Memory CD4+ T-cell–mediated protection depends on PNAS PLUS secondary effectors that are distinct from and superior to primary effectors

Tara 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°) and against lethal challenge (8–10), and studies demonstrating memory memory cell-derived secondary (2°) CD4+ T-cell effectors contrib- CD4+ T-cell protection in mice deficient for CD8+ TandBcells ute to protective recall responses is unclear. Here, we compare suggest that an important helper-independent protective contribu- these effectors directly after influenza A virus infection. Both de- tion of memory CD4+ T cells may be mediated directly by the 2° velop with similar kinetics, but 2° effectors accumulate in greater effectors (11–13). In addition, IAV-specific 1° effector (7, 10) and number in the infected lung and are the critical component of memory (14, 15) CD4+ T cells isolated from the lung and from memory CD4+ T-cell–mediated protection against influenza A vi- secondary lymphoid organs (SLO) display distinct functional and rus, independent of earlier-acting memory-cell helper functions. phenotypic characteristics. Whether 2° effectors comprise a simi- Phenotypic, functional, and transcriptome analyses indicate that larly heterogeneous population is unknown. This issue is important, 2° effectors share organ-specific expression patterns with 1° effec- because recent studies show that lung-resident memory CD4+ tors but are more multifunctional, with more multicytokine (IFN- T cells provide greater protection against IAV than SLO-resident γ+/IL-2+/TNF+)-producing cells and contain follicular helper T-cell memory cells (16). Thus, a more comprehensive understanding of populations not only in the spleen and draining lymph nodes organ-specific heterogeneity within responding CD4+ T-cell pools but also in the lung. In addition, they express more CD127 and may provide clues about the critical attributes of the most protective IMMUNOLOGY NKG2A but less ICOS and Lag-3 than 1° effectors and express CD4+ T cells that could be generated by vaccination. higher levels of several genes associated with survival and migra- We find that although both populations develop and peak with tion. Targeting two differentially expressed molecules, NKG2A similar kinetics, the 2° effectors accumulate in greater numbers in and Lag-3, reveals differential regulation of 1° and 2° effector the lung, the primary site of infection. We show that the generation functions during pathogen challenge. of the 2° effectors is the critical component of protective immunity mediated by memory CD4+ T cells against IAV and that 2° cytokines | viral infection | immune regulation effectors are superior to 1° effectors in mediating viral clearance. We demonstrate that 2° effectors contain more cells producing nalyses of the mechanisms underlying memory CD4+ T-cell– TNF and/or IL-2 together with IFN-γ and fewer cells producing IL- Amediated protection have focused largely on their earlier 10 than do 1° effectors. In addition, we identify several phenotypic provision of help as compared with naive cells (1), although it markers that distinguish the two effector populations from + also is appreciated that highly activated secondary CD4 T-cell each another. (hereafter, 2°) effectors develop after the re-expansion of memory To define the differences between 1° and 2° effectors further, we populations (2). Studies also suggest that optimal protection pro- analyzed gene expression by microarray. The 1° and 2° effectors + vided by T helper type 1 (TH1)-like memory CD4 Tcellscor- recovered from both lung and SLO display a high degree of relates with the capacity to produce multiple cytokines, including shared, organ-specific specialization. However, 2° effectors are γ γ IFN- ,TNF,andIL-2,ratherthanIFN- alone (3). Whether 2° less compartmentalized, as evidenced by a wider distribution of effectors derived from protective memory CD4+ T cells retain this follicular helper T (TFH) cells. Furthermore, we identify a short phenotype, the extent to which 2° effectors contribute to the list of genes that distinguish 1° and 2° effectors and that could be protection mediated by memory CD4+ T cells, and whether and + involved in controlling the greater expansion and more pluripo- how 2° effectors differ from primary CD4 T-cell (hereafter, 1°) tent functions of 2° effectors. Finally, we demonstrate the specific effectors are unclear. + regulation of 1° or 2° effector cytokine production by blocking the Comparison of 1° and 2° CD4 T-cell effectors within mixed populations is difficult. The higher proportion of antigen-specific differentially expressed surface proteins NKG2A and Lag-3. These findings define pathways that explain, in part, the functional memory cells as compared with naive cells complicates quantitative + analysis, and the maintenance of very few antigen-specificcellsin superiority of the memory CD4 T-cell response. general (4) precludes rigorous analysis ex vivo of the effectors to which 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, phenotypic Author contributions: T.M.S., K.K.M., and S.L.S. designed research; T.M.S., K.K.M., and discrimination alone between highly activated effectors and cells Y.K. performed research; L.M.B. contributed new reagents/analytic tools; T.M.S. and that have divided only once or twice is not reliable, because effectors K.K.M. analyzed data; and T.M.S., K.K.M., and S.L.S. wrote the paper. responding in different organs can express different surface phe- The authors declare no conflict of interest. notypes (7). To overcome these obstacles, we transferred equal *This Direct Submission article had a prearranged editor. + numbers of naive and memory HNT TcR transgenic CD4 Tcells Data deposition: The data reported in this paper have been deposited in the Gene Ex- recognizing the A/PR8/34 (PR8) strain of influenza A virus (IAV) to pression Omnibus (GEO) database, www.ncbi.nlm.nih.gov/geo (accession no. GSE40230). unprimed hosts and then challenged with PR8 to compare directly 1T.M.S. and K.K.M. contributed equally to this work. the generation, function, and protective capacity of 1° and 2To whom correspondence may be addressed. E-mail: [email protected], kai. 2° effectors. [email protected], or [email protected]. IAV infection presents a compelling model for addressing these This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. questions. The transfer of 1° effectors to unprimed mice can protect 1073/pnas.1205894109/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1205894109 PNAS Early Edition | 1of10 Downloaded by guest on October 1, 2021 Results activation of memory cells, but 4 d postinfection (dpi) the expan- Generation of 1° and 2° Effectors in Vivo. To generate comparable sion of naive and memory donors was similar (Fig. 1B). All donors effectors from naive and memory precursors, we transferred equal reached similar peak numbers in SLO by 7 dpi, but those arising numbers of naive or memory HNT CD4+ T cells to Thy-disparate from memory precursors accumulated in greater number in the hosts and infected the hosts with PR8. We used both in vivo-primed lung (Fig. 1B). A similar pattern was observed when precursors and reisolated (in vivo PR8 memory) and in vitro-generated T 1 were reduced up to 100-fold, with in vivo- and in vitro-derived H memory cells giving rise to about five times and 10 times more cells memory cells, allowing the investigation of effectors arising from in the lung, respectively, than naive donors (Fig. 1C); this result heterogeneous memory cells resulting from in vivo priming and fi fi con rms that the kinetics and magnitude of expansion are in- from populations with de ned polarization (17). Upon transfer dependent of precursor frequency. to uninfected mice, similar numbers of naive and memory HNT + By 7 dpi, virtually all donor cells recovered from the lung were CD4 T cells were initially present in all organs analyzed and both effectors as defined by their having undergone five or more decayed with identical kinetics (Fig. S1), arguing against differences divisions based on loss of carboxyfluorescein succinimidyl ester in initial trafficking or in survival of donor cells after adoptive (CFSE), as were >80% of cells arising from naive and >90% of transfer contributing to the results reported here. cells arising from memory donors in SLO. We titrated the + Unlike naive cells, memory CD4 T cells are poised for rapid number of donor cells and found that at lower numbers trans- secretion of cytokines (18). In agreement with studies using in ferred (≤2 × 106) nearly all naive and memory donors developed vitro-generated memory cells (19), in vivo PR8 memory cells up- into effectors in SLO and lung (Fig. 1D). Together Fig. 1 C and regulated CD69 1–2 d earlier in the lung and draining lymph nodes D indicate that both naive and memory cells give rise to highly (dLN) than did naive cells (Fig. 1A), indicating a more rapid divided 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 with

500 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) CD69 expression 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 donor

cells. 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 asterisks represent 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 from mice 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-primed 7 mice and were labeled with CFSE. Then 1 × 10 cells were transferred to Thy-disparate hosts, and the hosts then were challenged with 500 EID50 PR8. (F)At 7 dpi, donors that had divided at least five times were enumerated. n = 5 mice per group. All error bars represent the SD.

2of10 | www.pnas.org/cgi/doi/10.1073/pnas.1205894109 Strutt et al. Downloaded by guest on October 1, 2021 input (Fig. 1C). It is notable that, despite the earlier activation of had equivalently enhanced germinal center B-cell formation (Fig. PNAS PLUS memory cells (Fig. 1A), the kinetics of development in response 2B) and PR8-specific IgG (Fig. 2C) at 8 dpi, indicating that de- to PR8 in SLO were similar in 1° and 2° effectors, but the pletion at 5 dpi did not effect helper function. However, the magnitude of the 2° effector response increased markedly in the recipients depleted of memory HNT cells did not survive lethal lung. This enhanced accumulation of 2° effectors in the lung was challenge (Fig. 2D), although they survived longer than the seen in all experiments. recipients of naive HNT cells (Fig. 2D). This result stresses the To determine whether polyclonal memory CD4+ T cells would necessity of late-acting events and also is consistent with some show a similar enhanced effector response in the lung, we trans- protective contribution from earlier-acting memory cell functions ferred CFSE-labeled polyclonal CD4+ T cells from unprimed or (19). These results suggest that 2° effectors are key contributors to PR8-primed mice (enriched for PR8 memory) to new hosts and memory CD4+ T-cell–mediated protection against IAV. infected the hosts with PR8 (Fig. 1E). At 7 dpi, effectors were enumerated by gating on donors that had divided five times or Secondary Effectors Are Superior to 1° Effectors in Mediating Protection more. Effectors derived from donors containing memory cells Against IAV. The protection provided by 2° but not 1° effectors reached levels about fourfold higher in SLO, likely reflecting the described above could be caused largely by the differences in the increased proportion of precursor IAV-specific cells (Fig. 1F). magnitude of the response, as suggested by studies correlating Strikingly, cells from primed animals gave rise to more than 20-fold increased protection against IAV with increasing numbers of more effectors in the lung, about five times higher than the ratio in transferred 1° HNT effectors (8). Alternatively, 2° effectors may SLO (Fig. 1F). This result is consistent with observations using mediate superior protection through functional differences that equal numbers of naive and memory monoclonal CD4+ T cells distinguish them from 1° effectors. To establish that 2° effectors showing that the response in the lung is five- to 10-fold higher for 2° indeed are capable of enhanced protection as compared with 1° effectors than for 1° effectors. The enhanced ability of memory effectors and to investigate the possible reasons for the differ- CD4+ T cells to give rise to effectors in the lung following IAV ence, we isolated effectors from recipients of naive or memory infection suggests that this feature of memory may contribute to HNT cells on 7 dpi that had been infected with PR8. We trans- enhanced protection. ferred equal numbers of isolated 1° or 2° effectors directly to unprimed hosts and challenged the hosts with a lethal dose of Secondary Effectors Are Critical for Memory CD4+ T-Cell Protection. PR8 (Fig. 3A) against which 5 × 106 in vitro-generated 1° HNT We next analyzed the protective efficacy of the 2° effectors. effectors are required to protect unprimed hosts and to promote Memory CD4+ T cells regulate innate immunity during the initial enhanced viral clearance at 4 dpi (8). Consistent with previous 6 days of IAV infection (19) and provide help for the enhanced studies, 5 × 10 1° or 2° in vivo-generated effectors rescued hosts IMMUNOLOGY antibody production that is evident by 7 dpi (20). We reasoned that (Fig. 3B). However, when 2.5 × 106 cells were transferred, only 2° these functions would occur by 5 dpi, but, as described above, we effectors provided protection and enhanced viral control (Fig. 3 B found that 2° effectors enter the lung only at 6 dpi and peak at 7–8 and C). These results demonstrate that, in addition to their en- dpi (Fig. 1). Therefore we transferred 5 × 106 memory cells [the hanced representation in the lung, 2° effectors are superior to 1° number of memory cells found to protect reliably against lethal effectors in mediating protection as evaluated per cell input. PR8 challenge (12)] or an equal number of control naive cells to The previous experiments utilized effectors pooled from lung and unprimed Thy-disparate hosts and then selectively depleted SLO. Recent studies suggest that CD4+ T cells isolated from the donors at 5 dpi, leaving earlier functions of memory cells intact lung provide improved protection compared with those in SLO (16). but depleting 2° effectors before they finished differentiating and As the pooled 2° effectors contain proportionally more cells isolated trafficked to the lung (Fig. 2A). Treatment led to efficient removal from the lung compared with 1° effectors (see Fig. 1B), we trans- of donor cells (Fig. 2A). Mice receiving memory cells but not naive ferred 2.5 × 106 1° or 2° effectors isolated only from the lung to HNT cells and treated with control or Thy1.2-depleting antibody unprimed 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 transferred

to 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 antibody on 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 shown for one of two independent experiments. All error bars represent the SD.

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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 were

transferred 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 reisolated from 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 were 6 infected with 10,000 EID50 PR8. (B) Survival (n = 10 per group) and (C) viral titers from mice receiving no cells or 2.5 × 10 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.

We again observed that 2° effector protection was significantly en- We next infected mice with 50, 500, or 5,000 egg infectious doses hanced compared with 1° effector protection (Fig. 3D), although 1° (EID50) PR8 to determine whether the magnitude of infection effectors from the lung provided far greater protection than the would impact effector cytokine production. No differences in viral pooled 1° effectors (compare Fig. 3 D and B). Perhaps surprisingly, burden were observed in mice initially receiving 2 × 106 naive or equivalent numbers of 1° and 2° effectors were observed in the lung memory cells and infected with the same dose of PR8. Pulmonary at 4 dpi (Fig. 3E), and both populations exhibited a highly activated titers at 7 dpi were proportional to the challenge dose (Fig. 4D), phenotype (Fig. 3F). This result suggests that 2° effectors have consistent with previous studies (21). Interestingly, the proportion enhanced per cell function as compared with 1° effectors respond- of IL-2+IFN-γ+ cells did decrease significantly with increasing ing in the same organ. challenge dose (Fig. 4E). This was most notable for 2° effectors in the dLN, where the frequency of IL-2+IFN-γ+ cells detected at fi Cytokine Pro les of 1° and 2° Effectors. Many T-cell effector functions 50 > 500 > 5,000 EID50 challenge (P < 0.005 and 0.05, re- are mediated by cytokines made after TcR triggering. To examine spectively). However, regardless of the challenge dose, 2° effectors whether 1° and 2° effectors make different patterns or levels of contained more IL-2+IFN-γ+ cells than did 1° effectors. We ana- cytokines, we used intracellular cytokine staining (ICCS) to assess lyzed cytokine production throughout the peak effector phase (6–8 the coproduction of the dominant cytokines seen in the IAV re- dpi) to rule out the possibility that the difference was only transient sponse, namely, IL-2, IFN-γ, and TNF. The 2° effectors derived or that kinetic patterns in cytokine production differed in 1° and 2° from both in vitro and in vivo memory cells and recovered from effectors. We observed similar IFN-γ+IL2+ and IFN-γ+TNF+ cells either SLO or lung at 7 dpi contained higher proportions of throughout (Fig. 4F). Thus, 2° effectors consistently contain IL-2+IFN-γ+ (Fig. 4A), TNF+IFN-γ+,andIL-2+IFN-γ+TNF+ a higher proportion of double and triple cytokine producing cells. (Fig. S2) cells than did 1° effectors. Dilution of precursors up to To evaluate other key cytokines produced by T cells during 100-fold did not alter the proportion of IL-2+IFN-γ+ 1° or 2° pathogen challenge, we next assessed IL-10 and IL-17. We have effectors substantially (Fig. 4B). Higher frequencies of IL- shown that IL-10 production by 1° CD4+ T-cell effectors can 2+IFN-γ+ donors were also detected in effectors derived from impede protection against IAV, whereas IL-17+ T cells can polyclonal CD4 T cells from PR8-primed (enriched for 2° contribute to viral clearance (9, 22). We concentrated on lung- effectors) versus from naive mice (Fig. 4C), for which loss of resident effectors, because the expression of both IL-10 and IL- CFSE was used to gate IAV-specificeffectors(Fig. S3). These 17 from CD4+ T cells is restricted largely to the lung during IAV observations imply that enhanced multicytokine production is infection, with production of both IL-10 and IL-17 peaking at a general feature of 2° effectors. 7 dpi (9). A significantly lower fraction of 2° effectors, generated

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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 per group. (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-resident effectors. 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 surface markers. 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.

from either in vitro or in vivo memory cells, produced IL-10 (Fig. tinguished 1° from 2° effectors within the organs (Fig. 4H and Fig. 4G), consistent with the lack of IL-10 observed in our earlier S4A). The 2° effectors expressed increased CD127 (IL-7Rα)[a studies of memory CD4+ T-cell responses against IAV (9). A marker that is up-regulated on memory as compared with naive reciprocal pattern was found for IL-17 production. The 2° precursors (17) and that is necessary for CD4+ T-cell survival] effectors generated from in vivo PR8-primed memory cells and higher levels of NKG2A/C/E [a marker that has been asso- produced IL-17. However, virtually no IL-17 was detected from ciated with enhanced T-cell proliferative capacity and cytokine 1° effectors or from 2° effectors generated from in vitro TH1 production (23, 24)]. In contrast, ICOS, whose higher expression memory cells (Fig. 4G), suggesting that the TH1-polarizing con- has been linked with IL-10 production (25), was decreased on 2° ditions in vitro suppress subsequent TH17 differentiation. To- effectors in the lung, correlating well with enhanced IL-10 pro- gether, as is consistent with the superior protective capacity of 2° duction by 1° effectors. The 1° effectors found in the lung also effectors, these results indicate that 2° effectors are more capable expressed more of the inhibitory receptor Lag-3. These pheno- of producing a set of cytokines implicated in viral protection and typic distinctions were not affected by challenge dose or by produce less of the inhibitory cytokine IL-10. whether the 2° effectors were derived from in vitro- or in vivo- generated memory cells (Fig. S4 B and C). These shifts in the Phenotypic Profiles of 1° and 2° Effectors. We next screened effec- expression of phenotypic markers that have been associated with tors for expression of a broad panel of surface proteins to see if function provide additional clues about which cellular pathways we could identify any reproducible differences between 1° and 2° may be regulated differentially in 1° and 2° effectors subsets; effectors. Although cells in the lung and in the SLO differentially however, the changes in levels are sufficiently modest that they expressed many markers associated with functional potential, alone are not likely to be useful for definitively distinguishing 1° consistent with previous studies (7), only a few key markers dis- from 2° effectors in mixed populations in vivo.

Strutt et al. PNAS Early Edition | 5of10 Downloaded by guest on October 1, 2021 fi Organ-Speci c Gene-Expression Analysis of 1° and 2° Effectors. To analyzed 1° and 2° effectors at 7 dpi for TFH cells by FACS-based assess further the magnitude of differences between 1° and 2° expression of CXCR5, PD-1, and Bcl-6 (Fig. 5C). We used in effectors and to look for unexpected genes that might be expressed vitro TH1 memory precursors to rule out the possibility that in differentially, we compared transcriptomes using whole-genome vivo-primed PR8 memory cells used for microarray analysis microarrays. Effectors in the lung express a distinct and apparently contained preexisting TFH cells that could account for the dif- more highly differentiated phenotype than those in SLO, suggesting fering patterns of TFH gene expression. Both 1° and 2° effectors that organ-specific differences could obscure a global analysis. contained phenotypically defined TFH cells in SLO, but only 2° Therefore we separately analyzed effectors isolated from the fi fi effectors contained signi cant TFH cells in the lung (Fig. 5 D and spleen, dLN, and lung. We sort-puri ed 1° and 2° effectors from E). Similarly, we observed T in polyclonal 2° effector pop- recipients of naive or in vivo PR8-primed memory HNT cells, re- FH ulations in the lung during heterosubtypic challenge, but no TFH spectively (Fig. S5). As predicted, and as is consistent with pheno- were observed in 1° polyclonal lung effectors (Fig. S6). Thus, typic and functional distinctions among organs, a number of genes although 1° and 2° effectors in different organs express tran- that were shared by 1° and 2° effectors were expressed differentially scriptomes indicative of organ-specific heterogeneity, 2° effectors among organs. For example, 1° and 2° effectors recovered from the lung shared about 200 genes that were differentially expressed in express unique gene patterns that, together with enhanced effectors in SLO (Fig. 5A and Dataset S1). This cohort of shared multicytokine production, indicate broader and less-localized lung-enriched effector genes is enriched for immune-response, cy- functional capacity. tokine, chemokine, and defense-response pathways, as shown by Database for Annotation, Visualization and Integrated Discovery Primary vs. 2° Effector Gene Expression. We next focused on the genes (DAVID) analysis (Fig. 5A), suggesting that 1° and 2° effectors in that were expressed differentially in 1° and 2° effectors. A heatmap the lung share a spectrum of functions (likely including direct an- summarizing these genes by ANOVA analysis is shown in Fig. 6A. tiviral effector functions) and that 1° and 2° effectors in SLO have Pair-wise comparisons of 1° and 2° effector transcriptomes within other discrete functions (e.g., helper activities). each organ revealed differential expression of only a modest To test this hypothesis, we analyzed whether SLO-resident number of genes (about 150). Strikingly, very few of the genes effectors were enriched for TFH-associated genes compared with expressed differentially between 1° and 2° effectors were common lung effectors. Interestingly, only 1° effectors fit this pattern (Fig. between the spleen, dLN, and lung, and no genes were differen- 5B), suggesting either that 2° effectors in SLO do not express tially regulated in all three organs (Fig. 6B), again indicating a high fi these TFH-associated genes or that 2° effectors in all organs ex- level of organ-speci c specialization. When subjected to DAVID press TFH signature genes. To test these two possibilities, we analysis, 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 prepared for 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 the

dLN 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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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 per group. (B) Venn diagram of the number of genes differentially expressed in 1° and 2° effectors within spleen, dLN, and lung and differentially expressed genes 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 bars represent the SD.

between 1° and 2° effectors fall into functional annotation path- mirroring surface expression of NKG2A/C/E (Fig. 4G). We ways such as regulation of transcription, metabolism, cell growth, confirmed higher expression of Klrc1 in 2° effectors by RT-PCR and migration (Fig. 6C). In Dataset S2, we show the genes that (Fig. 6E) but found equal expression of Klrc2-3, encoding define the greatest differences between 1° and 2° effectors. A co- NKG2C/E, in both effector populations (Fig. 6E), indicating that hort of some of the most compelling of these genes and their increased NKG2A/C/E staining on 2° effectors is caused largely functional pathways are shown in Fig. 6D. Their differential ex- by changes in the expression of NKG2A. pression was validated by RT-PCR (Fig. 6E). These genes could Administration of blocking antibody against NKG2A/C/E (20d5) provide clues to the mechanisms responsible for the unique phe- on 4–6 dpi to mice that had received memory HNT cells (Fig. 7A) notype and function of 2° effectors responding to IAV (see below resulted in efficient in vivo blockade at 7 dpi as compared with and Discussion). administration of an isotype control (Fig. 7B). At 7 dpi, donor cells were enumerated and analyzed for cytokine production by ICCS. Differential Regulation of Function in 1° and 2° Effectors. To evalu- To help rule out an indirect effect of 20d5 treatment on CD4+ ate whether some of the distinctions identified above correlate T-cell responses through the regulation of other cell types with functions that are likely to be relevant in protective efficacy, expressing NKG2A, we also analyzed 1° effector responses from we tested whether the multicytokine production patterns of 1° recipients of naive HNT cells, which express reduced levels of and 2° effectors could be modified by targeting two differentially NKG2A (Fig. 6E), that were treated with 20d5 or isotype anti- expressed molecules for which blocking antibodies are available. body. The absolute numbers of 1° or 2° effectors present in the We first tested whether blocking a target preferentially expressed dLN and lung was not affected by 20d5 treatment (Fig. 7C). by 2° effectors would alter their cytokine production but not that However, treatment did reduce the frequency and number of of 1° effectors. We chose Klrc1, encoding NKG2A, because its TNF+IFN-γ+ 2° effectors in both the dLN and lung (Fig. 7D)and expression was up-regulated by 2° effectors in the lung and dLN, significantly reduced the mean fluorescence intensity (MFI) of

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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 as depicted (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 cells as 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 staining of dLN 1° effectors. n = 5 per group. Results are shown for one of two independent experiments. All error bars represent the SD.

TNF in 2° effectors (Fig. 7 E and F). Treatment had no effect on were not affected by treatment (Fig. 7 I and J). These results confirm IL-2 production by 2° effectors (Fig. 7E). Importantly, 20d5 the functional relevance of these two molecules identified in our treatment had no effect on cytokine production by 1° effectors analyses and suggest that 1° and 2° effector responses are regulated (Fig. 7D); this observation supports a selective role for NKG2A differently. Not surprisingly, these treatments show only partial signaling in regulating 2° but not 1° effector function in vivo. effects on modulating functional potential, suggesting that the en- We used the same approach to investigate if targeting a molecule hanced properties of 2° effectors also are regulated by changes in preferentially expressed by 1° effectors could affect their function genes other than NKG2A and Lag3. selectively. We focused on Lag3 because of its role in modulating T-cell responses in vivo (26). Although Lag3 expression was higher Discussion on 1° than on 2° effectors, as determined by FACS (Fig. 4G), it was Overall, these studies lead to three striking conclusions. First, 2° not expressed differentially in our microarray analysis. This result CD4+ T-cell effectors are distinct from and are functionally su- might reflect differential posttranscriptional regulation in 1° and 2° perior to 1° effectors and mediate better protection against lethal effectors, an hypothesis supported by the intracellular stores of IAV infection. The 2° effector response was temporally separable Lag3 in activated CD4+ T cells and by its rapid and tightly con- from helper activities mediated by memory cells, occurring after trolled surface translocation (27). 5 dpi, when help already had been delivered. The loss of pro- Mice receiving naive or memory HNT cells were treated at 4–6 tection observed upon depletion of transferred memory CD4+ dpi with Lag3-blocking antibody (C9B7W) (Fig. 7G), resulting in T cells, even though help was unchanged, suggests that the en- efficient blockade at 7 dpi (Fig. 7H). Strikingly, C9B7W-treated hanced abilities of 2° effectors as compared with 1° effectors are mice contained higher absolute numbers of 1° effectors at 7 dpi (Fig. a major component of the protection conferred by memory CD4+ 7I), and roughly twofold higher numbers of IL-2+IFN-γ+ (Fig. 7 J T cells. Our results thus provide a basis for understanding the and K) cells in the lung and dLN and a similar increase in protection mediated by memory CD4+ T cells in B-cell–deficient, TNF+IFN-γ+ cells (Fig. 7 J and K). In contrast, 2° effector responses CD8+ T-cell–deficient, and even lymphocyte-deficient hosts

8of10 | www.pnas.org/cgi/doi/10.1073/pnas.1205894109 Strutt et al. Downloaded by guest on October 1, 2021 challenged with IAV (11, 12). Second, we find that effectors in the (32) and dampening of cellular proliferation (lrig3) (33), as is PNAS PLUS spleen, dLN, and lung are strikingly different from one another, consistent with the increased magnitude of 2° effector responses. suggesting that they are specialized to perform unique functions at Similarly, the higher expression in 1° effectors of genes such as different sites. We also find that 2° effectors are more multi- satb2, a negative regulator of CD127 expression (34), and functional, regardless of location, as evidenced by their enhanced pcdh10, which can potentiate apoptosis (35), also is consistent production of multiple inflammatory cytokines and their wider with increased numbers of 2° effectors at the site of infection. In distribution of TFH cells, which are seen in the lung in the 2° but addition, 2° effectors express higher levels of gpr12, gpr63, rgs3, not the 1° effector response. This finding indicates that CD4+ T and tiam1, all of which are associated with chemotaxis and mi- cells responding to pathogen challenge follow multiple, separate gration (36–38) and also might be involved in the greater accu- developmental patterns and raises the possibility that unique cues mulation of 2° effectors in the lung. Other genes, such as eif4gI [a in distinct environments might coordinate effector specialization. translation initiation factor (39)], araf [a potential modulator of Third, we demonstrate distinct regulation of 1° and 2° effector TcR signaling (40)], and rab18 [involved in the ER secretory function in vivo. Our results underscore the unique character of 2° pathway (41)] also could regulate or mediate aspects of the su- effectors but also suggest that the regulation of the enhanced perior functional capabilities of 2° effectors. Further studies will function of 2° effectors is complex, involving multiple pathways. be needed to evaluate how each of these pathways affects the It perhaps is unexpected that the array of cytokines produced by generation, migration, and function of CD4+ T-cell effectors. individual CD4+ T cells broadens instead of becoming more spe- Despite these indications that multiple genes contribute to the cialized with further exposure to antigen and with division. We had more potent 2° effector response, we found that blocking NKG2A considered the possibility that 2° effectors might be more uniform on 2° effectors decreased the number of cells that secrete both among organs, because the process of epigenetic remodeling is TNF and IFN-γ, but the same treatment had little effect on 1° thought to change future expression by increasing the ease with effector responses. Similarly, blockade of Lag3 specifically en- which certain cytokines are expressed and by silencing others (28). hanced cytokine production by 1° effectors but not by 2° effectors. Instead, in agreement with studies addressing gene expression in Although these studies cannot formally rule out indirect effects of repeatedly stimulated memory T cells (29), our results suggest that treatment through other cellular populations expressing NKG2A + even well-polarized TH1 memory cells making a restricted cytokine or Lag3 on CD4 T-cell function, the comprehensive analysis profile (30) can differentiate further to make multiple cytokines at presented here provides insights into the differential regulation 1° higher levels. These findings suggest that the transition to the and 2° effector responses and provides a compelling integrated resting memory state may reset certain aspects of a CD4+ T cell’s and unbiased picture of organ-specific differences in T-cell ef-

response potential, a hypothesis supported by the nearly identical fector function. We suggest that the differences between 1° and 2° IMMUNOLOGY gene expression in naive and memory cells (17). effectors identified here bear further investigation to determine which genes are most important for the superior protection me- Our analysis of 1° and 2° effectors recovered from different + organs revealed striking similarities, as well as differences, in diated by memory CD4 Tcells.Defining the functionally rele- their transcriptomes. For example, 1° and 2° effectors isolated vant molecules and pathways will allow the definition of from the lung both expressed high levels of genes associated with mechanisms that contribute to the superior efficacy of T-cell antiviral responses, such as IFN-γ, and numerous chemokines memory. In addition, the genes we identified as being differentially and chemokine receptors. Our data are consistent with the hy- regulated in 1° and 2° effectors are good candidates for targets that pothesis that effectors in the lung are important for protection might be manipulated to increase the potency of T-cell effectors in against IAV through direct mediation of viral clearance. How IAV and also in other pathogen and therapeutic settings. CD4+ T-cell effectors combat IAV is not fully understood, but Our studies also provide an integrated and unbiased snapshot fi our studies suggest that individual protective mechanisms, in- of organ-speci c differences in T-cell effector function that de- cluding perforin-dependent killing of infected cells and pro- serve further analysis. How this specialization is achieved is not duction of IFN-γ, become more or less important depending on yet known. It will be interesting to investigate whether some of fi the context of infection (8, 12). In contrast, the expression of the determination occurs because of organ-speci c micro- environments, cells, and factors and how much is a result of the TFH-associated genes in 1° effectors was restricted to SLO, but 2° effectors contained substantial T populations in all organs selective recruitment of predestined subsets that develop in the FH same peripheral location. tested. It is interesting to speculate that TFH activity from 2° + effectors in the lung during recall responses against IAV might Often, the most important contribution from memory CD4 fi T cells during recall challenge is the provision of help, leading to be important in providing ef cient help for local B-cell antibody + production when inducible bronchus-associated lymphoid tissue accelerated B-cell and CD8 T-cell responses (42). However, under certain circumstances, such as those presented here, the is present (31). The dramatic distinctions seen at the gene-ex- + pression level among effectors in lung, spleen, and dLN suggest critical protective contribution from memory CD4 T cells results from the action of 2° effectors. Thus, the further defini- that the distribution of the TFH subset in 1° effectors likely fi tion of the protective contributions from 2° effectors and the represents only one set of organ-speci c distinctions and that fi other subsets also might be found preferentially in different sites. pathways responsible for their ef cacy could provide important Indeed we find that CD4+ T cells with cytotoxic activity gener- new correlates of vaccine-induced protection against important ated by IAV are restricted almost exclusively to the lung (10). human pathogens. Thus, correlates of protective T cells may differ in different Materials and Methods organs as a result of the simultaneous generation of effector + subsets specialized for different roles at different sites. Moreover Mice. Naive CD4 T cells were obtained from 5- to 8-wk-old HNT.Thy1.1/ – the depletion studies indicate that the helper functions occur Thy1.2 mice on a BALB/c background recognizing amino acids 126 138 earlier in SLO, whereas the peak of effector responses, which (HNTNGVTAACSHE) of PR8 HA (43). Recipients of cell transfers were BALB/c or BALB/c.Thy1.1 mice that were at least 8 wk old. Mice were obtained from correlates with viral clearance, occurs later in the lung. Thus, the breeding facility at Trudeau Institute or the University of Massachusetts a temporal distinction also must be considered in identifying Medical School. All experimental animal procedures were conducted in ac- correlates of protection. fi cordance with the Trudeau Institute or the University of Massachusetts Perhaps it is not surprising that we have identi ed only a rel- Medical School Animal Care and Use Committee guidelines. atively small number of genes, about 450, that are expressed differentially in 1° and 2° effectors. This cohort of genes includes Naive CD4+ T-Cell Isolation, Memory Generation, and Adoptive Transfer. Naive many that are involved in regulating apoptosis, signaling path- CD4+ T cells were obtained from pooled spleen and lymph nodes as previously ways, translation, cell migration, chemokine signaling, and me- described (7). Resulting cells were routinely >97% Vβ8.3+ and expressed tabolism. For example, 2° effectors expressed lower levels of a characteristic naive phenotype (small size, CD62Lhi, CD44lo, and CD25lo). In genes associated with the suppression of DNA replication (setd8) some experiments, CD4+ T cells were CFSE labeled, as previously described (44).

Strutt et al. PNAS Early Edition | 9of10 Downloaded by guest on October 1, 2021 + TH1-polarized memory CD4 T cells were generated in vitro as previously fused by injecting 10 mL of PBS into the left ventricle of the heart. Lungs, spleen, described (17). In vivo PR8-primed memory cells were generated and reiso- and dLN were prepared into single-cell suspensions by mechanical disruption of lated as described (19) by transferring naive HNT cells to nude hosts that organs and passage through a nylon membrane. Cell suspensions were washed,

then were infected with PR8 and allowed to recover at least 30 d before resuspended in FACS buffer (PBS plus 0.5% BSA and 0.02% NaN3), and in- reisolation of donor CD4+ T cells from SLO and lung. Similarly, polyclonal cubated on ice with 1 μg anti-FcR (2.4G2) followed by saturating concen- memory CD4+ T cells were isolated from SLO and lungs of mice that had trations of fluorochrome-labeled antibodies. Further details can be found in SI been primed with PR8 at least 30 d previously. Materials and Methods. Intracellular staining for cytokine expression was All donor CD4+ T cells were adoptively transferred in 200 μL PBS by i.v. performed as previously described (7). FACS analysis was performed using injection. In some experiments mice were treated i.p. with 1 mg of either a FACS Scan or LSR II (BD Biosciences) and FlowJo (Tree Star) software. – anti Thy1.2-depleting antibody (30-H12) (Bio X Cell) or with an isotype 1° and 2° effectors that had undergone at least five divisions by day 7 control. In further experiments, mice were treated i.p. as indicated with 0.5 postinfection were sort-purified from the spleen, dLN, and lung of recipi- mg of Rat IgG2a (20d5; eBioscience) directed against NKG2A/C/E or 0.5 mg of ents, and total mRNA was isolated (Qiagen) for microarray analysis. Further Rat IgG1 (C9B7W; Bio X Cell) or with the appropriate isotype controls. details can be found in SI Materials and Methods.

Virus and Infections. PR8 virus was produced in the allantoic cavity of em- Statistical Analysis. Unpaired, two-tailed Student’s t tests, ∝ = 0.05, were used ’ bryonated hen eggs from virus stocks originating at St. Jude Children s to assess whether the means of two normally distributed groups differed fl Hospital. Mice were infected intranasally under light iso urane anesthesia significantly. The Welch correction was applied when variances were found μ (Webster Veterinary Supply) with stated doses of virus in 50 L PBS (500 to differ. One-way ANOVA with Bonferroni’s multiple comparison posttest EID50 = 0.1 LD50 and 10,000 EID50 =2LD50). Viral infection was performed on was used to compare multiple means. the same day as cell transfer. Viral titer was assessed by PA copy number, and PR8-specific serum IgG titers were assessed as previously described (45). ACKNOWLEDGMENTS. We thank Drs. R. Dutton and A. Cooper for helpful discussions. This work was supported by funds from National Institutes of Tissue Preparation, Flow Cytometry, and Microarray Data Analysis. Mice were Health Grants AI-46530 (to S.L.S. and L.M.B.), AI-076534 (to S.L.S.), and NS- euthanized at different time points after virus infection, and lungs were per- 061014 (to Cory Teuscher) and from the Trudeau Institute.

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