NKG2C/E Marks the Unique Cytotoxic CD4 T Cell Subset, ThCTL, Generated by Influenza Infection

Nikki B. Marshall, Allen M. Vong, Priyadharshini Devarajan, Matthew D. Brauner, Yi Kuang, Ribhu Nayar, Elizabeth A. Schutten, Catherine H. Castonguay, Leslie J. Berg, Stephen L. Nutt and Susan L. Swain
J Immunol February 1, 2017, 198 (3) 1142-1155; DOI: https://doi.org/10.4049/jimmunol.1601297

Abstract

CD4 T cells can differentiate into multiple effector subsets, including ThCTL that mediate MHC class II–restricted cytotoxicity. Although CD4 T cell–mediated cytotoxicity has been reported in multiple viral infections, their characteristics and the factors regulating their generation are unclear, in part due to a lack of a signature marker. We show in this article that, in mice, NKG2C/E identifies the ThCTL that develop in the lung during influenza A virus infection. ThCTL express the NKG2X/CD94 complex, in particular the NKG2C/E isoforms. NKG2C/E+ ThCTL are part of the lung CD4 effector population, and they mediate influenza A virus–specific cytotoxic activity. The phenotype of NKG2C/E+ ThCTL indicates they are highly activated effectors expressing high levels of binding to P-selectin, T-bet, and Blimp-1, and that more of them secrete IFN-γ and readily degranulate than non-ThCTL. ThCTL also express more cytotoxicity-associated genes including perforin and granzymes, and fewer genes associated with recirculation and memory. They are found only at the site of infection and not in other peripheral sites. These data suggest ThCTL are marked by the expression of NKG2C/E and represent a unique CD4 effector population specialized for cytotoxicity.

This article is featured in In This Issue, p.977

Introduction

Activated CD4 T cells have the potential to differentiate into unique effector subsets tailored to respond to various pathogens. During viral infection, CD4 effectors can become specialized to help Ab responses, to secrete effector cytokines, and to mediate inflammation. These distinct activities are mediated by functionally and phenotypically distinct subsets that develop simultaneously, but in distinct sites and whose generation requires unique instructive signals from distinct APCs (1) and the microenvironment (2–4). We have studied the cytotoxic subset of CD4 T cells, which we call ThCTL (5), that are found in the lung after influenza A virus (IAV) infection. Although much is understood about how naive CD4 T cells differentiate into Th1 (6), T follicular helper (TFH) (7, 8), and Th17 (9), less is known about how ThCTL are generated and how they become restricted to what seems to be sites of infection.

ThCTL are found in response to multiple viral infections including lymphocytic choriomeningitis virus (LCMV) (10), poxvirus (11), γ-herpesvirus-68 (12), CMV (13), as well as IAV (14, 15). After intranasal IAV infection in mice, they are found in the lungs but are absent from secondary lymphoid organs (SLOs) (14), whereas in infections during which virus replicates in other sites, cytotoxic CD4 are reported in those sites (11). Our work and that of others have shown ThCTL generated against IAV infection in mice can lyse infected cells through a perforin-dependent mechanism likely using granular exocytosis (14, 16). They can protect against IAV infection on their own and in synergy with anti-IAV Ab to combat a lethal challenge of IAV (14, 15, 17). Monoclonal ThCTL effectively kill enough infected targets to drive the generation of IAV variants in vivo (17). In other studies, it has been shown that ThCTL correlate with better protection against IAV infection in humans (18) and predict better disease outcome in HIV (19). These potent functions highlight the importance of understanding how ThCTL are generated to design effective vaccines to best harness their potential.

ThCTL have unique requirements for generation. In vitro, generation of cytotoxic CD4 effectors requires IL-2, but no known polarizing cytokines such as IL-4, IL-12, IFN-γ, TGF-β, or IL-6 (20), indicating that a novel pathway regulates their generation. Recent studies show that activating CD137 (4-1BB) (21), CD134 (OX-40) (22), or both (23) can enhance the generation of cytotoxic CD4 T cells that mediate tumor rejection. Further work in an influenza model has demonstrated that ThCTL development and/or function depends on expression of the Blimp-1 transcriptional repressor and type I IFN (16), also consistent with an activated effector phenotype.

Viral infections are very effective at activating cytotoxic CD8 T cells (CTL) that play a key role in clearing virus by killing infected cells. However, viruses often employ evasion mechanisms that prevent CD8 CTL clearance, by downregulating MHC class II (MHC-II) expression and presentation of Ag (24). MHC-II–restricted cytotoxicity offers an alternate mechanism for host recognition and clearance of virally infected cells. Indeed, several groups have reported key roles for MHC-II–restricted cytotoxicity during infection with IAV (25), LCMV (26), Sendai virus (27), and γ-herpesvirus-68 (28). This supports the efficacy of ThCTL killing, especially given the fact that multiple arms of the immune system often work both independently and in synergy to promote clearance of IAV (17, 25, 29, 30) and other viruses (31).

Most studies have defined ThCTL by their ability to mediate MHC-II–restricted cytotoxicity or by their expression of high levels of granzyme B (GrB). Although cytotoxicity is the defining feature of ThCTL, the use of functional assays as a marker precludes the phenotyping and tracking of these cells. The serine protease GrB is commonly used to mark cytotoxic cells because among cytotoxic effectors, levels of expression of GrB correlate with cytotoxicity (20). However, noncytotoxic cells can also express GrB that mediates granular exocytosis for cytokine secretion (32); thus, it is not a reliable indicator of ThCTL. Another commonly used marker is CD107a (LAMP-1) that identifies cells that have recently degranulated. Identifying ThCTL by staining with CD107a requires stimulating cells in vitro before labeling the cells and then determining whether expression is related to cytotoxic function (33). Therefore, CD107a is not useful as a signature phenotypic marker. This lack of a cell surface marker to identify ThCTL has prevented the study of their phenotype and functions and of the pathways that regulate their development. Thus, a phenotypic surface marker that identifies unmanipulated ThCTL would greatly facilitate these further analyses.

In this article, we show that expression of NKG2A/C/E, collectively termed NKG2X (34), identified by Ab clone 20d5 (35), marks cytotoxic ThCTL in IAV-infected mice. We show that NKG2X+ effectors express high levels of Blimp-1, and that expression of this transcription factor is required for optimum CD4 effector differentiation to cytotoxic cells in the lung. However, ThCTL do not require NKG2X expression for their MHC-II–restricted cytotoxicity of conventional targets. ThCTL have a phenotype consistent with highly activated effector CD4 T cells, and we confirm their localization to the site of infection and show they are not found in other tissue sites. The ThCTL effectors are poised to secrete IFN-γ and to degranulate, and they express higher levels of multiple genes associated with increased cytotoxicity than other lung CD4 effectors, and lower levels of genes associated with memory, other CD4 subsets, and recirculation. Thus, we conclude that ThCTL represent a phenotypically and functionally unique cytotoxic subset of CD4 effectors generated at the site of acute viral infection.

Materials and Methods

Mice

BALB/cByJ, C57BL/6 (B6), and B6.PL-Thy1a/CyJ (B6.Thy1.1) mice were obtained from The Jackson Laboratory. Prdm1fl/fl mice were originally received from Dr. Alexander Tarakhovsky (The Rockefeller University, New York, NY) and were crossed with Cd4-cre+ (Blimp-1 conditional knockout [CKO]) (36). Prdm1gfp/+ (Blimp-1 GFP) knock-in mice were originally obtained from S.L.N. (The Walter and Eliza Hall Institute of Medical Research, VIC, Australia). Blimp-1 CKO OT-II cells were obtained by crossing Prdm1fl/fl Cd4-cre+ mice with OT-II TcR transgenic (Tg) mice. B6.OT-II.Thy1.1.Hcst−/−/Tyrobp−/− (OT-II.Thy1.1 DAP10/12 knockout [KO]) cells were obtained by crossing Hcst−/−/Tyrobp−/− mice (kindly provided by Dr. Toshiyuki Takai, Tohoku University, and Dr. Lewis Lanier, University of California, San Francisco) with OT-II.Thy1.1 TcR Tg mice. H2-t23−/− (Qa-1 KO) cells were kindly provided by Dr. Harvey Cantor (Dana Farber Cancer Institute). HNT mice express a TcR recognizing aa 126–138 of A/Puerto Rico/8/34 (PR8, H1N1) hemagglutinin (HA), and OT-II mice express a TcR recognizing aa 323–339 of chicken OVA. OT-II.Thy1.1 TcR Tg and HNT.Thy1.1 TcR Tg mice were obtained from the animal breeding facility at Trudeau Institute or University of Massachusetts Medical School (UMMS). SMARTA TcR Tg mice (kindly provided by Dr. Raymond Welsh, UMMS) express a TcR recognizing LCMV epitope gp61–80. All mice were at least 8 wk old at the time of infection. Naive CD4+ cells were obtained from 5- to 8-wk-old mice. Experimental animal procedures were conducted in accordance with the UMMS Animal Care and Use Committee guidelines.

Naive cell isolation

Naive T cells from TcR Tg mice were obtained from cell suspensions prepared from pooled spleen and lymph nodes as previously described (25). Resulting TcR Tg cells were routinely >97% TcR+ and expressed a characteristic naive phenotype (small size, CD62Lhi, CD44lo, and CD25lo). Purified CD4 cells (0.5–3 × 106) were adoptively transferred in 200 μl of PBS by i.v. injection and tracked by expression of congenic markers. Host mice were subsequently infected with virus.

Virus stocks and infections

Mouse-adapted influenza viruses A/Puerto Rico/8/34 (A/PR8; H1N1) originating from stocks at St. Jude Children’s Hospital and A/Puerto Rico/8/34-OVA323–339 (A/PR8-OVAII; H1N1) (kindly provided by Dr. Peter Doherty) were grown in the allantoic cavity of embryonated hen eggs at the Trudeau Institute. Mice were infected intranasally (i.n.) under light isoflurane anesthesia (Piramal Healthcare) with virus in 50 μl of PBS. Mice received a 0.3 LD50 dose of IAV. Mice that received adoptively transferred T cells were infected on the same day as cell transfer. For LCMV experiments, mice were infected with 5 × 104 PFU of LCMV Armstrong strain i.p.

Cell preparation from various organs or tissues

At time points indicated after virus infection, mice were euthanized and lungs, spleen, and draining mediastinal lymph node (dLN) were harvested and single-cell suspensions prepared by mechanical disruption of organs and passage through nylon mesh. For some experiments, mice were euthanized and bronchioalveolar lavage (BAL) was also isolated. Then peripheral blood was collected with cardiac puncture and mice were perfused with 10 ml of PBS, and the lungs, dLN, spleen, kidney, and liver were taken. The lungs, kidney, and liver were digested with collagenase P (Roche) and passed through a nylon membrane. Kidney and liver cell suspensions were layered over Percoll (GE Healthcare), and the interface between the 40 and 70% layers was collected.

Flow cytometry and sorting

Cell suspensions were washed, resuspended in FACS buffer (PBS + 0.5% BSA and 0.01% sodium azide; Sigma-Aldrich), and incubated on ice with 1 μg of anti-FcR (2.4G2) followed by incubation with combinations of fluorochrome-labeled Abs for surface staining: anti-CD4 (GK1.5, RM4-4, RM4-5), CD27 (LG.7F9), CD44 (IM7), CD90.1 (Thy1.1, OX-7, and HIS51), CD103 (2E7), CD150 (SLAM, TC15-12F12.2), CD152 (CTLA-4, UC10-4B9), CD160 (CNX46-3), CD183 (CXCR3, CXCR3-173), CD184 (CXCR4, 2B11), CD186 (CXCR6, SA051D1), CD191 (CCR1, 643854), CD192 (CCR2, SA203G11), CD194 (CCR4, 2G12), CD195 (CCR5, HM-CCR5), CD223 (Lag-3, C9B7W), CD244 (2B4, ebio244F4), CD272 (BTLA, 6F7), CD279 (PD-1, 29F.1A12), CD314 (NKG2D, CX5), CD335 (NKp46, 29A1.4), CD366 (Tim-3, 8B.2C12), CX3CR1 (SA011F11), KLRG1 (2F1), Ly-49A (A1), MHC-II (I-A/I-E, M5/114), NKG2A/C/E (20d5), NKG2A (16a11), NK1.1 (PK136). Abs were purchased from eBioscience, BioLegend, BD Biosciences, and R&D Systems. Dead cells were excluded using Live/Dead Fixable Amine Dye (Invitrogen). For P-selectin binding, cells were incubated with mouse P-selectin-IgG fusion protein (BD Biosciences), washed, and detected with fluorescent goat anti-human IgG (Jackson Immunoresearch) secondary Ab. For NKG2A and NKG2A/C/E costaining, cells were incubated with anti-NKG2A first, washed, and then incubated with anti-NKG2A/C/E to avoid steric blocking. For tetramer staining, cells were stained for 1 h at 37°C with fluorochrome-conjugated I-Ab–nucleoprotein (NP)311–325 tetramer obtained from the National Institutes of Health tetramer facility before surface marker staining.

For intracellular cytokine staining, donor lung CD4 T cells were isolated with CD90.1 MACS enrichment and were cultured for 4 h with peptide-pulsed activated B cells (LPS and dextran sulfate), brefeldin A (Sigma), monensin (BD GolgiStop), and anti-CD107a (LAMP-1, 1D4B; BioLegend). Cells were then surface stained and fixed for 20 min in 4% paraformaldehyde followed by permeabilization for 15 min by 0.1% saponin buffer (PBS plus 1% FBS, 0.1% NaN3, and 0.1% saponin; Sigma-Aldrich) and stained with anti–IFN-γ (XMG1.2; eBioscience) for 20 min. GrB (GB11; Thermo Fisher) expression was determined by intracellular staining directly ex vivo. For transcription factor staining, cells were fixed and permeabilized according to the manufacturer’s protocol (eBioscience) and then stained with labeled anti-Tbet (4B10), anti-Eomes (Dan11mag), anti-Foxp3 (FJK-16s), anti-Bcl6 (K112-91), anti-ROR(γ)t (B2D), anti–Gata-3 (TWAJ), and anti-ThPOK (2POK) (eBioscience and BD Biosciences).

All flow cytometry results were acquired using LSRII flow cytometers (BD Biosciences) and analyzed with FlowJo (Tree Star) analysis software.

For flow sorting, lungs were pooled and target cells were enriched by magnetic enrichment (MACS; Miltenyi Biotec) for CD90.1 according to the manufacturer’s protocol. Enriched cells were then stained to isolate NKG2A/C/E+ or NKG2A/C/E− of CD4+ CD90.1+ CD8a− NK1.1− I-Ab− cells using the FACSAria cell sorter (BD Biosciences). We routinely get 75–90% purity of NKG2A/C/E+ effectors and >95% purity for the NKG2A/C/E− effectors. For SMARTA effectors, we achieved >45% purity of NKG2A/C/E+ effectors and >90% purity of NKG2A/C/E− effectors.

Cytotoxic assays

For ex vivo cytotoxic assays, effector cells were isolated either through magnetic enrichment (MACS; Miltenyi Biotec) or by flow sorting from pooled lungs of IAV-infected mice. Effectors were preincubated with anti-CD178 (Fasl, MFL3; eBioscience). Target cells were generated 2 d prior by stimulating CD19 MACS-enriched spleen cells with 25 μg/ml LPS and 25 μg/ml dextran sulfate. Targets were separated into two fractions (targets and bystanders) and labeled with either 1 or 0.4 μM of dye (CFSE or CellTrace Violet; Thermo Fisher). Targets were pulsed with cognate peptide at 5 μM for 1 h at 37°C, including OVA323–339 (ISQAVHAAHAEINEAGR), HA126–138 (HNTNGVTAACSHE), NP311–325 (QVYSLIRPNENPAHK) (37), NP216–230 (RIAYERMCNILKGKF) (14), or gp67–80 (IYKGVYQFKSVEFD) (10), all from New England Peptide. Targets and bystanders were mixed at a 1:1 ratio and cocultured with effectors for 4 h at 37°C and 5% CO2. In some cases, anti–MHC-II Ab (M5/114; BioXCell) was added at 20 μg/ml or anti-NKG2A/C/E (20d5; eBioscience) was added at 10 μg/ml. Plates were then harvested, washed, and stained with Annexin V, 7-aminoactinomycin D (Sytox), or Live/Dead Amine (all Thermo Fisher). Specific killing was calculated as 100 × (1 − [live targets/live bystanders normalized to no effector control wells]). Peptide pulsing and cytotoxic assays were done in complete RPMI media (RPMI 1640 containing 7.5% FBS, 2 mM of l-glutamine, 50 μM of 2-ME, 100 IU of penicillin, 100 μg/ml streptomycin, and 10 mM of HEPES).

For in vivo cytotoxic assays, CD90.2-depleted spleens (MACS) were split into targets and bystanders and labeled and pulsed with peptide like described earlier. Targets and bystanders were mixed at a 1:1 ratio and injected i.v. into host mice. Eighteen hours later, mice were sacrificed and cells from the lungs were isolated for staining. Specific killing was calculated as 100 × (1 − [live MHC-II+ targets/live MHC-II+ bystanders normalized to the ratio found in uninfected mice]).

Intravenous labeling

A total of 2.5 μg of anti-CD4 clone (clone RM4-5) was injected i.v. into infected host mice. Mice were euthanized 3–5 min after injection and harvested quickly. Peripheral blood was taken via cardiac puncture, and the mouse was perfused with 10 ml of PBS. Organs were taken and single-cell suspensions were stained with anti-CD4 (clone RM4-4) as described. Staining of peripheral blood showed CD4+ (clone RM4-4) cells were also >95% CD4 (clone RM4-5) positive, indicating successful i.v. labeling.

Real-time PCR and microarray analysis

Isolated cell populations from flow sorting were immediately placed in RNA cell protect (Qiagen) and frozen at −80°C until extraction. RNA was extracted (Qiagen), and complementary RNAs were labeled and hybridized onto Affymetrix 2.0 ST arrays according to manufacturer’s protocols. Data were normalized with the robust multiarray average algorithm and log transformed using Affymetrix expression console. The p values were generated using unpaired ANOVA in Affymetrix Transcriptome Analysis Console 3.0. Heat maps were generated using Gene-E (The Broad Institute). Data for microarray analysis were deposited in the Gene Expression Omnibus (https://www.ncbi.nlm.nih.gov/geo/) under accession number GSE89634. Differential expression of selected genes was validated by reverse transcribing RNA and amplifying using TaqMan gene expression assays (Thermo Fisher). The fold increase in expression of NKG2A/C/E+ relative to NKG2A/C/E− was determined with the “2−ΔΔCT” method.

Statistical analysis

Unpaired, two-tailed Student t tests with an ∝ = 0.05 were used to assess whether the means of two normally distributed groups differed significantly. The Welch correction was applied when variances were found to differ. Paired analysis was done when comparing populations within the same mouse. Two-way ANOVA analyses were performed on log-transformed data with Fisher’s least significant difference. Analyses were performed using Prism GraphPad or JMP software (SAS Institute). Significance is indicated as *p < 0.05, **p < 0.005, ***p < 0.001, ****p < 0.0001. All error bars represent the SD.

Results

Using Blimp-1 to identify ThCTL markers

Because previous studies suggested that Blimp-1 is required during IAV infection for the generation of CD4 effectors with cytotoxic function (16), we reasoned that comparing the phenotype of wild type (WT) versus Blimp-1–deficient CD4 effectors might reveal candidate markers of ThCTL. We adoptively transferred either naive WT (a mix of either B6 or Prdm1+/+Cd4-cre+ mice) or Prdm1fl/flCd4-cre+ (Blimp-1 CKO) (36) OT-II Tg cells to B6.Thy1.1 mice and infected them with the recombinant A/PR8-OVAII virus, which generates a robust CD4 effector response in the lung, draining lymph node, and spleen (38). We have shown previously that ThCTL are found in the lung, but not SLOs, of IAV-infected mice (14). Therefore, we isolated CD4 effectors based on their congenic marker Thy1.2 from the lungs 8 d postinfection (dpi) and titrated them in an ex vivo flow cytometry–based cytotoxicity assay, which confirmed that T cell–intrinsic Blimp-1 was required for optimal induction of cytotoxic CD4 effectors from donor OT-II cells (Fig. 1A). We also found that polyclonal CD4 effectors from Blimp-1 CKO mice infected with A/PR8 demonstrated impaired ex vivo cytotoxicity against targets pulsed with IAV peptides compared with WT at a ratio of 20 effectors to one target (Fig. 1B). The polyclonal Blimp-1 CKO effectors at 8 dpi were also defective compared with WT in in vivo IAV MHC-II–restricted killing of activated T-depleted splenocyte targets pulsed with IAV-derived peptides (Fig. 1C). Because Blimp-1 was required for optimum development of ThCTL function, we reasoned that it might also be required for their differentiation, so we asked what difference in CD4 effector surface phenotype would be seen when Blimp-1 was absent. At the peak of the CD4 effector response in the lung, 8 dpi, Blimp-1 CKO CD4 effectors had reduced expression of several markers associated with effector T cells compared with WT. Lung CD4 effectors from Blimp-1 CKO mice expressed lower levels of PD-1 and CD27 (Fig. 1D, 1E). We also measured the ability of lung CD4 effectors to bind P-selectin via PSGL-1, a phenotype characteristic of effector T cells that are able to enter infected tissue sites (39). Blimp-1 CKO CD4 lung effectors bound significantly less P-selectin than WT CD4 (Fig. 1F). Taken together, these data indicate that the CD4 effectors generated in the absence of Blimp-1 express lower levels of markers associated with highly differentiated effector T cells and ThCTL, which supports the overall hypothesis that ThCTL are highly differentiated.

FIGURE 1.
FIGURE 1.

Blimp-1 is required for ThCTL differentiation. (A) WT (circle) or Blimp-1 CKO (square) naive OT-II CD4 cells were transferred into B6.Thy1.1 mice and infected with A/PR8-OVAII. Eight dpi, OT-II effectors were isolated from pooled lungs and assayed for ex vivo cytotoxicity against peptide-pulsed targets (representative of two independent experiments, n = 5 mice per group each). Significance is shown between WT and Blimp-1 CKO OT-II. (B) CD4 cells isolated from pooled lungs (n = 10–15 mice) from WT or Blimp-1 CKO mice infected with A/PR8 at 8 dpi was assayed for ex vivo cytotoxicity (pooled data of two independent experiments). (C) In vivo killing of peptide-pulsed targets in WT or Blimp-1 CKO mice infected with A/PR8 at 8 dpi (n = 5 mice). Right, Representative flow plot of targets (CFSEloMHC-II+) and bystander cells (CFSEhiMHC-II+). WT or Blimp-1 CKO mice were infected with A/PR8 and the phenotype of lung CD4 cells assayed at 8 dpi for (D) PD-1, (E) CD27, or (F) binding to P-selectin (n = 3–5 mice, representative of two independent experiments). (G) Representative histograms of GFP expression of A/PR8-infected Prdm1gfp/+ (open) or Prdm1+/+ (filled) mice at 8 dpi, gated on CD4 from the lung (left), draining lymph node (middle), or spleen (right). (H) Representative flow plot of NKG2A/C/E and Blimp-1 expression of CD4 cells from Prdm1gfp/+ mice infected with A/PR8 at 8 dpi. (I) WT or Blimp-1 CKO mice were infected with A/PR8, and the percentage of CD4 T cells expressing NKG2A/C/E on 8 dpi was quantified. (J) Same experimental setup as in (A), representative flow plot of NKG2A/C/E expression on WT or Blimp-1 CKO OT-II lung CD4 cells at 8 dpi. (K) Percent NKG2A/C/E+ of OT-II effectors recovered from the lung, dLN, and spleen. (L) Numbers of NKG2A/C/E OT-II effectors recovered from the lung. (J–L) n = 3–4 mice per group, representative of two independent experiments, (K) Pooled data. Error bars represent SD, and significant differences were determined with two-way ANOVA and Fisher’s least significant difference test (A and B) and unpaired two-tailed Student t tests (C–L), where α = 0.05, *p < 0.05, **p < 0.005, ***p < 0.001, ****p < 0.0001.

We next measured the expression of Blimp-1 in Prdm1gfp/+ (Blimp-1) reporter mice post A/PR8 infection. We found high Blimp-1 was expressed by CD4 effectors exclusively in the lung and not by other CD4 effectors in the SLOs, which belong to other subsets (Fig. 1G), indicating that high Blimp-1 correlates with the observed pattern of ThCTL and cytotoxicity that is found exclusively in the lung post IAV infection (14, 16). We searched for markers that costained with Blimp-1 expression. Because cytotoxic CD4 T cells in humans can express NKG2C (40) and highly polarized in vitro Th1 effectors also can express the NKG2 complex (41), we examined the expression of NKG2X (indicating expression of any of the forms NKG2A, NKG2C, or NKG2E recognized by the 20d5 Ab). The majority of the NKG2X+ CD4 effectors expressed Blimp-1 (Fig. 1H). Thus, we hypothesized that NKG2X might be a signature marker for the ThCTL subset. To test whether Blimp-1 was required for optimum generation of CD4 effectors expressing NKG2X, we infected either WT or Blimp-1 CKO mice and found Blimp-1 CKO polyclonal CD4 lung effectors expressed significantly less NKG2X than WT effectors (Fig. 1I). Moreover, at 8 d post A/PR8-OVAII infection, donor Blimp-1 CKO OT-II lung effectors also expressed reduced NKG2X compared with WT effectors (Fig. 1J, 1K), suggesting the reduced NKG2X is due to a T cell–intrinsic loss of Blimp-1. We also saw a reduction in total NKG2X+ OT-II recovered from the lungs (Fig. 1L). Importantly, the NKG2X+ Blimp-1 CKO OT-II cells were not found in the dLN or spleen (Fig. 1K), suggesting they were not stuck in the dLN as has been found for CD8 T cells lacking Blimp-1 (42). We also note the lung exclusivity of the expression of NKG2X (Fig. 1G) correlates with Blimp-1 tissue expression and the tissue restriction of CD4 cytotoxicity. Thus, NKG2X marks a population of T cell effectors that express Blimp-1 at the effector stage and require Blimp-1 for their optimal generation in or migration to the lung, and thus we postulate that NKG2X is a marker for ThCTL.

NKG2X marks cytotoxic ThCTL

To directly test whether NKG2X expression and ThCTL cytotoxic activity are associated, we sorted lung CD4 effector cells generated from congenically marked OT-II.Thy1.1 donor cells at 8 dpi for expression of NKG2X and compared the ability of positive and negative sorted effectors to kill targets pulsed with specific peptide. The donor NKG2X+ OT-II effectors were effective killers of MHC-II–restricted OVA323–339 peptide-pulsed targets even at low E:T ratios, whereas the NKG2X negative donor OT-II effectors had no detectable peptide-specific cytotoxic activity (Fig. 2A), confirming that the ThCTL activity was exclusively in the subpopulation of lung CD4 effector expressing NKG2X. To rule out the possibility that NKG2X marks cytotoxic ThCTL of only one Tg TcR, we also examined the cytotoxic activity of HNT TcR Tg CD4 effectors that recognize HA126–138 of A/PR8 in the context of I-Ad (43) in BALB/c mice. In this case, naive congenically marked HNT.Thy1.1 CD4 T cells were adoptively transferred into BALB/c mice infected with A/PR8. A subset of the donor HNT effectors in the lung expressed NKG2X as shown in Fig. 3A. HNT CD4 effectors were then sorted after 8 dpi and as with OT-II cells, the ThCTL cytotoxic activity was only in the NKG2X+ population (Fig. 2B). This population also coexpressed CD94, the known coreceptor for NKG2X. In at least five separate experiments, the cytotoxic activity was found only in the NKG2X-expressing CD4 effectors. Taken together, these data strongly suggest that NKG2X is a reliable signature marker of cytotoxic CD4 effectors in the lung of IAV-infected hosts. In the subsequent experiments, we will refer to NKG2X+ CD4 effectors as ThCTL.

FIGURE 2.
FIGURE 2.

NKG2A/C/E marks cytotoxic ThCTL in the lung. (A) Naive OT-II.Thy1.1 CD4 T cells were adoptively transferred into B6 mice and infected with A/PR8-OVAII. At 8 dpi, lung donor OT-II cells were isolated and sorted based on NKG2A/C/E expression. Top left, Sample flow plot of sorted purities of NKG2A/C/E− OT-II (top) and NKG2A/C/E+ OT-II (bottom); both are gated on donor OT-II cells (CD90.1+CD4+). Right, NKG2A/C/E+ OT-II (open circles) and NKG2A/C/E− OT-II (closed circles) CD4 T cells were assayed in triplicate for cytotoxicity against peptide-pulsed targets. Anti–MHC-II Ab was also added to NKGA/C/E+ OT-II (open triangle) or NKG2A/C/E− OT-II (closed triangle), one well per E:T ratio. Significance is between NKG2A/C/E+ versus NKG2A/C/E− OT-II. Bottom, Representative flow plots from the cytotoxicity assay, gated on CellTrace+ cells, targets (CellTracehi) and bystanders (CellTracelo), after 4-h incubation with NKG2A/C/E+ OT-II effectors (left), NKG2A/C/E− OT-II effectors (middle), and NKG2A/C/E+ OT-II effectors with anti–MHC-II Ab (right). CD4 T cell–sorted lungs of n = 10 mice and pooled each time, representative of two experiments. (B) Naive HNT.Thy1.1 CD4 T cells were adoptively transferred into BALB/c mice and infected with A/PR8. At 8 dpi, donor CD4 cells were isolated from lungs and sorted based on NKG2A/C/E expression. NKG2A/C/E+ HNT (open circles) and NKG2A/C/E− HNT (closed circles) CD4 T cells were assayed in triplicate for cytotoxicity against peptide-pulsed targets. The E:T ratio of 0.1:1 are single wells. Data are representative of two independent experiments. Error bars represent SD, and significant differences were determined with two-way ANOVA and Fisher’s least significant differences test, where α = 0.05, **p < 0.005, ***p < 0.001.

FIGURE 3.
FIGURE 3.

ThCTL express the NKG2C/E isoforms, which is not required for cytotoxicity. (A) Representative plots showing CD94/NKG2A/C/E coexpression on lung CD4+ cells harvested from mice 8 d post A/PR8 infection. Cells were isolated from either infected or uninfected B6 mice (left two panels), or infected BALB/c mice (right two panels). HNT indicates HNT effectors cells after adoptive transfer of naive HNT.Thy1.1 CD4 into mice with subsequent infection, gated on CD4+CD90.1+. (B) Representative plots of NKG2A and NKG2A/C/E expression, gated on NK1.1 (left) or OT-II CD4 T cells (middle) from lungs 8 d post A/PR8-OVAII infection. Right, Quantification of the percent NKG2C/E+NKG2A+ of the indicated populations (representative of two independent experiments, n = 5 mice each). (C) Naive OT-II.Thy1.1 CD4 cells were adoptively transferred into B6 hosts and then infected with A/PR8-OVAII. OT-II.Thy1.1 CD4 effectors from the lungs or spleen 8 dpi were isolated and assayed for ex vivo cytotoxicity against peptide-pulsed B6 targets (WT) or Qa-1 KO targets (CD4 T cells sorted from pooled lungs n = 10 mice or pooled spleens n = 5 mice, representative of two independent experiments). (D) Same OT-II effector generation as in (C); then OT-II.Thy1.1 CD4 T cells isolated from the lungs of 8 dpi mice were assayed for ex vivo cytotoxicity against peptide-pulsed targets in the presence of anti-NKG2A/C/E or isotype control Ab (10 μg/ml). CD4 T cells were isolated from pooled lungs of n = 5–10 mice, or pooled spleens of n = 2–5 mice, representative of two independent experiments. (E) WT or Qa-1 KO targets were transferred into infected host mice generated similarly like in Fig. 3C. Eighteen hours posttransfer, the ratio of live targets versus bystanders was quantified with flow cytometry. Targets generated from pooled mice are n = 2 each, and data are pooled from two independent experiments with n = 4–5 mice each. (F) WT (closed squares) or DAP10/12 KO (closed circles) OT-II.Thy1.1 naive CD4 T cells were adoptively transferred into B6 hosts, and mice were infected with A/PR8-OVAII. Donor OT-II cells were isolated 8 dpi and assayed for ex vivo cytotoxicity against peptide-pulsed target cells. Anti–MHC-II Ab was added at 20 μg/ml with WT (open squares) or DAP10/12 KO (open circles) OT-II CD4 T cells. CD4 T cells isolated from pooled lungs of n = 5 mice per group, representative of two independent experiments. Error bars represent SD, and significant differences were determined with unpaired two-tailed Student t tests, where α = 0.05, **p < 0.005, ***p < 0.001.

NKG2C or NKG2E, but not NKG2A, are on ThCTL, but they do not affect the cytotoxic activity of ThCTL against conventional targets

NKG2X receptors have been found to play a role in modulating cytotoxicity of NK cells (44, 45) and CD8 T cells (34, 46), but their role in CD4 function is less well studied. Because NKG2X forms a heterodimer with CD94, we stained CD94 on B6, BALB/c, and HNT lung effectors. Indeed, the effector CD4 T cells generated to IAV coexpress NKG2X and CD94 (Fig. 3A). NKG2 has three isoforms; C and E are activating receptors, whereas the A isoform is an inhibiting receptor for NK cytotoxicity, and all three are recognized by the 20d5 Ab (35) that we use to identify ThCTL. To determine the ThCTL, expression of NKG2A versus C/E isoforms in the lung, we stained cells with Ab clone 16a11, a specific Ab that recognizes only the A isoform in B6 mice as well as the pan-specific 20d5 Ab. We also stained for NK cells (NK1.1+) as a positive control for NKG2A expression. The majority of 20d5+ NK cells, staining with NK1.1, also expressed NKG2A. In contrast, only a few of the lung donor OT-II effectors, including those in the NKG2X+ population, expressed NKG2A, and they did so at low levels. This suggests that ThCTL express mainly the NKG2C and NKG2E activating receptors (Fig. 3B), and we will from this point describe ThCTL in B6 mice as NKG2C/E+.

We reasoned that engaging the NKG2C/E receptors might costimulate ThCTL killing of MHC-II targets. The primary known ligand for NKG2X receptor is the nonclassical MHC-I molecule Qa-1 (44, 45). To probe whether the NKG2X was playing a role in the killing of conventional targets, we assayed the ability of ThCTL to kill either WT targets or those lacking Qa-1 in an ex vivo cytotoxicity assay. The ThCTL effectors lysed WT and H2-t23−/− (Qa-1 KO) (45) target cells derived from activated T-depleted spleen equally well (Fig. 3C). As a second approach to determine whether the NKG2C/E receptor is involved in induction or delivery of cytotoxicity, we added the clone 20d5 Ab to block NKG2A/C/E receptor interactions with cognate ligands (35) in the ex vivo cytotoxic assay. Blocking NKG2A/C/E also did not affect ex vivo MHC-II–specific cytotoxicity (Fig. 3D). A third approach was to determine whether in vivo killing of target cells was also similarly affected by their lack of Qa-1 expression. We isolated target cells from the spleens of either WT or Qa-1 KO mice and adoptively transferred them into host mice with OT-II CD4 effectors and assayed their in vivo cytotoxicity. We found no significant difference between WT and Qa-1 KO target cells in ability to be killed in a peptide-specific manner (Fig. 3E). Because the NKG2X complexes signal through the adaptors DAP10 and DAP12 (47), we next asked whether the ability of ThCTL to lyse conventional target cells was lost or reduced when ThCTL were generated from CD4 T cells deficient in both DAP10 and DAP12 adaptors, Hcst−/−/Tyrobp−/− (DAP10/12 KO) (48). Congenically marked, OT-II.Thy1.1 ThCTL were derived by transfer of naive CD4 from OT-II.Thy1.1 or OT-II.Thy1.1.DAP10/12 KO donors into B6 mice. Hosts were then infected with IAV. When compared in ex vivo cytotoxicity assays, the lysis of target cells by WT and KO effectors was equivalent (Fig. 3F). In contrast, as expected, blocking MHC-II interactions abolished the cytotoxic activity of both WT and DAP10/12 KO effectors. This suggests that neither the generation of ThCTL nor their ability to kill conventional targets depends on the activating signals from NKG2C/E. Taken together, we conclude that ThCTL express NKG2C/E and that this expression is strongly correlated with their ThCTL function, but that the NKG2X receptors are not directly involved in the cytotoxic process when conventional targets are used. Thus, we found no evidence that any additional MHC, other than the MHC-II restriction element seen by the OT-II TcR, was needed on conventional target cells for effective ThCTL killing.

ThCTL represent a highly activated lung effector CD4 subset

To test whether a subset of endogenous Ag-specific polyclonal effectors express NKG2C/E, we isolated CD4 T cells from lungs of IAV-infected B6 mice and incubated the cells with I-Ab tetramers loaded with IAV NP311–325 peptide. As in previous polyclonal experiments, a fraction of the I-Ab-NP311–325–specific lung CD4 effectors expressed NKG2C/E (Fig. 4A). NKG2C/E+ CD4 effectors were ∼20% of the lung CD4 effector population, similar to what we see in the donor Tg models using OT-II and HNT naive CD4 transfer and in polyclonal effectors in BALB/c mice (Fig. 3A, 3B).

FIGURE 4.
FIGURE 4.

ThCTL are a highly activated lung effector subset. (A) B6 mice were infected with A/PR8-OVAII, and lungs were harvested 9 dpi. NP311–325 tetramer staining of NKG2A/C/E+ ThCTL (right) compared with control CLIP-loaded tetramer (left) (representative of at least two independent experiments, n = 3–5 each). (B) Kinetics of NKG2A/C/E expression on lung CD4 T cells control or A/PR8-infected B6 mice. (C) B6 mice were infected with A/PR8-OVAII, and lungs were harvested 9 dpi. Phenotyping analysis of lung CD4 T cells gated on NKG2A/C/E+ (solid black line), NKG2A/C/E− (tinted gray), or CD44lo (dashed line). Histograms are normalized to the mode of each population. NKG2A/C/E+ and NKG2A/C/E− cells were gated on either CD44hiCD4+ cells (CXCR6 and SLAM) or NP311–325 tetramer+CD4+ (remainder of the plots). Representative of two to three independent experiments, n = 3–5 mice each. (D) Phenotype kinetics of CD4 T cells isolated from the lungs of B6 mice at 9, 14, and 22 dpi with A/PR8-OVAII. NKG2A/C/E+ or NKG2A/C/E− CD4 T cell expression of selected markers are shown. All plots are on gated NP311–325 tetramer+CD4+, except for CXCR6 and SLAM, which are gated on CD44hiCD4+ cells. GrB and T-bet are representative of at least two experiments, and the remainder of the graphs are pooled from two experiments; n = 4–5 mice each. Error bars represent SD, and significant differences were determined with paired two-tailed Student t tests, where α = 0.05, **p < 0.005, ****p < 0.0001.

Using NKG2C/E to identify ThCTL, we analyzed the kinetics of ThCTL generation and compared the phenotype of NKG2C/E+-gated ThCTL and NKG2C/E−-gated, non-ThCTL lung effectors. When naive B6 mice were infected with A/PR8 and the expression of NKG2X was measured 4, 6, 8, 11, and 13 dpi, NKG2C/E-expressing ThCTL peaked around 8–9 dpi and declined thereafter (Fig. 4B), consistent with the peak total effector CD4 response in the lung and with studies of OT-II and HNT transfers (38, 49). Our laboratory has found that there is extensive interorgan heterogeneity between CD4 effectors in different sites (49, 50), so to focus on the cytotoxic population, we compared the phenotype of ThCTL versus non-ThCTL effectors from only the lung at 8–9 dpi (Fig. 4C, 4D). Although ThCTL (NKG2C/E+) had higher levels of GrB, non-ThCTL (NKG2C/E−) also expresses a high level compared with naive cells, confirming the necessity of using markers in addition to GrB to identify ThCTL. The lung CD4 effectors, both positive and negative for NXG2C/E, are highly activated as indicated by forward scatter and high levels of activation markers CD27, CTLA-4, GrB, and SLAM, an activation marker that is absent on TFH cells (51). Lung effectors express low levels of exhaustion markers BTLA, Lag-3, CD160, and Tim-3 (Supplemental Fig. 1), consistent with high effector function, but express high PD-1, a checkpoint regulator on T cells that is also expressed by many other activated T cells including TFH (1). This pattern connotes high levels of activation and cytotoxicity indicators without most of the signature exhaustion markers (excepting PD-1) that are often associated with terminal differentiation.

We measured various chemokine receptors and found that CD4 lung effectors, both ThCTL and non-ThCTL, express CCR5, CXCR6, and CXCR3, but not CCR1, CCR2, CCR4, CXCR4, and CX3CR1 (Fig. 4C, Supplemental Fig. 1). CCR5 and CXCR3 are associated with migration of activated T cells the lung (52, 53), whereas CXCR6 may be important for trafficking to inflamed sites (54). In most cases, the NKG2C/E+ ThCTL and non-ThCTL had similar chemokine receptor expression patterns except that ThCTL uniformly express high levels of CXCR6, whereas a fraction of non-ThCTL were negative (Fig. 4C). This pattern of chemokine receptor expression suggests ThCTL and some other lung effectors may be preferentially recruited to particular sites in infected lung tissues where the ligand for CXCR6, CXCL16, is present. Consistent with this idea, ThCTL also had more uniformly high binding to P-selectin-Fc compared with non-ThCTL. P-selectin binding to its ligands is proposed to promote lung CD4 effector entry into specialized peripheral sites (39). The chemokine and adhesion receptor phenotypes of lung effectors, and the enhanced P-selectin binding and CXCR6 levels on ThCTL suggest they may be recruited to a unique tissue niche, which would be consistent with their proposed role of killing IAV-infected epithelial cells.

To further evaluate ThCTL versus noncytotoxic lung CD4 effectors, we stained for transcription factors expressed by T cells. The ThCTL expressed more T-bet protein than non-ThCTL lung effectors, consistent with the general requirement of T-bet for effector and cytotoxicity in IAV infection (Fig. 4C, 4D) (16). This contrasted with the fact that the NKG2X+ ThCTL were negative for Eomes, Bcl6, and Foxp3 (Fig. 4C) and other transcription factors (Supplemental Fig. 1). Foxp3+ Tregs can also be cytotoxic (55); however, ThCTL are Foxp3−. Type 1 regulatory cells lack Foxp3 expression as well; however, these cells often express high levels of Lag-3 (56, 57), which ThCTL are low for at the peak of the infection. The lack of transcription factors associated with the other major CD4 subsets suggests that few, if any, of the ThCTL have differentiated down other polarized cytokine pathways including those expressed by Th2 (GATA-3 expressing), TFH (Bcl6-expressing), Th17 (RORγt), or Foxp3+ Tregs.

We followed the contraction of CD4 effectors post influenza infection at day 14 postinfection and their resulting formation of an early memory pool at day 22 postinfection (Fig. 4D). CD4 T cells rapidly become resting, so day 22 represents a memory time point (50, 58, 59). The percent and numbers of NKG2C/E+ ThCTL at 2–3 wk (Fig. 4D) suggest that ThCTL CD4 cells are likely retained at the same rate as other lung subsets in this memory pool. Phenotypic analysis of NKG2C/E-expressing and negative cells shows that many effector-associated markers show reduced expression from the peak at 9–14 and 22 d, including CD27, CTLA-4, GrB, PD-1, binding to P-selectin, and T-bet. In contrast, memory markers CD127 and CXCR3 increase with time. Differences in expression of SLAM, CXCR6, and binding to P-selectin persist, with ThCTL still expressing higher levels through day 22, suggesting they retain stable programming into memory.

ThCTL localize to the site of infection

After their generation in the SLOs at ∼6 dpi with IAV, CD4 effectors traffic throughout the body (60) much like CD8 T cell effectors and memory cells (61, 62), and become widely dispersed (63). To analyze in more detail sites to which CD4 effectors and in particular ThCTL accumulate, we transferred naive, congenically marked OT-II.Thy1.1 CD4 T cells to B6 hosts, infected with A/PR8-OVAII, and recovered donor OT-II effectors from various organs at 8 dpi. We found detectable numbers of donor CD4 effectors in all the organs we harvested, confirming that they migrate widely and become dispersed to most tissue sites throughout the body (Fig. 5A, 5B). After i.n. IAV infection, the total effector CD4 cells were found in largest numbers in the spleen and lung (Fig. 5B). To track ThCTL, we stained donor effectors for NKG2X. ThCTL were found only in the lung and the BAL (Fig. 5A, 5C, 5D), and were absent from the liver and blood. There were also significant numbers of ThCTL in the spleen, which had high numbers of total effectors, but they were a low fraction of that population (Fig. 5C, 5D). When mice are infected i.n. with A/PR8, the virus replicates only in lung epithelial cells (64), suggesting that ThCTL are restricted to the site of infection and/or viral replication. This observation is also consistent with their proposed function of killing infected targets. To further ask whether ThCTL are associated with vascular sites or are to be found in the parenchyma less accessible to the blood, where tissue resident cells reside, we labeled CD4 T cells in vivo with fluorescently labeled mAbs introduced i.v. (65). We found ThCTL in both the i.v.-shielded and i.v.-labeled portion of CD4 effectors in the lung with a slight enrichment in the shielded fraction (Fig. 5E). These data suggest that ThCTL can be found in both vascular and nonvascular associated sites in the lung, with more in the shielded sites.

FIGURE 5.
FIGURE 5.

ThCTL localize to the lung, the site of IAV infection. (A) Naive OT-II.Thy1.1 CD4 T cells were adoptively transferred into B6 mice that were then infected with A/PR8-OVAII. Representative staining of NKG2A/C/E expression on OT-II CD4 T cells recovered. Quantification of (B) donor OT-II CD4 T cells recovered from various organs; (C) percent NKG2A/C/E expression on OT-II CD4 T cells and (D) the number of NKG2A/C/E+ OT-II CD4 T cells recovered from indicated organs. Pooled from two independent experiments, n = 3–5 mice each. (E) At 8 dpi, mice were i.v. labeled with anti-CD4 Ab and quickly harvested. Left, Representative flow plot of lung CD4 T cells. Right, Quantification of the proportion of i.v. labeled or shielded CD4 T cells expressing NKG2A/C/E. Pooled from three independent experiments, n = 3–4 mice each. (F) Naive SMARTA CD4 T cells were adoptively transferred into congenically marked B6.Thy1.1 mice and then subsequently infected with LCMV Armstrong. At 8 dpi, spleens were harvested and stained for ThCTL. Representative staining is shown of NKG2A/C/E expression on donor and host cells in the spleen along with uninfected mouse as control. (G) Quantification of the percent donor or host CD4 T cells expressing NKG2A/C/E. (H) CXCR6 expression on gated NKG2A/C/E positive or negative SMARTA CD4 cells in the spleen. (I) GrB expression on gated NKG2A/C/E positive or negative SMARTA CD4 cells in the spleen. (F–H) Data are pooled of two independent experiments with n = 3–5 mice each. (I) Data are of one experiment with n = 3 mice. Error bars represent SD, and significant differences were determined with paired two-tailed Student t tests, where α = 0.05, *p < 0.05, **p < 0.005.

Because ThCTL appear to be restricted to the site of infection, we also looked at CD4 effectors generated against LCMV to test whether we could find ThCTL in the spleen, a main site of LCMV replication (66). We transferred naive CD4 cells from SMARTA TcR Tg mice into congenically labeled naive hosts and subsequently infected them with LCMV Armstrong. On 8 dpi, we harvested the spleen and assayed for ThCTL formation by staining for NKG2X. In contrast with what we saw after i.n. infection with IAV, we found NKG2X+ CD4 effectors in both the SMARTA and host populations in the spleen (Fig. 5F, 5G). These data suggest ThCTL can form in the spleen when the virus is also replicating in that organ. Further phenotyping shows that the splenic ThCTL generated by LCMV administered i.p. are also high in CXCR6 and GrB expression (Fig. 5H, 5I, Supplemental Fig. 2A–C), similar to the ThCTL found restricted to the lung after i.n. IAV infection (Fig. 4C). We sorted NKG2A/C/E+ cells from spleen at 8 dpi with LCMV and found they are enriched for cytotoxic activity against targets expressing the gp67–80 peptide seen by SMARTA TcR Tg, and this increase is blocked by Ab to MHC-II (Supplemental Fig. 2D), further supporting the hypothesis that ThCTL of similar phenotype, including expression of NKG2A/C/E, are generated by multiple viral infections in sites where the virus replicates.

ThCTL secrete IFN-γ and degranulate in response to Ag stimulation

ThCTL exhibit cytotoxic function, but it is unclear whether they also secrete IFN-γ, a cytokine that contributes to CD4 effector T cell–protective function (17). We showed previously that ThCTL do not require IFN-γ for their function or generation (15). To evaluate IFN-γ production, we stimulated OT-II.Thy1.1 effector cells isolated from the lung at 8 dpi with OVA323–339–pulsed APCs, and measured intracellular IFN-γ expression in NKG2C/E-positive and -negative populations. Notably, a high proportion of ThCTL (>70%) secreted IFN-γ, and they produced more IFN-γ on a per-cell basis than the non-ThCTL (Fig. 6A–C). To compare their relative dose response with TcR stimulation for IFN-γ production, we stimulated ThCTL and non-ThCTL with a range of peptide doses, using activated splenic APCs. We found that, compared with the non-ThCTL, more ThCTL secreted more IFN-γ over several logs of peptide dose (Fig. 6A–C), suggesting ThCTL are likely somewhat more activated and hence better poised to carry out effector function at lower density of peptide than other lung CD4 effectors.

FIGURE 6.
FIGURE 6.

ThCTL show increased IFN-γ secretion and degranulation compared with the non-ThCTL lung CD4 T cells. Lung OT-II.Thy1.1 CD4 T cells were isolated 8 dpi and stimulated with peptide-pulsed activated B cells. (A) Representative staining plots of IFN-γ and CD107a expression of donor cells after 10−5 M OT-II peptide stimulation. (B) Quantification of the percent IFN-γ–producing OT-II CD4 T cells gated on NKG2A/C/E+ (open circles) or NKG2A/C/E− (closed circles). (C) Median fluorescence values of IFN-γ, gated on IFN-γ+ cells of indicated populations. (D) Quantification of percent CD107a+ cells. (E) Median fluorescence values of CD107a, gated on CD107a+ cells. CD4 T cells isolated from pooled lungs, representative of two independent experiments; n = 5 mice each.

We also assessed the degranulation of lung CD4 effectors by staining for CD107a. Both cytotoxic cells and cells secreting cytokines are known to degranulate (33). ThCTL and non-ThCTL populations degranulated in a peptide dose-dependent manner as measured by induction of CD107a expression. A greater proportion of ThCTL expressed more CD107a compared with non-ThCTL lung effectors across a wide range of peptide doses (Fig. 6D, 6E), consistent with their cytotoxic and higher cytokine production potential. Because those CD4 lung effectors not expressing NKG2C/E have no cytotoxic activity (Fig. 2A, 2B) despite having substantial CD107a expression, this confirms that, like GrB, CD107a alone is insufficient to identify ThCTL. Thus, in addition to possessing all the cytotoxic activity, the ThCTL, defined by NKG2C/E expression, are more readily stimulated to secrete IFN-γ and degranulate than are other lung effectors.

ThCTL upregulate genes associated with cytotoxic function and downregulate genes associated with recirculation

To further characterize ThCTL, we compared gene expression profiles of isolated ThCTL with the non-ThCTL population in the lung. Congenically marked HNT.Thy1.1 lung effectors were flow sorted at 7 dpi from BALB/c mice based on NKG2A/C/E expression, and their RNA was isolated for microarray analysis. Importantly, both populations were isolated from the same organ, ruling out any tissue-dependent differences, and both populations have the same TcR, ruling out differences in avidity. Comparing the gene expression profiles revealed that only a limited number of genes differed between the two populations, with 58 genes significantly different (p < 0.05) using a 2-fold relative change cutoff (Fig. 7A). Additionally shown is a heat map of selected genes associated with cytotoxicity and Th differentiation (67) that did not meet the criteria of significance or 2-fold relative change. The ThCTL population was enriched in expression of genes associated with cytotoxic function including genes encoding perforin (Prf1) and granzymes a, c, and f (Gzma, Gzmc, and Gzmf). Tbx21 was upregulated, consistent with the higher levels of T-bet protein in ThCTL (Fig. 4C, 4D). Id2, a transcription factor associated with effector function in CD8 T cells (68, 69), was also upregulated. Prdm1 was expressed equally in both groups, suggesting Blimp-1 may be required for both ThCTL and non-ThCTL lung effectors. Notably, genes associated with recirculation such as Klf2, Ccr7, and S1p1r (70–72) were downregulated, suggesting ThCTL are not part of the recirculating pool, but instead remain in the lung at least during the peak of the CD4 response when this analysis was done. This may explain the lack of ThCTL found in other peripheral sites. ThCTL also had lower expression of memory-associated genes Tcf7, Id3 (63, 67, 73), and Il2, which we find is required for the transition to memory (38). This pattern is consistent with a highly differentiated effector phenotype, because non-ThCTL are also full-fledged effectors that may be suppressing genes that would drive the cells to rest and progress to memory.

FIGURE 7.
FIGURE 7.

ThCTL express effector genes and suppress circulation and memory genes compared with non-ThCTL. (A) Naive HNT.Thy1.1 CD4 T cells were adoptively transferred into BALB/cByJ mice that were then infected with A/PR8. Left, Microarray heat map showing gene expression profiles of NKG2A/C/E+ or NKG2A/C/E− HNT.Thy1.1 CD4 T cells sorted from pooled lungs at 7 dpi. Right, Additional genes associated with cytotoxicity and Th differentiation. Microarray ran twice with n = 10 mice each time, indicated as 1 and 2. (B) Naive OT-II.Thy1.1 CD4 T cells were adoptively transferred into B6 mice that were then infected with A/PR8-OVAII. Fold change in expression of indicated genes from quantitative real-time PCR expressed by NKG2A/C/E+ over NKG2A/C/E− CD4 T cells 8 dpi. Data shown are pooled from three independent experiments, with RNA from NKG2A/C/E+ or NKG2A/C/E− OT-II CD4 T cells sorted from pooled lungs; n = 10 mice in each experiment.

We used quantitative RT-PCR to analyze the difference in expression of selected genes in NKG2X-sorted OT-II.Thy1.1 donor effectors to confirm that the differences in expression were also found in another TcR Tg in B6 mice (Fig. 7B). We noted that by qRT-PCR the NKG2X+ lung effectors expressed less mRNA for CCR7 and CXCR5 relative to the non-ThCTL lung effectors, further distinguishing them. Taken together, these data suggest that ThCTL express a cytotoxic effector program compared with other lung effectors, and that they express high levels of effector-associated and low levels of resting cell–associated genes and low levels of genes associated with recirculation. This gene expression pattern emphasizes the unique identity of ThCTL compared with other CD4 subsets in the lung.

Discussion

Viral infection induces the generation of a remarkably heterogeneous population of CD4 effectors composed of multiple subsets, each possessing an array of distinct effector functions that play multiple roles in combating pathogens. The functionally distinct subsets generated can be characterized by different polarizing cytokines, by distinct patterns of transcription factors, and by their migration to different locations. CD4 T cells with cytotoxic function, ThCTL, are less well characterized than most CD4 subsets, but their role is likely to become more appreciated as their unique features and functions are uncovered. There is growing appreciation that cytotoxic CD4 T cells play important roles in clearing viral pathogens in humans, including IAV (18), HIV (19), and Dengue virus (74). They have been shown to be important for tumor rejection (21, 22) and protective immunity to viral infections in mice, indicating they warrant further study of this critical subset. Studies of ThCTL have been hampered by the lack of a signature marker to identify the subset.

Our results in this study establish that ThCTL generated by IAV infection in mice can be identified with a surface marker NKG2A/C/E, collectively called NKG2X. We use this marker to thoroughly characterize ThCTL by phenotype, function, gene expression, and localization. These studies reveal that ThCTL are a unique subset of CD4 T cells that share multiple aspects of a program of cytotoxic effector function with CD8 CTL. Of particular interest is their restriction to the lung and their status as highly activated and functional effectors with strong cytotoxic activity in the lung and BAL.

Staining analysis indicates that ThCTL express mainly NKG2C/E isoforms of the NKG2X family. We show that NKG2C/E is strongly correlated with the cytotoxic function of CD4 T cells and serves as a reliable marker superior to other phenotypes used previously including GrB (16) and CD107a (75), which although expressed by ThCTL, also are present on many non-ThCTL effectors in the lung (Figs. 4, 6). Using NKG2C/E expression to identify ThCTL and compare them with non-ThCTL in the lung, we found ThCTL express a highly activated effector phenotype including high levels of T-bet and Blimp-1 and of multiple indicators of cytotoxic function including high levels of GrB protein and the CD107a degranulation marker, as well as granzymes and perforin genes. Functionally, they secrete high levels of IFN-γ in response to low peptide dose and express low levels of inhibitory receptors CD160, Lag-3, BTLA, and Tim-3.

ThCTL are found restricted to the lung, including the BAL, but are not found in other tissue sites after i.n. IAV infection. Consistent with this finding, ThCTL have increased expression of molecules associated with lung retention including the glycosylated PSGL-1 adhesion molecule that binds P-selectin, CXCR3, CCR5, and CXCR6, whereas having low levels of CCR7, CXCR5, and S1p1r. Other CD4 effectors in the IAV-infected lung share many of these properties, suggesting a common pathway for effector CD4 T cells that accumulate in sites of infection and viral replication.

It was disappointing that we could find no evidence that NKG2C/E itself is needed for ThCTL differentiation or killing of the targets we tested. It is possible that the putative targets involved in protection, lung epithelial cells infected with IAV, have a distinct requirement for lysis that does require this pathway and we are investigating this possibility. It is also possible that one or more other NK receptor family members are also expressed by ThCTL and are necessary or play redundant roles in cytotoxicity of IAV-infected targets. Another possibility is that NKG2X receptors are needed in some other as yet unidentified context.

Because IAV generates a heterogeneous population of effector CD4 T cells, the fact that all the ThCTL activity was found in the NKG2C/E+ population indicates that only a fraction of the CD4 effector population in the lung are ThCTL. This suggests that unique signals or programs are in place during CD4 effector differentiation to guide and promote ThCTL generation over other alternate subset pathways, such as non-ThCTL in lung and TFH in follicular sites. Like ThCTL, the TFH represent a tissue-restricted subset, but they contrast sharply with ThCTL, developing in the follicular environment of the SLOs and expressing distinct signature surface markers such as high CXCR5, and they require Bcl-6, but not Blimp-1 (76). Exactly how these functionally defined subsets (ThCTL and TFH) relate to the cytokine-polarized subsets such as Th1, Th2, and Th17 remains unclear at this time, but the cytokine-polarized subsets appear somewhat earlier during the immune response and are generally more widely dispersed, consistent with the idea that the functionally defined ThCTL and TFH subsets require further differentiation. This is indeed shown to be the case with TFH, which require multiple interactions with Ag-presenting specific B cells first at the border of the follicles and subsequently in the germinal centers to develop into the germinal center-TFH that then drive B cell isotype switching and somatic hypermutation (1). We favor the hypothesis that ThCTL require some form of additional differentiation from CD4 effectors that most likely occurs in the infected tissue, in this case, the lung.

The lack of expression of transcription factors associated with Th1, Th2, Th17, and Foxp3+ Treg subsets seems most compatible with the idea that ThCTL arise from either nonpolarized Th0 or Th1 precursors. Investigators examining ThCTL in other settings have identified suppression of Thpok (67) and the increase of Eomes (21–23) transcription factors as key mediators driving ThCTL in the gut and in a tumor site, respectively. These organs may present a different inflammatory environment than that of an IAV-infected lung. IAV infection induces a strong systemic Th1-polarizing environment, and we found that ThCTL expressed a Th1 cytokine profile with more T-bet than the non-ThCTL lung effectors. This could suggest they are derived from Th1-polarized cells or that multiple CD4 subsets with distinct function share T-bet expression and IFN-γ production. We have shown previously that ThCTL can be generated independently of IFN-γ (14) and in vitro are optimally generated under Th0 conditions (20), suggesting the cytotoxic program and Th1 cytokine production may be independently regulated.

Our results agree with those of Sun and colleagues (16), who suggested Blimp-1 is required for ThCTL. We found intrinsic Blimp-1 is required broadly for several features of ThCTL in addition to cytotoxicity. Thus, we speculate that ThCTL require unique combinations of transcription factors that drive cytotoxic function as well as retention at the site of infection.

ThCTL control the level of IAV and contribute to protection against lethal IAV challenge. They can synergize with Ab to protect naive hosts from lethal doses of IAV (15). All of these activities have been shown previously to be perforin dependent. Thus, we assume that the key function of ThCTL is to kill infected lung epithelial cells. Although normally MHC-II−, IAV-infected lung epithelial cells express MHC-II (14). Indeed, compared with other lung effectors, ThCTL express high levels of perforin and granzyme. There is a broad array of effector CD4 mechanisms of viral control that develop after IAV infection, and when the response is deconstructed it is clear that at least one of these is IFN-γ mediated (15), so ThCTL may contribute to clearing infected cells through both cytotoxic and IFN-γ pathways. Although it might seem redundant to mount both CD8 and CD4 cytotoxic cells, we point out that viruses have evolved multiple evasion strategies to escape CD8 cytotoxicity (24), including mechanisms to downregulate MHC-I. Having CD4 cytotoxic cells provides a way to subvert such strategies. Indeed, in CMV-based vector-vaccinated animals, the immune system targets HLA-E–restricted responses as a possible mechanism to counter pathogen immune evasion mechanisms like blocking class I–restricted killing (77), supporting the need for countering viral evasion strategies that target MHC-I expression.

In summary, the data presented in this article suggest NKG2C/E can be used as a signature marker for ThCTL, and that using this to characterize ThCTL reveals their unique surface phenotype, gene expression profile, and lung/site infection-restricted location, and strongly supports the concept that they represent a unique lineage of CD4 T cells. The fact that many of these properties and phenotypes optimally require Blimp-1 suggests the transcriptional repressor plays a central role in specifying their development. The data showing that they are poised to degranulate and can secrete IFN-γ when restimulated with lower peptide dose, combined with their signature ability to kill targets via perforin and that their killing is restricted only by MHC-II, altogether support their ability to perform unique roles in combating viruses and tumors. We predict further identification of the mechanisms involved in their generation will yield further insights into their unique protective functions and additional clues as to how we can harness their potential activities by vaccination or in immune therapies for tumors.

Disclosures

The authors have no financial conflicts of interest.

Acknowledgments

We thank Dr. Harvey Cantor, Dr. Lewis Lanier, Dr. Toshiyuki Takai, and Dr. Jianmei Leavenworth for reagents and mice. We thank the UMMS flow cytometry core for help with cell sorting and the UMMS genomics core for help with the microarray. We thank all members of the Swain Dutton laboratory for helpful discussions throughout.

Footnotes

  • This work was supported by National Institutes of Health Grants P01AI046539 and R01AI118820 (to S.L.S.), U19AI109858 (to Dr. Raymond M. Welsh), and T32-AI 007349 (to A.M.V.).

  • The online version of this article contains supplemental material.

  • Abbreviations used in this article:

    A/PR8
    A/Puerto Rico/8/34
    A/PR8-OVAII
    A/Puerto Rico/8/34-OVA323–339
    B6
    C57BL/6
    BAL
    bronchioalveolar lavage
    CKO
    conditional knock out
    dLN
    draining mediastinal lymph node
    dpi
    day postinfection
    GrB
    granzyme B
    HA
    hemagglutinin
    IAV
    influenza A virus
    i.n.
    intranasally
    KO
    knockout
    LCMV
    lymphocytic choriomeningitis virus
    MHC-II
    MHC class II
    NP
    nucleoprotein
    SLO
    secondary lymphoid organ
    TFH
    T follicular helper
    Tg
    transgenic
    UMMS
    University of Massachusetts Medical School
    WT
    wild type.

  • Received July 26, 2016.
  • Accepted December 5, 2016.

References

  1. ↵
    1. Choi Y. S.,
    2. R. Kageyama,
    3. D. Eto,
    4. T. C. Escobar,
    5. R. J. Johnston,
    6. L. Monticelli,
    7. C. Lao,
    8. S. Crotty
    . 2011. ICOS receptor instructs T follicular helper cell versus effector cell differentiation via induction of the transcriptional repressor Bcl6. Immunity 34: 932–946.
    OpenUrlCrossRefPubMed
  2. ↵
    1. Johnston R. J.,
    2. Y. S. Choi,
    3. J. A. Diamond,
    4. J. A. Yang,
    5. S. Crotty
    . 2012. STAT5 is a potent negative regulator of TFH cell differentiation. J. Exp. Med. 209: 243–250.
    OpenUrlAbstract/FREE Full Text
    1. Harker J. A.,
    2. G. M. Lewis,
    3. L. Mack,
    4. E. I. Zuniga
    . 2011. Late interleukin-6 escalates T follicular helper cell responses and controls a chronic viral infection. Science 334: 825–829.
    OpenUrlAbstract/FREE Full Text
  4. ↵
    1. Nurieva R. I.,
    2. Y. Chung,
    3. D. Hwang,
    4. X. O. Yang,
    5. H. S. Kang,
    6. L. Ma,
    7. Y. H. Wang,
    8. S. S. Watowich,
    9. A. M. Jetten,
    10. Q. Tian,
    11. C. Dong
    . 2008. Generation of T follicular helper cells is mediated by interleukin-21 but independent of T helper 1, 2, or 17 cell lineages. Immunity 29: 138–149.
    OpenUrlCrossRefPubMed
  5. ↵
    1. Marshall N. B.,
    2. S. L. Swain
    . 2011. Cytotoxic CD4 T cells in antiviral immunity. J. Biomed. Biotechnol. 2011: 954602.
    OpenUrlCrossRefPubMed
  6. ↵
    1. Pepper M.,
    2. A. J. Pagán,
    3. B. Z. Igyártó,
    4. J. J. Taylor,
    5. M. K. Jenkins
    . 2011. Opposing signals from the Bcl6 transcription factor and the interleukin-2 receptor generate T helper 1 central and effector memory cells. Immunity 35: 583–595.
    OpenUrlCrossRefPubMed
  7. ↵
    1. Johnston R. J.,
    2. A. C. Poholek,
    3. D. DiToro,
    4. I. Yusuf,
    5. D. Eto,
    6. B. Barnett,
    7. A. L. Dent,
    8. J. Craft,
    9. S. Crotty
    . 2009. Bcl6 and Blimp-1 are reciprocal and antagonistic regulators of T follicular helper cell differentiation. Science 325: 1006–1010.
    OpenUrlAbstract/FREE Full Text
  8. ↵
    1. Nurieva R. I.,
    2. Y. Chung,
    3. G. J. Martinez,
    4. X. O. Yang,
    5. S. Tanaka,
    6. T. D. Matskevitch,
    7. Y. H. Wang,
    8. C. Dong
    . 2009. Bcl6 mediates the development of T follicular helper cells. Science 325: 1001–1005.
    OpenUrlAbstract/FREE Full Text
  9. ↵
    1. Korn T.,
    2. E. Bettelli,
    3. M. Oukka,
    4. V. K. Kuchroo
    . 2009. IL-17 and Th17 cells. Annu. Rev. Immunol. 27: 485–517.
    OpenUrlCrossRefPubMed
  10. ↵
    1. Jellison E. R.,
    2. S. K. Kim,
    3. R. M. Welsh
    . 2005. Cutting edge: MHC class II-restricted killing in vivo during viral infection. J. Immunol. 174: 614–618.
    OpenUrlAbstract/FREE Full Text
  11. ↵
    1. Fang M.,
    2. N. A. Siciliano,
    3. A. R. Hersperger,
    4. F. Roscoe,
    5. A. Hu,
    6. X. Ma,
    7. A. R. Shamsedeen,
    8. L. C. Eisenlohr,
    9. L. J. Sigal
    . 2012. Perforin-dependent CD4+ T-cell cytotoxicity contributes to control a murine poxvirus infection. Proc. Natl. Acad. Sci. USA 109: 9983–9988.
    OpenUrlAbstract/FREE Full Text
  12. ↵
    1. Stuller K. A.,
    2. E. Flaño
    . 2009. CD4 T cells mediate killing during persistent gammaherpesvirus 68 infection. J. Virol. 83: 4700–4703.
    OpenUrlAbstract/FREE Full Text
  13. ↵
    1. Verma S.,
    2. D. Weiskopf,
    3. A. Gupta,
    4. B. McDonald,
    5. B. Peters,
    6. A. Sette,
    7. C. A. Benedict
    . 2015. Cytomegalovirus-specific CD4 T cells are cytolytic and mediate vaccine protection. J. Virol. 90: 650–658.
    OpenUrl
  14. ↵
    1. Brown D. M.,
    2. S. Lee,
    3. Mde. L. Garcia-Hernandez,
    4. S. L. Swain
    . 2012. Multifunctional CD4 cells expressing gamma interferon and perforin mediate protection against lethal influenza virus infection. J. Virol. 86: 6792–6803.
    OpenUrlAbstract/FREE Full Text
  15. ↵
    1. Brown D. M.,
    2. A. M. Dilzer,
    3. D. L. Meents,
    4. S. L. Swain
    . 2006. CD4 T cell-mediated protection from lethal influenza: perforin and antibody-mediated mechanisms give a one-two punch. J. Immunol. 177: 2888–2898.
    OpenUrlAbstract/FREE Full Text
  16. ↵
    1. Hua L.,
    2. S. Yao,
    3. D. Pham,
    4. L. Jiang,
    5. J. Wright,
    6. D. Sawant,
    7. A. L. Dent,
    8. T. J. Braciale,
    9. M. H. Kaplan,
    10. J. Sun
    . 2013. Cytokine-dependent induction of CD4+ T cells with cytotoxic potential during influenza virus infection. J. Virol. 87: 11884–11893.
    OpenUrlAbstract/FREE Full Text
  17. ↵
    1. McKinstry K. K.,
    2. T. M. Strutt,
    3. Y. Kuang,
    4. D. M. Brown,
    5. S. Sell,
    6. R. W. Dutton,
    7. S. L. Swain
    . 2012. Memory CD4+ T cells protect against influenza through multiple synergizing mechanisms. J. Clin. Invest. 122: 2847–2856.
    OpenUrlCrossRefPubMed
  18. ↵
    1. Wilkinson T. M.,
    2. C. K. Li,
    3. C. S. Chui,
    4. A. K. Huang,
    5. M. Perkins,
    6. J. C. Liebner,
    7. R. Lambkin-Williams,
    8. A. Gilbert,
    9. J. Oxford,
    10. B. Nicholas,
    11. et al
    . 2012. Preexisting influenza-specific CD4+ T cells correlate with disease protection against influenza challenge in humans. Nat. Med. 18: 274–280.
    OpenUrlCrossRefPubMed
  19. ↵
    1. Soghoian D. Z.,
    2. H. Jessen,
    3. M. Flanders,
    4. K. Sierra-Davidson,
    5. S. Cutler,
    6. T. Pertel,
    7. S. Ranasinghe,
    8. M. Lindqvist,
    9. I. Davis,
    10. K. Lane,
    11. et al
    . 2012. HIV-specific cytolytic CD4 T cell responses during acute HIV infection predict disease outcome. Sci. Transl. Med. 4: 123ra25.
    OpenUrlAbstract/FREE Full Text
  20. ↵
    1. Brown D. M.,
    2. C. Kamperschroer,
    3. A. M. Dilzer,
    4. D. M. Roberts,
    5. S. L. Swain
    . 2009. IL-2 and antigen dose differentially regulate perforin- and FasL-mediated cytolytic activity in antigen specific CD4+ T cells. Cell. Immunol. 257: 69–79.
    OpenUrlCrossRefPubMed
  21. ↵
    1. Curran M. A.,
    2. T. L. Geiger,
    3. W. Montalvo,
    4. M. Kim,
    5. S. L. Reiner,
    6. A. Al-Shamkhani,
    7. J. C. Sun,
    8. J. P. Allison
    . 2013. Systemic 4-1BB activation induces a novel T cell phenotype driven by high expression of Eomesodermin. J. Exp. Med. 210: 743–755.
    OpenUrlAbstract/FREE Full Text
  22. ↵
    1. Hirschhorn-Cymerman D.,
    2. S. Budhu,
    3. S. Kitano,
    4. C. Liu,
    5. F. Zhao,
    6. H. Zhong,
    7. A. M. Lesokhin,
    8. F. Avogadri-Connors,
    9. J. Yuan,
    10. Y. Li,
    11. et al
    . 2012. Induction of tumoricidal function in CD4+ T cells is associated with concomitant memory and terminally differentiated phenotype. J. Exp. Med. 209: 2113–2126.
    OpenUrlAbstract/FREE Full Text
  23. ↵
    1. Qui H. Z.,
    2. A. T. Hagymasi,
    3. S. Bandyopadhyay,
    4. M.-C. St Rose,
    5. R. Ramanarasimhaiah,
    6. A. Ménoret,
    7. R. S. Mittler,
    8. S. M. Gordon,
    9. S. L. Reiner,
    10. A. T. Vella,
    11. A. J. Adler
    . 2011. CD134 plus CD137 dual costimulation induces Eomesodermin in CD4 T cells to program cytotoxic Th1 differentiation. J. Immunol. 187: 3555–3564.
    OpenUrlAbstract/FREE Full Text
  24. ↵
    1. Petersen J. L.,
    2. C. R. Morris,
    3. J. C. Solheim
    . 2003. Virus evasion of MHC class I molecule presentation. J. Immunol. 171: 4473–4478.
    OpenUrlFREE Full Text
  25. ↵
    1. Eichelberger M.,
    2. W. Allan,
    3. M. Zijlstra,
    4. R. Jaenisch,
    5. P. C. Doherty
    . 1991. Clearance of influenza virus respiratory infection in mice lacking class I major histocompatibility complex-restricted CD8+ T cells. J. Exp. Med. 174: 875–880.
    OpenUrlAbstract/FREE Full Text
  26. ↵
    1. Muller D.,
    2. B. H. Koller,
    3. J. L. Whitton,
    4. K. E. LaPan,
    5. K. K. Brigman,
    6. J. A. Frelinger
    . 1992. LCMV-specific, class II-restricted cytotoxic T cells in beta 2-microglobulin-deficient mice. Science 255: 1576–1578.
    OpenUrlAbstract/FREE Full Text
  27. ↵
    1. Hou S.,
    2. P. C. Doherty,
    3. M. Zijlstra,
    4. R. Jaenisch,
    5. J. M. Katz
    . 1992. Delayed clearance of Sendai virus in mice lacking class I MHC-restricted CD8+ T cells. J. Immunol. 149: 1319–1325.
    OpenUrlAbstract
  28. ↵
    1. Freeman M. L.,
    2. C. E. Burkum,
    3. T. Cookenham,
    4. A. D. Roberts,
    5. K. G. Lanzer,
    6. G. E. Huston,
    7. M. K. Jensen,
    8. J. Sidney,
    9. B. Peters,
    10. J. E. Kohlmeier,
    11. et al
    . 2014. CD4 T cells specific for a latency-associated γ-herpesvirus epitope are polyfunctional and cytotoxic. J. Immunol. 193: 5827–5834.
    OpenUrlAbstract/FREE Full Text
  29. ↵
    1. Topham D. J.,
    2. P. C. Doherty
    . 1998. Clearance of an influenza A virus by CD4+ T cells is inefficient in the absence of B cells. J. Virol. 72: 882–885.
    OpenUrlAbstract/FREE Full Text
  30. ↵
    1. Mozdzanowska K.,
    2. M. Furchner,
    3. K. Maiese,
    4. W. Gerhard
    . 1997. CD4+ T cells are ineffective in clearing a pulmonary infection with influenza type A virus in the absence of B cells. Virology 239: 217–225.
    OpenUrlCrossRefPubMed
  31. ↵
    1. Swain S. L.,
    2. K. K. McKinstry,
    3. T. M. Strutt
    . 2012. Expanding roles for CD4+ T cells in immunity to viruses. Nat. Rev. Immunol. 12: 136–148.
    OpenUrlCrossRefPubMed
  32. ↵
    1. Afonina I. S.,
    2. S. P. Cullen,
    3. S. J. Martin
    . 2010. Cytotoxic and non-cytotoxic roles of the CTL/NK protease granzyme B. Immunol. Rev. 235: 105–116.
    OpenUrlCrossRefPubMed
  33. ↵
    1. Wolint P.,
    2. M. R. Betts,
    3. R. A. Koup,
    4. A. Oxenius
    . 2004. Immediate cytotoxicity but not degranulation distinguishes effector and memory subsets of CD8+ T cells. J. Exp. Med. 199: 925–936.
    OpenUrlAbstract/FREE Full Text
  34. ↵
    1. Rapaport A. S.,
    2. J. Schriewer,
    3. S. Gilfillan,
    4. E. Hembrador,
    5. R. Crump,
    6. B. F. Plougastel,
    7. Y. Wang,
    8. G. Le Friec,
    9. J. Gao,
    10. M. Cella,
    11. et al
    . 2015. The inhibitory receptor NKG2A sustains virus-specific CD8+ T cells in response to a lethal poxvirus infection. Immunity 43: 1112–1124.
    OpenUrl
  35. ↵
    1. Vance R. E.,
    2. A. M. Jamieson,
    3. D. H. Raulet
    . 1999. Recognition of the class Ib molecule Qa-1(b) by putative activating receptors CD94/NKG2C and CD94/NKG2E on mouse natural killer cells. J. Exp. Med. 190: 1801–1812.
    OpenUrlAbstract/FREE Full Text
  36. ↵
    1. Shin H. M.,
    2. V. N. Kapoor,
    3. T. Guan,
    4. S. M. Kaech,
    5. R. M. Welsh,
    6. L. J. Berg
    . 2013. Epigenetic modifications induced by Blimp-1 regulate CD8+ T cell memory progression during acute virus infection. Immunity 39: 661–675.
    OpenUrlCrossRefPubMed
  37. ↵
    1. Crowe S. R.,
    2. S. C. Miller,
    3. D. M. Brown,
    4. P. S. Adams,
    5. R. W. Dutton,
    6. A. G. Harmsen,
    7. F. E. Lund,
    8. T. D. Randall,
    9. S. L. Swain,
    10. D. L. Woodland
    . 2006. Uneven distribution of MHC class II epitopes within the influenza virus. Vaccine 24: 457–467.
    OpenUrlCrossRefPubMed
  38. ↵
    1. McKinstry K. K.,
    2. T. M. Strutt,
    3. B. Bautista,
    4. W. Zhang,
    5. Y. Kuang,
    6. A. M. Cooper,
    7. S. L. Swain
    . 2014. Effector CD4 T-cell transition to memory requires late cognate interactions that induce autocrine IL-2. Nat. Commun. 5: 5377.
    OpenUrlCrossRefPubMed
  39. ↵
    1. Haddad W.,
    2. C. J. Cooper,
    3. Z. Zhang,
    4. J. B. Brown,
    5. Y. Zhu,
    6. A. Issekutz,
    7. I. Fuss,
    8. H. O. Lee,
    9. G. S. Kansas,
    10. T. A. Barrett
    . 2003. P-selectin and P-selectin glycoprotein ligand 1 are major determinants for Th1 cell recruitment to nonlymphoid effector sites in the intestinal lamina propria. J. Exp. Med. 198: 369–377.
    OpenUrlAbstract/FREE Full Text
  40. ↵
    1. Zaguia F.,
    2. P. Saikali,
    3. S. Ludwin,
    4. J. Newcombe,
    5. D. Beauseigle,
    6. E. McCrea,
    7. P. Duquette,
    8. A. Prat,
    9. J. P. Antel,
    10. N. Arbour
    . 2013. Cytotoxic NKG2C+ CD4 T cells target oligodendrocytes in multiple sclerosis. J. Immunol. 190: 2510–2518.
    OpenUrlAbstract/FREE Full Text
  41. ↵
    1. Meyers J. H.,
    2. A. Ryu,
    3. L. Monney,
    4. K. Nguyen,
    5. E. A. Greenfield,
    6. G. J. Freeman,
    7. V. K. Kuchroo
    . 2002. Cutting edge: CD94/NKG2 is expressed on Th1 but not Th2 cells and costimulates Th1 effector functions. J. Immunol. 169: 5382–5386.
    OpenUrlAbstract/FREE Full Text
  42. ↵
    1. Kallies A.,
    2. A. Xin,
    3. G. T. Belz,
    4. S. L. Nutt
    . 2009. Blimp-1 transcription factor is required for the differentiation of effector CD8(+) T cells and memory responses. Immunity 31: 283–295.
    OpenUrlCrossRefPubMed
  43. ↵
    1. Scott B.,
    2. R. Liblau,
    3. S. Degermann,
    4. L. A. Marconi,
    5. L. Ogata,
    6. A. J. Caton,
    7. H. O. McDevitt,
    8. D. Lo
    . 1994. A role for non-MHC genetic polymorphism in susceptibility to spontaneous autoimmunity. Immunity 1: 73–83.
    OpenUrlCrossRefPubMed
  44. ↵
    1. Vance R. E.,
    2. J. R. Kraft,
    3. J. D. Altman,
    4. P. E. Jensen,
    5. D. H. Raulet
    . 1998. Mouse CD94/NKG2A is a natural killer cell receptor for the nonclassical major histocompatibility complex (MHC) class I molecule Qa-1(b). J. Exp. Med. 188: 1841–1848.
    OpenUrlAbstract/FREE Full Text
  45. ↵
    1. Lu L.,
    2. K. Ikizawa,
    3. D. Hu,
    4. M. B. F. Werneck,
    5. K. W. Wucherpfennig,
    6. H. Cantor
    . 2007. Regulation of activated CD4+ T cells by NK cells via the Qa-1-NKG2A inhibitory pathway. Immunity 26: 593–604.
    OpenUrlCrossRefPubMed
  46. ↵
    1. Moser J. M.,
    2. J. Gibbs,
    3. P. E. Jensen,
    4. A. E. Lukacher
    . 2002. CD94-NKG2A receptors regulate antiviral CD8(+) T cell responses. Nat. Immunol. 3: 189–195.
    OpenUrlCrossRefPubMed
  47. ↵
    1. Saether P. C.,
    2. S. E. Hoelsbrekken,
    3. S. Fossum,
    4. E. Dissen
    . 2011. Rat and mouse CD94 associate directly with the activating transmembrane adaptor proteins DAP12 and DAP10 and activate NK cell cytotoxicity. J. Immunol. 187: 6365–6373.
    OpenUrlAbstract/FREE Full Text
  48. ↵
    1. Inui M.,
    2. Y. Kikuchi,
    3. N. Aoki,
    4. S. Endo,
    5. T. Maeda,
    6. A. Sugahara-Tobinai,
    7. S. Fujimura,
    8. A. Nakamura,
    9. A. Kumanogoh,
    10. M. Colonna,
    11. T. Takai
    . 2009. Signal adaptor DAP10 associates with MDL-1 and triggers osteoclastogenesis in cooperation with DAP12. Proc. Natl. Acad. Sci. USA 106: 4816–4821.
    OpenUrlAbstract/FREE Full Text
  49. ↵
    1. Strutt T. M.,
    2. K. K. McKinstry,
    3. Y. Kuang,
    4. L. M. Bradley,
    5. S. L. Swain
    . 2012. Memory CD4+ T-cell-mediated protection depends on secondary effectors that are distinct from and superior to primary effectors. Proc. Natl. Acad. Sci. USA 109: E2551–E2560.
    OpenUrlAbstract/FREE Full Text
  50. ↵
    1. Román E.,
    2. E. Miller,
    3. A. Harmsen,
    4. J. Wiley,
    5. U. H. Von Andrian,
    6. G. Huston,
    7. S. L. Swain
    . 2002. CD4 effector T cell subsets in the response to influenza: heterogeneity, migration, and function. J. Exp. Med. 196: 957–968.
    OpenUrlAbstract/FREE Full Text
  51. ↵
    1. Yusuf I.,
    2. R. Kageyama,
    3. L. Monticelli,
    4. R. J. Johnston,
    5. D. Ditoro,
    6. K. Hansen,
    7. B. Barnett,
    8. S. Crotty
    . 2010. Germinal center T follicular helper cell IL-4 production is dependent on signaling lymphocytic activation molecule receptor (CD150). J. Immunol. 185: 190–202.
    OpenUrlAbstract/FREE Full Text
  52. ↵
    1. Kohlmeier J. E.,
    2. S. C. Miller,
    3. J. Smith,
    4. B. Lu,
    5. C. Gerard,
    6. T. Cookenham,
    7. A. D. Roberts,
    8. D. L. Woodland
    . 2008. The chemokine receptor CCR5 plays a key role in the early memory CD8+ T cell response to respiratory virus infections. Immunity 29: 101–113.
    OpenUrlCrossRefPubMed
  53. ↵
    1. Kohlmeier J. E.,
    2. T. Cookenham,
    3. S. C. Miller,
    4. A. D. Roberts,
    5. J. P. Christensen,
    6. A. R. Thomsen,
    7. D. L. Woodland
    . 2009. CXCR3 directs antigen-specific effector CD4+ T cell migration to the lung during parainfluenza virus infection. J. Immunol. 183: 4378–4384.
    OpenUrlAbstract/FREE Full Text
  54. ↵
    1. Kim C. H.,
    2. E. J. Kunkel,
    3. J. Boisvert,
    4. B. Johnston,
    5. J. J. Campbell,
    6. M. C. Genovese,
    7. H. B. Greenberg,
    8. E. C. Butcher
    . 2001. Bonzo/CXCR6 expression defines type 1-polarized T-cell subsets with extralymphoid tissue homing potential. J. Clin. Invest. 107: 595–601.
    OpenUrlCrossRefPubMed
  55. ↵
    1. Cao X.,
    2. S. F. Cai,
    3. T. A. Fehniger,
    4. J. Song,
    5. L. I. Collins,
    6. D. R. Piwnica-Worms,
    7. T. J. Ley
    . 2007. Granzyme B and perforin are important for regulatory T cell-mediated suppression of tumor clearance. Immunity 27: 635–646.
    OpenUrlCrossRefPubMed
  56. ↵
    1. Yao Y.,
    2. J. Vent-Schmidt,
    3. M. D. McGeough,
    4. M. Wong,
    5. H. M. Hoffman,
    6. T. S. Steiner,
    7. M. K. Levings
    . 2015. Tr1 cells, but not Foxp3+ regulatory T cells, suppress NLRP3 inflammasome activation via an IL-10-dependent mechanism. J. Immunol. 195: 488–497.
    OpenUrlAbstract/FREE Full Text
  57. ↵
    1. Gagliani N.,
    2. C. F. Magnani,
    3. S. Huber,
    4. M. E. Gianolini,
    5. M. Pala,
    6. P. Licona-Limon,
    7. B. Guo,
    8. D. R. Herbert,
    9. A. Bulfone,
    10. F. Trentini,
    11. et al
    . 2013. Coexpression of CD49b and LAG-3 identifies human and mouse T regulatory type 1 cells. Nat. Med. 19: 739–746.
    OpenUrlCrossRefPubMed
  58. ↵
    1. McKinstry K. K.,
    2. S. Golech,
    3. W. H. Lee,
    4. G. Huston,
    5. N. P. Weng,
    6. S. L. Swain
    . 2007. Rapid default transition of CD4 T cell effectors to functional memory cells. J. Exp. Med. 204: 2199–2211.
    OpenUrlAbstract/FREE Full Text
  59. ↵
    1. Bautista B. L.,
    2. P. Devarajan,
    3. K. K. McKinstry,
    4. T. M. Strutt,
    5. A. M. Vong,
    6. M. C. Jones,
    7. Y. Kuang,
    8. D. Mott,
    9. S. L. Swain
    . 2016. Short-lived antigen recognition but not viral infection at a defined checkpoint programs effector CD4 T cells to become protective memory. J. Immunol. 197: 3936–3949.
    OpenUrlAbstract/FREE Full Text
  60. ↵
    1. Agrewala J. N.,
    2. D. M. Brown,
    3. N. M. Lepak,
    4. D. Duso,
    5. G. Huston,
    6. S. L. Swain
    . 2007. Unique ability of activated CD4+ T cells but not rested effectors to migrate to non-lymphoid sites in the absence of inflammation. J. Biol. Chem. 282: 6106–6115.
    OpenUrlAbstract/FREE Full Text
  61. ↵
    1. Masopust D.,
    2. V. Vezys,
    3. E. J. Usherwood,
    4. L. S. Cauley,
    5. S. Olson,
    6. A. L. Marzo,
    7. R. L. Ward,
    8. D. L. Woodland,
    9. L. Lefrançois
    . 2004. Activated primary and memory CD8 T cells migrate to nonlymphoid tissues regardless of site of activation or tissue of origin. J. Immunol. 172: 4875–4882.
    OpenUrlAbstract/FREE Full Text
  62. ↵
    1. Masopust D.,
    2. V. Vezys,
    3. A. L. Marzo,
    4. L. Lefrançois
    . 2001. Preferential localization of effector memory cells in nonlymphoid tissue. Science 291: 2413–2417.
    OpenUrlAbstract/FREE Full Text
  63. ↵
    1. Marshall D. R.,
    2. S. J. Turner,
    3. G. T. Belz,
    4. S. Wingo,
    5. S. Andreansky,
    6. M. Y. Sangster,
    7. J. M. Riberdy,
    8. T. Liu,
    9. M. Tan,
    10. P. C. Doherty
    . 2001. Measuring the diaspora for virus-specific CD8+ T cells. Proc. Natl. Acad. Sci. USA 98: 6313–6318.
    OpenUrlAbstract/FREE Full Text
  64. ↵
    1. Helft J.,
    2. B. Manicassamy,
    3. P. Guermonprez,
    4. D. Hashimoto,
    5. A. Silvin,
    6. J. Agudo,
    7. B. D. Brown,
    8. M. Schmolke,
    9. J. C. Miller,
    10. M. Leboeuf,
    11. et al
    . 2012. Cross-presenting CD103+ dendritic cells are protected from influenza virus infection. J. Clin. Invest. 122: 4037–4047.
    OpenUrlCrossRefPubMed
  65. ↵
    1. Turner D. L.,
    2. K. L. Bickham,
    3. J. J. Thome,
    4. C. Y. Kim,
    5. F. D’Ovidio,
    6. E. J. Wherry,
    7. D. L. Farber
    . 2014. Lung niches for the generation and maintenance of tissue-resident memory T cells. Mucosal Immunol. 7: 501–510.
    OpenUrlCrossRefPubMed
  66. ↵
    1. Olson M. R.,
    2. D. S. McDermott,
    3. S. M. Varga
    . 2012. The initial draining lymph node primes the bulk of the CD8 T cell response and influences memory T cell trafficking after a systemic viral infection. PLoS Pathog. 8: e1003054.
    OpenUrlCrossRefPubMed
  67. ↵
    1. Mucida D.,
    2. M. M. Husain,
    3. S. Muroi,
    4. F. van Wijk,
    5. R. Shinnakasu,
    6. Y. Naoe,
    7. B. S. Reis,
    8. Y. Huang,
    9. F. Lambolez,
    10. M. Docherty,
    11. et al
    . 2013. Transcriptional reprogramming of mature CD4+ helper T cells generates distinct MHC class II-restricted cytotoxic T lymphocytes. Nat. Immunol. 14: 281–289.
    OpenUrlCrossRefPubMed
  68. ↵
    1. Cannarile M. A.,
    2. N. A. Lind,
    3. R. Rivera,
    4. A. D. Sheridan,
    5. K. A. Camfield,
    6. B. B. Wu,
    7. K. P. Cheung,
    8. Z. Ding,
    9. A. W. Goldrath
    . 2006. Transcriptional regulator Id2 mediates CD8+ T cell immunity. Nat. Immunol. 7: 1317–1325.
    OpenUrlCrossRefPubMed
  69. ↵
    1. Yang C. Y.,
    2. J. A. Best,
    3. J. Knell,
    4. E. Yang,
    5. A. D. Sheridan,
    6. A. K. Jesionek,
    7. H. S. Li,
    8. R. R. Rivera,
    9. K. C. Lind,
    10. L. M. D’Cruz,
    11. et al
    . 2011. The transcriptional regulators Id2 and Id3 control the formation of distinct memory CD8+ T cell subsets. Nat. Immunol. 12: 1221–1229.
    OpenUrlCrossRefPubMed
  70. ↵
    1. Carlson C. M.,
    2. B. T. Endrizzi,
    3. J. Wu,
    4. X. Ding,
    5. M. A. Weinreich,
    6. E. R. Walsh,
    7. M. A. Wani,
    8. J. B. Lingrel,
    9. K. A. Hogquist,
    10. S. C. Jameson
    . 2006. Kruppel-like factor 2 regulates thymocyte and T-cell migration. Nature 442: 299–302.
    OpenUrlCrossRefPubMed
    1. Skon C. N.,
    2. J.-Y. Lee,
    3. K. G. Anderson,
    4. D. Masopust,
    5. K. A. Hogquist,
    6. S. C. Jameson
    . 2013. Transcriptional downregulation of S1pr1 is required for the establishment of resident memory CD8+ T cells. Nat. Immunol. 14: 1285–1293.
    OpenUrlCrossRefPubMed
  72. ↵
    1. Lee J.-Y.,
    2. C. N. Skon,
    3. Y. J. Lee,
    4. S. Oh,
    5. J. J. Taylor,
    6. D. Malhotra,
    7. M. K. Jenkins,
    8. M. G. Rosenfeld,
    9. K. A. Hogquist,
    10. S. C. Jameson
    . 2015. The transcription factor KLF2 restrains CD4+ T follicular helper cell differentiation. Immunity 42: 252–264.
    OpenUrlCrossRefPubMed
  73. ↵
    1. Zhou X.,
    2. S. Yu,
    3. D.-M. Zhao,
    4. J. T. Harty,
    5. V. P. Badovinac,
    6. H.-H. Xue
    . 2010. Differentiation and persistence of memory CD8(+) T cells depend on T cell factor 1. Immunity 33: 229–240.
    OpenUrlCrossRefPubMed
  74. ↵
    1. Weiskopf D.,
    2. D. J. Bangs,
    3. J. Sidney,
    4. R. V. Kolla,
    5. A. D. De Silva,
    6. A. M. de Silva,
    7. S. Crotty,
    8. B. Peters,
    9. A. Sette
    . 2015. Dengue virus infection elicits highly polarized CX3CR1+ cytotoxic CD4+ T cells associated with protective immunity. Proc. Natl. Acad. Sci. USA 112: E4256–E4263.
    OpenUrlAbstract/FREE Full Text
  75. ↵
    1. Workman A. M.,
    2. A. K. Jacobs,
    3. A. J. Vogel,
    4. S. Condon,
    5. D. M. Brown
    . 2014. Inflammation enhances IL-2 driven differentiation of cytolytic CD4 T cells. PLoS One 9: e89010.
    OpenUrlCrossRefPubMed
  76. ↵
    1. Crotty S.
    2014. T follicular helper cell differentiation, function, and roles in disease. Immunity 41: 529–542.
    OpenUrlCrossRefPubMed
  77. ↵
    1. Hansen S. G.,
    2. H. L. Wu,
    3. B. J. Burwitz,
    4. C. M. Hughes,
    5. K. B. Hammond,
    6. A. B. Ventura,
    7. J. S. Reed,
    8. R. M. Gilbride,
    9. E. Ainslie,
    10. D. W. Morrow,
    11. et al
    . 2016. Broadly targeted CD8+ T cell responses restricted by major histocompatibility complex E. Science 351: 714–720.
    OpenUrlAbstract/FREE Full Text
Back to top

In this issue

Print
Download PDF
Article Alerts
Email Article
Citation Tools

Jump to section