzol (Invitrogen). RNA from sorted 14), CD69 (H1.2F3), CD71 (RI7217), CD98 (RL3889) IFN-g (XMG1.2), cells was isolated as described previously (42). One to 20 ng high-quality and TCRb (H57-597) (all BioLegend); CD25 (PC61), T-bet (eBio4B10), total RNA (RNA integrity number. 7; 2100 Bioanalyzer; Agilent Tech- and Eomes (DAN11MAG) (all eBioscience), Ki67-Al488 (B56), Vb5 nologies) was amplified using the two-cycle MessageAmp II aRNA Am- (MR9-4) from BD, Va2 (CL7213F) from Cedarlane Laboratories, and plification Kit (Ambion). Amplified aRNA (10 mg) was further processed Vb3 (a gift from N. Asinovski, C. Benoist, and D. Mathis, Boston, MA). using the Message Amp II-Biotin Enhanced Kit (Ambion) and hybridized Single-cell suspensions from lymphoid organs were prepared by me- on Affymetrix Mouse Genome 430 2.0 arrays in a GeneChip Hybridization chanical dispersion. Erythrocytes were removed from splenocyte sus- Oven 645, washed, and stained with a GeneChip Fluidics Station 450 and pensions by centrifugation through a Ficoll cushion (LSM1077, PAA, or scanned with a GeneChip Scanner 3000. Three biological replicates of lympholyte M; Cedarlane Laboratories). All cytokine stainings were per- stimulated and control CD4+ and CD8+ T cell samples at days 2 and 5 were formed after a 4-h incubation with 20 ng/ml PMA and 1 mg/ml ionomycin, analyzed as indicated in Figs. 2 and 6 (with two exceptions; 28 samples with 10 mg/ml brefeldin A (all Sigma-Aldrich) included for the last 2 h. altogether). The data were analyzed with modules of the Genepattern The Journal of Immunology 3 package (Broad Institute, Boston, MA; Ref. 44). Primary .cel files were cells in dtg-O animals. When we restricted the transgenes to BM- normalized with the ExpressionFileCreator module implementing RMA derivedAPCsindtg-O→ wild-type (wt) radiation chimeras, and redundant probe sets collapsed with the CollapseDataset module. One background and residual OVA presentation was extin- hundred and twenty-eight gene probes for B cell–specific (45) and XY 257–264 chromosome–encoded genes were removed. Data were visualized with the guished so that OT1 T cells did only proliferate in dox-treated Multiplot (volcano and fold change [FC]/FC plots), HierarchicalCluster- recipients but not in chimeras that had been treated 3 d before or ingViewer and HeatMapViewer modules, nearest neighbors clustering not at all (Fig. 1B). was done with the Gene-E module of GenePattern. Regression lines in To directly compare the effective half-lives of the two epitopes Fig. 6 were calculated in Matlab (Mathworks). in the steady state, we treated dtg-M and -O → WT chimeras with Statistical analysis dox for 24 h and transferred the respective T cells 4–0 d later. We Cellular data were analyzed using the Prism 5.0 software (GraphPad, San used the CFSE dilution of transferred T cells to estimate the Diego, CA). disappearance of pMHC complexes as far as they can be detected by naive TCR-transgenic T cells. In this sense, we estimated the Accession numbers epitopes’ relative survival time to about a 1 d for Ek/MCC, as The microarray data have been deposited in the Gene Expression Omnibus observed previously in nonchimeric dtg-M animals (28) and 2 d database (http://www.ncbi.nlm.nih.gov/gds) under the accession number for Kb/OVA (Supplemental Fig. 1), likely because of the slow GSE49063. dissociation of this peptide (46). Results Comparable activation of AND and OT1 T cell activation Inducible Ag presentation of MHC class II and I epitopes in vitro

For comparing CD4+ and CD8+ T cell responses, we used dtg We asked next whether CD4+ and CD8+ T cells responded differ- Downloaded from mouse models where DCs present antigenic epitopes under tet- or ently to transient signals. Because of the “leakiness” of the dtg-O dox-dependent control of the Ii-rTA transgene described previ- system, we decided to prime the T cells in vitro by surface-bound ously (28). Its combination with the tet-inducible modified Ii mAbs. Thereby, MHC class differences of tissue distribution, cell MCC transgene (dtg-M for brevity) allows for the induction of an biology, and stability as well as differential imprecision of the tet epitope consisting of H-2Ek and an MCC peptide to which AND systems are circumvented: because the anti-CD3 and -CD28 + CD4 T cells respond vigorously (Fig. 1A, 1B). To generate mice mAbs trigger T cells independently of abTCR and coreceptor http://www.jimmunol.org/ b with controllable presentation of the H-2K /OVA257–264 complex, avidities, this system may be better suited for revealing differences a transcriptional unit consisting of a signal sequence, the epitope, between the two T cell types. Following 2 d of stimulation, AND stop codons and a splicing substrate was put under the control of and OT1 T cells exhibited no significant differences as far as TCR tet-responsive regulatory elements (TSO) and introduced into the downregulation and the activation markers CD25, CD44, CD62L, germ line of B6 animals (Fig. 1A). This minigene targets the CD69, CD71, and CD98 were concerned; the only notable dif- OVA257–264 peptide directly to the ER in a proteasome- and TAP- ference was the slightly lower TCR expression levels on the OT1 independent fashion and excludes indirect presentation of trans- cells before and after stimulation (Fig. 2A), a finding that also ferred OVA protein by other APCs. TSO+ founder animals were applied to polyclonal B6 T cells (data not shown). We also noted bred to the Ii-rTA line. dtg-O offspring displayed dox-inducible a difference of CD5 expression between the subsets (Supplemental by guest on October 7, 2021 OVA257–264 presentation in vivo as evidenced by proliferation of Fig. 2), in agreement with previous work on higher tonic TCR CFSE-labeled OT1 TCR transgenic T cells (Fig. 1B). However, signaling (47) and ensuing CD5 expression by CD4+ T cells (48, in contrast to dtg-M recipients, we observed proliferation in the 49). Following incubation, the cells had become blasts and had absence of dox in the drinking water and 3 d after turn-off, sug- initiated proliferation because they had divided once on average gesting unregulated TSO expression in at least some MHC class I+ (Fig. 2B). To find out how similar the cells were in terms of their

FIGURE 1. Inducible Ag Presentation of MHC class II and I–restricted epitopes in vivo. (A) Scheme of double transgenics for tet-inducible epitope expression. See the first paragraph of Results for details. (B) Performance of the dtg systems. Congenically marked CFSE-labeled AND and OT1 TCR transgenic LN cells were transferred into dtg-M, dtg-O animals and 6-wk previously prepared dtg-O → WT BM chimeras and were analyzed in LNs and spleen (SPL) 3.5 d later. The recipients were left untreated or were fed dox for 24 h, starting on day 24or21 as indicated. Shown are the transferred CD4+ or CD8+ T cell populations. Dox treatment of the dtg-O transgene combination led to uncontrolled gene expression in non–BM-derived cells. The data are representative of three independent experiments. 4 DIFFERENTIAL Ag DEPENDENCY OF T CELL SUBSETS

gene transcription at this point, we analyzed their transcriptome with microarrays. Both cell types had induced ∼1400 genes at least 2-fold (Fig. 2C, upper panels). The high accuracy of the reciprocal projections of these gene sets between the T cell subsets, evidenced by infinitesimal p values, indicated the high degree of similarity of the two induced transcriptomes at this time point. Overall, these data indicated that the culture conditions activated both T cell subsets comparably as far as protein, proliferative, and transcrip- tional parameters can tell and are in agreement with recent data on subset signaling and transriptomes (50). CD8+ but not CD4+ T cells keep dividing after a transient stimulus To address whether CD4+ and CD8+ T cells exhibit different pro- liferative capacities following a transient TCR stimulus, MACS- sorted AND and OT1 T cells were cultured in the absence or presence of anti-CD3 and -CD28 mAbs, CFSE-labeled 2 d later, and transferred into congenic Ag-free (condition 2) or -expressing (condition 3) recipients. Cells cultured without the mAbs and transferred into nontransgenics served as controls (condition 1; Downloaded from Fig. 3A). When the LN cells were analyzed 3 d later, AND T cells had divided once or twice upon withdrawal of the stimulus, whereas OT1 T cells had continued to divide further (mean 6 SEM: 1.5 6 0.4 versus 4.0 6 0.4 divisions). Because lymphocyte egress from LNs is regulated specifically, the recipients’ spleens

also were analyzed, and a similar difference was found (1.3 6 0.2 http://www.jimmunol.org/ versus 4.3 6 0.4). Interestingly, the OT1 T cells had divided al- most as much as in the presence of persistent Kb/OVA presentation (Fig. 3B). To test whether the observations could be reproduced for cells with a diverse TCR repertoire, we used nontransgenic CD4+ and CD8+ T cells from B6 animals under the conditions 1 and 2 de- scribed above and found again the CD4/CD8 split in LN (1.8 6 0.4 versus 3.8 6 0.4) and spleen (1.6 6 0.4 versus 3.6 6 0.5; Fig. 3C). This could also be reproduced with cells from BALB/c by guest on October 7, 2021 animals (Fig. 3D), indicating that our findings were not confined to particular TCR transgenics or genetic backgrounds. Because the proliferation data were indicative of an increased propensity for programming in CD8+ Tcells,wecomparedthe IFN-g secretion by AND and OT1 cells under conditions of tran- sient and continued TCR triggering. Although both cell types produced the cytokine in response to the persistent signal, only OT1 but not AND cells could be stained for IFN-g following the tran- sient stimulus (Fig. 3E). Furthermore, OVA257–264-pulsed target cells were efficiently killed by OT1 cells after both transient and continued stimuli (Fig. 3F). These data indicated that CD8+ Tef- fector cell differentiation can occur following a signal of limited duration. It is conceivable that CD8+ T cells continue their proliferation beyond the initial priming by interactions with self-pMHC com- plexes, like under the homeostatic conditions of the steady state and in the case of lymphopenia-driven proliferation. Therefore, we used recipients that were deleted of their b2m genes and hence lack MHC class I surface expression but were replete with a transgene expressed in thymic cortical epithelium to allow for FIGURE 2. Comparable activation marker expression of AND and OT1 the selection of a full T cell compartment, thus reining in + + T cells in vitro. TCR transgenic CD4 AND and CD8 OT1 T cells were lymphopenia-driven proliferation of adoptively transferred CD8+ magnetically sorted and cultured separately in medium (unstim.) or with T cells. Recipients carrying an additional DC-targeted b mtrans- plate-bound anti-CD3 and anti-CD28 mAbs (stim.) and analyzed 2 d later. 2 A gene served as positive controls (40). However, when 2-d- ( ) Expression of activation markers with unstained controls (gray) and + data compiled from three to six separate experiments (open symbols: stimulated CD8 T cells were transferred into such Ag-free hosts, unstimulated; filled symbols: stimulated). *p , 0.05, ***p , 0.001. (B) CFSE analysis after 2 d of culture. (C) Transcriptome comparison of stimulated and unstimulated AND and OT1 T cells. Induced genes with an numbers indicated (top panels). These genes are projected on the reciprocal

FC . 2, and p , 0.01 is depicted in red (AND) and blue (OT1) and their data sets with the number of genes whose log10FC is #0 as indicated. The Journal of Immunology 5 Downloaded from http://www.jimmunol.org/ by guest on October 7, 2021

FIGURE 3. Differential Ag requirements of CD4+ and CD8+ T cells upon abrupt removal of the TCR stimulus. (A) Experimental outline. Sorted AND and OT1 T cells were cultured in the absence (condition 1) or presence of anti-CD3 and -CD28 mAbs for 2 d, then labeled with CFSE, transferred into congenic Ag-free (condition 1 and 2) or -expressing (condition 3) recipients, and analyzed 3 d later. (B) CFSE dilution of congenically identified AND and OT1 T cells from LN and spleen (SPL) cells with the proliferation index N given in each panel. Shown are representative (top) and cumulative data (bottom) from 10 (LN) and 13 (SPL) independent experiments. (C and D) Results with polyclonal CD4+ and CD8+ T cells from B6 (C) and BALB/c (D) animals, treated as depicted in (A) without condition 3. (E) IFN-g production by transiently and persistently AND and OT1 T cells (line) with isotype controls (gray). The bottom panel shows compiled data from 8 (AND) and 10 (OT1) independent experiments; the bars represent means 6 SEM. (F) In vivo cytotoxicity of transiently and continuously stimulated OT1 T cells. Sorted OT1 T cells were stimulated and transferred as in (A) but without CFSE labeling. On d 5, congeneic OVA257–264-coated target cells were added and analyzed 1 d later. Data in the bottom panel are compiled from three independent experiments. 0/0 0/0 (G) OT1 T cells were treated as in (A) with two additional recipients: 2b, b2m ;K14-b2m;CD11c-b2m animals, 2c, b2m ;K14-b2m recipients. Data are from three independent experiments. The bars indicate means. *p , 0.05, **p , 0.01, ***p , 0.001 determined with an unpaired two-tailed Student t test. they proliferated, regardless of MHC class I expression on pe- are involved in late CD8+ T cell proliferation (Supplemental ripheral cells (Fig. 3G). These data indicated that CD8+ Tcells Fig. 3). were able to proliferate beyond the priming phase independently + of any TCR signals delivered by self-pMHC complexes. Ag-independent CD4 T cell proliferation is not affected by Th Likewise, Ab-mediated blocking of LFA-1–dependent T cell subtype, coinhibiton, inflammation, or memory cell differentiation clusters (51) did not interfere with the Ag-independent prolifera- We asked next whether the difference in Ag-independent prolifer- tion phase of OT-1 cells, making it unlikely that T-T synapses ation between CD4+ and CD8+ T cells was caused by specific inhibition 6 DIFFERENTIAL Ag DEPENDENCY OF T CELL SUBSETS of the CD4+ T cells. Th1/Th2-polarizing conditions in the priming cytokines including IL-12 and IFN-a (54).However,CD4+ T cells cultures did not affect the impeded proliferation of AND cells in Ag- were not triggered to undergo additional divisions in MCMV-infected free hosts, excluding subset-specific cytokines as mediators (Fig. 4A). recipients (Fig. 4D), indicating that cytokines produced by the Coinhibitory molecules like CTLA-4 and PD-1 interfere with recipients’ cells do not contribute to the expansion of naive T cells T cell priming and there is evidence that they transmit the initial following initial Ag encounter. T cell migratory stop signal and thus regulate time and/or strength To assess the effect of precursor frequency in our system, we of the priming interaction between T cells and DCs (52, 53). To lowered the number of transferred AND cells stepwise down to investigate whether these molecules block Ag-independent pro- 62,500 per recipient and did not detect any effect on proliferation liferation of CD4+ T cells, we transferred 2-d-primed CFSE-labeled (data not shown). The results excluded mutual T cell inhibition as AND and OT1 T cells into Ag-free hosts, which had received a mechanism of blunted CD4+ T cell proliferation and confirmed blocking mAbs against CTLA-4, PD-1, or PD-L1 (Fig. 4B). None earlier data on the issue obtained by different methods (31). of these treatments enhanced AND T cell division, indicating that We next asked whether CD4+ T cells acquire the capacity to CD4+ T cells were not specifically blocked by any of these mol- proliferate in an Ag-independent way upon their differentiation ecules. To test whether this also applied to the priming phase of into memory cells. We therefore generated rested effector cells, the response, we transferred naive AND T cells into transiently which resemble memory cells in their transcriptome, physiology, dox-treated dtg-M recipients, as described for Fig. 1D, and and function (43, 55), and tested whether their response to tran- injected an anti–CTLA-4 mAb at the same time. In addition, we sient Ag presentation, as described for Supplemental Fig. 1, differs used animals lacking the gene encoding PD-L1 (Cd274) to assess from naive cells. This was not the case, making it unlikely that the effect of both pathways being effectively blocked. The ap- CD4+ memory cells proliferate independently of Ag in secondary plication of the mAb did not reveal any difference to controls, responses (32). In summary, our data exclude coinhibition and the Downloaded from even in recipients lacking PD-L1 (Fig. 4C). Thus, CD4+ T cell lack of proinflammatory cytokines as blocks of Ag-independent proliferation is not specifically held in check by cell-extrinsic proliferation of CD4+ T cells and show that memory cells do not coinhibition. acquire this function, suggesting it is a lineage-specific trait. We then tested whether the increased production of inflamma- tory cytokines may trigger T cell expansion in an Ag-independent Direct comparison of cell cycle status way by transferring prestimulated CD4+ and CD8+ T cells into Although CFSE dilution records the division history of a cell http://www.jimmunol.org/ animals that were or were not infected by a high dose of MCMV population, direct cell cycle analyses visualize proliferation at the 2 d before. This virus sets off the release of multiple proinflammatory time of dissection. Therefore, we quantified the percentages of by guest on October 7, 2021

FIGURE 4. CD4+ T cells cannot be driven into Ag-independent proliferation. (A) Th-type polarization does not affect proliferative behavior. AND CD4+ T cells were stimulated in vitro as in Fig. 3 but also under Th1 and Th2 conditions, CFSE labeled after 2 d, transferred, and the proliferative index N was determined in LN (top panel) and spleen cells (bottom panel). (B) Coinhibition blockade does not affect CD4+ T cell proliferation. Control (cond. 1) and 2-d-primed AND T cells were CFSE labeled and transferred into Ag-free (2) and -expressing recipients (3), with mAbs interfering with CTLA4, PD-L1, and PD1 and control mAb given i.p. upon transfer. The proliferative index N was analyzed in the spleen 3 d later. Data are compiled from 11 (AND) and 12 (OT1) independent experiments. (C) CD4+ T cell proliferation in the absence of PD-L1. Naive CFSE-labeled AND T cells were transferred into WT or Cd274o/o dtg-M recipients that were untreated or fed with dox for 1 d or continuously, with the indicated animals also injected with anti–CTLA-4. Proliferative index N was analyzed in the spleen 3 d later. Data are from nine independent experiments. (D) MCMV-induced inflammation by itself does not support CD4+ T cell proliferation. Polyclonal B6 control (cond. 1) and 2-d-primed T cells were CFSE labeled and transferred into wt recipients that had been infected with a high dose of MCMV 2 d earlier. CFSE dilution of transferred cells was analyzed in the spleen 3 d later. Data are from three in- dependent experiments. (E) Memory-like CD4+ T cells do not acquire the capacity to proliferate Ag-independently. Naive (left panels) and rested effector (RE) AND T cells were CFSE labeled and transferred into dtg-M recipients treated with dox for 24 h and continuously. Lymph node analyses showed similar results in all experiments. Data in the bottom panels are from four independent experiments. The bars indicate means. **p , 0.01, ***p , 0.001 determined with an unpaired two-tailed Student t test. The Journal of Immunology 7

AND and OT1 T cells in S and G2-M phases by intracellular DAPI belong in the Ag-independent category, whereas the expression stains at several time points (Fig. 5A). Following 2 d of stimula- of Grzmb, Grzmk, Irf4,andIrf8 is rather Ag-dependent in both tion, similar fractions of the two cell types were in cell cycle CD4+ AND and CD8+ OT1 T cells (Fig. 6A, 6B). Of note, the (AND: 43.1 6 4.2; OT1: 41.5 6 3.3%). However, after 7 d of group of genes expressed Ag independently by OT1 but not TCR triggering, the OT1 cells showed a significantly higher per- AND T cells include cell cycle-associated Cdc25b, Cdc26, centage of cycling cells (7.6 6 0.5%) than AND T cells (2.3 6 Cdc34,andCdc37. Both Cdc25 and Cdc34 are regulators of 0.3%), suggesting that CD8+ T cells are susceptible to TCR sig- proliferation for which specific inhibitors have identified that nals for longer periods of time (Fig. 5A). block cell cycle progression of tumor cells (57, 58). Tbx21 The Ki67 protein is associated with proliferation in many cell encoding T-bet, the transcription factor regulating CTL dif- types and is a reliable though indirect probe for division. Following ferentiation, also falls into this category (Fig. 6B, 6C). Ac- 2 d of stimulation, both AND and OT1 cells express it, and both cordingly, T-bet protein is expressed in a more Ag-independent T cell types lose it upon withdrawal of the stimulus with a t1/2 of way in OT1 T cells than it is in AND T cells (Fig. 6D). In sum- days (Fig. 5B, upper panels). The AND T cells, however, lose this mary, these data show a differential Ag-dependency of the tran- marker despite persisting Ag presentation, whereas the OT1 cells scriptional landscape in AND and OT1 T cells and support the maintain it at high levels (Fig. 5B, lower panels). These data in- notion of the CD8+ T cells’ propensity to divide and differentiate dicate that CD8+ T cells are able to translate TCR signals into cell following an Ag pulse of limited duration. division for longer periods of time than CD4+ T cells, a fact that Stabilization of pMHC complexes upon DC activation in vivo may well contribute to the better overall expansion of CD8+ + + T cells. Our data also indicate that Ki67 is a protein with a survival The main difference between CD4 and CD8 T cells is their re- time of several days and not necessarily an authentic reporter of spective MHC class II and I ligands. In view of our finding that Downloaded from + + momentary cell division. Similar findings have been reported re- CD4 and CD8 T cells differ in their temporal requirements of cently for noncycling blood monocytes that maintain Ki67 ex- TCR signals, we asked whether the kinetics of Ag presentation pression from their precursor stage in the BM (56). differs for their respective MHC ligands on DCs, the main APCs for priming. Because MHC class II molecules are stabilized on the cell Gene expression following transient and persistent stimulation surface upon DC activation by downregulated oligo-ubiquitination

To assess how continued TCR signaling affects the overall tran- of class II b-chains (59, 60) by membrane-associated RING-CH– http://www.jimmunol.org/ scriptome of the two cell types, we transferred 2-d-stimulated AND like E3 ligases (61, 62) in vitro (63, 64) and in vivo (42, 65), we and OT1 T cells into Ag-free or Ag-expressing dtg-M and -O hosts, tested whether DC activation affects molecules of both MHC respectively, and sorted them 3 d later for microarray analysis. The classes similarly. We activated dtg-M and -O DCs in vivo by FC/FC plots in Fig. 6A compare the cells’ transcriptomes in re- treatment with a stimulatory CD40 mAb and fed the animals with sponse to transient (abscissa) and persistent (ordinate) TCR trig- dox for 24 h subsequently. Three days after turn-off, CFSE- gering to their respective controls. There are evidently more genes labeled AND and OT1 cells were transferred as probes for sta- expressed in a stimulus-independent way in the CD8+ OT1 cells bilized pMHC complexes. Fig. 7 shows that AND but not OT1 compared with the CD4+ AND T cells. This is reflected in the T cells detected remaining pMHC complexes. We have shown trend lines’ slopes differing by a factor of ∼2 (Fig. 6A). The previously that this proliferation of AND T cells was not caused by guest on October 7, 2021 expressed genes can be grouped as Ag-independent and close to by increased costimulation (42). These findings indicate that the b the x = y diagonal on the one hand and rather Ag-dependent turnover of K /Ova257–264 complexes is not affected by DC mat- aligning around the y = 1 horizontal on the other hand. As noted uration in such a way that OT1 cells might detect them 3 d after in a previous analysis on day 3 (42), Ctla2, Ctla4,andCxcr3 dox turn-off. Our data are in agreement with results obtained with

FIGURE 5. Cell cycle status in comparison. 2-d-activated AND and OT1 T cells were transferred into recipients expressing the respective Ags (top panels) or not (bottom panels) and analyzed for DNA quantity (A) and Ki67 expression 2, 5, and 7 d after the beginning of priming (B). Congenically identified live singlets are shown. Gray tracings depict isotype control stains. The panels on the right depict means from three independent experiments. *p , 0.05, **p , 0.01, ***p , 0.001 determined with an unpaired two-tailed Student t test. 8 DIFFERENTIAL Ag DEPENDENCY OF T CELL SUBSETS Downloaded from http://www.jimmunol.org/ by guest on October 7, 2021

FIGURE 6. Transcriptome analyses of transiently and continuously stimulated AND and OT1 T cells. (A) Comparison of transcripts in AND (left) and OT1 T cells (right) that were transiently (y-axis) and continuously (x-axis) stimulated as depicted in Fig. 3A. Depicted is the FC in comparison with unstimulated and transferred cells. Red lines indicate the regression lines with slope p = 0.27 and correlation r2 = 0.14 for AND and p = 0.46 and r2 = 0.44 for OT1. (B) Relative gene expression of selected genes on days 2 and 5. (C) Compilation of 140 genes expressed in an Ag-independent manner in OT1 cells with selected genes indicated on the right. The complete list and expression values are listed in Supplemental Table I. (D) T-bet protein expression in transiently and continuously stimulated AND and OT1 cells. Numbers indicate the mean fluorescence intensity (MFI) values of T-bet and isotype control stains (shaded). The panels on the right depict data from five independent experiments. **p , 0.01 determined with an unpaired two-tailed Student t test. human DCs in vitro that showed no extension of bulk MHC class I executed in a way to circumvent confounding differences like half-lives upon LPS treatment (63, 66) and a 3-fold increase by direct APC identity, Ag processing, and cell biology of MHC class I and influenza virus infection (66). Recent data, obtained with the mini- II proteins on the one hand, and the T cells’ differential costim- mally invasive technique of heavy water labeling, found a 2-fold ulatory, adhesion, and third-signal cytokine requirements and CD4/8 increase of MHC class I, but a more than 10-fold increase of MHC coreceptor affinities on the other hand. Although CD8+ T cells do class II half lives in activated DCs (R. Busch, personal communica- not necessarily keep dividing following a short antigenic pulse, tion). We conclude that our in vivo findings with two TCR/pMHC they can be pushed to do so, whereas CD4+ T cells cannot. combinations are in agreement with previous in vitro data on bulk Transiently stimulated CD8+ T cells are fully functional as they MHC protein turnover rates. The differential Ag dependency of CD4+ differentiate into cytotoxic and IFN-g–secreting cells, are inde- and CD8+ T cells might thus be reflected by their respective pMHC pendent of self-pMHC complexes and are therefore truly “on ligands’ distinct susceptibility to regulation of their stability. autopilot” (11). CD4+ T cells could not be brought into an Ag-independent phase of proliferation by coinhibition blockade Discussion and inflammatory conditions, are in S phase of the cell cycle Our comparison showed cell-intrinsic differences of proliferation and express the proliferation marker Ki67 for shorter periods of between the CD4+ and CD8+ T cell subsets. The experiments were time and show a gene expression profile upon Ag withdrawal The Journal of Immunology 9

class discrimination of the presentation pathways is breached by cross-presentation of exogenous Ags by class I and autophagy of cytosolic Ags for presentation by class II molecules. The half-lives of the MHC molecules, however, are controlled by distinct ubiq- uitination pathways. The membrane-associated RING-CH family E3 ubiquitin ligases affect not only several aspects of the MHC class II pathway, such as its master transcription factor CIITA, the peptide exchange by DM, class II trafficking and t1/2, but also costimulatory molecules such as CD80 and CD86. In activated DCs, class II and CD80/86 ubiquitination is reduced, allowing for high surface expression of the molecules necessary for efficient + CD4 T cell priming (59, 60). In contrast, the t1/2 of bulk MHC class I molecules is not extended .3-fold on LPS-activated DCs (63, 66; R. Busch, personal communication), with perhaps indi- vidual pMHC complexes varying over a wide range (76), and ubiquitination by viral E3 ligases that targets them for destruction has been merely described as a mechanism of immune evasion. A second difference between class I and class II molecules affecting their presentation kinetics is the source of Ag: While for class II whole proteins are internalized and degraded in the Downloaded from endosomal compartment, most of the substrates for the class I pathway are newly synthesized polypeptides and defective ri- bosomal products (77). Accordingly, in a side-by-side comparison of presentation kinetics with precisely regulated Ag expression, the presentation via MHC class I correlated closely with active gene transcription and that of class II with the stability of mature viral http://www.jimmunol.org/ FIGURE 7. Stabilization of pMHC complexes upon DC activation proteins (78). It is thus tempting to speculate that the capability of in vivo. Dtg-M (left) and dtg-O → B6 chimeras (right) were treated with CD8+ T cells to respond to a short Ag pulse might reflect transient dox (black box, 1 d) or regular drinking water (gray) and treated with PBS viral gene transcription and its preferred feeding of the class I or anti-CD40 as indicated. CFSE-labeled T cells were transferred as Ag pathway.ThefactthatCD4+ T cells respond much longer to residual probes and their proliferation analyzed 3 d later. Data in the bottom panel Ag following viral clearance than CD8+ T cells do also indicates that are compiled from three and four experiments with one or two animals per antiviral CD8+ T cell responses depend on viral transcription (79, 80). treatment. **p , 0.01 determined with an unpaired Student t test. Further evidence for the importance of extended Ag presenta- tion for CD4+ immunity has been generated by targeting Ags to by guest on October 7, 2021 clearly distinct from their CD8+ counterparts. It is likely that our DCs by mAbs of different half-lives: CD4+ T cell immunity and results underestimate the differences described as the T cells were Abs can be raised in the absence of adjuvants given an extended t1/2 stimulated and transferred separately. If anything, CD4+ Tcellsassist of the immunizing mAb (81). This finding suggests necessary APCs in Ag presentation to CD8+ Tcells,whereasCD8+ T cells refinements of our understanding of DCs in immunity and toler- rather limit APC availability for their CD4+ counterparts (30, 32, 33). ance (82) with important implications for peptide vaccination (83). We discuss below how these findings have intriguing parallels in In summary we have shown that CD4+ and CD8+ T cells differ the thymic differentiation of the two subsets, migratory behavior of in their cell-intrinsic capacities to proliferate beyond an initial naive and memory cells, differential importance of immunization antigenic stimulus. As CD4+ T cells direct (“help”) other immune parameters, and, importantly, correlate with differential stability of cells toward effector functions they appear to be under tighter MHC class I and II complexes and their respective Ag sources. control of Ag presentation than CD8+ T cells whose main func- The TCR-guided differentiation of CD4+CD8+ precursors to tions IFN-g production and cytotoxicity unravel even under Ag- single-positive mature thymocytes is influenced by the kinetics of free conditions. This difference might be reflected in differences the TCR signals to achieve the match of MHC class reactivity with of Ag source for and stability of presentation by their respective coreceptor expression: A transient signal favors the differentiation MHC protein classes. of CD8+ cells, whereas CD4+ ones require longer signals (67–69). Although the molecular details are not yet known, it is tempting to Acknowledgments speculate that thymic assortment and peripheral response mode We thank A. Kollar and S. Pentz for technical assistance, A. Bol and share a molecular basis. The theme of higher dependency on MHC- W. Mertl for animal husbandry, B. Arnold, H.-G. Rammensee, C. Benoist, derived signals extends to naive CD4+ T cells that traverse LNs in D. Mathis, and T. Brocker for reagents or mice, C. Guo for Matlab-based an MHC-dependent manner and adjust CD5 levels to TCR affinity, calculations, R. Busch for sharing unpublished data, J. Johnson and whereas CD8+ T cells do not (49, 70). In addition, the CD4+ T T. Brocker for discussions, and A. Erlebacher and E. Huseby for comments memory cells recirculate more freely through tissues, while their on the manuscript. CD8+ counterparts are confined in an Ag-independent way at the infection site (71, 72). These findings suggest that the CD4+ T cell Disclosures lineage is marked for TCR-guided plasticity, whereas its CD8+ The authors have no financial conflicts of interest. counterpart responds in a more predetermined way. + + The key difference between CD4 and CD8 T cells is their References MHC ligands and their tissue distribution (73). It is understood 1. Yoon, H., T. S. Kim, and T. J. Braciale. 2010. The cell cycle time of CD8+ T cells that both T cell subsets are primed best by activated DCs, although responding in vivo is controlled by the type of antigenic stimulus. PLoS One 5: not necessarily by the same subsets (74, 75). In DCs, the classical e15423. 10 DIFFERENTIAL Ag DEPENDENCY OF T CELL SUBSETS

2. Jenkins, M. K., and J. J. Moon. 2012. The role of naive T cell precursor fre- dendritic cell licensing and CD8+ T cell cross-priming. J. Immunol. 181: 3067– quency and recruitment in dictating immune response magnitude. J. Immunol. 3076. 188: 4135–4140. 31. Yarke, C. A., S. L. Dalheimer, N. Zhang, D. M. Catron, M. K. Jenkins, and 3. Zhang, N., and M. J. Bevan. 2011. CD8+ T cells: foot soldiers of the immune D. L. Mueller. 2008. Proliferating CD4+ T cells undergo immediate growth arrest system. Immunity 35: 161–168. upon cessation of TCR signaling in vivo. J. Immunol. 180: 156–162. 4. Hafalla, J. C., G. Sano, L. H. Carvalho, A. Morrot, and F. Zavala. 2002. Short- 32. Ravkov, E. V., and M. A. Williams. 2009. The magnitude of CD4+ T cell recall term antigen presentation and single clonal burst limit the magnitude of the responses is controlled by the duration of the secondary stimulus. J. Immunol. CD8+ T cell responses to malaria liver stages. Proc. Natl. Acad. Sci. USA 99: 183: 2382–2389. 11819–11824. 33. Ma, J. Z., S. N. Lim, J. S. Qin, J. Yang, N. Enomoto, C. Ruedl, and F. Ronchese. 5. Kaech, S. M., and R. Ahmed. 2001. Memory CD8+ T cell differentiation: initial 2012. Murine CD4+ T cell responses are inhibited by cytotoxic T cell-mediated antigen encounter triggers a developmental program in naı¨ve cells. Nat. Immu- killing of dendritic cells and are restored by antigen transfer. PLoS One 7: nol. 2: 415–422. e37481. 6. Mercado, R., S. Vijh, S. E. Allen, K. Kerksiek, I. M. Pilip, and E. G. Pamer. 34. Jatzek, A., M. Marie Tejera, E. H. Plisch, M. L. Fero, and M. Suresh. 2013. T- 2000. Early programming of T cell populations responding to bacterial infection. cell intrinsic and extrinsic mechanisms of p27Kip1 in the regulation of CD8 T-cell J. Immunol. 165: 6833–6839. memory. Immunol. Cell Biol. 91: 120–129. 7. van Stipdonk, M. J., G. Hardenberg, M. S. Bijker, E. E. Lemmens, N. M. Droin, 35. Foulds, K. E., L. A. Zenewicz, D. J. Shedlock, J. Jiang, A. E. Troy, and H. Shen. + D. R. Green, and S. P. Schoenberger. 2003. Dynamic programming of CD8 2002. Cutting edge: CD4 and CD8 T cells are intrinsically different in their T lymphocyte responses. Nat. Immunol. 4: 361–365. proliferative responses. J. Immunol. 168: 1528–1532. 8. Williams, M. A., and M. J. Bevan. 2004. Shortening the infectious period does 36. Seder, R. A., and R. Ahmed. 2003. Similarities and differences in CD4+ and not alter expansion of CD8 T cells but diminishes their capacity to differentiate CD8+ effector and memory T cell generation. Nat. Immunol. 4: 835–842. into memory cells. J. Immunol. 173: 6694–6702. 37. Kaye, J., M. L. Hsu, M. E. Sauron, S. C. Jameson, N. R. Gascoigne, and 9. Wong, P., and E. G. Pamer. 2004. Disparate in vitro and in vivo requirements for S. M. Hedrick. 1989. Selective development of CD4+ T cells in transgenic mice IL-2 during antigen-independent CD8 T cell expansion. J. Immunol. 172: 2171– expressing a class II MHC-restricted antigen receptor. Nature 341: 746–749. 2176. 38. Hogquist, K. A., S. C. Jameson, W. R. Heath, J. L. Howard, M. J. Bevan, and 10. Starbeck-Miller, G. R., H. H. Xue, and J. T. Harty. 2014. IL-12 and type I in- F. R. Carbone. 1994. T cell receptor antagonist peptides induce positive selec- terferon prolong the division of activated CD8 T cells by maintaining high- tion. Cell 76: 17–27. Downloaded from affinity IL-2 signaling in vivo. J. Exp. Med. 211: 105–120. 39. Yamashiro, H., N. Hozumi, and N. Nakano. 2002. Development of CD25+ 11. Bevan, M. J., and P. J. Fink. 2001. The CD8 response on autopilot. Nat. Immunol. T cells secreting transforming growth factor-b1 by altered peptide ligands 2: 381–382. expressed as self-antigens. Int. Immunol. 14: 857–865. 12. Masopust, D., S. M. Kaech, E. J. Wherry, and R. Ahmed. 2004. The role of 40. Gruber, A., M. A. Cannarile, C. Cheminay, C. Ried, P. Marconi, G. Ha¨cker, and programming in memory T-cell development. Curr. Opin. Immunol. 16: 217– T. Brocker. 2010. Parenchymal cells critically curtail cytotoxic T-cell responses 225. by inducing Bim-mediated apoptosis. Eur. J. Immunol. 40: 966–975. 13. Blair, D. A., D. L. Turner, T. O. Bose, Q. M. Pham, K. R. Bouchard, 41. Dong, H., G. Zhu, K. Tamada, D. B. Flies, J. M. van Deursen, and L. Chen. 2004. K. J. Williams, J. P. McAleer, L. S. Cauley, A. T. Vella, and L. Lefranc¸ois. 2011. B7-H1 determines accumulation and deletion of intrahepatic CD8+ http://www.jimmunol.org/ Duration of antigen availability influences the expansion and memory differ- T lymphocytes. Immunity 20: 327–336. entiation of T cells. J. Immunol. 187: 2310–2321. 42. Obst, R., H. M. van Santen, R. Melamed, A. O. Kamphorst, C. Benoist, and 14. Cockburn, I. A., Y. C. Chen, M. G. Overstreet, J. R. Lees, N. van Rooijen, D. Mathis. 2007. Sustained antigen presentation can promote an immunogenic D. L. Farber, and F. Zavala. 2010. Prolonged antigen presentation is required for T cell response, like dendritic cell activation. Proc. Natl. Acad. Sci. USA 104: optimal CD8+ T cell responses against malaria liver stage parasites. PLoS 15460–15465. Pathog. 6: e1000877. 43. McKinstry, K. K., S. Golech, W. H. Lee, G. Huston, N. P. Weng, and S. L. Swain. 15. Curtsinger, J. M., C. M. Johnson, and M. F. Mescher. 2003. CD8 T cell clonal 2007. Rapid default transition of CD4 T cell effectors to functional memory expansion and development of effector function require prolonged exposure to cells. J. Exp. Med. 204: 2199–2211. antigen, costimulation, and signal 3 cytokine. J. Immunol. 171: 5165–5171. 44. Reich, M., T. Liefeld, J. Gould, J. Lerner, P. Tamayo, and J. P. Mesirov. 2006. 16. Kang, S. S., J. Herz, J. V. Kim, D. Nayak, P. Stewart-Hutchinson, M. L. Dustin, GenePattern 2.0. Nat. Genet. 38: 500–501. and D. B. McGavern. 2011. Migration of cytotoxic lymphocytes in cell cycle 45. Painter, M. W., S. Davis, R. R. Hardy, D. Mathis, and C. Benoist; Immunological permits local MHC I-dependent control of division at sites of viral infection. J.

Genome Project Consortium. 2011. Transcriptomes of the B and T lineages by guest on October 7, 2021 Exp. Med. 208: 747–759. compared by multiplatform microarray profiling. J. Immunol. 186: 3047–3057. 17. Prlic, M., G. Hernandez-Hoyos, and M. J. Bevan. 2006. Duration of the initial 46. Eisen, H. N., X. H. Hou, C. Shen, K. Wang, V. K. Tanguturi, C. Smith, TCR stimulus controls the magnitude but not functionality of the CD8+ T cell K. Kozyrytska, L. Nambiar, C. A. McKinley, J. Chen, and R. J. Cohen. 2012. response. J. Exp. Med. 203: 2135–2143. Promiscuous binding of extracellular peptides to cell surface class I MHC pro- 18. Shaulov, A., and K. Murali-Krishna. 2008. CD8 T cell expansion and memory differentiation are facilitated by simultaneous and sustained exposure to anti- tein. Proc. Natl. Acad. Sci. USA 109: 4580–4585. 47. Moran, A. E., K. L. Holzapfel, Y. Xing, N. R. Cunningham, J. S. Maltzman, genic and inflammatory milieu. J. Immunol. 180: 1131–1138. 19. Storni, T., C. Ruedl, W. A. Renner, and M. F. Bachmann. 2003. Innate immunity J. Punt, and K. A. Hogquist. 2011. T cell receptor signal strength in Treg and together with duration of antigen persistence regulate effector T cell induction. J. iNKT cell development demonstrated by a novel fluorescent reporter mouse. J. Immunol. 171: 795–801. Exp. Med. 208: 1279–1289. 48. Cho, J. H., H. O. Kim, C. D. Surh, and J. Sprent. 2010. T cell receptor-dependent 20. Tewari, K., J. Walent, J. Svaren, R. Zamoyska, and M. Suresh. 2006. Differential + requirement for Lck during primary and memory CD8+ T cell responses. Proc. regulation of lipid rafts controls naive CD8 T cell homeostasis. Immunity 32: Natl. Acad. Sci. USA 103: 16388–16393. 214–226. 21. Tseng, K. E., C. Y. Chung, W. S. H’ng, and S. L. Wang. 2009. Early infection 49. Mandl, J. N., J. P. Monteiro, N. Vrisekoop, and R. N. Germain. 2013. T cell- termination affects number of CD8+ memory T cells and protective capacities in positive selection uses self-ligand binding strength to optimize repertoire rec- listeria monocytogenes-infected mice upon rechallenge. J. Immunol. 182: 4590– ognition of foreign antigens. Immunity 38: 263–274. 4600. 50. Mingueneau, M., T. Kreslavsky, D. Gray, T. Heng, R. Cruse, J. Ericson, 22. Baje´noff, M., O. Wurtz, and S. Guerder. 2002. Repeated antigen exposure is S. Bendall, M. H. Spitzer, G. P. Nolan, K. Kobayashi, et al. 2013. The tran- necessary for the differentiation, but not the initial proliferation, of naive CD4+ scriptional landscape of ab T cell differentiation. Nat. Immunol. 14: 619–632. T cells. J. Immunol. 168: 1723–1729. 51. Ge´rard, A., O. Khan, P. Beemiller, E. Oswald, J. Hu, M. Matloubian, and 23. Lee, W. T., G. Pasos, L. Cecchini, and J. N. Mittler. 2002. Continued antigen M. F. Krummel. 2013. Secondary T cell-T cell synaptic interactions drive the + stimulation is not required during CD4+ T cell clonal expansion. J. Immunol. differentiation of protective CD8 T cells. Nat. Immunol. 14: 356–363. 168: 1682–1689. 52. Fife, B. T., K. E. Pauken, T. N. Eagar, T. Obu, J. Wu, Q. Tang, M. Azuma, 24. Iezzi, G., K. Karjalainen, and A. Lanzavecchia. 1998. The duration of antigenic M. F. Krummel, and J. A. Bluestone. 2009. Interactions between PD-1 and PD- stimulation determines the fate of naive and effector T cells. Immunity 8: 89–95. L1 promote tolerance by blocking the TCR-induced stop signal. Nat. Immunol. 25. Gett, A. V., F. Sallusto, A. Lanzavecchia, and J. Geginat. 2003. T cell fitness 10: 1185–1192. determined by signal strength. Nat. Immunol. 4: 355–360. 53. Schneider, H., J. Downey, A. Smith, B. H. Zinselmeyer, C. Rush, J. M. Brewer, 26. Huppa, J. B., M. Gleimer, C. Sumen, and M. M. Davis. 2003. Continuous T cell B. Wei, N. Hogg, P. Garside, and C. E. Rudd. 2006. Reversal of the TCR stop receptor signaling required for synapse maintenance and full effector potential. signal by CTLA-4. Science 313: 1972–1975. Nat. Immunol. 4: 749–755. 54. Krug, A., A. R. French, W. Barchet, J. A. Fischer, A. Dzionek, J. T. Pingel, 27. Schrum, A. G., and L. A. Turka. 2002. The proliferative capacity of individual M. M. Orihuela, S. Akira, W. M. Yokoyama, and M. Colonna. 2004. TLR9- naive CD4+ T cells is amplified by prolonged T cell antigen receptor triggering. dependent recognition of MCMV by IPC and DC generates coordinated cytokine J. Exp. Med. 196: 793–803. responses that activate antiviral NK cell function. Immunity 21: 107–119. 28. Obst, R., H. M. van Santen, D. Mathis, and C. Benoist. 2005. Antigen persistence 55. McKinstry, K. K., T. M. Strutt, Y. Kuang, D. M. Brown, S. Sell, R. W. Dutton, is required throughout the expansion phase of a CD4+ T cell response. J. Exp. and S. L. Swain. 2012. Memory CD4+ T cells protect against influenza through Med. 201: 1555–1565. multiple synergizing mechanisms. J. Clin. Invest. 122: 2847–2856. 29. Celli, S., F. Lemaıˆtre, and P. Bousso. 2007. Real-time manipulation of T cell- 56. Hettinger, J., D. M. Richards, J. Hansson, M. M. Barra, A. C. Joschko, dendritic cell interactions in vivo reveals the importance of prolonged contacts J. Krijgsveld, and M. Feuerer. 2013. Origin of monocytes and macrophages in for CD4+ T cell activation. Immunity 27: 625–634. a committed progenitor. Nat. Immunol. 14: 821–830. 30. Jusforgues-Saklani, H., M. Uhl, N. Blache`re, F. Lemaıˆtre, O. Lantz, P. Bousso, 57. Ceccarelli, D. F., X. Tang, B. Pelletier, S. Orlicky, W. Xie, V. Plantevin, D. Braun, J. J. Moon, and M. L. Albert. 2008. Antigen persistence is required for D. Neculai, Y. C. Chou, A. Ogunjimi, A. Al-Hakim, et al. 2011. An allosteric The Journal of Immunology 11

inhibitor of the human Cdc34 ubiquitin-conjugating enzyme. Cell 145: 1075– 71. Gebhardt, T., P. G. Whitney, A. Zaid, L. K. Mackay, A. G. Brooks, W. R. Heath, 1087. F. R. Carbone, and S. N. Mueller. 2011. Different patterns of peripheral mi- 58. Lavecchia, A., C. Di Giovanni, and E. Novellino. 2012. CDC25 phosphatase gration by memory CD4+ and CD8+ T cells. Nature 477: 216–219. inhibitors: an update. Mini Rev. Med. Chem. 12: 62–73. 72. Mackay, L. K., A. T. Stock, J. Z. Ma, C. M. Jones, S. J. Kent, S. N. Mueller, 59. Shin, J. S., M. Ebersold, M. Pypaert, L. Delamarre, A. Hartley, and I. Mellman. W. R. Heath, F. R. Carbone, and T. Gebhardt. 2012. Long-lived epithelial im- 2006. Surface expression of MHC class II in dendritic cells is controlled by munity by tissue-resident memory T (TRM) cells in the absence of persisting regulated ubiquitination. Nature 444: 115–118. local antigen presentation. Proc. Natl. Acad. Sci. USA 109: 7037–7042. 60. van Niel, G., R. Wubbolts, T. Ten Broeke, S. I. Buschow, F. A. Ossendorp, 73. Klein, J. 1986. Tissue Distribution. In Natural History of the Major Histocom- C. J. Melief, G. Raposo, B. W. van Balkom, and W. Stoorvogel. 2006. Dendritic patibility Complex. John Wiley & Sons, New York, NY, p. 152–175. cells regulate exposure of MHC class II at their plasma membrane by oligou- 74. Dudziak, D., A. O. Kamphorst, G. F. Heidkamp, V. R. Buchholz, biquitination. Immunity 25: 885–894. C. Trumpfheller, S. Yamazaki, C. Cheong, K. Liu, H. W. Lee, C. G. Park, et al. 61. De Gassart, A., V. Camosseto, J. Thibodeau, M. Ceppi, N. Catalan, P. Pierre, and 2007. Differential antigen processing by dendritic cell subsets in vivo. Science E. Gatti. 2008. MHC class II stabilization at the surface of human dendritic cells is the result of maturation-dependent MARCH I down-regulation. Proc. Natl. 315: 107–111. Acad. Sci. USA 105: 3491–3496. 75. Vander Lugt, B., A. A. Khan, J. A. Hackney, S. Agrawal, J. Lesch, M. Zhou, 62. Walseng, E., K. Furuta, B. Bosch, K. A. Weih, Y. Matsuki, O. Bakke, S. Ishido, W. P. Lee, S. Park, M. Xu, J. Devoss, et al. 2014. Transcriptional programming and P. A. Roche. 2010. Ubiquitination regulates MHC class II-peptide complex of dendritic cells for enhanced MHC class II antigen presentation. Nat. Immunol. retention and degradation in dendritic cells. Proc. Natl. Acad. Sci. USA 107: 15: 161–167. 20465–20470. 76. Zehn, D., C. J. Cohen, Y. Reiter, and P. Walden. 2004. Extended presentation of 63. Cella, M., A. Engering, V. Pinet, J. Pieters, and A. Lanzavecchia. 1997. In- specific MHC-peptide complexes by mature dendritic cells compared to other flammatory stimuli induce accumulation of MHC class II complexes on dendritic types of antigen-presenting cells. Eur. J. Immunol. 34: 1551–1560. cells. Nature 388: 782–787. 77. Yewdell, J. W. 2011. DRiPs solidify: progress in understanding endogenous 64. Pierre, P., S. J. Turley, E. Gatti, M. Hull, J. Meltzer, A. Mirza, K. Inaba, MHC class I antigen processing. Trends Immunol. 32: 548–558. R. M. Steinman, and I. Mellman. 1997. Developmental regulation of MHC class 78. Mackay, L. K., H. M. Long, J. M. Brooks, G. S. Taylor, C. S. Leung, A. Chen, II transport in mouse dendritic cells. Nature 388: 787–792. F. Wang, and A. B. Rickinson. 2009. T cell detection of a B-cell tropic virus 65. Singh, N. J., M. Cox, and R. H. Schwartz. 2007. TLR ligands differentially infection: newly-synthesised versus mature viral proteins as antigen sources for Downloaded from modulate T cell responses to acute and chronic antigen presentation. J. Immunol. CD4 and CD8 epitope display. PLoS Pathog. 5: e1000699. 179: 7999–8008. 79. Kim, T. S., M. M. Hufford, J. Sun, Y. X. Fu, and T. J. Braciale. 2010. Antigen 66. Cella, M., M. Salio, Y. Sakakibara, H. Langen, I. Julkunen, and A. Lanzavecchia. persistence and the control of local T cell memory by migrant respiratory den- 1999. Maturation, activation, and protection of dendritic cells induced by dritic cells after acute virus infection. J. Exp. Med. 207: 1161–1172. double-stranded RNA. J. Exp. Med. 189: 821–829. 80. Misumi, I., M. Alirezaei, B. Eam, M. A. Su, J. L. Whitton, and J. K. Whitmire. 67. Yasutomo, K., C. Doyle, L. Miele, C. Fuchs, and R. N. Germain. 2000. The + 2013. Differential T cell responses to residual viral antigen prolong CD4 Tcell duration of antigen receptor signalling determines CD4+ versus CD8+ T-cell contraction following the resolution of infection. J. Immunol. 191: 5655–5668. lineage fate. Nature 404: 506–510. 81. Lahoud, M. H., F. Ahmet, S. Kitsoulis, S. S. Wan, D. Vremec, C. N. Lee,

68. Liu, X., and R. Bosselut. 2004. Duration of TCR signaling controls CD4-CD8 http://www.jimmunol.org/ lineage differentiation in vivo. Nat. Immunol. 5: 280–288. B. Phipson, W. Shi, G. K. Smyth, A. M. Lew, et al. 2011. Targeting antigen to 69. Saini, M., C. Sinclair, D. Marshall, M. Tolaini, S. Sakaguchi, and B. Seddon. mouse dendritic cells via Clec9A induces potent CD4 T cell responses biased 2010. Regulation of Zap70 expression during thymocyte development enables toward a follicular helper phenotype. J. Immunol. 187: 842–850. temporal separation of CD4 and CD8 repertoire selection at different signaling 82. Caminschi, I., and K. Shortman. 2012. Boosting antibody responses by targeting thresholds. Sci. Signal. 3: ra23. antigens to dendritic cells. Trends Immunol. 33: 71–77. 70. Mandl, J. N., R. Liou, F. Klauschen, N. Vrisekoop, J. P. Monteiro, A. J. Yates, 83. Walter, S., T. Weinschenk, A. Stenzl, R. Zdrojowy, A. Pluzanska, C. Szczylik, A. Y. Huang, and R. N. Germain. 2012. Quantification of lymph node transit M. Staehler, W. Brugger, P. Y. Dietrich, R. Mendrzyk, et al. 2012. Multipeptide times reveals differences in antigen surveillance strategies of naive CD4+ and immune response to cancer vaccine IMA901 after single-dose cyclophospha- CD8+ T cells. Proc. Natl. Acad. Sci. USA 109: 18036–18041. mide associates with longer patient survival. Nat. Med. 18: 1254–1261. by guest on October 7, 2021 Supplemental Data

Differential kinetics of antigen-dependency of CD4+ and CD8+ T cells

Hannah Rabenstein, Anne C. Behrendt, Joachim W. Ellwart, Ronald Naumann,

Marion Horsch, Johannes Beckers, and Reinhard Obst

A B

AND OT1 (dtg-M B10.BR) (dtg-O B6)

LN SPL LN SPL

transfer

AND OT1 (dtg-M wt) (dtg-O wt)

none

- 4 - 3 - 2 - 1 cont. [d before transfer] 24 h dox treatment 0 2 4 6 0 2 4 6 N N

CFSE

Supplemental Figure 1. Estimating pMHC class II and I survival in the steady state. (A) Dtg-M → B10.BR (left) and dtg-O → B6 chimeras (right) were treated with dox (black box represents one day) or regular drinking water (grey) as indicated and were transferred with CFSE-labeled AND (left) or OT1 T cells (right). Lymph node and spleen cells were analyzed 3.5 days later. Shown are CD4+CD45.1+ AND (left) and CD8+CD45.1+ OT1 cells (right). (B) Mean values ± SEM from 3 independent experiments. Shown is the proliferative index N, as described in Materials and Methods.

H-2b H-2d H-2k + CD4 + CD8

CD5 CD5 CD5

10 *** ** * *** *** *** 60 ) 3 40 1 CV 20 gMFI (10

0.1 0 4+ 8+ 4+ 8+ 4+ 8+ 4+ 8+ 4+ 8+ 4+ 8+ b d k b d k H-2 H-2

Supplemental Figure 2. CD5 expression by CD4+ and CD8+ T cells. CD5 levels were determined on CD4+ (top) and CD8+ (bottom panels) polyclonal splenocytes from C57BL/6, BALB/c and B10.BR animals, with isotype control stains in grey. Shown below are geometric mean fluorescence intensities (left) and coeffients of variation (right). P- values were determined by a paired two-tailed Student's t test. Shown are results from 3 to 5 (B6) animals per group.

2

A AND OT1 B

OT1

- - SPL IC -LFA-1 4.6 4.2 2 IC

5.6 5.2

A-1 3 F -CD3/CD28 -L CFSE

Supplemental Figure 3. LFA-1 blockade does not interfere antigen-free proliferation of CD8+ T cells. 5x104 congenically marked and sorted AND and OT-1 T cells were stimulated with anti-CD3 and anti-CD28 mAbs in presence of 10 µg/m isotype control (IC) or anti-LFA-1 (M17.4) mAbs. Medium and antibodies were exchanged after 24 h. (A) Appearance of cultures after 2 days. The anti-LFA-1 mAb blocks cluster formation observable in CD8+ T cell cultures only (10 x magnification). (B) OT1 T cells treated with anti-LFA-1 in the culture were then transferred into antigen-free (condition 2) oder -expressing animals (condition 3). Mice were injected with 200 µg anti- LFA-1 i.p. three times 12 h apart beginning 4 h before T cell transfer. CFSE dilution in spleen CD8+CD90.1+ cells shown.

3

Suppl. Table 1. Genes expressed in OT1 CD8+ T cells following transient and persistent stimulation.

AND CD4+ OT1 CD8+ Transient / None Persistent / None Transient / None Persistent / None FC P FC P FC P FC P 1 0610007P06RIK RIKEN cDNA 0610007P06 gene 0,628 0,0986338 2,002 0,0148288 2,198 0,0015367 3,009 0,0121480 2 0610040D20RIK RIKEN cDNA 0610040D20 gene 0,188 0,0018070 1,340 0,0072275 7,842 0,0002713 8,594 0,0003873 3 1110002E23RIK RIKEN cDNA 1110002E23 gene 0,417 0,0079481 1,458 0,0337212 2,110 0,0084425 2,835 0,0005643 4 2400010G15RIK RIKEN cDNA 2400010G15 gene 0,402 0,0405370 0,837 0,4144009 3,524 0,0000665 2,743 0,0004197 5 3300001G02RIK RIKEN cDNA 3300001G02 gene 0,561 0,0519504 1,962 0,0377822 2,780 0,0020283 4,198 0,0034701 6 5430432M24RIK RIKEN cDNA 5430432M24 gene 0,785 0,1889825 1,924 0,0186402 2,356 0,0009699 3,052 0,0042386 7 AA407659 expressed sequence AA407659 1,144 0,3918731 2,548 0,0111372 3,037 0,0014297 2,315 0,0011770 8 AA536717 expressed sequence AA536717 0,784 0,3676597 2,598 0,0054256 2,252 0,0058058 2,428 0,0071108 9 ACADL acyl-Coenzyme A dehydrogenase, long-chain 0,480 0,0021080 1,717 0,0041404 3,166 0,0000383 3,792 0,0013628 10 ADPRH ADP-ribosylarginine hydrolase 0,720 0,0043056 2,044 0,0078273 2,710 0,0000472 3,069 0,0056677 11 ADRBK1 adrenergic receptor kinase, beta 1 0,601 0,0431489 1,465 0,0945056 3,328 0,0000104 2,642 0,0000051 12 AI586015 expressed sequence AI586015 1,109 0,6807473 1,047 0,7282164 2,600 0,0005956 2,465 0,0486390 13 AL033326 expressed sequence AL033326 0,595 0,0278526 1,004 0,9741718 2,497 0,0013455 1,818 0,0043192 14 AP2S1 adaptor-related protein complex 2, sigma 1 subunit 0,565 0,0140101 1,963 0,0140617 3,135 0,0009994 3,060 0,0003742 15 AP3S1 adaptor-related protein complex 3, sigma 1 subunit 0,253 0,0054170 2,023 0,0393948 5,041 0,0000524 9,253 0,0009163 16 APEH acylpeptide hydrolase 0,515 0,0230626 1,354 0,0878666 2,838 0,0007522 2,859 0,0000319 17 ARHGAP18 Rho GTPase activating protein 18 1,464 0,2046362 2,490 0,0052344 2,226 0,0095740 2,367 0,0167968 18 ARPC4 actin related protein 2/3 complex, subunit 4 0,572 0,1070435 2,070 0,0035226 2,486 0,0021706 2,503 0,0004780 19 ASNA1 arsA (bacterial) arsenite transporter, ATP-binding, homolog 1 0,291 0,0041143 1,241 0,0928547 2,803 0,0002717 4,715 0,0016968 20 ASRGL1 asparaginase like 1 0,528 0,0334046 1,750 0,0127659 2,893 0,0000466 2,805 0,0082013 21 AU022870 expressed sequence AU022870 0,822 0,0607645 1,164 0,2381919 1,701 0,0007439 2,478 0,0000660 22 BAG1 Bcl2-associated athanogene 1 0,641 0,0019032 1,414 0,0352889 2,779 0,0017273 3,183 0,0003320 23 BAIAP3 BAI1-associated protein 3 1,161 0,4456121 7,802 0,0021188 1,267 0,0286191 0,840 0,2626742 24 BATF basic leucine zipper transcription factor, ATF-like 0,973 0,8917724 2,471 0,0099193 3,228 0,0002724 3,339 0,0059074 25 C19ORF20 chromosome 19 open reading frame 20 0,584 0,0121264 1,292 0,0092584 2,741 0,0104152 2,028 0,0085368 26 CAPZB capping protein (actin filament) muscle Z-line, beta 0,375 0,0133908 1,644 0,0093796 3,383 0,0005600 3,151 0,0001609 27 CASP1 caspase 1 6,602 0,0006882 5,008 0,0093407 3,131 0,0003402 2,439 0,0011774 28 CASP4 caspase 4, apoptosis-related cysteine peptidase 8,189 0,0642046 8,398 0,0198369 3,380 0,0026260 5,399 0,0033052 29 CCDC104 coiled-coil domain containing 104 0,608 0,0756204 1,359 0,0394534 2,143 0,0008484 2,661 0,0013820 30 CCNK cyclin K 0,248 0,0062080 0,934 0,5391072 3,621 0,0025944 3,467 0,0001328 31 CD8A /// LOC636147 /// LOC669166 CD8 antigen, alpha chain 1,402 0,0316350 1,036 0,6509475 1,989 0,0011950 1,758 0,0035755 32 CD48 CD48 antigen 1,424 0,1283036 2,058 0,0179245 1,516 0,0020840 1,840 0,0057512 33 CD99 CD99 antigen 0,855 0,1803642 2,481 0,0780230 3,245 0,0019429 3,478 0,0001020 34 CDC25B cell division cycle 25 homolog B (S. cerevisiae) 0,620 0,3134251 8,376 0,0199282 5,248 0,0000294 7,452 0,0232935 35 CDC26 cell division cycle 26 0,381 0,0249501 0,878 0,2916489 2,535 0,0045600 2,413 0,0016101 36 CDC34 cell division cycle 34 homolog (S. cerevisiae) 0,563 0,0012880 1,579 0,0056705 2,074 0,0011623 3,665 0,0039828 37 CDC37 cell division cycle 37 homolog (S. cerevisiae) 0,287 0,0008074 1,053 0,1453844 2,398 0,0005381 3,471 0,0013266 38 CLDND1 claudin domain containing 1 0,811 0,6534181 1,060 0,7806546 2,557 0,0003707 9,126 0,0372850 39 COTL1 coactosin-like 1 (Dictyostelium) 0,435 0,0040875 1,445 0,0917199 2,517 0,0008758 2,265 0,0022433 40 COX5A cytochrome c oxidase, subunit Va 0,445 0,0059818 1,329 0,0031840 2,088 0,0002320 2,212 0,0000159 41 CTLA2A cytotoxic T lymphocyte-associated protein 2 alpha 16,692 0,1652282 21,734 0,0219259 6,920 0,0038255 5,028 0,0029347 42 CTLA2B cytotoxic T lymphocyte-associated protein 2 beta 24,680 0,0001730 36,954 0,0117827 7,675 0,0015060 8,739 0,0034017 43 CTSD cathepsin D 0,646 0,1957573 2,094 0,0459957 2,328 0,0003872 1,982 0,0012369 44 CYB5R4 cytochrome b5 reductase 4 0,967 0,8559305 1,685 0,0291436 2,052 0,0066172 2,015 0,0006115 dodecenoyl-Coenzyme A delta isomerase (3,2 trans-enoyl-Coenyme A 45 DCI 0,517 0,0249267 0,697 0,0203863 2,779 0,0002101 2,779 0,0006440 isomerase) 46 DDEF1 development and differentiation enhancing 0,591 0,0209052 1,745 0,0088483 3,627 0,0003661 3,873 0,0055377 47 DDX41 DEAD (Asp-Glu-Ala-Asp) box polypeptide 41 0,672 0,0895948 1,114 0,5441812 1,927 0,0007280 1,975 0,0006769 48 DENR Density-regulated protein 0,628 0,0192512 1,220 0,2855328 2,051 0,0011155 2,078 0,0000152 49 DNAJC15 DnaJ (Hsp40) homolog, subfamily C, member 15 1,090 0,2530549 3,108 0,0080454 4,040 0,0000859 2,410 0,0041174 50 DOCK9 /// LOC670309 dedicator of cytokinesis 9 0,696 0,4458324 1,459 0,2509652 3,745 0,0004018 2,423 0,0000681 51 DTX3 deltex 3 homolog (Drosophila) 0,727 0,1267485 1,484 0,0105760 3,469 0,0001995 2,058 0,0070271 52 DUSP2 dual specificity phosphatase 2 0,669 0,2222826 1,158 0,2600101 2,472 0,0003638 2,462 0,0007913 53 EFHD2 EF hand domain containing 2 0,724 0,0879999 2,087 0,0040999 1,909 0,0015117 2,363 0,0055083 54 ELK3 ELK3, member of ETS oncogene family 0,776 0,4533028 1,262 0,0131179 2,229 0,0004561 2,764 0,0311709 55 EXOC8 exocyst complex component 8 0,656 0,2622138 1,374 0,0001078 2,244 0,0001751 2,144 0,0002780 56 FAM62A family with sequence similarity 62 (C2 domain containing), member A 0,737 0,0152998 1,209 0,0585985 1,349 0,0336679 1,302 0,0036586 57 FBXO31 F-box only protein 31 0,562 0,0399151 0,948 0,6198037 2,051 0,0040813 2,126 0,0039050 58 FKBP1A FK506 binding protein 1a 0,593 0,0294955 1,330 0,0268334 2,505 0,0000311 3,370 0,0000765 59 GADD45GIP1 growth arrest and DNA-damage-inducible, gamma interacting protein 1 0,744 0,0610668 1,143 0,4492350 2,205 0,0044862 2,618 0,0033654 60 GAS8 growth arrest specific 8 0,900 0,1323674 1,315 0,0093795 2,026 0,0005401 2,082 0,0008122 61 GATA3 GATA binding protein 3 0,724 0,1206519 0,773 0,2650718 3,299 0,0119875 2,647 0,0013252 62 GEM GTP binding protein (gene overexpressed in skeletal muscle) 3,768 0,0041632 18,431 0,0524139 4,981 0,0000555 6,671 0,0009361 63 GIMAP6 GTPase, IMAP family member 6 0,524 0,0511782 1,227 0,3197702 4,462 0,0000778 2,104 0,0253445 64 GLRX2 glutaredoxin 2 (thioltransferase) 0,478 0,1634833 1,365 0,3586311 2,414 0,0021644 2,405 0,0011495 65 GPX7 glutathione peroxidase 7 1,012 0,9537640 1,181 0,3899496 1,959 0,0045120 2,554 0,0031772 66 HCLS1 hematopoietic cell specific Lyn substrate 1 0,273 0,0315742 0,900 0,5182840 2,329 0,0082793 2,799 0,0120139 67 HRAS v-Ha-ras Harvey rat sarcoma viral oncogene homolog 0,354 0,0125395 2,181 0,0064802 3,383 0,0002694 5,435 0,0059524 68 HSDL2 hydroxysteroid dehydrogenase like 2 0,617 0,0355437 1,053 0,6159259 1,911 0,0020126 2,138 0,0001681 69 ICOS inducible T-cell co-stimulator 0,583 0,1020674 3,623 0,0074079 3,827 0,0097914 4,594 0,0016967 70 IL2RB interleukin-2 receptor, beta chain 1,479 0,4048202 1,767 0,1433570 2,727 0,0021813 3,401 0,0296962 71 IRAK1 interleukin-1 receptor-associated kinase 1 0,774 0,3241485 0,782 0,1824445 1,721 0,0164967 1,507 0,0456560 72 ITGB1BP1 integrin beta 1 binding protein 1 0,559 0,0033079 1,761 0,0303010 4,276 0,0003536 4,163 0,0003256 73 JAK3 Janus kinase 3 0,441 0,0311532 2,141 0,0352221 2,830 0,0016013 3,396 0,0011130 74 KCNAB2 potassium voltage-gated channel, shaker-related subfamily, beta member 2 0,505 0,0805728 1,564 0,1141672 2,299 0,0008660 2,227 0,0427925 75 KLRC1 /// KLRC2 killer cell lectin-like receptor subfamily C, member 1 & 2 1,218 0,1952786 4,379 0,0328902 10,227 0,0000786 16,896 0,0035114 76 KLRK1 killer cell lectin-like receptor subfamily K, member 1 0,772 0,0495002 1,187 0,0629132 7,475 0,0002740 7,772 0,0023882 77 LAG3 lymphocyte-activation gene 3 1,150 0,2920882 5,284 0,0473986 2,393 0,0021756 4,351 0,0098381 78 LGALS9 lectin, galactose binding, soluble 9 0,197 0,0355950 1,362 0,2780756 6,930 0,0043790 7,851 0,0008007 79 LSP1 lymphocyte specific 1 0,982 0,9074709 1,653 0,0484620 2,036 0,0001971 1,569 0,0196895 80 MAGEE1 melanoma antigen, family E, 1 0,602 0,0412070 1,313 0,3627002 2,106 0,0132478 2,514 0,0095777 81 MAP2K5 mitogen activated protein kinase kinase 5 0,437 0,0103302 0,993 0,9540420 2,795 0,0000715 2,210 0,0004817 82 MAP2K6 mitogen activated protein kinase kinase 6 0,424 0,0275473 1,048 0,8311227 2,885 0,0068656 2,308 0,0010642 83 MAPK3 mitogen activated protein kinase 3 0,349 0,0113709 0,806 0,2965211 3,312 0,0031613 3,072 0,0006545 84 MAPK6 mitogen-activated protein kinase 6 0,291 0,0599430 2,429 0,0342753 3,539 0,0028930 9,851 0,0000894 85 MARCH5 membrane-associated ring finger (C3HC4) 5 0,473 0,0012822 1,289 0,0327969 2,518 0,0006571 3,078 0,0001051 86 MAT2B methionine adenosyltransferase II, beta 0,324 0,0174432 0,789 0,1408280 4,890 0,0002786 3,236 0,0005753 87 MGAT2 mannoside acetylglucosaminyltransferase 2 0,554 0,0096264 1,430 0,0024196 2,253 0,0051783 3,127 0,0013975 88 MNS1 meiosis-specific nuclear structural protein 1 0,879 0,3163405 3,168 0,0275122 2,392 0,0001628 3,579 0,0002093 89 MTCH1 mitochondrial carrier homolog 1 (C. elegans) 0,237 0,0051248 1,444 0,1474826 2,810 0,0074644 5,509 0,0145621 90 MYO1F myosin IF 2,405 0,1618509 3,865 0,0064190 1,440 0,0131535 1,273 0,2665556 91 NAT2 N-acetyltransferase 2 (arylamine N-acetyltransferase) 0,657 0,0770729 1,251 0,2784016 3,033 0,0000181 2,528 0,0037329 92 NCDN neurochondrin 0,415 0,0074062 0,951 0,4552033 2,436 0,0042022 2,313 0,0013497 93 NKG7 natural killer cell group 7 sequence 5,311 0,0021460 50,751 0,0003489 1,373 0,0011710 1,402 0,0234863 94 NQO2 NAD(P)H dehydrogenase, quinone 2 0,946 0,8407635 3,975 0,0006378 3,615 0,0054050 4,266 0,0144224 95 NRP1 neuropilin 1 4,169 0,0274333 8,047 0,0136199 10,249 0,0036856 13,189 0,0260623 96 OGDH oxoglutarate dehydrogenase (lipoamide) 0,428 0,0068757 0,970 0,7278726 2,120 0,0041099 2,753 0,0017054 97 PGLYRP1 peptidoglycan recognition protein 1 0,954 0,8691309 3,853 0,0007081 6,029 0,0031057 6,090 0,0038590 98 PLEKHJ1 pleckstrin homology domain containing, family J member 1 0,149 0,0013538 1,183 0,2489805 6,831 0,0000930 7,531 0,0009388 99 PLEKHQ1 pleckstrin homology domain containing, family Q member 1 0,670 0,0198672 1,559 0,0160888 2,081 0,0043702 3,035 0,0033587 100 PRKCA protein kinase C, alpha 0,466 0,0179521 2,983 0,0064035 3,178 0,0002826 5,593 0,0005460 101 PSMA4 proteasome (prosome, macropain) subunit, alpha type 4 0,433 0,0007867 1,328 0,0836201 2,990 0,0001356 4,802 0,0000364 102 PTPMT1 protein tyrosine phosphatase, mitochondrial 1 0,487 0,0055086 1,019 0,7905401 3,625 0,0004605 4,053 0,0005310 103 RDH12 retinol dehydrogenase 12 0,860 0,0297531 1,195 0,5221518 3,392 0,0020385 2,037 0,0191086 104 REEP5 receptor accessory protein 5 0,242 0,0021171 1,415 0,0322999 6,280 0,0000906 9,536 0,0003031 105 RPRC1 arginine/proline rich coiled-coil 1 0,538 0,0093826 1,419 0,0494708 1,949 0,0003699 2,182 0,0009404 106 RPS6KB2 ribosomal protein S6 kinase, polypeptide 2 0,404 0,0000524 1,149 0,2720657 2,234 0,0011210 2,298 0,0025650 107 S100A10 S100 calcium binding protein A10 (calpactin) 1,065 0,8404380 2,790 0,0024133 2,752 0,0003238 2,396 0,0032712 108 S100A6 S100 calcium binding protein A6 (calcyclin) 6,676 0,1413221 24,453 0,0079592 7,003 0,0185086 4,035 0,0327121 109 SAP18 Sin3-associated polypeptide 18 0,356 0,0262385 1,170 0,2705837 3,648 0,0005591 4,638 0,0000232 110 SDHD succinate dehydrogenase complex, subunit D, integral membrane protein 0,518 0,0056201 1,522 0,0158351 2,551 0,0003177 4,280 0,0033190 111 SERPINB6A serine (or cysteine) peptidase inhibitor, clade B, member 6a 1,124 0,1875568 1,749 0,0191469 2,718 0,0009290 3,517 0,0002569 112 SERPINB6B serine (or cysteine) peptidase inhibitor, clade B, member 6b 4,669 0,0627706 9,265 0,0004340 2,748 0,0021114 4,611 0,0031465 splicing factor proline/glutamine rich (polypyrimidine tract binding protein 113 SFPQ 0,962 0,9190587 1,735 0,0129785 2,806 0,0029983 2,389 0,0316598 associated) 114 SH2D1A SH2 domain protein 1A 0,774 0,0937629 2,261 0,0070233 3,970 0,0037161 3,309 0,0000497 115 SH2D2A SH2 domain protein 2A 0,990 0,5765315 2,090 0,0243104 2,496 0,0000326 3,307 0,0015837 116 SIPA1 signal-induced proliferation associated gene 1 0,637 0,0172557 1,751 0,0111710 2,603 0,0017627 2,840 0,0010399 117 SLC35C1 solute carrier family 35, member C1 0,278 0,0032500 0,865 0,2538316 3,704 0,0027920 2,541 0,0095292 118 SLC39A6 solute carrier family 39 (metal ion transporter), member 6 0,556 0,0845959 1,187 0,3734503 3,026 0,0000107 2,499 0,0013645 spermine synthase /// similar to Spermine synthase (Spermidine 119 SMS /// LOC671878 0,538 0,0098808 1,294 0,0156410 2,355 0,0002506 3,292 0,0012839 aminopropyltransferase) (SPMSY) 120 SNRP1C U1 small nuclear ribonucleoprotein 1C 0,539 0,0171702 1,361 0,0094597 2,299 0,0020799 2,856 0,0001133 121 SP100 nuclear antigen Sp100 0,939 0,3776663 1,295 0,1693104 2,879 0,0000899 2,003 0,0173709 122 TBX21 T-box 21 4,812 0,0303684 35,688 0,0012453 7,939 0,0000605 10,449 0,0475983 123 THRAP4 thyroid hormone receptor associated protein 4 0,406 0,0166600 1,066 0,7691442 2,575 0,0071196 2,685 0,0009798 124 THY1 thymus cell antigen 1, theta 0,608 0,0718362 1,340 0,3848261 3,026 0,0141800 2,755 0,0082450 125 TIMM8B translocase of inner mitochondrial membrane 8 homolog b (yeast) 0,624 0,0698086 1,249 0,0116593 2,334 0,0086598 3,333 0,0002065 126 TPM1 tropomyosin 1, alpha 0,525 0,0081689 1,544 0,0402712 2,125 0,0003876 2,856 0,0174562 127 TRAF1 Tnf receptor-associated factor 1 0,794 0,4613669 1,379 0,2751810 4,664 0,0001529 2,959 0,0145390 128 TRP53RK /// 2810408M09RIK Trp53 regulating kinase /// RIKEN cDNA 2810408M09 gene 0,476 0,0358696 1,300 0,0128003 2,094 0,0019463 3,594 0,0029032 129 TTC7B tetratricopeptide repeat domain 7B 0,561 0,0945932 1,225 0,2853397 2,449 0,0051200 2,928 0,0004201 130 TXN1 thioredoxin 1 0,492 0,0391432 1,946 0,0061432 2,892 0,0003839 4,044 0,0016046 131 UCP2 uncoupling protein 2 (mitochondrial, proton carrier) 0,365 0,0186746 2,049 0,1014855 2,992 0,0256691 4,184 0,0002177 132 UQCR ubiquinol-cytochrome c reductase (6.4kD) subunit 0,542 0,0269642 1,210 0,0892475 2,121 0,0351099 2,269 0,0001665 133 VDAC2 voltage-dependent anion channel 2 0,390 0,0063229 1,354 0,0016885 2,295 0,0001205 3,497 0,0010850 134 WDR79 WD repeat domain 79 0,542 0,0013731 1,193 0,2127547 2,350 0,0000870 2,790 0,0000324 135 WDSUB1 WD repeat, SAM and U-box domain containing 1 0,543 0,0533604 1,938 0,0254710 2,977 0,0172149 4,205 0,0000905 136 XRN2 5'-3' exoribonuclease 2 0,395 0,0188382 0,914 0,4748404 2,290 0,0029992 1,838 0,0008758 137 YIPF1 Yip1 domain family, member 1 0,612 0,0355019 1,545 0,0312137 2,279 0,0000430 2,577 0,0015931 138 ZAP70 zeta-chain (TCR) associated protein kinase 0,435 0,0065199 1,497 0,0350371 2,476 0,0007320 2,689 0,0054887 139 ZDHHC16 zinc finger, DHHC domain containing 16 0,576 0,0147593 1,568 0,0459080 2,104 0,0007866 2,720 0,0011775 140 ZYX zyxin 0,804 0,4666501 2,013 0,0041272 3,406 0,0000115 2,350 0,0000054

Supplemental Table I. Genes expressed by OT1 CD8+ T cells following transient and persistent stimulation.

Cells were analyzed as described for Figure 6. Given is the fold change (FC) in comparison to unstimulated control cells and the P-values as determined by Student's t test.

4