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 AC B D EF 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.