Short-lived Antigen Recognition but not Viral Infection at a Defined Checkpoint Programs Effector CD4 T Cells to become Protective Memory

Abstract

While memory CD4 T cells are critical for effective immunity to pathogens, the mechanisms underlying their generation are still poorly defined. We find that following murine influenza infection, most effector CD4 T cells undergo apoptosis unless they encounter cognate Ag at a defined stage near the peak of effector generation. Ag recognition at this “memory checkpoint” blocks default apoptosis and programs their transition to long-lived memory. Strikingly, we find that viral infection is not required, as memory formation can be restored by the addition of short-lived, Ag-pulsed APC at this checkpoint. The resulting memory CD4 T cells express an enhanced memory phenotype, have increased cytokine production, and provide protection against lethal influenza infection. Finally, we find that memory CD4 T cell formation following cold-adapted influenza vaccination is boosted when Ag is administered during this checkpoint. These findings imply that persistence of viral Ag presentation into the effector phase is the key factor that determines the efficiency of memory generation. We also suggest that administering Ag at this checkpoint may improve vaccine efficacy.

Introduction

While much progress has been made in defining the early activation events required for the generation of effector CD4 T cell subsets, the pathways that drive a cohort of effector T cells to successfully transition to a memory state remain poorly defined. It is unclear to what extent programming during initial cognate interaction of T cells with antigen-presenting cells (APC) determines the fate of effector T cells and if later signals affect memory generation.

Various models defining the role of antigen (Ag) in effector and memory differentiation have been proposed. Some suggest that the initial interaction with Ag/APC is sufficient to program a cohort of T cells to become memory and further exposure to Ag and inflammation drive terminal differentiation of effector T cells (1–5). In contrast, other studies suggest that late Ag enhances the function but not the number of memory CD8 T cells (6, 7). It has been shown that CD4 T cells require more Ag stimulation for effector and memory generation than do CD8 T cells, but most of these analyses have been limited to the priming phase of the response (8–11). Other studies have concluded that while prolonged Ag stimulation can enhance effector CD4 T cell proliferation, it is deleterious to memory formation (12), and continuous Ag stimulation may drive CD4 T cells to a state of reduced responsiveness (13, 14). In vivo, responding T cells disengage from APC 24 h after initial interaction, engaging in few APC contacts during the last phase of priming (9, 15). Thus, it remains unclear how often responding CD4 T cells encounter Ag after the initial priming phase of the response and if later Ag exposure impacts memory generation.

During a response to a live pathogen, it would be advantageous for the quality and quantity of the effector and memory response to be determined at the effector stage when the immune system could sense whether there is still a threat. In earlier studies, we found that in vitro-generated effector CD4 T cells are programmed to undergo activation-induced cell death, but can be rescued by addition of IL-2 and TGFβ (16). In an in vivo model of influenza A virus (IAV) infection, we recently found that autocrine IL-2 production by effector CD4 T cells during a defined memory checkpoint (days 5–7) of the response was essential to promote survival and memory formation (17). These findings help explain studies with lymphocytic choriomeningitis virus, where IL-2 complexes added late in the response promoted memory formation (18). Since IL-2 production is typically induced by cognate Ag recognition, we investigate here whether the interaction of effector CD4 T cells with APC during this checkpoint is the key event that drives them to make IL-2, to survive, and to differentiate into long-lasting memory cells. A defined stage of effector CD4 T cell development, where CD4 effector fate is determined by cognate Ag interaction, would suggest a new paradigm in which the formation of memory depends on a cohort of cells being selected by persisting Ag to become memory cells. If Ag presentation during the effector phase determines memory, it might explain why most vaccines lacking live organisms induce much less durable immunity than infection. Here we ask whether late recognition of Ag on APC is the signal that initiates the transition of CD4 effectors to memory cells at this “memory checkpoint” and we define the key parameters that are involved.

In most in vivo studies heretofore, it has not been possible to define the necessary timing and duration of the signals needed for the rescue of effectors from apoptosis and exessive contraction. Additionally, as T cell reach the effector stage, the roles that ongoing infection play in promoting memory have not been definitively examined in an in vivo model of infection. Defining these elements is critical for rational vaccine design.

To address these gaps in our understanding, we use a well-defined model of IAV infection to determine the role that Ag presentation and ongoing infection, during the effector phase, play in shaping memory CD4 T cell formation. IAV induces a highly protective memory CD4 T cell population that synergizes with B cells and CD8 T cells to provide protection from challenge with supralethal viral doses (19–22). The response thus epitomizes successful memory CD4 T cell generation in response to infection and is therefore well-suited to reveal the mechanisms involved in effective memory generation.

We find that effector CD4 T cells, induced by IAV infection, require cognate Ag recognition at 6 days post infection (dpi) for continued expansion, survival, and all but a minor fraction of memory generation. In well-controlled adoptive transfer models, we find that Ag/APC encounter at the effector stage (6 dpi) enhances the recovery of memory cells in secondary lymphoid organs (SLO) and in the lung at least 10 to 100-fold. Notably, other infection-induced effects, such as inflammation, are not required for this increased memory generation. Effector T cells, exposed to Ag/APC for as little as 2 d, expressed higher levels of memory-associated molecules CD25 (IL-2Rα), B cell lymphoma 6 (Bcl-6), CD127 (IL-7Rα), and CXCR3. The memory cells generated by Ag encounter between 6–8 dpi had enhanced ability to make cytokines and provided better protection against a lethal dose of IAV than those that were not exposed to checkpoint Ag. Moreover, in a cold-adapted vaccine model, we found very little Ag presentation during this late checkpoint, but when additional Ag/APC were introduced at this time, memory CD4 T cell formation was enhanced. This suggests that low levels of Ag presentation from 6–8 dpi may limit vaccine efficacy of vaccines that do not provide high levels of persisting Ag. These findings imply that whether pathogen infection persists into the effector stage determines effector fate by supplying late Ag/APC that are needed to program memory formation and that interventions to achieve this need not involve long-lived infection and its potentially deleterious effects.

Materials and Methods

Mice

Naïve CD4 T cells were isolated from OT-II.Thy1.1^+/−, OT-II.Nr4a1^eGFP.Thy1.1^+/−, OT-II.Bcl2l11^+/−, or OT-II.Osb.eGFP mice bred at the UMMS breeding facility. Hosts were B6 male mice ordered from Jackson Laboratories (JAX). Nr4a1^eGFP mice originally obtained from JAX and bred at the UMMS breeding facility were also used. Mice used in experiments were 8–12 wk of age. The Institutional Animal Care and Use Committee of the University of Massachusetts Medical School approved all animal procedures.

Viral Stocks, Infections, and Immunizations

For all influenza viral infections described, mice were lightly anesthetized with isoflurane (Piramal Healthcare) before intranasal infection with 50μL of virus diluted in PBS. Influenza A PR8-OVA_II and PR8 (H1N1) viruses were produced in the allantoic cavity of embryonated hen eggs from stock obtained from Dr. Peter Doherty (PR8-OVA_II) of St. Jude Children’s Hospital (PR8). A sublethal dose of 0.3LD₅₀ was used. Protection experiments were performed using a lethal dose of 2LD₅₀. Cold-adapted, attenuated ca.A/Alaska/72/CR9 (H3N2) was originally supplied by S. Epstein (NIH, Bethesda, MD) then grown at the Trudeau Institute (23). Mice were immunized with 2500 TCID₅₀ ca.Alaska, a dose shown to elicit T cell mediated protection (23). Influenza A/Philippines/2/82/x-79 (H3N2) was supplied by S. Epstein (NIH, Bethesda, MD). Mice were infected with 100 PFU.

Naïve CD4 T cell isolation and effector generation in primary hosts

Spleens and peripheral LNs were harvested from 6–10 wk old TcR transgenic or WT mice. Resting cells were enriched using a percoll gradient. CD4 T cells were then isolated using CD4 MACS beads (Miltenyi). Naive CD4 T cells were washed twice, resuspended in PBS, and a total of 3×10⁵–5×10⁵ cells were transferred by i.v. injection into hosts. Hosts were infected with PR8-OVA_II on the same day.

Isolation of 6 dpi effector CD4 T cells

Spleen and DLN (Lung Draining Lymph Nodes) were harvested from B6 mice on 6 d after PR8-OVA_II infection. Cell suspensions were pooled and donor cells were isolated by either Thy1.1 or CD4 MACS isolation (Miltenyi). Cells were resuspended in PBS and 1–4×10⁶ effector cells were transferred by i.v. injection to hosts. All steps were conducted at RT (except for one 15 m incubation at 4°C) to maintain effector phenotype. This minimal protocol ensures that effector cells are only out of mice for 2.5 h.

Bone Marrow Dendritic Cell preparation

Bone marrow was harvested from B6 mice and washed with RPMI including 1% FBS. Cells were plated at 7–8×10⁶ cells/mL in RPMI with 7.5% FBS including 10ng/ml GMCSF. After 7 d, cells were harvested and CD11c⁺ cells were isolated by MACS. Purified cells were then re-plated at 2×10⁶ cells/ml and stimulated with PolyI:C at 10μg/ml for 1 d in culture and used as dendritic cells (DC). DC were harvested, pulsed with 10μM OVA_II or NP_311-325-peptide at 37°C for 1 h with shaking. Cells were resuspended in PBS and 3–5×10⁵ cells per mouse were injected i.v.

PR8-infected splenic APC preparation and in vitro culture

Spleens from PR8-infected B6 mice were harvested at 6 dpi. Cell suspensions were pooled and washed with RPMI containing 1% FBS. Cells were depleted of Thy1.2⁺ cells using MACS beads. Cells were irradiated with 3000 Rads. This APC population was then co-cultured with isolated 6 dpi effectors at a ratio of 5:1 APC:T-cell. OVA_II or NP_311-325 peptide was added to culture at 0.5μM. IL-7 was added to cultures at 0.1ng/ml (a concentration that does not promote proliferation). All blocking antibodies were used at 10μg/ml.

APC for protection experiment

Spleen cells were harvested from uninfected B6 mice. Thy1.2⁺ cells were depleted using MACS beads. The Thy1.2-depleted fraction was then plated at 3×10⁶ cells/ml in RPMI containing 7.5% FBS and 10ng/ml LPS and 10ng/ml dextran sulfate. After 2 d in culture, these activated APC enriched cells were harvested and pulsed with 10μM OVA_II peptide at 37°C with shaking for 1 h. APC were transferred to hosts with 6 dpi effectors at a ratio of 1:1.

Histology

B6 mice were infected with 0.3LD₅₀ PR8 or PR8-OVA_II. Lungs were harvested at 6 dpi and fixed in 10% buffered formalin. 10μm sections were taken and stained with hematoxylin and eosin (H&E) stain. Lungs were scored as follows: (1) Healthy looking bronchioles with consolidation and mononuclear infiltrates comprising under 5% of the lung. (2) Mild bronchiolitis with consolidation and mononuclear infiltrates comprising over 5% of the lung. (3) Moderate bronchiolitis with consolidation and mononuclear infiltrates comprising equal to or greater than 15% of the lung. (4) Moderate bronchiolitis with consolidation and mononuclear infiltrates comprising equal to or greater than 25% of the lung. (5) Severe bronchiolitis with consolidation and mononuclear infiltrates comprising over 50% of the lung. Scoring was done blind and four sections of each lung were scored and the average is presented.

Viral Titers

Viral titers of PR8 or PR8-OVA_II-infected lungs were determined by quantification of viral RNA. Whole lungs were homogenized in TRIsol/Chloroform (Sigma-Aldrich) and RNA was extracted using the VWR E.Z.N.A kit and Turbo DNA-free kit (Thermofisher). 2.0μg of RNA was reverse transcribed into cDNA using the High Capacity cDNA reverse transcription kit (Thermofisher). Quantitative PCR was performed to amplify the acidic polymerase (PA) gene using the Bio-Rad CFX96 Realtime PCR system with 50ng of cDNA per reaction. The following primers and probe were used: forward primer: 5′-CGGTCCAAATTCCTGCTGA-3′; reverse primer: 5′CATTGGGTTCCTTCCATCCA-3′; probe: 5′-6-FAM-CCAAGTCATGAAG GAGAGGGAATACCGCT-3′. Data were analyzed using the CFX Manager Software Version 20 (Bio-Rad). A standard curve generated using a PA-containing plasmid obtained from Dr. Rob Webster at St. Jude’s Children’s Research Hospital was used to calculate the PA gene copy number per 50 ng of cDNA. This was used to calculate the total PA copy number per lung.

Flow Cytometry and Cytokine Staining

For cytokine staining, total splenoyctes were stimulated with PMA and Ionomycin for 4 h at 37°C. Brefeldin A (10μg/ml) was added after 2 h of stimulation. Following a surface stain, cells were fixed in 4% paraformaldehyde and permeablized in 0.1% saponin for 30 min at 4°C. Cytokines were then stained for 30 min at 4°C. Bim, Bcl-2, Ki67, Bcl-6, and T-bet were stained using the eBioscience Foxp3 staining buffer kit following manufacturer’s recommendations. Bim Ab was stained with a fluorescent Goat α-Rabbit Ab from Invitrogen. Host IA^b-NP_311-325-specific CD4 T cells were stained with the IA^b-NP_311-325-APC tetramer obtained from the NIH Tetramer Core Facility. All antibodies were obtained from eBioscience except anti-Bim (Cell Signaling) and anti-Bcl-6-PE (BD biosciences). Gating strategy includes gating on single cells, lymphocytes, and live cells distinguished by Invitrogen Live/Dead cell viability dye. Samples were run on LSRII instruments (BD biosciences) and analysis was done using Flowjo (Tree Star) analysis software.

Statistical Analysis

Groups of at least 3 mice were used for all experiments to ensure sufficient power. MFI in all graphs is median fluorescence intensity. For analysis comparing more than 2 samples, a one-way ANOVA analysis was conducted with GraphPad prism software. To compare 2 samples an unpaired, two-tailed student’s t-test was conducted with GraphPad prism software. All data was included unless found to be a significant outlier using the Grubb’s test (ESD method) available through GraphPad prism software. Welch’s correction was applied when the standard deviations were unequal. Significance is indicated by * = P<0.05, ** = P<0.01, *** = P<0.001, **** = P<0.0001.

Results

Antigen recognition at the effector phase of the CD4 T cell response is limited

To determine when responding CD4 T cells encounter Ag in vivo following IAV infection, we crossed OT-II.Thy1.1^+/− (Ovalbumin_323-339 (OVA_II)-specific TCR transgenic mice) to Nr4a1^eGFP (Nur77^GFP) mice that transiently express GFP following TCR stimulation (24, 25). To evaluate the feasibility of using Nur77^GFP as an indicator of recent Ag-induced TCR stimulation in effector T cells, we isolated CD4 T cells from Nur77^GFP mice and stimulated them in vitro. GFP expression was rapidly induced and remained high with continued TCR stimulation (Fig. 1A), but was significantly reduced within 24 h following removal of stimulation (Fig. 1B). Additionally, GFP was rapidly re-expressed following secondary exposure to Ag (Fig. 1C) and did not decrease with division (Fig. 1D).

FIGURE 1. Antigen recognition at the effector phase of the CD4 T cell response is limited.

(A and B) CD4 T cells were isolated from Nur77^GFP mice and cultured with anti-CD3 + anti-CD28 either continuously for 4d (A) or removed from stimulation at 48h and re-plated (B). GFP expression was determined by flow cytometry. (C) OT-II.Nur77^GFP.Thy1.1^+/− cells were stimulated in vitro with irradiated OVA_II-pulsed APCs for 2 d with 5ng/mL of IL-2, then rested for 3d in culture. Cells were then re-stimulated in culture with OVA_II-pulsed APC for 2 d. (D) Nur77^GFP CD4 T cells were labelled with cell trace violet (CTV) and stimulated with anti-CD3 + anti-CD28 for 3d in culture. (E) 5×10⁵ naïve OT-II.Nur77^GFP were transferred to B6 mice. Mice were infected with PR8-OVA_II. Lung, spleen, and DLN were harvested at various time points and GFP expression of donor cells was analyzed. (F) Kinetics of GFP expression by OT-II.Nur77^GFP cells during PR8-OVA_II infection in B6 mice (top) and of NP_311-325 tetramer⁺ cells during PR8-OVA_II infection of Nur77^GFP mice (bottom). (A–D) Representative data, n=6, 2 experiments. (E–F) Pooled data, n=12–16, 4 experiments, mean ± SD.

To determine the kinetics of IAV Ag recognition in vivo, we transferred naïve OT-II.Nur77^GFP.Thy1.1^+/− cells to C57BL/6J (B6) mice and infected with a sublethal dose of A/PuertoRico/8/34-Ovalbumin_323-339 (PR8-OVA_II) (Fig. 1E, 1F). As expected, during priming (3 dpi) most cells were GFP⁺ indicating recent Ag exposure. However, by 5 dpi, only a fraction of effector CD4 T cells had recently encountered Ag and by 9 dpi (the peak of the lung effector T cell response) very few cells express GFP (Fig. 1E, 1F). This was true of OT-II cells as well as polyclonal host cells following IAV infection of Nur77^GFP mice (Fig. 1F). Thus, effector CD4 T cells only intermittently respond to cognate antigen in vivo and the Ag recognition that does occur, is mostly limited to just before the peak of T cell effector response corresponding to the memory checkpoint. These findings suggest that Ag recognition at the effector stage could act to select a limited number of effectors to become memory.

Ag recognition at the effector phase is required for memory formation

We next asked if Ag recognition during the effector phase had any effect on memory generation. For this, we did a sequential adoptive transfer experiment as described in Figure 2A. We first transferred naïve OT-II.Thy1.1^+/− cells to B6 mice and infected with a sublethal dose of PR8-OVA_II. At 6 dpi donor OT-II.Thy1.1^+/− effector cells were isolated from the SLO of IAV infected hosts. These 6 dpi effectors were fully activated (Supplemental Fig. 1). Donor cells were transferred into 3 groups of recipients, also infected 6 days previously with PR8-OVA_II (Ag and virus), PR8 (virus without Ag), or no virus (Fig. 2A). We did not include lung effector T cells, since they are more likely to have recently encountered Ag (Fig. 1). The kinetics of endogenous CD4 T cell responses to PR8-OVA_II and PR8 viruses are the same (Supplemental Fig. 2A) indicating that with respect to factors that govern the CD4 T cell response, these viruses are very similar. Additionally, both viral titer kinetics and lung pathology are similar in PR8-OVA_II and in PR8-infected mice (Supplemental Fig. 2B–2D).

FIGURE 2. Ag recognition at the effector phase is required for memory formation.

(A–G) 5×10⁵ naïve OT-II.Nur77^GFP.Thy1.1^+/− were transferred to B6 hosts. Hosts were infected with PR8-OVA_II. On the same day, groups of B6 mice were either infected with PR8-OVA_II or PR8. On 6 dpi, donor OT-II cells were isolated from the spleen and DLN of infected mice. 2×10⁶ 6 dpi effectors were transferred to the PR8-OVA_II, PR8, or uninfected hosts. (A) Experimental schematic. (B) Representative flow cytometry plot gated on live cells at 7 dpt. (C) Quantification of donor cell recovery at 7 dpt. (D) Kinetics of donor cell recovery. The spleen (shown) was representative of the lung and DLN (Supplementary Fig. 3). (E) Quantification of cells harvested 53 dpt. (F–G) Forward scatter (F) and ICOS and PD-1 expression (G) of donor OT-II transferred to PR8-OVA_II infected hosts either 2 or 8 dpt. (H) Recovery of 14 dpi effectors isolated as in (A) and transferred to kinetically matched PR8-OVA_II, PR8-infected, or uninfected hosts. Analysis was at 7 dpt. All data is representative, n=3–5 each, 3 experiments, mean ± SD.

We enumerated donor cells in the lung, spleen, and lung draining lymph nodes (DLN) at 3, 7, and 14 days post-transfer (dpt). At 7 dpt, there were 60–200x more donor OT-II cells in the lung, 15–30X more in the spleen, and 80–400X more in the DLN of PR8-OVA_II-infected hosts compared to PR8-infected or uninfected hosts which were equally poor in supporting donor cell recovery (Fig. 2B, 2C). In PR8-OVA_II hosts, donor OT-II numbers peaked at 3 dpt (9 dpi) and then contracted slowly over the subsequent 12 d (Fig. 2D, Supplementary Fig. 3) mimicking the endogenous CD4 T cell response (26). However, in PR8-infected and uninfected hosts, donor cells underwent a sharp, immediate contraction and by 14 dpt (20 dpi) were reduced to close to the limit of detection (Fig. 2D, Supplementary Fig. 3). A highly significant difference in memory recovery was still seen at 53 dpt (Fig. 2E). These results imply that re-exposure to Ag at or after 6 dpi is necessary to maximize the effector CD4 T cell response, prevent excessive contraction, and generate a long-lived memory population, and that infection without Ag has little if any impact on memory formation.

Some have reported that late Ag promotes increased effector expansion but leads to exacerbated contraction, resulting in fewer or similar numbers of long-lived memory cells (6, 12). To determine if the increased number of donor cells in the PR8-OVA_II-infected hosts was the result of an extended expansion of short-lived effectors, we assayed the size and phenotype of donor cells 2 and 8 dpt (Fig 2F, 2G). At 2 dpt, the donor cells where large in size (Fig. 2F), with high expression of effector markers ICOS and PD-1 (Fig. 2G), but by 8 dpt they were small (Fig. 2F) and had downregulated ICOS and PD-1 (Fig. 2G). Thus, by 8 dpt donor cells no longer had an effector phenotype and had mostly transitioned to resting cells.

To test if the ability of Ag recognition to promote memory is transient or instead persists to later time-points, we isolated donor OT-II effectors at 14 dpi instead of 6 dpi and transferred them to kinetically-matched PR8-OVA_II, PR8, or uninfected hosts. The presence of Ag in the hosts had little or no impact on recovery of these 14 dpi donor cells (Fig. 2H) indicating that at this time, CD4 T cells are no longer dependent on Ag recognition. We therefore postulate that the checkpoint is defined by a unique differentiation state of effectors during which they require cognate interaction for survival, but after which their fate has mostly been determined so that they no longer require TCR triggering.

A short duration of Ag presentation at the checkpoint is sufficient to restore memory formation

Given that re-encounter with Ag was required at 6 dpi but not 14 dpi, we tested whether a short exposure of donor cells to Ag might be sufficient to induce memory formation. We transferred 6 dpi OT-II donor cells to PR8-infected mice and asked if intravenously injected bone marrow-derived dendritic cells pulsed with OVA_II-peptide (DC-OVA_II) during this memory checkpoint would be sufficient to restore memory formation. We found that these DC present Ag for no longer than 2 d after transfer in vivo by tracking their ability to induce proliferation of naïve OT-II cells (Supplemental Fig. 4A, 4B). The Ag signal provided by DC-OVA_II did not appear to be excessive as OT-II cell numbers in groups with DC-OVA_II were similar to those seen in hosts infected with PR8-OVA_II at 2 dpt (Supplemental Fig. 4C). Strikingly, the donor cells transferred to PR8-infected hosts that received DC-OVA_II were recovered at similar levels as those transferred to PR8-OVA_II-infected hosts out to 14 dpt (Fig. 3A, 3B). This indicates that encounter with cognate Ag for 48 h or less, starting at 6 dpi, was sufficient to prevent excessive contraction and promote memory formation.

FIGURE 3. A short duration of Ag presentation at the checkpoint is sufficient to restore memory formation.

(A–B) 5×10⁵ naïve OT-II.Nur77^GFP.Thy1.1^+/− were transferred to B6 mice. Mice were infected with PR8-OVA_II. At 6 dpi, donor OT-II cells were isolated as described in Figure 2A, and transferred to kinetically-matched PR8-OVA_II or PR8 infected hosts along with 0.5×10⁶ BMDCs either pulsed with 10μM OVA_II-peptide or not. (A) Donor cell recovery was assayed at 7 dpt. (B) Kinetics of cell recovery, spleen (shown) was representative of lung and DLN. In a variation using uninfected hosts (C–D), 6 dpi effectors were isolated as in (A) then transferred to mice that were either infected with PR8 (6 dpi) or uninfected, along with BMDC that were pulsed with OVA_II-peptide or not. (C) Cell recovery was assayed 7 dpt. (D) PD-1 and ICOS expression of donor cells at 7 dpt in the spleen, similar phenotypic changes occur in the lung. All data is representative, n=3–5 each, 2–3 experiments, mean ± SD. (E–G) Comparison of memory cells generated following transfer of naïve OT-II at day 0, or transfer of 6 dpi OT-II effectors into PR8-OVA_II-infected, PR8-infected with DC-OVA_II, or uninfected hosts with DC-OVA_II. Memory cells harvested 14 dpt (20 dpi) were compared to 6 dpi effectors generated in vivo. (E) Representative flow cytometry plots showing intracellular cytokine staining of IFNγ and TNFα following 4 h of PMA + Ionomycin stimulation in memory or 6 dpi effector OT-II cells. (F) Quantification of IFNγ⁺TNFα⁺, IFNγ⁺TNFα⁺IL-2⁺, and CD127 MFI of memory or effector OT-II populations in the spleen. (G) Quantification of Trm via CD69 expression (Lung), Th1 via IFNγ production (Spleen), and Tfh/Tcm via CXCR5 expression (DLN) of memory or effector OT-II cells. (Other organs shown in Supplemental Fig. 4D, 4E). Data is pooled, n=6–8, 2 experiments, mean ± SD.

Virus infection is not required at the memory checkpoint

To determine if viral infection itself is important in promoting memory formation, other than providing Ag presentation, we tested whether adding DC-OVA_II would similarly increase memory formation in uninfected hosts. Strikingly, DC-OVA_II strongly promoted donor recovery to a similar extent in PR8-infected and uninfected hosts (Fig. 3C). These data, combined with Figure 1 in which there was no difference in memory formation following transfer of 6 dpi effectors into PR8-infected and uninfected hosts, suggests that aspects of infection other than providing Ag presentation, such as induction of lung inflammation, have no discernable impact at the memory checkpoint. The DC we used were activated so infection-induced viral-sensing pathways may be needed to activate in situ APC. The donor OT-II effectors exposed to Ag either in PR8-infected or uninfected hosts down-regulated effector molecules PD-1 and ICOS by 7 dpt (Fig. 3D), suggesting their loss of effector phenotype.

We further examined if memory formation occurred normally when late Ag was provided by short-lived DC-OVA_II. We directly compared OT-II memory generated when naïve cells were transferred on day 0 and left in the same initial host (No Eff. Trans.) or when 6 dpi OT-II effectors were isolated and transferred to kinetically-matched PR8-OVA_II-infected, PR8-infected with DC-OVA_II, or uninfected hosts with DC-OVA_II (Eff. Trans.). To highlight the changes that distinguish memory cells from effectors, we included 6 dpi OT-II effectors for comparison.

One functionally important characteristic of memory cells is their ability to produce multiple cytokines upon re-stimulation (19). We found that the memory cells generated following transfer (either to hosts with virally produced Ag or with short-lived Ag provided by DC-OVA_II) had regained the ability to produce multiple cytokines to a similar extent as those generated without transfer (Fig. 3E–3F). Additionally, memory cells generated both with and without transfer had upregulated the critical memory marker CD127 that is necessary for their persistence (Fig. 3F, Supplemental Fig. 4D). Interestingly, when comparing memory cells generated in PR8-infected or uninfected hosts with late transfer of DC-OVA_II, there was a decrease in CD127 expression in the uninfected hosts (Fig. 3F, Supplemental Fig. 4D). This suggests that although systemic virus-induced inflammation may not be needed for memory cell numbers, function, or subset differentiation, it may be that virus-induced inflammation is required for full CD127 upregulation. However, the equivalent recovery of memory cells argues that DC-OVA_II exposure induced sufficient levels of CD127 for persistence.

We next examined CD4 memory subset differentiation. The tissue resident memory (T_rm) population identified by CD69 expression (27, 28) in the lung was similar with and without transfer (Fig. 3G). IFNγ production, an indicator of Th1 differentiation (29, 30), was also produced to a similar extent in all memory groups (Fig. 3G). CXCR5 has been shown to mark a memory subset that is thought to be T follicular helper (Tfh) or central memory (Tcm)-like (31, 32). CXCR5 expression was also similar among all memory groups (Fig. 3G, Supplemental Fig. 4E). Therefore, the limited Ag provided by DC-OVA_II at 6 dpi is sufficient to generate canonical memory formation.

Effector CD4 T cells require Ag recognition at the checkpoint for continued proliferation

Some studies suggest that effector CD4 T cell division is programmed by initial Ag encounter (33, 34), while others suggest that CD4 T cells do not undergo such “autopilot” proliferation after 2 d of stimulation during priming (10), but it remains unclear if they acquire this ability later during infection. To determine if division past 6 dpi depends on Ag recognition, we labeled isolated 6 dpi effectors with CFSE, transferred to hosts with and without Ag, and assayed dilution of dye at 3 dpt. Only donor cells in hosts with Ag divided more than once (Fig. 4A). To determine if this proliferation was an artifact of the transfer system, we used Ki67 staining to compare the proliferation of donor OT-II cells to that of endogenous IA^b-NP_311-325-specific host cells in PR8-OVA_II-infected hosts. We found that at 2 dpt there was a similar percentage of proliferating donor and hosts cells, and by 8 dpt neither were undergoing division, a pattern seen in the lung, spleen, and DLN (Fig. 4B, 4C). Thus, division after 6 dpi is Ag-dependent, short-lived, and followed by the transition to non-dividing cells within a week. This also illustrates that the kinetics of proliferation of the transferred donor cells mimics that of the endogenous host CD4 T cell response to live IAV.

FIGURE 4. Effector CD4 T cells require Ag recognition at the checkpoint for continued proliferation.

(A) 6 dpi effectors were isolated, stained with CFSE, and transferred to kinetically matched PR8-OVA_II or PR8-infected or uninfected hosts as in Figure 2A. CFSE dilution was determined 3 dpt in the spleen, similar results seen in the lung. (B) Same experimental approach as in Figure 2A. Ki67 expression of donor OT-II and NP_311-325 tetramer positive host cells was determined at indicated time points following donor cell transfer. Representative flow cytometry plot shown of the spleen. (C) Quantification of Ki67 staining in the lung, spleen, and DLN at 2 dpt (8 dpi) and 8 dpt (13 dpi). Representative data, n=3–5 each, 2 independent experiments, mean ± SD.

Ag recognition at the effector phase promotes survival of CD4 T cells

After viral clearance, most effector T cells undergo apoptosis leading to contraction, while a cohort survives to become memory. This suggests that avoiding apoptosis is a key step in the transition to memory. We propose that a cohort of effector CD4 T cells recognize Ag/APC which drives them to make and respond to IL-2 which drives their survival and supports their transition to memory (17). We evaluated several components of this hypothesis.

To test if Ag recognition at the checkpoint promoted enhanced survival of effector CD4 T cells, we transferred naive OT-II.Nur77^GFP cells to hosts and infected with PR8-OVA_II. At 7 dpi, donors that had seen Ag during the first 1–2 d of the checkpoint are GFP⁺ while those that did not, are GFP⁻. We analyzed donor CD4 T cells from the lung, spleen, and DLN directly ex vivo, gating on GFP⁺ and GFP⁻ cells. To detect cell death directly ex vivo we measured 7-Aminoactinomycin D (7-AAD) staining (Fig. 5A). In each organ, 7-AAD staining was significantly greater in GFP⁻ cells than in GFP⁺ cells, indicating that more effector cells that recognized Ag between 5–6 dpi survived than those that did not recently encounter Ag.

FIGURE 5. Ag recognition at the checkpoint promotes survival via downregulation of Bim and induction of IL-2.

(A) OT-II.Nur77^GFP.Thy1.1^+/− naïve T cells were transferred to B6 mice followed by infection with PR8-OVA_II. Lung, spleen, and DLN were harvested 7 dpi. Left: Representative plot of GFP⁺ vs. GFP⁻ OT-II.Nur77^GFP cells in the spleen. Right: 7-AAD⁺ of GFP⁺ vs. GFP⁻ OT-II Nur77^GFP cells on 7 dpi. (B–F) OT-II 6 dpi effectors were co-cultured with irradiated APC pulsed with OVA_II-peptide or not. (C–D) Kinetics of OT-II cell recovery (C) or CD127 expression (D) of OT-II cells. (E) 7-AAD staining of OT-II cells after 2 d. Left: Representative staining. Right: Quantification of 7AAD⁺ cells. (F) Bim expression of OT-II cells after 2 d +/− Ag. (G–H) WT GFP or Bcl2l11^+/− OT-II cells were mixed at a 1:1 ratio and co-transferred into B6.Thy1.1^+/− mice followed by infection with PR8-OVA_II. 6 dpi effectors were isolated and cultured with APC +/− OVA_II. (G) The percentage of WT and Bcl2l11^+/− OT-II was determined after 14d. (H) Bim staining of WT GFP or Bcl2l11^+/− OT-II cells after 2 d +/− Ag/APC. (I) 6 dpi OT-II cells were co-cultured with APC +/− OVA_II-peptide, after 4 h IL-2 production was assayed by intracellular staining. (J–L) 6 dpi effectors were co-cultured with APC without Ag, with Ag, or with Ag plus αCD25 + αCD122. Cells were stained with CTV. (J) Cell recovery was determined after 6 d. (K) Dilution of CTV after 2 d. (L) 7-AAD⁺ after 14 d. All data is representative, n=3–4 each, 3 experiments, mean ± SD.

To further dissect the mechanisms involved in this survival, we developed an in vitro model to better control the signals that the effectors receive. We isolated 6 dpi OT-II effectors and co-cultured them with T-depleted splenocytes isolated from PR8-infected mice, a physiologically relevant APC, either with or without OVA_II peptide. To mimic the short-term Ag presentation that occurs in vivo, we irradiated the APC, ensuring Ag presentation was restricted to the first 2 d of culture (Fig. 5B) (5). In this model, we found Ag enhanced cell recovery (Fig. 5C) and also promoted expression of CD127, which is strongly associated with memory cell survival (17) (Fig. 5D). After 2 d of culture, effector CD4 T cells exposed to Ag had decreased 7-AAD staining, indicating reduced cell death (Fig. 5E), as well as reduced levels of Bim, a pro-apoptotic protein known to mediate death during T cell contraction (35, 36) (Fig. 5F). Thus, instead of inducing widespread cell death, Ag presentation to in vivo-generated 6 dpi effectors, drove effector survival.

Ex vivo recognition of Ag enhances survival via down-regulation of Bim

We tested if the reduced level of Bim seen in the Ag-exposed effectors was responsible for their increased survival. We co-transferred WT GFP Bcl2l11^+/+ or Bcl2l11^+/− [which express half the WT levels of Bim (37)] OT-II cells mixed at a 1:1 ratio into B6.Thy1.1^+/− mice and infected with PR8-OVA_II. We harvested total effector CD4 T cells at 6 dpi and stimulated them ex vivo with APC with or without OVA_II-peptide, and determined which donor cells preferentially survived after 14 d. When no Ag was present in vitro, the Bcl2l11^+/− OT-II cells survived much better than WT OT-II cells (Fig. 5G), implicating the high levels of Bim in the death/contraction of the 6 dpi effectors. In contrast, in the presence of Ag, the Bcl2l11^+/− OT-II and WT OT-II cells survived comparably (Fig. 5G), consistent with the hypothesis that Ag acts to counteract apoptosis by causing Bim reduction. Indeed, in the absence of Ag, the Bcl2l11^+/− OT-II cells expressed less Bim than WT, but with Ag, Bim levels were similar (Fig. 5H). This supports the hypothesis that Ag recognition by effectors at the checkpoint acts in part through reduction of Bim expression, which prevents short-term apoptosis and promotes survival.

The pro-survival effects of Ag recognition at the checkpoint are IL-2 dependent

Since our previous studies found that autocrine IL-2 was required for CD4 effector survival (17), we tested whether Ag stimulation of 6 dpi effectors ex vivo would promote IL-2 production and if that IL-2 was necessary for enhanced survival. Indeed, the ex vivo effector CD4 T cells produced IL-2 only when cultured with Ag/APC (Fig. 5I). We cultured 6 dpi effectors with APC, Ag/APC, or Ag/APC plus Ab specific for both CD25 (IL-2Rα) and CD122 (IL-2Rβ) to block IL-2 function. The exposure to Ag/APC enhanced donor cell recovery after 6 d, and blocking IL-2 signaling reduced that recovery (Fig. 5J). Notably, blocking IL-2 only slightly inhibited Ag/APC-induced proliferation (Fig. 5K), but dramatically increased cell death as measured by 7-AAD staining (Fig. 5L). Thus in vitro Ag/APC stimulation of 6 dpi effectors induces IL-2 production that prevents apoptosis and enhances the survival necessary for memory formation. Partial effects seen on cell recovery (Fig. 5J) and cell proliferation (Fig. 5K) imply that factors beyond IL-2 also play a role in the effects of Ag seen at the memory checkpoint.

Ag recognition at the checkpoint promotes expression of a memory phenotype

Since Ag/APC exposure of 6 dpi effectors at the checkpoint promotes formation of a larger cohort of memory cells, as opposed to driving terminal differentiation, we asked if it also promoted expression of known memory-associated markers. We transferred OT-II.Nur77^GFP.Thy1.1^+/− naive cells to hosts, infected with PR8-OVA_II, and harvested lung, spleen, and DLN at 7 dpi. We analyzed donor GFP⁺ cells (recent Ag exposure) vs. GFP⁻ cells (no recent Ag exposure). The GFP⁺ cells expressed higher levels of CD25 at 5–7 dpi in the lung compared to GFP⁻ cells (Fig. 6A) consistent with the role of IL-2 postulated above. The 7 dpi GFP⁺ cells in lung, spleen and DLN also expressed higher levels of Bcl-6, a transcription factor implicated in memory formation (38, 39) (Fig. 6B), while expression of T-bet, thought to promote terminal differentiation (40–42) was equivalent (Fig. 6C). We also cultured both OT-II donors and polyclonal effector CD4 T cells, with APC pulsed with OVA_II or NP_311-325 as respective cognate peptides. After 2 d, Ag/APC had induced both OT-II and NP₃₁₁⁺ cells to upregulate CD25 and Bcl-6 compared to culture with APC alone (Fig. 6D, 6E). STAT3 signaling promotes the transcription of Bcl-6 and may promote memory formation (43, 44). We found exposure of 6 dpi CD4 effectors to Ag/APC significantly increased phosphorylated STAT3 (pSTAT3) after 4 h (Fig. 6F). The ability of Ag/APC to rapidly upregulate these memory signature proteins seen both in vivo and in vitro, provides further evidence that Ag recognition at the checkpoint drives effectors to express a comprehensive program that initiates and carries out their transition to memory cells.

FIGURE 6. Ag recognition at the checkpoint promotes expression of a memory phenotype.

(A–C) 5×10⁵ naïve OT-II Nur77^GFP cells were transferred to B6 hosts followed by infection with PR8-OVA_II. Lung, spleen and DLN were harvested and OT-II Nur77^GFP effectors were analyzed for expression of memory markers by flow cytometry after gating on GFP⁺ and GFP⁻ populations. (A) CD25 expression of GFP⁺ vs. GFP⁻ OT-II.Nur77^GFP cells in the lung. (B–C) Bcl-6 (B) or T-bet (C) expression of GFP⁺ vs. GFP⁻ OT-II.Nur77^GFP cells on 7 dpi. (D–F) 6 dpi OT-II or total effector CD4 T cells were generated in vivo and cultured ex vivo with activated APC alone or in the presence of OVA_II-peptide or the NP_311-325-peptide. (D) CD25 and (E) Bcl-6 expression were assayed after 2 d. (F) pSTAT3 was assayed on OT-II cells after 4h of culture using Phosflow. All data is representative, n=3–4 each, 3 experiments, mean ± SD.

Memory cells generated by cognate interactions at the checkpoint have enhanced phenotype, function, and protective ability

In the transfer model (Fig. 2–4) 6 dpi effector cells transferred to hosts without Ag underwent extensive contraction and were often at or below the limit of detection within 7 dpt. This low number of memory cells in hosts without Ag hampered our ability to determine the long-term phenotypic and functional differences between memory cells generated with or without Ag at the checkpoint. To increase the recovery of memory cells that develop without Ag at the checkpoint, we cultured in vivo-generated effector CD4 T cells with or without Ag for 2 d in vitro (as in Fig. 5), transferred equal numbers of each to uninfected mice, and allowed the cells to transition to memory for 7 d. In vivo, 3 d without Ag is sufficient for effector CD4 T cells to become virtually identical to memory (45). We then assayed cell recovery, phenotype, and cytokine production. In vivo, 3 d without Ag is sufficient for effector CD4 T cells to become virtually identical to memory (45).

As expected, the donor cells that had been exposed to Ag/APC in vitro, formed a significantly larger memory population after transfer to uninfected hosts even though their numbers were equivalent at the time of transfer, with 18-fold more in lung and 5-fold more in spleen (Fig. 7A). This indicates that the 2 d in vitro exposure to Ag was sufficient to confer significantly greater survival. Compared to APC without Ag, the donor effector cells exposed to Ag/APC in vitro, expressed increased levels of CD127 and CXCR3, a memory marker needed for homing and protective function (46, 47) (Fig. 7B). Moreover, they secreted more IFNγ and had a higher frequency of IFNγ/TNFα double producers after restimulation (Fig. 7C, 7D). These results indicate that even short-term Ag recognition at the 6 dpi checkpoint results in both a much larger, and a functionally superior memory population.

FIGURE 7. Memory cells generated by cognate interactions at the checkpoint have enhanced protective ability.

(A–D) 6 dpt OT-II in vivo-generated effectors and PR8-activated APC were co-cultured ex vivo as described in Figure 4. After 2 d, live cells were isolated using lympholyte and 2×10⁶ cells were transferred to uninfected B6 mice. Representative data, n=3–4 each, 3 experiments, mean ± SD. (A) Cell recovery was determined 7 dpt in the lung and the spleen of host mice. (B) CD127 and CXCR3 expression was assayed in the spleen 7 dpt. (C) Representative flow cytometry plots of intracellular cytokine staining of cells harvested from the spleen 7 dpt and re-stimulated for 4h with PMA + Ionomycin. (D) Quantification of IFNγ⁺ and IFNγ⁺TNFα⁺ donor cells. (E) 6 dpi OT-II effector cells were generated in vivo as described in Figure 1 and transferred to uninfected mice along with either OVA_II-pulsed APC (APC-OVA_II) or unpulsed APC. One group of mice received OVA_II-pulsed APC alone (no effectors). Another group received 5×10⁵ naïve OT-II cells. After 2–3 wk, hosts were challenged with 2LD₅₀ PR8-OVA_II. The survival of mice was plotted. Pooled data, n=14–15, 3 experiments. Significance for Figure 5E was determined using the Log-rank (Mantel-Cox) test.

To evaluate if the differences in memory formation with or without Ag at the checkpoint would lead to differences in protection against a lethal challenge of IAV, we transferred 6 dpi OT-II effectors to uninfected B6 mice along with OVA_II-pulsed or un-pulsed APC. To account for a potential host naïve CD4 T cell response, we included a group of mice that received OVA_II-pulsed APC without transfer of 6 dpi effectors. We also included a group that received naïve OT-II cells to control for the possibility that a similar number of naïve donor OT-II could provide enhanced protection. Hosts were rested for 2–3 wk to ensure memory generation, and then challenged with a lethal dose of PR8-OVA_II.

Despite the fact that the only memory cells in the hosts were the donor 6 dpi effectors, the hosts that received effectors plus APC-OVA_II were mostly protected against lethal infection (12/15), whereas those that received naïve OT-II, OT-II 6 dpi effectors without Ag, or APC-OVA_II alone were largely unprotected (Fig. 7E). Thus, providing effectors with only short-term in vivo Ag stimulation at the checkpoint drove the formation of protective memory cells. Since the hosts were not previously infected, we conclude that short-term Ag stimulation by activated APC, without any viral infection, is sufficient to promote the transition of 6 dpi effectors to become protective memory.

Ag during the checkpoint boosts memory in a cold-adapted vaccine model

Our findings establish a checkpoint that occurs at 6–8 dpi following IAV infection where Ag recognition drives functional memory CD4 T cell formation. Since many standard vaccinations likely do not induce the persistent levels of Ag that live virus does, we postulate that memory CD4 T cell formation following vaccination is normally constrained by a lack of Ag at the checkpoint. Therefore, the addition of Ag/APC at this time may enhance vaccine-induced memory. To test this premise, we immunized with a live attenuated, cold-adapted (ca) influenza vaccine (ca.IAV). Replication of ca.IAV is limited to the upper respiratory tract, potentially limiting the duration of Ag presentation. The ca.IAV vaccines have been shown to induce enhanced T cell responses when compared to inactivated vaccines (48). To determine if Ag persisted into the memory checkpoint following ca.IAV inoculation, we immunized Nur77^GFP mice with ca.A/Alaska/6/77CR29 (ca.Alaska) and measured GFP expression in immunization-induced effector T cells. Our earlier studies showed that ca.Alaska induces a strong heterosubtypic response to PR8 and that the NP_311-325 is a dominant CD4 epitope shared between these two viruses (23). At 7 dpi, effector NP_311-325-specific cells expressed no GFP after ca.Alaska immunization indicating no recent Ag recognition (Fig. 8A), while in mice infected with PR8, or a non-ca H3N2 strain (A/Philippines/2/82/x-79), a cohort of NP_311-325⁺ cells were GFP⁺, indicating recent Ag recognition in the live infections.

FIGURE 8. Ag during the checkpoint boosts memory in a cold-adapted vaccine model.

(A) Nur77^GFP expression of CD4⁺CD44^hi NP_311-325 tetramer⁺ cells or CD4⁺CD44^lo naïve cells in the lung on day 7 following PR8 infection, Philippines infection, or ca.Alaska immunization. (B) Experimental approach; B6 mice were infected with 2500 TCID₅₀ ca.Alaska intranasally. 6 d later, 2×10⁶ NP_311-325-pulsed APC were added via intravenous injection. (C) Quantification of NP_311-325 tetramer⁺ memory CD4⁺CD44^hi T cells in the lung, spleen, and DLN 33–44 d following immunization. Representative FACS plots shown, 2–4 independent experiments. Pooled cell recovery data, 3 independent experiments, n=4–5 each, mean ± SD.

To determine if the addition of Ag during the checkpoint could boost memory following ca.Alaska immunization, we added NP_311-325-pulsed APC at 6 dpi to ca.Alaska-immunized mice and assayed memory CD4 T cell formation by enumerating NP_311-325 tetramer positive cells after 33–44 dpi (Fig. 8B). We found significantly more NP_311-325⁺ CD4 T cells in the lung and spleen, although there was no difference in the small number of donors found in the DLN (Fig. 8C). This finding suggests that the memory checkpoint exists for effectors generated by attenuated as well as live WT influenza infection. Additionally, it shows that the introduction of Ag/APC at the checkpoint can promote effector CD4 T cells induced by attenuated virus immunization to form more memory without the need for persisting live virus.

Discussion

Cognate interactions during initial priming of naive CD4 T cells affect many aspects of the T cell response, including effector and, some suggest, memory differentiation (4, 5, 49). Our findings here using a sequential transfer model show unequivocally that Ag recognition is again required at the effector phase to drive mature effector CD4 T cells to become memory. This implies that continuing Ag presentation indicative of pathogen persistence is required for optimum generation of CD4 memory. Our studies define a clear-cut checkpoint during the effector phase, lay out the timing of events that occur, define key pathways that are involved, and show that the memory cells generated are protective.

We show that primary effector CD4 T cells must engage in cognate interactions with Ag/APC at a checkpoint from 6–8 dpi. While the IL-2 dependent checkpoint is 5–7 dpi for primed CD4 effectors in our earlier study (17) here we used primary cells which seem to have a slightly delayed kinetics. This fate-determining effector:Ag/APC interaction induces a limited expansion of effectors, depends on IL-2 production, and lead to increased survival mediated in part by a down-regulation of Bim. Importantly, although Ag persistence during the checkpoint would normally depend on continued live pathogen, our transfer model revealed that a short-lived Ag-bearing APC population acting for as little as 2 d at the checkpoint is sufficient for strong memory generation that confers protection and is phenotypically and functionally similar to memory cells generated during viral infection.

We find Ag recognition at the memory checkpoint initiates a program of memory-associated changes that results in a larger, long-lived memory population with increased CD127 and CXCR3 expression as well as increased cytokine production. Our Nur77^GFP experiments highlight that the early signaling events that occur following Ag recognition at the checkpoint include an upregulation of CD25, Bcl-6, and pSTAT3. CD25 expression is generally heterogeneous at the effector time point and one recent study found that CD25^hi effector T cells present late in the response preferentially form memory (50). Increased expression of the IL-2 receptor ensures the cells that encountered late Ag would effectively use the autocrine IL-2 required for memory formation (17). Bcl-6 was recently shown to promote the metabolic switch required for memory formation (38). Ag at the memory checkpoint may therefore serve to selectively upregulate Bcl-6 late in the response as cells destined to become memory must transition to a self-renewing, resting population. The regulatory effect of Bcl-6 in Th1 cells has been shown to reflect the relative levels of Bcl-6 and T-bet (51). Since no significant increase in T-bet occurred following late Ag stimulation, even a modest increase in Bcl-6 expression may tip the balance in favor of a Bcl-6 mediated gene expression program. The main known promoter of Bcl-6 expression is pSTAT3, which was also induced. Future work will determine whether TCR stimulation alone promotes Bcl-6 transcription or if a STAT3-inducing cytokine either produced by the responding T cell or the APC is responsible for increased Bcl-6 expression.

The effects of exposure to Ag/APC from 6–8 dpi are dramatic, leading to differences in memory recovery that sometimes exceed 100-fold, so there is little question that cognate Ag recognition is critical for transition of effectors to memory. This suggests that effectors generated by live IAV infection reach a stage of differentiation where they will die by default programmed death unless they are rescued by Ag/APC interaction. The donor OT-II CD4 T cells we followed have a single, high affinity receptor, so it could be argued that less vigorous infections or T cells with less avid receptors might generate effectors with a less differentiated phenotype that do not reach the default death stage. We point out however that the polyclonal response of NP_311-325⁺ cells behaves similarly in our studies and that ca.Alaska IAV also produces effectors that form enhanced memory when they recognize Ag at the checkpoint. Additionally, it is important to consider that less differentiated effectors will have divided fewer times and will be present in much lower numbers, so their potential contribution to the memory pool is limited. Many viral infections follow a similar time-course of virus accumulation and effector CD4 T cell generation as are seen in the IAV response, so we predict the checkpoint will be broadly applicable to other acute viral responses.

While the vast majority of studies that find no role for late Ag in memory T cell formation were CD8⁺ T cell studies (2, 52–54), some have shown similar results for CD4⁺ T cells during Listeria Monocytogenes (L. monocytogenes) and Vesicular stomatitis virus (VSV) infection (3, 12). In the L. monocytogenes study, ablation of Ag after 48 h via antibiotic treatment had no effect on effector or memory cell numbers. However, a contradictory study demonstrates that truncating L. monocytogenes infection significantly reduces CD4⁺ memory formation (55). Additionally, since both studies used antibiotics to truncate infection, it cannot be ruled out that lingering Ag presentation may have been present. The VSV study is quite elegant in its use of antibodies directed against a specific epitope in the context of MHC Class II. In this study, the authors found that blocking Ag 24 h after infection had no impact on memory formation. However, this study also found differences in the Ag dependency of CD8⁺ T cells responding to VSV versus IAV suggesting that memory requirements for these two pathogens may differ. Additionally, studies using in vitro generated effector T cells find no role for late Ag (5, 56). However, our unpublished findings suggest that in vitro generated effectors do not mimic the kinetics of endogenous T cell expansion and contraction when transferred to infected mice. We propose that such in vitro generated effectors, though useful for some studies, do not adequately mimic effectors generated in vivo by viral infection in terms of their requirements for memory T cell formation.

A requirement of Ag recognition at the effector stage to generate CD4 T cell memory makes teleological sense. First, it would ensure that a substantial memory population is only formed when Ag, indicating a continuing threat, persists. Generation of memory would be undesirable if the pathogen were rapidly cleared. Second, the additional round of Ag-dependent selection may help select a memory pool with greater multi-functionality. Many recent studies have demonstrated that memory CD4 T cells retain a significant level of the differentiation acquired during the effector phase (31, 32, 57, 58). An intriguing hypothesis is that, via the memory checkpoint described here, this late Ag interaction may be responsible for the selection and formation of more specialized subsets of effectors that become memory cells particularly tailored to combat the given pathogen upon re-encounter.

It is well established that live infections (and vaccines mimicking them) generate the best immunity (small pox and others) while newer vaccines containing purified proteins with little or no adjuvant induce weak T cell immunity (48, 59). Our results suggest that one key reason such vaccines may generate poor memory is because they do not induce sufficient persisting Ag presentation during the checkpoint. We tested this using ca.IAV since the attenuated virus initially has the properties of live vaccine as it replicates in the cooler upper respiratory tract, but then fails to replicate in the warmer environment of the lower respiratory tract, and is therefore short-lived. Indeed, by 7 dpi after ca.IAV there was no evidence of Ag presentation and when we introduced Ag/APC at 6 dpi after ca.IAV vaccination it significantly improved memory CD4 T cell generation. This suggests that strategies to provide Ag/APC at a relevant checkpoint for each vaccine may often enhance memory CD4 T cell formation. Indeed in another scenario, an early “boost” strategy efficiently promoted CD8 T cell memory (60). Importantly since we find no need for live virus at the checkpoint, our findings also suggest that an optimal vaccine response could be achieved without the destructive inflammation caused by replicating live virus or systemic adjuvants. Although the exact timing and optimum approach for providing Ag at the checkpoint may need to be tailored to the specific vaccine, we anticipate such an approach could be developed to improve vaccines in humans.