Memory CD4+ T-cell–mediated protection depends onsecondary effectors that are distinct from and superiorto primary effectorsTara M. Strutta,1,2, K. Kai McKinstrya,1,2, Yi Kuanga, Linda M. Bradleyb, and Susan L. Swaina,2

aDepartment of Pathology, University of Massachusetts Medical School, Worcester, MA 01655; and bInfectious and Inflammatory Diseases Center, Sanford-Burnham Institute for Medical Research, La Jolla, CA 92037

Edited* by Robert L. Coffman, Dynavax Technologies, Berkeley, CA, and approved August 3, 2012 (received for review April 9, 2012)

Whether differences between naive cell-derived primary (1°) andmemory cell-derived secondary (2°) CD4+ T-cell effectors contrib-ute to protective recall responses is unclear. Here, we comparethese effectors directly after influenza A virus infection. Both de-velop with similar kinetics, but 2° effectors accumulate in greaternumber in the infected lung and are the critical component ofmemory CD4+ T-cell–mediated protection against influenza A vi-rus, independent of earlier-acting memory-cell helper functions.Phenotypic, functional, and transcriptome analyses indicate that2° effectors share organ-specific expression patterns with 1° effec-tors but are more multifunctional, with more multicytokine (IFN-γ+/IL-2+/TNF+)-producing cells and contain follicular helper T-cellpopulations not only in the spleen and draining lymph nodesbut also in the lung. In addition, they express more CD127 andNKG2A but less ICOS and Lag-3 than 1° effectors and expresshigher levels of several genes associated with survival and migra-tion. Targeting two differentially expressed molecules, NKG2Aand Lag-3, reveals differential regulation of 1° and 2° effectorfunctions during pathogen challenge.

cytokines | viral infection | immune regulation

Analyses of the mechanisms underlying memory CD4+ T-cell–mediated protection have focused largely on their earlier

provision of help as compared with naive cells (1), although italso is appreciated that highly activated secondary CD4+ T-cell(hereafter, 2°) effectors develop after the re-expansion of memorypopulations (2). Studies also suggest that optimal protection pro-vided by T helper type 1 (TH1)-like memory CD4+ T cells cor-relates with the capacity to produce multiple cytokines, includingIFN-γ, TNF, and IL-2, rather than IFN-γ alone (3). Whether 2°effectors derived from protective memory CD4+ T cells retain thisphenotype, the extent to which 2° effectors contribute to theprotection mediated by memory CD4+ T cells, and whether andhow 2° effectors differ from primary CD4+ T-cell (hereafter, 1°)effectors are unclear.Comparison of 1° and 2° CD4+ T-cell effectors within mixed

populations is difficult. The higher proportion of antigen-specificmemory cells as compared with naive cells complicates quantitativeanalysis, and the maintenance of very few antigen-specific cells ingeneral (4) precludes rigorous analysis ex vivo of the effectors towhich they give rise. Polyclonal naive and memory T-cell pop-ulations also may differ in repertoire and T-cell receptor (TcR)affinity (5, 6), further complicating comparisons. Finally, phenotypicdiscrimination alone between highly activated effectors and cellsthat have divided only once or twice is not reliable, because effectorsresponding in different organs can express different surface phe-notypes (7). To overcome these obstacles, we transferred equalnumbers of naive and memory HNT TcR transgenic CD4+ T cellsrecognizing theA/PR8/34 (PR8) strain of influenzaAvirus (IAV) tounprimed hosts and then challenged with PR8 to compare directlythe generation, function, and protective capacity of 1° and2° effectors.IAV infection presents a compelling model for addressing these

questions. The transfer of 1° effectors to unprimedmice can protect

against lethal challenge (8–10), and studies demonstrating memoryCD4+ T-cell protection in mice deficient for CD8+ T and B cellssuggest that an important helper-independent protective contribu-tion of memory CD4+ T cells may be mediated directly by the 2°effectors (11–13). In addition, IAV-specific 1° effector (7, 10) andmemory (14, 15) CD4+ T cells isolated from the lung and fromsecondary lymphoid organs (SLO) display distinct functional andphenotypic characteristics. Whether 2° effectors comprise a simi-larly heterogeneous population is unknown. This issue is important,because recent studies show that lung-resident memory CD4+

T cells provide greater protection against IAV than SLO-residentmemory cells (16). Thus, a more comprehensive understanding oforgan-specific heterogeneity within responding CD4+ T-cell poolsmay provide clues about the critical attributes of themost protectiveCD4+ T cells that could be generated by vaccination.We find that although both populations develop and peak with

similar kinetics, the 2° effectors accumulate in greater numbers inthe lung, the primary site of infection.We show that the generationof the 2° effectors is the critical component of protective immunitymediated by memory CD4+ T cells against IAV and that 2°effectors are superior to 1° effectors in mediating viral clearance.We demonstrate that 2° effectors contain more cells producingTNF and/or IL-2 togetherwith IFN-γ and fewer cells producing IL-10 than do 1° effectors. In addition, we identify several phenotypicmarkers that distinguish the two effector populations fromeach another.To define the differences between 1° and 2° effectors further, we

analyzed gene expression by microarray. The 1° and 2° effectorsrecovered from both lung and SLO display a high degree ofshared, organ-specific specialization. However, 2° effectors areless compartmentalized, as evidenced by a wider distribution offollicular helper T (TFH) cells. Furthermore, we identify a shortlist of genes that distinguish 1° and 2° effectors and that could beinvolved in controlling the greater expansion and more pluripo-tent functions of 2° effectors. Finally, we demonstrate the specificregulation of 1° or 2° effector cytokine production by blocking thedifferentially expressed surface proteins NKG2A and Lag-3.These findings define pathways that explain, in part, the functionalsuperiority of the memory CD4+ T-cell response.

Author contributions: T.M.S., K.K.M., and S.L.S. designed research; T.M.S., K.K.M., andY.K. performed research; L.M.B. contributed new reagents/analytic tools; T.M.S. andK.K.M. analyzed data; and T.M.S., K.K.M., and S.L.S. wrote the paper.

The authors declare no conflict of interest.

*This Direct Submission article had a prearranged editor.

Data deposition: The data reported in this paper have been deposited in the Gene Ex-pression Omnibus (GEO) database, www.ncbi.nlm.nih.gov/geo (accession no. GSE40230).1T.M.S. and K.K.M. contributed equally to this work.2To whom correspondence may be addressed. E-mail: tara.strutt@umassmed.edu, kai.mckinstry@umassmed.edu, or Susan.Swain@umassmed.edu.

See Author Summary on page 15095 (volume 109, number 38).

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1205894109/-/DCSupplemental.

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ResultsGeneration of 1° and 2° Effectors in Vivo. To generate comparableeffectors from naive and memory precursors, we transferred equalnumbers of naive or memory HNT CD4+ T cells to Thy-disparatehosts and infected the hosts with PR8.We used both in vivo-primedand reisolated (in vivo PR8 memory) and in vitro-generated TH1memory cells, allowing the investigation of effectors arising fromheterogeneous memory cells resulting from in vivo priming andfrom populations with defined polarization (17). Upon transferto uninfected mice, similar numbers of naive and memory HNTCD4+ T cells were initially present in all organs analyzed and bothdecayedwith identical kinetics (Fig. S1), arguing against differencesin initial trafficking or in survival of donor cells after adoptivetransfer contributing to the results reported here.Unlike naive cells, memory CD4+ T cells are poised for rapid

secretion of cytokines (18). In agreement with studies using invitro-generated memory cells (19), in vivo PR8 memory cells up-regulated CD69 1–2 d earlier in the lung and draining lymph nodes(dLN) than did naive cells (Fig. 1A), indicating a more rapid

activation of memory cells, but 4 d postinfection (dpi) the expan-sion of naive and memory donors was similar (Fig. 1B). All donorsreached similar peak numbers in SLO by 7 dpi, but those arisingfrom memory precursors accumulated in greater number in thelung (Fig. 1B). A similar pattern was observed when precursorswere reduced up to 100-fold, with in vivo- and in vitro-derivedmemory cells giving rise to about five times and 10 timesmore cellsin the lung, respectively, than naive donors (Fig. 1C); this resultconfirms that the kinetics and magnitude of expansion are in-dependent of precursor frequency.By 7 dpi, virtually all donor cells recovered from the lung were

effectors as defined by their having undergone five or moredivisions based on loss of carboxyfluorescein succinimidyl ester(CFSE), as were >80% of cells arising from naive and >90% ofcells arising from memory donors in SLO. We titrated thenumber of donor cells and found that at lower numbers trans-ferred (≤2 × 106) nearly all naive and memory donors developedinto effectors in SLO and lung (Fig. 1D). Together Fig. 1 C andD indicate that both naive and memory cells give rise to highlydivided effectors (Fig. 1D) whose number is proportional to

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Fig. 1. Generation of 1° and 2° effectors in vivo. Naive or memory HNT cells (5 × 106) were transferred to Thy-disparate hosts that then were infected with500 EID50 PR8. Spleen, dLN, and lungs were harvested on stated days (n = 5 mice per group on each day) and stained to visualize donor cells. (A) CD69expression on donor cells. (B) Number of donor cells detected. *P < 0.05. (C) Lung-resident donor cells at 7 dpi from mice receiving indicated number of donorcells. n = 4 mice per group. *P < 0.05, ***P < 0.001, and ****P < 0.0001. Upper asterisks represent in vitro TH1 memory cells vs. naive cells; lower asterisksrepresent in vivo PR8 memory cells vs. naive cells). (D) Proportion of 1° and 2° effectors present in the dLN at 7 dpi (as determined by loss of CFSE) derived frommice receiving the indicated number of precursors. n = 3–5 mice per group. (E) CD4+ T cells were isolated from the SLO and lung of unprimed or PR8-primedmice and were labeled with CFSE. Then 1 × 107 cells were transferred to Thy-disparate hosts, and the hosts then were challenged with 500 EID50 PR8. (F) At7 dpi, donors that had divided at least five times were enumerated. n = 5 mice per group. All error bars represent the SD.

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input (Fig. 1C). It is notable that, despite the earlier activation ofmemory cells (Fig. 1A), the kinetics of development in responseto PR8 in SLO were similar in 1° and 2° effectors, but themagnitude of the 2° effector response increased markedly in thelung. This enhanced accumulation of 2° effectors in the lung wasseen in all experiments.To determine whether polyclonal memory CD4+ T cells would

show a similar enhanced effector response in the lung, we trans-ferred CFSE-labeled polyclonal CD4+ T cells from unprimed orPR8-primed mice (enriched for PR8 memory) to new hosts andinfected the hosts with PR8 (Fig. 1E). At 7 dpi, effectors wereenumerated by gating on donors that had divided five times ormore. Effectors derived from donors containing memory cellsreached levels about fourfold higher in SLO, likely reflecting theincreased proportion of precursor IAV-specific cells (Fig. 1F).Strikingly, cells fromprimed animals gave rise tomore than 20-foldmore effectors in the lung, about five times higher than the ratio inSLO (Fig. 1F). This result is consistent with observations usingequal numbers of naive and memory monoclonal CD4+ T cellsshowing that the response in the lung is five- to 10-fold higher for 2°effectors than for 1° effectors. The enhanced ability of memoryCD4+ T cells to give rise to effectors in the lung following IAVinfection suggests that this feature of memory may contribute toenhanced protection.

Secondary Effectors Are Critical for Memory CD4+ T-Cell Protection.We next analyzed the protective efficacy of the 2° effectors.Memory CD4+ T cells regulate innate immunity during the initialdays of IAV infection (19) and provide help for the enhancedantibody production that is evident by 7 dpi (20).We reasoned thatthese functions would occur by 5 dpi, but, as described above, wefound that 2° effectors enter the lung only at 6 dpi and peak at 7–8dpi (Fig. 1). Therefore we transferred 5 × 106 memory cells [thenumber of memory cells found to protect reliably against lethalPR8 challenge (12)] or an equal number of control naive cells tounprimed Thy-disparate hosts and then selectively depleteddonors at 5 dpi, leaving earlier functions of memory cells intactbut depleting 2° effectors before they finished differentiating andtrafficked to the lung (Fig. 2A). Treatment led to efficient removalof donor cells (Fig. 2A). Mice receiving memory cells but not naiveHNT cells and treated with control or Thy1.2-depleting antibody

had equivalently enhanced germinal center B-cell formation (Fig.2B) and PR8-specific IgG (Fig. 2C) at 8 dpi, indicating that de-pletion at 5 dpi did not effect helper function. However, therecipients depleted of memory HNT cells did not survive lethalchallenge (Fig. 2D), although they survived longer than therecipients of naive HNT cells (Fig. 2D). This result stresses thenecessity of late-acting events and also is consistent with someprotective contribution from earlier-acting memory cell functions(19). These results suggest that 2° effectors are key contributors tomemory CD4+ T-cell–mediated protection against IAV.

Secondary Effectors Are Superior to 1° Effectors in Mediating ProtectionAgainst IAV. The protection provided by 2° but not 1° effectorsdescribed above could be caused largely by the differences in themagnitude of the response, as suggested by studies correlatingincreased protection against IAV with increasing numbers oftransferred 1° HNT effectors (8). Alternatively, 2° effectors maymediate superior protection through functional differences thatdistinguish them from 1° effectors. To establish that 2° effectorsindeed are capable of enhanced protection as compared with 1°effectors and to investigate the possible reasons for the differ-ence, we isolated effectors from recipients of naive or memoryHNT cells on 7 dpi that had been infected with PR8. We trans-ferred equal numbers of isolated 1° or 2° effectors directly tounprimed hosts and challenged the hosts with a lethal dose ofPR8 (Fig. 3A) against which 5 × 106 in vitro-generated 1° HNTeffectors are required to protect unprimed hosts and to promoteenhanced viral clearance at 4 dpi (8). Consistent with previousstudies, 5 × 106 1° or 2° in vivo-generated effectors rescued hosts(Fig. 3B). However, when 2.5 × 106 cells were transferred, only 2°effectors provided protection and enhanced viral control (Fig. 3 Band C). These results demonstrate that, in addition to their en-hanced representation in the lung, 2° effectors are superior to 1°effectors in mediating protection as evaluated per cell input.The previous experiments utilized effectors pooled from lung and

SLO. Recent studies suggest that CD4+ T cells isolated from thelung provide improved protection comparedwith those in SLO (16).As the pooled 2° effectors contain proportionallymore cells isolatedfrom the lung compared with 1° effectors (see Fig. 1B), we trans-ferred 2.5 × 106 1° or 2° effectors isolated only from the lung tounprimed hosts and challenged the hosts with a lethal dose of PR8.

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Fig. 2. The 2° effectors are critical for memory CD4+ T-cell–mediated protection. (A) Naive or in vitro-generated memory HNT cells (5 × 106) were transferredto Thy-disparate hosts, and the hosts then were infected with a 10,000 EID50 of PR8 and treated with either donor cell depleting or isotype control antibodyon day 5 with representative staining of dLN on day 6. (B) Absolute numbers of germinal center (GC) B cells present in spleen and dLN at 8 dpi. (C) Serum PR8-specific IgG levels were determined from three to five mice per group. (D) Survival of experimental groups described in A. n = 5 per group. Results are shownfor one of two independent experiments. All error bars represent the SD.

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We again observed that 2° effector protection was significantly en-hanced compared with 1° effector protection (Fig. 3D), although 1°effectors from the lung provided far greater protection than thepooled 1° effectors (compare Fig. 3 D and B). Perhaps surprisingly,equivalent numbers of 1° and 2° effectors were observed in the lungat 4 dpi (Fig. 3E), and both populations exhibited a highly activatedphenotype (Fig. 3F). This result suggests that 2° effectors haveenhanced per cell function as compared with 1° effectors respond-ing in the same organ.

Cytokine Profiles of 1° and 2° Effectors.Many T-cell effector functionsare mediated by cytokines made after TcR triggering. To examinewhether 1° and 2° effectors make different patterns or levels ofcytokines, we used intracellular cytokine staining (ICCS) to assessthe coproduction of the dominant cytokines seen in the IAV re-sponse, namely, IL-2, IFN-γ, and TNF. The 2° effectors derivedfrom both in vitro and in vivo memory cells and recovered fromeither SLO or lung at 7 dpi contained higher proportions ofIL-2+IFN-γ+ (Fig. 4A), TNF+IFN-γ+, and IL-2+IFN-γ+TNF+

(Fig. S2) cells than did 1° effectors. Dilution of precursors up to100-fold did not alter the proportion of IL-2+IFN-γ+ 1° or 2°effectors substantially (Fig. 4B). Higher frequencies of IL-2+IFN-γ+ donors were also detected in effectors derived frompolyclonal CD4 T cells from PR8-primed (enriched for 2°effectors) versus from naive mice (Fig. 4C), for which loss ofCFSE was used to gate IAV-specific effectors (Fig. S3). Theseobservations imply that enhanced multicytokine production isa general feature of 2° effectors.

We next infected mice with 50, 500, or 5,000 egg infectious doses(EID50) PR8 to determine whether the magnitude of infectionwould impact effector cytokine production. No differences in viralburden were observed in mice initially receiving 2 × 106 naive ormemory cells and infected with the same dose of PR8. Pulmonarytiters at 7 dpi were proportional to the challenge dose (Fig. 4D),consistent with previous studies (21). Interestingly, the proportionof IL-2+IFN-γ+ cells did decrease significantly with increasingchallenge dose (Fig. 4E). This was most notable for 2° effectors inthe dLN, where the frequency of IL-2+IFN-γ+ cells detected at50 > 500 > 5,000 EID50 challenge (P < 0.005 and 0.05, re-spectively). However, regardless of the challenge dose, 2° effectorscontained more IL-2+IFN-γ+ cells than did 1° effectors. We ana-lyzed cytokine production throughout the peak effector phase (6–8dpi) to rule out the possibility that the difference was only transientor that kinetic patterns in cytokine production differed in 1° and 2°effectors.We observed similar IFN-γ+IL2+ and IFN-γ+TNF+ cellsthroughout (Fig. 4F). Thus, 2° effectors consistently containa higher proportion of double and triple cytokine producing cells.To evaluate other key cytokines produced by T cells during

pathogen challenge, we next assessed IL-10 and IL-17. We haveshown that IL-10 production by 1° CD4+ T-cell effectors canimpede protection against IAV, whereas IL-17+ T cells cancontribute to viral clearance (9, 22). We concentrated on lung-resident effectors, because the expression of both IL-10 and IL-17 from CD4+ T cells is restricted largely to the lung during IAVinfection, with production of both IL-10 and IL-17 peaking at7 dpi (9). A significantly lower fraction of 2° effectors, generated

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Fig. 3. Secondary effectors are superior to 1° effectors in mediating protection against lethal infection. (A) Naive or in vitro-generated memory HNT cells weretransferred to Thy-disparate hosts that then were infected with 500 EID50 PR8 to generate 1° or 2° effectors, respectively. The 1° and 2° effectors were reisolatedfrom SLO and lung at 7 dpi and were pooled, and equal numbers of each (5 or 2.5 × 106) were transferred to unprimed BALB/c hosts. The hosts then wereinfected with 10,000 EID50 PR8. (B) Survival (n = 10 per group) and (C) viral titers from mice receiving no cells or 2.5 × 106 donor cells at 4 dpi (n = 5 per group).Results are shown for one of two independent experiments. (D) Survival of mice receiving no cells or 2.5 × 106 1° or 2° effectors isolated from the lung only. n =10 per group. (E) At 4 dpi, donor cells in the lung were enumerated. n = 5 per group. (F) Representative staining of donor cells at 4 dpi for forward scatter (FSC),CD69, and CD25 expression as compared with host CD4+ T cells (results are shown for one of two independent experiments). All error bars represent the SD.

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from either in vitro or in vivo memory cells, produced IL-10 (Fig.4G), consistent with the lack of IL-10 observed in our earlierstudies of memory CD4+ T-cell responses against IAV (9). Areciprocal pattern was found for IL-17 production. The 2°effectors generated from in vivo PR8-primed memory cellsproduced IL-17. However, virtually no IL-17 was detected from1° effectors or from 2° effectors generated from in vitro TH1memory cells (Fig. 4G), suggesting that the TH1-polarizing con-ditions in vitro suppress subsequent TH17 differentiation. To-gether, as is consistent with the superior protective capacity of 2°effectors, these results indicate that 2° effectors are more capableof producing a set of cytokines implicated in viral protection andproduce less of the inhibitory cytokine IL-10.

Phenotypic Profiles of 1° and 2° Effectors. We next screened effec-tors for expression of a broad panel of surface proteins to see ifwe could identify any reproducible differences between 1° and 2°effectors. Although cells in the lung and in the SLO differentiallyexpressed many markers associated with functional potential,consistent with previous studies (7), only a few key markers dis-

tinguished 1° from 2° effectors within the organs (Fig. 4H and Fig.S4A). The 2° effectors expressed increased CD127 (IL-7Rα) [amarker that is up-regulated on memory as compared with naiveprecursors (17) and that is necessary for CD4+ T-cell survival]and higher levels of NKG2A/C/E [a marker that has been asso-ciated with enhanced T-cell proliferative capacity and cytokineproduction (23, 24)]. In contrast, ICOS, whose higher expressionhas been linked with IL-10 production (25), was decreased on 2°effectors in the lung, correlating well with enhanced IL-10 pro-duction by 1° effectors. The 1° effectors found in the lung alsoexpressed more of the inhibitory receptor Lag-3. These pheno-typic distinctions were not affected by challenge dose or bywhether the 2° effectors were derived from in vitro- or in vivo-generated memory cells (Fig. S4 B and C). These shifts in theexpression of phenotypic markers that have been associated withfunction provide additional clues about which cellular pathwaysmay be regulated differentially in 1° and 2° effectors subsets;however, the changes in levels are sufficiently modest that theyalone are not likely to be useful for definitively distinguishing 1°from 2° effectors in mixed populations in vivo.

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Fig. 4. Cytokine production and differential surface marker expression by 1° and 2° effectors. Naive or memory HNT cells (2 × 106) were transferred to Thy-disparate hosts, and the hosts then were infected with 500 EID50 PR8. (A) The percent of effectors coproducing IFN-γ and IL-2 as determined by ICCS at 7 dpi.(B) Coproduction of IFN-γ and IL-2 from effectors in dLNs at 7 dpi from mice initially receiving the indicated number of donor cells. n = 3 mice per group. *P <0.05, **P < 0.005, ***P < 0.001. (C) Percentage of IFN-γ+IL-2+ effectors at 8 dpi from mice receiving naive or PR8-primed bulk polyclonal CD4 T cells. n = 4 pergroup. (D) Viral titers (n = 5 per group) and (E) percentage of IFN-γ+IL-2+ HNT effectors following infection with indicated doses of PR8 (n = 4 per group). (F)Dual cytokine-producing 1° and 2° effectors responding in the dLN were determined on stated days. (G) IL-10 and IL-17 production from lung-residenteffectors. n = 3 mice per group. **P < 0.005, ***P < 0.001. (H) At 7 dpi, 1° and 2° effectors from the dLN and lung were analyzed for the indicated surfacemarkers. Representative histograms are shown as well as MFI from five mice per group. *P < 0.05, ***P < 0.001. All error bars represent the SD.

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Organ-Specific Gene-Expression Analysis of 1° and 2° Effectors. Toassess further the magnitude of differences between 1° and 2°effectors and to look for unexpected genes that might be expresseddifferentially, we compared transcriptomes using whole-genomemicroarrays. Effectors in the lung express a distinct and apparentlymore highly differentiated phenotype than those in SLO, suggestingthat organ-specific differences could obscure a global analysis.Therefore we separately analyzed effectors isolated from thespleen, dLN, and lung. We sort-purified 1° and 2° effectors fromrecipients of naive or in vivo PR8-primed memory HNT cells, re-spectively (Fig. S5). As predicted, and as is consistent with pheno-typic and functional distinctions among organs, a number of genesthat were shared by 1° and 2° effectors were expressed differentiallyamong organs. For example, 1° and 2° effectors recovered from thelung shared about 200 genes that were differentially expressed ineffectors in SLO (Fig. 5A and Dataset S1). This cohort of sharedlung-enriched effector genes is enriched for immune-response, cy-tokine, chemokine, and defense-response pathways, as shown byDatabase for Annotation, Visualization and Integrated Discovery(DAVID) analysis (Fig. 5A), suggesting that 1° and 2° effectors inthe lung share a spectrum of functions (likely including direct an-tiviral effector functions) and that 1° and 2° effectors in SLO haveother discrete functions (e.g., helper activities).To test this hypothesis, we analyzed whether SLO-resident

effectors were enriched for TFH-associated genes compared withlung effectors. Interestingly, only 1° effectors fit this pattern (Fig.5B), suggesting either that 2° effectors in SLO do not expressthese TFH-associated genes or that 2° effectors in all organs ex-press TFH signature genes. To test these two possibilities, we

analyzed 1° and 2° effectors at 7 dpi for TFH cells by FACS-basedexpression of CXCR5, PD-1, and Bcl-6 (Fig. 5C). We used invitro TH1 memory precursors to rule out the possibility that invivo-primed PR8 memory cells used for microarray analysiscontained preexisting TFH cells that could account for the dif-fering patterns of TFH gene expression. Both 1° and 2° effectorscontained phenotypically defined TFH cells in SLO, but only 2°effectors contained significant TFH cells in the lung (Fig. 5 D andE). Similarly, we observed TFH in polyclonal 2° effector pop-ulations in the lung during heterosubtypic challenge, but no TFHwere observed in 1° polyclonal lung effectors (Fig. S6). Thus,although 1° and 2° effectors in different organs express tran-scriptomes indicative of organ-specific heterogeneity, 2° effectorsexpress unique gene patterns that, together with enhancedmulticytokine production, indicate broader and less-localizedfunctional capacity.

Primary vs. 2° Effector Gene Expression.Wenext focused on the genesthat were expressed differentially in 1° and 2° effectors. A heatmapsummarizing these genes by ANOVA analysis is shown in Fig. 6A.Pair-wise comparisons of 1° and 2° effector transcriptomes withineach organ revealed differential expression of only a modestnumber of genes (about 150). Strikingly, very few of the genesexpressed differentially between 1° and 2° effectors were commonbetween the spleen, dLN, and lung, and no genes were differen-tially regulated in all three organs (Fig. 6B), again indicating a highlevel of organ-specific specialization. When subjected to DAVIDanalysis, the genes that show significant (at least twofold) changes

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Fig. 5. Differential gene expression by 1° and 2° effectors in various organs. Organ-resident 1° and 2° effectors were sort-purified, and mRNA was preparedfor microarray analysis as described. (A) (Upper) Venn diagram of the number of genes differentially expressed in 1° and 2° effectors in the lung and in thedLN and spleen combined (SLO). (Lower) DAVID functional annotation enrichment on genes shared by 1° and 2° effectors in the lung or in SLO. (B) TFH-associated mRNA expression in 1° effectors relative to naive cells isolated from stated organs. Naive or in vitro memory HNT cells were transferred to Thy-disparate hosts; then the hosts were infected with 500 EID50 PR8, and at 7 dpi effectors were stained for the TFH-associated markers PD-1, CXCR5, and Bcl-6. (C)Representative staining and gating strategy. (D) The percentage and (E) the absolute number of TFH in 1° and 2° effectors in stated organs. n = 3 per group.Results are shown for one of three independent experiments. **P < 0.005, ***P < 0.001. All error bars represent the SD.

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between 1° and 2° effectors fall into functional annotation path-ways such as regulation of transcription, metabolism, cell growth,and migration (Fig. 6C). In Dataset S2, we show the genes thatdefine the greatest differences between 1° and 2° effectors. A co-hort of some of the most compelling of these genes and theirfunctional pathways are shown in Fig. 6D. Their differential ex-pression was validated by RT-PCR (Fig. 6E). These genes couldprovide clues to the mechanisms responsible for the unique phe-notype and function of 2° effectors responding to IAV (see belowand Discussion).

Differential Regulation of Function in 1° and 2° Effectors. To evalu-ate whether some of the distinctions identified above correlatewith functions that are likely to be relevant in protective efficacy,we tested whether the multicytokine production patterns of 1°and 2° effectors could be modified by targeting two differentiallyexpressed molecules for which blocking antibodies are available.We first tested whether blocking a target preferentially expressedby 2° effectors would alter their cytokine production but not thatof 1° effectors. We chose Klrc1, encoding NKG2A, because itsexpression was up-regulated by 2° effectors in the lung and dLN,

mirroring surface expression of NKG2A/C/E (Fig. 4G). Weconfirmed higher expression of Klrc1 in 2° effectors by RT-PCR(Fig. 6E) but found equal expression of Klrc2-3, encodingNKG2C/E, in both effector populations (Fig. 6E), indicating thatincreased NKG2A/C/E staining on 2° effectors is caused largelyby changes in the expression of NKG2A.Administration of blocking antibody against NKG2A/C/E (20d5)

on 4–6 dpi to mice that had received memory HNT cells (Fig. 7A)resulted in efficient in vivo blockade at 7 dpi as compared withadministration of an isotype control (Fig. 7B). At 7 dpi, donor cellswere enumerated and analyzed for cytokine production by ICCS.To help rule out an indirect effect of 20d5 treatment on CD4+

T-cell responses through the regulation of other cell typesexpressing NKG2A, we also analyzed 1° effector responses fromrecipients of naive HNT cells, which express reduced levels ofNKG2A (Fig. 6E), that were treated with 20d5 or isotype anti-body. The absolute numbers of 1° or 2° effectors present in thedLN and lung was not affected by 20d5 treatment (Fig. 7C).However, treatment did reduce the frequency and number ofTNF+IFN-γ+ 2° effectors in both the dLN and lung (Fig. 7D) andsignificantly reduced the mean fluorescence intensity (MFI) of

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Fig. 6. Genes expressed differentially in 1° and 2° effectors. (A) Heatmap showing signal strength for individual probes for all organs, n = 2–3 organs pergroup. (B) Venn diagram of the number of genes differentially expressed in 1° and 2° effectors within spleen, dLN, and lung and differentially expressedgenes shared by different organs. (C) DAVID functional annotation enrichment on genes differentially expressed in 1° and 2° effectors within organs. (D)Select genes within the enriched pathways. (E) PCR validation of differential gene expression in 1° and 2° effectors. n = 2–3 organs per group. Error barsrepresent the SD.

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TNF in 2° effectors (Fig. 7 E and F). Treatment had no effect onIL-2 production by 2° effectors (Fig. 7E). Importantly, 20d5treatment had no effect on cytokine production by 1° effectors(Fig. 7D); this observation supports a selective role for NKG2Asignaling in regulating 2° but not 1° effector function in vivo.Weused the same approach to investigate if targeting amolecule

preferentially expressed by 1° effectors could affect their functionselectively. We focused on Lag3 because of its role in modulatingT-cell responses in vivo (26). Although Lag3 expression was higheron 1° than on 2° effectors, as determined by FACS (Fig. 4G), it wasnot expressed differentially in our microarray analysis. This resultmight reflect differential posttranscriptional regulation in 1° and 2°effectors, an hypothesis supported by the intracellular stores ofLag3 in activated CD4+ T cells and by its rapid and tightly con-trolled surface translocation (27).Mice receiving naive or memory HNT cells were treated at 4–6

dpi with Lag3-blocking antibody (C9B7W) (Fig. 7G), resulting inefficient blockade at 7 dpi (Fig. 7H). Strikingly, C9B7W-treatedmice contained higher absolute numbers of 1° effectors at 7 dpi (Fig.7I), and roughly twofold higher numbers of IL-2+IFN-γ+ (Fig. 7 Jand K) cells in the lung and dLN and a similar increase inTNF+IFN-γ+ cells (Fig. 7 J andK). In contrast, 2° effector responses

were not affected by treatment (Fig. 7 I and J). These results confirmthe functional relevance of these two molecules identified in ouranalyses and suggest that 1° and 2° effector responses are regulateddifferently. Not surprisingly, these treatments show only partialeffects on modulating functional potential, suggesting that the en-hanced properties of 2° effectors also are regulated by changes ingenes other than NKG2A and Lag3.

DiscussionOverall, these studies lead to three striking conclusions. First, 2°CD4+ T-cell effectors are distinct from and are functionally su-perior to 1° effectors and mediate better protection against lethalIAV infection. The 2° effector response was temporally separablefrom helper activities mediated by memory cells, occurring after5 dpi, when help already had been delivered. The loss of pro-tection observed upon depletion of transferred memory CD4+

T cells, even though help was unchanged, suggests that the en-hanced abilities of 2° effectors as compared with 1° effectors area major component of the protection conferred by memory CD4+

T cells. Our results thus provide a basis for understanding theprotection mediated by memory CD4+ T cells in B-cell–deficient,CD8+ T-cell–deficient, and even lymphocyte-deficient hosts

A B G H

I JDC

E F K

Fig. 7. NKG2A and Lag3 blockade affect the cytokine production potential of 1° and 2° effectors. Naive or in vitro-generated memory HNT cells (2 × 106)were transferred to Thy-disparate hosts; then hosts were infected with 500 EID50 PR8, and at 4–6 dpi mice were treated with isotype antibody or 20d5 asdepicted (A). (B) Blockade of lung donor cell NKG2A/C/E expression. (C) The ratio of 1° and 2° effectors from the dLN and lungs at 7 dpi. n = 4 per group. (D)The number of IFN-γ+TNF+ effectors in the dLN and lung. n = 4 per group. Results are shown for one of two independent experiments. **P < 0.005. (E)Representative ICCS staining of dLN 2° effectors for IFN-γ+TNF+ and IFN-γ+IL-2+ cells. (F) Summary of 2° effector TNF+ MFI. **P < 0.005. (G) Mice received cellsas above and were treated with isotype antibody or C9B7W at 4–6 dpi. (H) Blockade of Lag3 expression on 1° effectors. (I) Number of effectors. *P < 0.05. (J)The ratio of IFN-γ+IL-2+ (filled circles) and IFN-γ+TNF+ cells (open circles) in the dLN and lung with or without C9B7W at 7 dpi. (K) Representative ICCS stainingof dLN 1° effectors. n = 5 per group. Results are shown for one of two independent experiments. All error bars represent the SD.

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challenged with IAV (11, 12). Second, we find that effectors in thespleen, dLN, and lung are strikingly different from one another,suggesting that they are specialized to perform unique functions atdifferent sites. We also find that 2° effectors are more multi-functional, regardless of location, as evidenced by their enhancedproduction of multiple inflammatory cytokines and their widerdistribution of TFH cells, which are seen in the lung in the 2° butnot the 1° effector response. This finding indicates that CD4+ Tcells responding to pathogen challenge follow multiple, separatedevelopmental patterns and raises the possibility that unique cuesin distinct environments might coordinate effector specialization.Third, we demonstrate distinct regulation of 1° and 2° effectorfunction in vivo. Our results underscore the unique character of 2°effectors but also suggest that the regulation of the enhancedfunction of 2° effectors is complex, involving multiple pathways.It perhaps is unexpected that the array of cytokines produced by

individual CD4+ T cells broadens instead of becoming more spe-cialized with further exposure to antigen and with division.We hadconsidered the possibility that 2° effectors might be more uniformamong organs, because the process of epigenetic remodeling isthought to change future expression by increasing the ease withwhich certain cytokines are expressed and by silencing others (28).Instead, in agreement with studies addressing gene expression inrepeatedly stimulatedmemory T cells (29), our results suggest thateven well-polarized TH1memory cells making a restricted cytokineprofile (30) can differentiate further to make multiple cytokines athigher levels. These findings suggest that the transition to theresting memory state may reset certain aspects of a CD4+ T cell’sresponse potential, a hypothesis supported by the nearly identicalgene expression in naive and memory cells (17).Our analysis of 1° and 2° effectors recovered from different

organs revealed striking similarities, as well as differences, intheir transcriptomes. For example, 1° and 2° effectors isolatedfrom the lung both expressed high levels of genes associated withantiviral responses, such as IFN-γ, and numerous chemokinesand chemokine receptors. Our data are consistent with the hy-pothesis that effectors in the lung are important for protectionagainst IAV through direct mediation of viral clearance. HowCD4+ T-cell effectors combat IAV is not fully understood, butour studies suggest that individual protective mechanisms, in-cluding perforin-dependent killing of infected cells and pro-duction of IFN-γ, become more or less important depending onthe context of infection (8, 12). In contrast, the expression ofTFH-associated genes in 1° effectors was restricted to SLO, but 2°effectors contained substantial TFH populations in all organstested. It is interesting to speculate that TFH activity from 2°effectors in the lung during recall responses against IAV mightbe important in providing efficient help for local B-cell antibodyproduction when inducible bronchus-associated lymphoid tissueis present (31). The dramatic distinctions seen at the gene-ex-pression level among effectors in lung, spleen, and dLN suggestthat the distribution of the TFH subset in 1° effectors likelyrepresents only one set of organ-specific distinctions and thatother subsets also might be found preferentially in different sites.Indeed we find that CD4+ T cells with cytotoxic activity gener-ated by IAV are restricted almost exclusively to the lung (10).Thus, correlates of protective T cells may differ in differentorgans as a result of the simultaneous generation of effectorsubsets specialized for different roles at different sites. Moreoverthe depletion studies indicate that the helper functions occurearlier in SLO, whereas the peak of effector responses, whichcorrelates with viral clearance, occurs later in the lung. Thus,a temporal distinction also must be considered in identifyingcorrelates of protection.Perhaps it is not surprising that we have identified only a rel-

atively small number of genes, about 450, that are expresseddifferentially in 1° and 2° effectors. This cohort of genes includesmany that are involved in regulating apoptosis, signaling path-ways, translation, cell migration, chemokine signaling, and me-tabolism. For example, 2° effectors expressed lower levels ofgenes associated with the suppression of DNA replication (setd8)

(32) and dampening of cellular proliferation (lrig3) (33), as isconsistent with the increased magnitude of 2° effector responses.Similarly, the higher expression in 1° effectors of genes such assatb2, a negative regulator of CD127 expression (34), andpcdh10, which can potentiate apoptosis (35), also is consistentwith increased numbers of 2° effectors at the site of infection. Inaddition, 2° effectors express higher levels of gpr12, gpr63, rgs3,and tiam1, all of which are associated with chemotaxis and mi-gration (36–38) and also might be involved in the greater accu-mulation of 2° effectors in the lung. Other genes, such as eif4gI [atranslation initiation factor (39)], araf [a potential modulator ofTcR signaling (40)], and rab18 [involved in the ER secretorypathway (41)] also could regulate or mediate aspects of the su-perior functional capabilities of 2° effectors. Further studies willbe needed to evaluate how each of these pathways affects thegeneration, migration, and function of CD4+ T-cell effectors.Despite these indications that multiple genes contribute to the

more potent 2° effector response, we found that blocking NKG2Aon 2° effectors decreased the number of cells that secrete bothTNF and IFN-γ, but the same treatment had little effect on 1°effector responses. Similarly, blockade of Lag3 specifically en-hanced cytokine production by 1° effectors but not by 2° effectors.Although these studies cannot formally rule out indirect effects oftreatment through other cellular populations expressing NKG2Aor Lag3 on CD4+ T-cell function, the comprehensive analysispresented here provides insights into the differential regulation 1°and 2° effector responses and provides a compelling integratedand unbiased picture of organ-specific differences in T-cell ef-fector function. We suggest that the differences between 1° and 2°effectors identified here bear further investigation to determinewhich genes are most important for the superior protection me-diated by memory CD4+ T cells. Defining the functionally rele-vant molecules and pathways will allow the definition ofmechanisms that contribute to the superior efficacy of T-cellmemory. In addition, the genes we identified as being differentiallyregulated in 1° and 2° effectors are good candidates for targets thatmight be manipulated to increase the potency of T-cell effectors inIAV and also in other pathogen and therapeutic settings.Our studies also provide an integrated and unbiased snapshot

of organ-specific differences in T-cell effector function that de-serve further analysis. How this specialization is achieved is notyet known. It will be interesting to investigate whether some ofthe determination occurs because of organ-specific micro-environments, cells, and factors and how much is a result of theselective recruitment of predestined subsets that develop in thesame peripheral location.Often, the most important contribution from memory CD4+

T cells during recall challenge is the provision of help, leading toaccelerated B-cell and CD8+ T-cell responses (42). However,under certain circumstances, such as those presented here, thecritical protective contribution from memory CD4+ T cellsresults from the action of 2° effectors. Thus, the further defini-tion of the protective contributions from 2° effectors and thepathways responsible for their efficacy could provide importantnew correlates of vaccine-induced protection against importanthuman pathogens.

Materials and MethodsMice. Naive CD4+ T cells were obtained from 5- to 8-wk-old HNT.Thy1.1/Thy1.2 mice on a BALB/c background recognizing amino acids 126–138(HNTNGVTAACSHE) of PR8 HA (43). Recipients of cell transfers were BALB/cor BALB/c.Thy1.1 mice that were at least 8 wk old. Mice were obtained fromthe breeding facility at Trudeau Institute or the University of MassachusettsMedical School. All experimental animal procedures were conducted in ac-cordance with the Trudeau Institute or the University of MassachusettsMedical School Animal Care and Use Committee guidelines.

Naive CD4+ T-Cell Isolation, Memory Generation, and Adoptive Transfer. NaiveCD4+ T cells were obtained from pooled spleen and lymph nodes as previouslydescribed (7). Resulting cells were routinely >97% Vβ8.3+ and expresseda characteristic naive phenotype (small size, CD62Lhi, CD44lo, and CD25lo). Insome experiments, CD4+ T cells were CFSE labeled, as previously described (44).

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TH1-polarized memory CD4+ T cells were generated in vitro as previouslydescribed (17). In vivo PR8-primed memory cells were generated and reiso-lated as described (19) by transferring naive HNT cells to nude hosts thatthen were infected with PR8 and allowed to recover at least 30 d beforereisolation of donor CD4+ T cells from SLO and lung. Similarly, polyclonalmemory CD4+ T cells were isolated from SLO and lungs of mice that hadbeen primed with PR8 at least 30 d previously.

All donor CD4+ T cells were adoptively transferred in 200 μL PBS by i.v.injection. In some experiments mice were treated i.p. with 1 mg of eitheranti–Thy1.2-depleting antibody (30-H12) (Bio X Cell) or with an isotypecontrol. In further experiments, mice were treated i.p. as indicated with 0.5mg of Rat IgG2a (20d5; eBioscience) directed against NKG2A/C/E or 0.5 mg ofRat IgG1 (C9B7W; Bio X Cell) or with the appropriate isotype controls.

Virus and Infections. PR8 virus was produced in the allantoic cavity of em-bryonated hen eggs from virus stocks originating at St. Jude Children’sHospital. Mice were infected intranasally under light isoflurane anesthesia(Webster Veterinary Supply) with stated doses of virus in 50 μL PBS (500EID50 = 0.1 LD50 and 10,000 EID50 = 2 LD50). Viral infection was performed onthe same day as cell transfer. Viral titer was assessed by PA copy number, andPR8-specific serum IgG titers were assessed as previously described (45).

Tissue Preparation, Flow Cytometry, and Microarray Data Analysis. Mice wereeuthanized at different time points after virus infection, and lungs were per-

fusedby injecting10mLofPBS into the left ventricleof theheart. Lungs, spleen,anddLNwereprepared into single-cell suspensionsbymechanical disruptionoforgans andpassage throughanylonmembrane.Cell suspensionswerewashed,resuspended in FACS buffer (PBS plus 0.5% BSA and 0.02% NaN3), and in-cubated on ice with 1 μg anti-FcR (2.4G2) followed by saturating concen-trations of fluorochrome-labeled antibodies. Further details can be found in SIMaterials and Methods. Intracellular staining for cytokine expression wasperformed as previously described (7). FACS analysis was performed usinga FACS Scan or LSR II (BD Biosciences) and FlowJo (Tree Star) software.

1° and 2° effectors that had undergone at least five divisions by day 7postinfection were sort-purified from the spleen, dLN, and lung of recipi-ents, and total mRNA was isolated (Qiagen) for microarray analysis. Furtherdetails can be found in SI Materials and Methods.

Statistical Analysis. Unpaired, two-tailed Student’s t tests, ∝ = 0.05, were usedto assess whether the means of two normally distributed groups differedsignificantly. The Welch correction was applied when variances were foundto differ. One-way ANOVA with Bonferroni’s multiple comparison posttestwas used to compare multiple means.

ACKNOWLEDGMENTS. We thank Drs. R. Dutton and A. Cooper for helpfuldiscussions. This work was supported by funds from National Institutes ofHealth Grants AI-46530 (to S.L.S. and L.M.B.), AI-076534 (to S.L.S.), and NS-061014 (to Cory Teuscher) and from the Trudeau Institute.

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