Influenza Vaccine-Induced CD4 Effectors Require Ag-Recognition at an Effector Checkpoint to Generate CD4 Lung Memory and Ab Production

Abstract

Previously, we discovered that influenza-generated CD4 effectors must recognize cognate antigen at a defined effector checkpoint to become memory cells. Ag recognition was also required for efficient protection against lethal influenza infection. To extend these findings, we investigated if vaccine-generated effectors would have the same requirement. We compared live infection with influenza to an inactivated whole influenza vaccine. Live infection provided strong, long-lasting Ag presentation that persisted through the effector phase. It stimulated effector generation, long-lived CD4 memory generation and robust generation of Ab-producing B cells. In contrast, immunization with an inactivated virus vaccine, even when enhanced by additional Ag-pulsed APC, presented Ag for three days or less and generated few CD4 memory cells or long-lived Ab-producing B cells. To test if checkpoint Ag addition would enhance this vaccine response, we immunized mice with inactivated vaccine and injected Ag-pulsed activated APC at the predicted effector checkpoint to provide Ag presentation to the effector CD4 T cells. This enhanced generation of CD4 memory, especially tissue-resident memory in the lung, long-lived bone marrow Ab-secreting cells and influenza-specific IgG Ab. All responses increased as we increased the density of peptide Ag on the APC to high levels. This suggests that CD4 effectors induced by inactivated vaccine, require high levels of cognate Ag recognition at the effector checkpoint to most efficiently become memory cells. Thus, we suggest that non-live vaccines will need to provide high levels of Ag recognition throughout the effector checkpoint to optimize CD4 memory generation.

Introduction.

Live infection with influenza and other viruses produces robust immune responses that generate long-lived CD4, CD8 and B cell memory that synergize to provide durable and long-lived protection (1–6). However, influenza and other single-stranded (ss) RNA viruses (e.g. HIV and corona viruses) rapidly accumulate mutations, leading to changes in viral antigens (Ag) and consequential escape from pre-existing memory immunity. Memory B cells and neutralizing antibodies (Abs) predominantly recognize determinants on hemagglutinin (HA) and neuraminidase (NA) coat protein antigens that differ between heterosubtypic influenza strains (e.g. H1N1, H3N2, H5N1). In contrast, memory T cells, both CD4 and CD8, recognize epitopes from core proteins, many of which are conserved in the influenza strains in almost all outbreaks (7) and thus can provide heterosubtypic protection. New influenza strains are generated by genetic recombination (heterosubtypic strains) as well as by rapid mutation including a lack of proofreading. Among other ssRNA viruses, coronaviruses accumulates mutations mostly through rapid replication (8), while HIV diversity is attributed in part to reverse transcription (9). Antibodies often drive selection of non-recognized variants, but more T cell epitopes are conserved (10, 11) and so T cells provide key targets for vaccine design. Memory T cells are heterogeneous, functionally diverse and able to provide protection through multiple synergizing mechanism (5). The different subsets of CD4 memory T cells have varying lifespans (12) and distinct distribution among lymphoid and tissue sites. Central memory cells (T_CM), defined by expression (or re-expression) of CD62L, are retained in the secondary lymphoid organs (spleen and dLN) (13), while effector memory cells (T_EM) continuously recirculate and tissue resident memory cells (T_RM) reside in the peripheral lymphoid and non-lymphoid tissues (3, 13–15). Compared to T_CM and T_EM, CD4 T_RM are more protective in part because they respond quickly at sites of entry to provide a first line of defense against pathogens (16). Influenza infection induces all these subsets, including a large population of lung-resident memory T cells (17). They clear virus and promote resolution and repair after infection by multiple mechanisms (5, 18). However, currently used influenza vaccines generate only modest titers of neutralizing Ab that are often short-lived, but little T cell immunity and thus they do not provide robust long-term or heterosubtypic protection (19–21), especially to new or mutated strains that develop each year or two.

Our previous studies (22, 23) showed that following live influenza infection CD4 effectors must recognize antigen again at a defined “checkpoint” in order to differentiate into memory cells. The checkpoint occurred during the time when effector accumulation was peaking, at 6–8 days post-infection (dpi). Primary effectors needed to be induced to produce and respond to IL-2, which acted to block their default apoptosis and induced longevity (22). The same checkpoint is defined by the time when activated antigen-presenting cells (Ag/*APC) and IL-2 are required (22, 23). The signals are delivered during the effector:Ag/*APC cognate interaction and without these signals effectors disappear, while with them, some can become memory cells. We now call this the “effector checkpoint” because during this time interval the effectors can follow multiple fates that include: 1) apoptosis resulting in contraction when they do not see Ag; 2) further differentiation into late functional effectors such as T follicular helpers (T_FH) and cytotoxic CD4 T cells (ThCTL); and 3) transition to resting memory cells in both the secondary lymphoid organs (SLO) and infected lung, both when signals from Ag recognition. Using an in vivo sequential transfer model, we showed that without Ag presentation at the checkpoint, the CD4 effectors did not become memory cells. The provision of Ag to CD4 effectors at the “checkpoint”, even delivered by peptide-pulsed activated APC, which presented Ag for only 48 hr, drove the effectors to differentiate into memory cells (19). With effector checkpoint cognate Ag recognition, memory generation increased 20–200 fold in spleen, lymph nodes and lungs indicating that the short cognate interaction was sufficient for effectors to avoid apoptosis and differentiate into memory (22). The requirement by CD4 effectors for Ag presentation through the checkpoint interval is consistent with other studies that suggested the differentiation of naïve CD4 T cells into memory cells requires prolonged Ag presentation (24).

Unlike viral infection, current inactivated, purified split-virion or subunit vaccines focus on inducing Ab to seasonal HA variants and are mostly ineffective at inducing strong long-lived T or B cell memory (19, 25, 26). Current influenza vaccine are based on three basic formulations including 1) whole inactivated influenza virus (WIV) which was the first influenza vaccine in 1936 and is still in use (27); 2) more purified split-virion or subunit vaccines which are used most extensively; and 3) cold-adapted live vaccines which were developed more recently (28) and which survive only a couple of days in the respiratory tract. Here, we use WIV since it contains all viral components including small amount of RNA, which provides PAMP (pathogen associated molecular patterns), and a wide range of epitope peptides for T cell recognition (7) and immunogens for B cell recognition (e.g. HA protein). In contrast, the purified split-virion or subunit vaccines are composed of isolated and enriched components or recombinant proteins (e.g. HA and NA) from multiple viral strains to induce broader Ab response, and T cell epitope peptides from internal viral proteins are minimized (29). These are given i.m. except for the cold-adapted FluMist, that is given as a nasal mist. In one case an adjuvant is added. Given the short longevity of proteins, even in those in oil-based adjuvants, it is likely, that vaccines do not present high amounts of Ag at the effector checkpoint and thus we postulate they are unlikely to efficiently drive CD4 memory differentiation or full effector differentiation. Here we ask if whole inactivated viral vaccine-induced CD4 effectors generated in mice do indeed require Ag recognition at an effector checkpoint to drive differentiation of effectors, so that they reach the lung and therein become memory cells. If so, it would imply changes in the delivery of vaccines are called for to provide the additional signals needed.

Our studies here show that WIV immunization leads naïve CD4 T cells to proliferate and accumulate, which peaks at 4–5 days, while the response induced by PR8 infection is more vigorous and persistent. We find WIV immunization presents Ag only during the first 3 days, suggesting that Ag presentation at the checkpoint interval does not occur. Even when we add, TLR agonist-activated APC along with WIV during priming (WIV+Ag/*APC), which we showed earlier could strikingly enhance CD4 responses (30, 31), Ag presentation is still short-lived and occurs effectively only during the first 3 days. In contrast, PR8 infection leads to a delayed but persistent Ag presentation. We find that addition of Ag-pulsed APC following WIV vaccines, at 5 dpt, increased the generation of donor memory cells and strongly enhanced lung donor T cells with a resident memory phenotype and modestly enhanced number of plasma cells secreting anti-PR8 antibodies (Ab) in the bone marrow. Thus, our study shows CD4 effectors, generated by a vaccine (WIV+Ag/*APC) require Ag recognition at an effector checkpoint to drive differentiate into memory cells, especially lung T_RM, supporting the concept that vaccine-induced effectors, like those generated by infection, have a checkpoint requiring Ag recognition that regulates memory generation. Our results also suggest that checkpoint Ag recognition by CD4 effectors also plays a role in supporting B cell responses. We discuss the implications for vaccine and the possible reasons why the impact of adding Ag/*APC at the checkpoint is not as dramatic as it was with effectors generated by live virus.

Materials and Methods

Mice.

Female and male 8-to-10 wk BALB/c, C57BL/6 and BALB/c.Thy1.1^+/+ mice were purchased from Jackson Laboratory. The transgenic DO11.10 (BALB/cJ-Tg (DO11.10) Tg (CARΔ−1)1Jdgr) mice were purchased from Taconic Biosciences (Rensselaer, NY). HNT TcR Tg.Thy1.1^+/− mice were generated by breeding BALB/c.Thy1.1^+/+ mice with HNT TcR Tg mice. NP TcR Tg mice were generated in a collaborative effort by Dr. Eric Huseby’s laboratory. FluNP TcR transgenic mice express CD4 T cell receptor that is specific for the MHCII-IA^b-restricted influenza NP_311–325 epitope (QVYSLIRPNENPAHK). Thy1.1^+/− mice were generated by breeding C57BL/6.Thy1.1^+/+ mice with NP TcR Tg mice. Details of this strain will be described in a manuscript in preparation. All the animals were maintained in the animal facility at University of Massachusetts Medical School (UMMS).

Viral infection and immunization.

Influenza A virus, A/PR8/34 (PR8, H1N1) was derived from a stock in Allen Harmsen’s Laboratory originally from David Morgan at Scripps Research Institute. It was passaged through BALB/c mice and grown in chicken eggs at the Trudeau Institute. This same batch of virus has been used in our previous studies (32). Formalin-inactivated influenza vaccine (WIV, purified and inactivated Influenza A/PR/8/34 (H1N1)) was purchased from Charles River (Material#10100782). PR8 infection was carried out by intranasal (i.n.) administration of virus at 0.3LD₅₀. Immunization with WIV was by intravenous (i.v.) injection of 2.5μg WIV. To enhance the CD4 T cell response, 3×10⁵ HNT/*BMDC (bone marrow derived dendritic cells), *BMDC pulsed with 12.5 μM HA_126–138 for 1hr (see below), was added with the WIV as in our previous study (30). The immunization strategy is referred to as WIV+Ag/*APC.

Ag/*APC generation.

For BMDC generation, bone marrow from BALB/c mice was harvested from femurs and tibias and a single cell suspension was generated. 8×10⁶ bone marrow cells were seeded in each Petri dish (#FB0875712, Fisher Scientific, Hampton, NH) and cultured in 10 ml T cell medium with GM-CSF (#576306, Biolegend, San Diego, CA) (at 10 ng/mL). On day 3, 10 ml medium was added. On day 5, the medium was refreshed by removing 10 mL culture and replacing with 10 ml fresh medium. On day 6, Poly (I:C) (LMW) (InvivoGen, San Diego, CA) and CpG ODN 1826 (5’-TCCATGACGTTCCTGACGTT-3’) (Integrated DNA Technologies, Inc. San Diego, CA) added to activate the cultured cells. On day 7, the cells were harvested and CD11c⁺ cells were isolated using the CD11c MicroBeads UltraPure for mouse (#130–108-338, Miltenyi Biotec, Bergisch Gladbach, Germany) and LS column (#130–042-401, Miltenyi Biotec, Bergisch Gladbach, Germany). These activated CD11c⁺ cells (*BMDC) were pulsed with the HNT epitope peptide, HA_126–138 (HNTNGVTAACSHE, New England Peptide, Gardner, MA), at the indicated concentrations (1.25 μM, 12.5 μM, 125 μM) for 1hr at 37⁰C. The HNT peptide-pulsed, activated BMDCs are referred to as Ag/*APC.

Lymphocyte isolation and naïve CD4 T cell preparation.

Spleen and prominent lymph nodes (LN) were harvested from HNT Thy1.1⁺ BALB/c TcR transgenic mice or NP Thy1.1⁺ TcR C57BL/6 Tg mice. Single-cell suspensions were generated by processing the tissues. Small resting cells were isolated by 40%, 53%, 62%, and 80% percoll (#17–0891-01, GE Healthcare Bio-Sciences, Pittsburgh, PA) density gradient centrifugation and collecting the cells between the 40% and 53% inner layers. Naïve CD4 T cells were isolated from those small resting cells by mouse CD4 (L3T4) MicroBeads (#130–117-043, Miltenyi Biotec, Bergisch Gladbach, Germany) and LS column (#130–042-401, Miltenyi Biotec, Bergisch Gladbach, Germany).

Transfer experiments.

For transfer experiments (Figs 3, 4, and 5 and Fig 6F-H), 10⁵ naïve Thy1.1⁺ HNT TcR Tg CD4 T cells were transferred into DO11.10 hosts, treated either with WIV+HNT/*BMDC (Ag/*APC) immunization or by infection with 0.3LD₅₀ PR8 1 day prior to treatment, −1 days post treatment (dpt). To study the impact of checkpoint Ag recognition on memory generation and T helper function, 3×10⁵ Ag/*APC were injected intravenously (i.v.) to the WIV+Ag/*APC immunized mice at 5 dpt. For the CD4 T cell kinetics study in Fig 1, naïve Thy1.1⁺ HNT TcR Tg CD4 T cells were labeled with CFSE by incubating the naïve CD4 T cells with 5μM CFSE (#C34554, Invitrogen, Carlsbad, CA) solution at 10⁷ cells/mL at 37⁰C for 20 min. 10⁵ CFSE labeled naïve CD4 T cells were injected i.v. on −1 days post-treatment (dpt) into the BALB/c mice given WIV+Ag/*APC or PR8 live virus. For the CD4 T cell kinetics study in Supplementary Figure 1 and Supplementary Figure 4, 10⁵ naïve Thy1.1⁺ NP TcR Tg CD4 T cells were i.v. injected on −1 dpt into C57BL/6 mice given WIV or PR8 live virus. For Supplementary Figure 4, *BMDC pulsed with NP_311–325 were used as Ag/*APC, to assess the impact of “checkpoint” Ag, 3×10⁵ of these Ag/*APC were i.v. injected to hosts at 5 dpt. For the Ag presentation kinetics studies (Figure 2, Supplementary Figure 2 and 3), 10⁶ CFSE labeled naïve Thy1.1⁺ HNT TcR Tg CD4 T cells were i.v. injected into treated BALB/c mice on 0, 3, 5, 7, and 11 dpt. After 2.5 days, spleen, lung dLN (draining LN)/mLN (mediastinal LN) and lung cells were harvested from the host mice. We gated on donor Thy1.1⁺ cells and determined the loss of CFSE dye indicating division induced by Ag presentation.

FIGURE 3. Impact of checkpoint Ag/*APC addition at 5 dpt on donor CD4 memory generation.

As in Fig 1 and 2, naïve HNT.Thy1.1 TcR Tg CD4 T cells were transferred into DO11.10 BALB/c hosts 1 day before immunization with WIV+Ag/*APC. On 5 dpt, Ag/*APC were i.v. injected (empty squares) or not injected (filled circles) into the immunized mice hosts to provide checkpoint Ag. At 35 dpt, Thy1.1⁺ CD4⁺ donor HNT memory cells were detected by FACS. A) Schematic diagram of the experiment. B) and C) The enumeration of Thy1.1⁺ CD4⁺ donor memory cells in spleen (B) and lung (C). The graphs show the frequency (left panel) as a % of spleen or lung CD4 T cells, and the total number per organ (right panel) of Thy1.1⁺ CD4⁺ donor memory cells on 35 dpt. D) Representative plots show the expression of CD44 and CD62L on Thy1.1⁺ CD4⁺ donor memory cells in spleen (upper panel) and in lung (lower panel) with or without Ag/*APC treatment on 5 dpt. E-F) The frequency (left panel) and total number (right panel) of CD62L^lo Thy1.1⁺ CD4⁺ donor memory cells in spleen (E) and lung (F). The results of 2 experiments were pooled. For each experiment, n≥4 mice were used in each group.

FIGURE 4. Impact of checkpoint Ag/*APC addition at the 5 dpt on donor CD4 T_RM generation.

Naïve HNT.Thy1.1 TcR Tg CD4 T cells were transferred into DO11.10 BALB/c hosts 1d before the initial WIV+*Ag/APC immunization (0 dpt). Additional Ag/*APC were injected i.v. into the immunized mice on 5 dpt (empty squares) or not (filled circles). On 35 dpt, Thy1.1⁺CD4⁺ memory donor cells in lung were detected by FACS and analyzed for expression of CD69, CXCR6 and for in vivo labeling with anti-CD4 Ab. (A and B) CD69 expression on Thy1.1⁺CD4⁺ donor memory cells in lung. A) Representative histograms show CD69 expression on the donor cells from lungs of individual mice. B) The frequency (left panel) and total number (right panel) of CD69⁺ donor cells in lung from individual mice. (C and D) CXCR6 expression on Thy1.1⁺CD4⁺ donor memory cells in lung. A) Representative histograms show CXCR6 expression on the donor cells from lungs of individual mice. B) The frequency (left panel) and total number (right panel) of CXCR6⁺ donor cells in lung from individual mice. (E-G) Accessibility to in vivo antibody labeling was used to distinguish T_RM from circulating memory donor cells in lung. C) Representative graph showing in vivo staining of cells in blood and donor memory cells in lung and spleen with anti-CD4 injected i.v. to determine accessible (circulating) and inaccessible (resident) T_RM. The frequency (left) and number (right) of cells per spleen D) and lung E) were determined. For CD69 and in vivo study, n≥4 mice were in each group. Data from two experiments were pooled. For CXCR6 study, n=4 mice were in each group. The experiment was repeated and showed the same pattern of results. The data of one experiment is shown.

FIGURE 5. Effect of checkpoint Ag/*APC on CD4 T cell helper activity.

As in other experiments, Naïve HNT.Thy1.1 TcR Tg CD4 T cells were transferred into BALB/c mice on −1 dpt. Mice were immunized with WIV+Ag/*APC on 0 dpt. Additional checkpoint Ag/*APC were i.v. injected or not into the immunized mice on 5 dpt. A-C) The impact of checkpoint Ag/*APC on ICOS expression. 2 days after the injection, ICOS expression on donor cells were detected by FACS. A) Representative histograms show ICOS expression on naïve donor cells (dashed line), donor cells received no Ag/*APC treatment on 5 dpt (solid line) and donor cells treated with Ag/*APC on 5 dpt (filled black). B) The mean fluorescence intensity (MFI) of ICOS on donor cells 2 days after the treatment with (empty squares) or without (filled circles) checkpoint Ag/*APC. C) The frequency (left) and quantity (right) of ICOS⁺ donor cells 2 days after the treatment with (empty squares) or without (filled circles) checkpoint Ag/*APC. D-F) The impact of checkpoint Ag/*APC on T_FH marker expression. D) Representative plots show Bcl6⁺CXCR5⁺ (upper panel) and Bcl-6⁺ICOS⁺ (lower panel) donor cells without (left) or with (middle) 5 dpt Ag/*APC treatment. The population in donor cells in PR8 infection (right) was used as control. E-F) The frequency (left) and number (right) of Bcl-6⁺CXCR5⁺ (E) and Bcl-6⁺ICOS⁺ (F) donor cells 2 days after treatment with (empty squares) or without (filled circles) checkpoint Ag/*APC. The experiment was repeated. 2 experiments showed similar results. The data from 1 experiment is shown. (G-I) T helper function on AbSC generation was assessed in the same model. G) The level of PR8 specific IgG in serum collected from individual mice from each group treated with (empty) or without (filled) checkpoint Ag/*APC was measured by ELISA at 35 dpt. H) and I) The frequency (left) and number (right) per organ of PR8-specific IgG secreting cells (AbSC) in spleen H) and BM I) were detected by ELISpot. Data from two experiments were pooled. For each experiment, n≥3 mice were in each group.

FIGURE 6. Impact of increased antigen density at the checkpoint on donor memory generation.

Naïve HNT.Thy1.1 TcR Tg CD4 T cells were transferred into DO11.10 BALB/c hosts on −1 dpt. Mice were immunized with WIV+Ag/*APC. On 5 dpt Ag/*APCs were injected i.v. into the immunized mice or not. The activated BMDC (*APC) were pulsed with 10-fold increasing concentrations of HNT peptide Ag. 1.25 μM (square), 12.5 μM (triangle) and 125 μM (upside down triangle). Immunized mice receiving no Ag/*APC were used as control (filled circle). On 35 dpt, Thy1.1⁺ CD4⁺ donor memory cells in spleens and lungs were detected by FACS. A). Schematic diagram of the experiment. B). The frequency (left) and the quantity (right) of Thy1.1⁺ CD4⁺ memory donor cells in spleen. C). The frequency (left) and the quantity (right) of Thy1.1⁺ CD4⁺ donor memory cells in lung. D). The generation of CD69⁺ donor memory cells in lung. E) The level of PR8-specific IgG in serum collected from individual mice from each group was measured by ELISA at 35 dpt. (control, Black. 1.25 μM, dark grey. 12.5 μM, blank. 125 μM, light grey.) For each experiment, n≥3 mice were in each group. Data from two experiments were pooled.

Figure 1. Kinetics of the CD4 T cell response: accumulation and division.

Isolated BALB/c.HNT.Thy1.1 TcR Tg naïve CD4 T cells were labeled with CFSE, and 10⁵ per mouse were adoptively transferred into BALB/c mice (n=4), 1 day before immunization with WIV vaccine (WIV+Ag/*APC) or PR8 infection. BALB/c hosts were injected intravenously (i.v.) on day 0, with WIV (2.5 μg) mixed with 3×10⁵ bone marrow-derived dendritic cells (BMDC) pulsed with HNT_126–138 (12.5μM). BMDC were activated with Poly I:C and CpG (*BMDC) (aka Ag/*APC) overnight then pulsed with HNT peptide. The control group of hosts was inoculated intranasally (i.n.) with A/PR8/34 (PR8, H1N1) influenza A virus at a sublethal dose, 0.3LD₅₀. A). Schematic diagram of the experimental design. B, D, F) Kinetics of CD4 T cell accumulation. Following WIV+Ag/*APC immunization (solid line, filled square) or PR8 infection (dashed line, empty circle), the number of donor Thy1.1⁺ CD4⁺ T cells in spleen (B), dLN (draining LN) for infection and mLN (mediastinal LN) for immunization (D) and lung (F) was determined at the indicated time points: 0, 2, 3, 5, 7 days post-treatment (dpt) using FACS analysis. C, E, G) Dynamics of CD4 T cell division. Following WIV+Ag/*APC immunization (left panel) and PR8 infection (right panel), the intensity of CFSE expression by Thy1.1⁺ donor CD4⁺ T cells in spleen (C), lung (E) and LN (G) was measured at 0, 2, 3, 5, 7 (dpt) by FACS analysis. A representative histogram from one individual is shown to indicate the extent of division within of the donor CD4 T cell population. Individual mice in each group (n=4) were analyzed at each indicated time points. The exact experiment was repeated twice. Data from one of the experiments is shown. Similar results were seen in other 2 experiments.

Figure 2. Kinetics of Ag presentation in spleen.

As in Fig. 1, BALB/c hosts were treated with WIV+Ag/*APC immunization, WIV immunization or PR8 infection on 0 dpt. Mice receiving no treatment (None) were used as negative controls. 10⁶ naïve CFSE-labeled HNT.Thy1.1 CD4 T cells (Detector cells) were injected i.v. into treated host mice on 0, 3, 5, 7, and 11 dpt. 2.5 days after HNT donor injection spleens, pooled LN and lung were harvested, and the intensity of CFSE dilution in the donor HNT,Thy1.1⁺ CD4⁺ detector cells was determined by FACS. A) Schematic diagram of this experimental design. B) Representative histograms showing CFSE fluorescence in Thy1.1⁺ donor cells harvested from the spleens of hosts from each group. The gate and percentage of the divided detector cells are indicated. C) The total number of the divided detector cells recovered from individual spleens of PR8-infected (empty), WIV+Ag/*APC immunized (filled black), WIV immunized (filled dark grey) and no treatment (filled light grey) mice. D) The number of the total donor cells in spleens of hosts that were PR8 infected (empty), WIV+Ag/*APC immunized (filled black), WIV immunized (filled dark grey) and untreated (filled light grey) mice. Mice (n=3) in each group at each indicated time points. Experiments were repeated twice. Data from one of the experiments is shown. Similar results were seen in other 2 experiments. For statistics, the data of the vaccine immunized groups was compared to that of the PR8 infected group. The data of WIV immunized group was compared to that of WIV+Ag/*APC immunized group.

Enzyme-linked immunosorbent assay (ELISA).

Corning® 96 Well EIA/RIA Assay Microplates (MilliporeSigma, Burlington, MA) were coated with WIV (Charles River) in ELISA coating buffer (0.05M Carbonate-Bicarbonate, pH9.6). Plates were blocked with bovine serum albumin (BSA) in PBS. Serial dilutions of serum were plated and incubated at 4⁰C overnight. Plates were washed with ELISA wash buffer (0.05% Tween20 in PBS) followed by incubation with HPR-conjugated goat anti-mouse IgG (Southern Biotech, Birmingham, AL). Plates were developed with the peroxidase substrate, o-Phenylenediamine (OPD) (MilliporeSigma, Burlington, MA). Optical density (OD) readings at 490 nm (OD490) obtained from serial dilution were analyzed by nonlinear regression. The endpoint titer was defined as the serum dilution resulting in an OD490 value equivalent to the cutoff value.

B cell enzyme-linked immunospot (ELISpot).

MultiScreen-HA filter plates (MilliporeSigma, Burlington, MA) were coated with WIV (Charles River) in ELISA coating buffer (0.05M Carbonate-Bicarbonate, pH9.6). Plates were blocked with bovine serum albumin (BSA) in PBS. Serial dilutions of splenocytes and BM cells were plated and incubated at 37⁰C overnight. Plates were washed with ELISA wash buffer (0.05% Tween20 in PBS) followed by incubation with HPR-conjugated goat anti-mouse IgG (Southern Biotech, Birmingham, AL). Plates were developed with AEC Substrate Set (BD Biosciences, San Jose, CA). PR8-specific IgG antibody secreting cell (AbSC) spots were quantified using the ImmunoSpot reader and the data was analyzed with ImmunoSpot® Software. (Cellular Technology Ltd., Cleveland, OH).

Flow cytometry and antibody reagents.

Spleens, lungs and LNs (dLN/mLN) were harvested from the infected, immunized or naïve mice. Single-cell suspensions were prepared, blocked with anti-Fc RII/III monoclonal antibody (Bio X cell, West Lebanon, NH) and live/dead stained using LIVE/DEAD™ Fixable Yellow Dead Cell Stain Kit (Invitrogen, Carlsbad, CA). Cells were then surface stained with antibodies: FITC-anti-mouse CD69 (clone H1.2F3), PE-Cy7-anti-mouse CD44 (clone IM7), PE-Cy-anti-mouse Thy1.1 (clone OX-7), APC-anti-mouse CXCR6 (clone SA051D1) and Alexa Fluor® 488 anti-mouse ICOS (clone C398.4A) were purchased from Biolegend (San Diego, CA); APC-anti-mouse Thy1.1 (clone HISS1), APC-efluor 780-anti-mouse CD4 (clone RM4–5), PerCP-eFluor 710-anti-mouse CXCR5 (clone SPRCL5), and PerCP-Cyanine5.5-anti-mouse CD62L (clone MEL-14) were purchased from ebioscience, Inc. (San Diego, CA); PerCP-Cy™ Rat anti-mouse CD44 (clone IM7), and PE-anti-mouse Bcl-6 (clone K112–91) was purchased from BD Biosciences (San Jose, CA). For the in vivo and ex vivo antibody labeling, Pacific Blue anti-mouse CD4 (clone RM4–4, Biolegend, San Diego, CA) was injected into the immunized mice. At 5 min after injection mice were euthanized with isoflurane (Patterson Veterinary Supply, Inc. Devens, MA) followed by cervical dislocation. Blood, spleens and lungs of the mice were harvested and the single cell suspensions were prepared. Cells from different tissues were stained as indicated above. A successful in vivo antibody labeling was assured by over 99% blood CD4 T cells accessible to in vivo antibodies as indicated in the published protocol (33). Stained samples were fixed with 1% formaldehyde (Thermo Fisher Scientific Inc., Waltham, MA). Data were acquired on a BD LSRII Fortessa instrument (BD Biosciences, San Jose, CA) and analyzed using the FlowJo software program (Tree Star, Ashland, OR).

Ethics Statement.

Experimental animal procedures were conducted in accordance with guidelines outlined by the Office of Laboratory Animal Welfare (OLAW), National Institute of Health, USA. Protocols were approved by the Institutional Animal Care and Use Committee of the University of Massachusetts Medical School (Worcester, MA).

Statistical analysis.

For statistical analysis, significance comparing the mean of two normally distributed groups was determined by the two-tailed unpaired student t-test. Significance comparing the mean of over 3 normally distributed groups was determined by one-way ANOVA with Bonferroni’s multiple comparison post-test. p values of <0.05 were considered significant. Error bars in figures represent the SEM. NS, not significant. *=P<0.05. **=P<0.01. ***=P<0.001. ****=P<0.0001. The results were analyzed by GraphPad Prism software (GraphPad Prism software, San Diego, CA).

Results

Vaccine induces an earlier and transient CD4 T cell response compared to live infection

We based our experimental design on the cell transfer model that we previously developed to evaluate the requirements for naïve CD4 T cell response to WIV immunization. To evaluate the development of CD4 effectors and memory, we transferred HNT TcR Tg CD4 T cells (BALB/c. HNT. Thy1.1) into BALB/c mice. We previously showed the naïve HNT CD4 T cells, whose TCR specifically recognizes the HA_126–138 peptide in the hemagglutinin of PR8 (H1) influenza, respond to PR8 infection in vivo (23, 32) and in vitro to HNT peptide-pulsed APC (31). We treated the host mice with WIV vaccine (i. v.) and for comparison infected with influenza (PR8) live virus. By following the proliferation and differentiation of donor cells, this model allows us to study both Ag presentation of the HNT epitope peptide following infection vs vaccination and the fate of naïve CD4 T cells after infection vs vaccine. We used our previously published strategy, WIV+Ag/*APC, as our standard “immunization” because adding exogenous Ag/*APC induced a more robust naïve CD4 T cell effector response of both young and aged CD4 T cells (30) and we want to ensure efficient initial naive CD4 effector generation. HNT peptide-pulsed BMDC were activated with poly I:C and CpG (Ag/*APC) since activated APCs express high levels of MHC and costimulatory ligands (CD80/86) that maximize naïve CD4 T cell response (30). The co-injection WIV/Ag-*APC strategy, induced greater donor CD4 T cell proliferation and differentiation, and greater PR8-specific IgG in a IL-6 dependent manner (30). In key experiments we compared responses to WIV alone. We know the Ag/*APC only present Ag for 48 hr or less (22), so we can readily evaluate the subsequent requirement for Ag recognition by adding Ag/*APC after 4–5 d.

To determine the kinetics of the vaccine-induced CD4 T cell response, we followed donor naïve BALB/c.HNT.Thy1.1 CD4 T cell recovery and division following WIV+Ag/*APC-immunized mice and compared it to that in PR8-infected mice. WIV+Ag/*APC immunization induced a rapid and vigorous expansion of CD4 T cells from 2 to 5 dpt in the spleen (Figure 1B), LN (Figure 1D) and lung (Figure 1F). CD4 donor cell number peaked on 4 or 5 dpt and then rapidly decreased in spleen (Figure 1B) and mLN (Figure 1D). We found fewer CD4 T effectors in the lung and their numbers were maintained at similar levels during 4–7 dpt (Figure 1F). In contrast to the vaccination, PR8 infection induced a delayed CD4 T cell response. After infection, donor CD4 T cells did not increase detectably until 3–4 dpt in dLN (Figure 1D) and even later (5 dpt) in spleen (Figure 1B). In earlier studies, we found PR8-induced donor HNT CD4 effector and polyclonal CD4 effector response peaked at 6–7 dpt in spleen, lung and dLN (15, 34) and stayed elevated through 10 dpt. Thus, WIV+Ag/*APC induced an earlier but transient CD4 T cell response, while live PR8 infection induced a later response that was sustained longer, consistent with our earlier studies (15, 34).

To determine if the addition of Ag/*APC to WIV would alter the kinetics of CD4 response to WIV immunization, we compared response kinetics of WIV alone (without the added Ag/*APC) and to live PR8 virus. We found the same pattern of the early and transient response in spleen, LN and lung in WIV-treated mice as with WIV+Ag/*APC-treated mice (Supplementary Figure 1), indicating that the addition of Ag*/APC did not alter the kinetics of CD4 response to WIV immunization.

To further evaluate when naïve CD4 T cell actually started proliferating following WIV immunization, we assessed donor CD4 T cell division by analyzing the loss of CFSE (Figure 1C, 1E and 1G). After immunization with WIV+Ag/*APC, donor cells were dividing by 2 dpt in the spleen and by 3 dpt in the LN and lung (Figure 1C, 1E and 1G). We compared this to WIV alone without Ag/*APC. After both WIV and WIV with Ag/*APC, detector cells had undergone>6–8 divisions in all organs by 3 dpt. In contrast, after PR8 infection, divided donor CD4 HNT cells were not detectable in spleen until 5 dpt (Figure 1C), in LN until 4 dpt (Figure 1E) and in lung until 5 dpt (Figure 1G). By 7 dpt, transferred cells in all 3 organs were fully divided. Thus, compared to PR8 infection, vaccine immunization, with or without extra Ag/*APC at 0 dpt, induced earlier but more transient donor CD4 T cell division that led to an earlier peak of effector accumulation and an early contraction.

Vaccine induces earlier but more transient Ag presentation

The earlier but more transient CD4 T cell response to vaccine immunization, suggests Ag presentation was short-lived. Thus, we determined when the donor CD4 T cells saw the Ag, as in earlier studies (32). We used CFSE-labeled naïve donor HNT CD4 T cells as detectors that lose the dye as they divide, to visualize ongoing Ag presentation. We transferred these detector cells into the immunized or infected hosts at 0, 3, 5, 7 and 11 dpt (Figure 2A), harvested them 2.5 days later, and assessed CFSE dilution of the donor CD4 T cells in spleen, LN and lung (Figure 2A). We quantitated the extent of Ag presentation at the indicated intervals (0–2.5, 3–5.5, 5–7.5, 7–9.5 and 11–13.5 dpt). In mice immunized with WIV+Ag/*APC, donor cells divided most extensively in the spleen when added at 0 dpt, while those added at 3 dpt or later showed much less division (loss of CFSE) (Figure 2B). Immunization with WIV alone showed a similar pattern of results, confirming that both the WIV alone and mixed with Ag/*APC, present little Ag after 3 dpt. In contrast, in PR8 infected mice, donor cells showed no evidence of Ag recognition during the 0–2.5 dpt interval, with no division of detector cells in spleen (Figure 2A), dLN or lung (Supplementary Figure 2A and 3A) when added at 0 dpt. Instead, PR8 infection caused donor division that was sustained at a higher level at intervals from day 3 on, continuing to days 5 −7, with less, but still detectable division at 11 dpt in spleen (Figure 2B and C), LN and lung (Supplementary Figure 2B,C and 3B,C). The sustained division lead to greater expansion by 7 dpt (Figure 1, Supplementary Figure 1).

We calculated the total donor cell recovery and the divided donor cells. In vaccine-immunized hosts, the highest recovery of both total and divided donor cells occurred when the detectors were added at 0 dpt, with the numbers decreasing when added at 3 dpt and thereafter (Figure 2C and 2D). Similar results were seen in LN and lung. (Supplementary Figure 2B,C and 3B,C) Thus, in the WIV+Ag/*APC or WIV alone immunized mice, the magnitude of Ag presentation, shown as both division and recovery of detectors, peaked before 2.5 dpt (Figure 2B) and declined abruptly after 3 dpt. In contrast, following PR8 infection, CFSE loss was delayed for at least 2–3 days, peaking at day 5, then gradually declining through days 7 and by 11 dpt in the spleen, dLN (Supplemental Figure 2), and lung (Supplemental Figure 3). Donor cells recovered in the lung at later times might be effector cells that recognized Ag and may have divided in spleen and LN and thereafter migrated to the lung, this set of results may not accurately reflect in situ Ag presentation in lung. Ag presentation induced by WIV alone, was of a somewhat lower magnitude, with significantly fewer divisions and lower recovery of detectors, but it followed similar kinetics as that induced by our standard WIV+Ag/*APC vaccine (Figure 2, and Supplementary Figure 2 and 3).

Together these data indicate that effective Ag presentation by either WIV+Ag/*APC or WIV immunization is early but transient, leading to early but robust CD4 T cell proliferation and generation of highly divided effectors peaking at 4–5 dpt, which then contract (Figure 1 and 2), while PR8 infection leads to a longer period of Ag presentation with effectors peaking at 5–7 dpt before the onset of contraction. This is consistent with the concept that even if WIV-generated effectors get extra help from Ag/*APC during priming, the CD4 donor effectors are not exposed to sufficiently strong Ag presentation at 4–5 dpt, when the response peaks, since high amounts of Ag are present for only 3 days. In contrast, PR8-induced effectors peak at 7 dpt and are exposed to Ag presentation from 3–11 dpt. Thus PR8-generated, but not WIV-generated CD4 effectors, can and do recognize Ag during the peak of their effector response which we earlier identified as a “checkpoint”, co-incident with the effector peak.

Addition of Ag/*APC at 5 dpt promotes more generation of total and CD62^lo memory CD4 T cells in lung than in spleen

Since Ag presentation signals induced by WIV+Ag/*APC vaccine had nearly disappeared by 3–5 dpt (Figure 1 and 2), we tested whether providing Ag/*APC exogenously at 5 dpt, at the time when effector numbers peak, would augment memory generation. We reasoned that at their peak, effectors are most committed to default apoptosis and require Ag/*APC to stimulate IL-2 that promotes their rescue, as suggested in our earlier studies (22, 23). As above, mice were immunized by WIV+Ag/*APC on 0 dpt and Ag/*APC, known to effectively provide Ag presentation signals that drive PR8-induced effectors to become CD4 memory generation effectors (22), were added at 5 dpt. To study effects of checkpoint Ag on both memory CD4 T cell generation and CD4 helper function, indicated by Ab production, we used our previously established reductionist model. We transferred donor HNT.Thy1.1 naïve CD4 T cells into the DO11.10 TCR hosts and followed the donor CD4 T cell response after WIV immunization. DO11.10 TCR hosts, bear a transgenic TCR specific for an influenza-irrelevant ovalbumin peptide (OVA_II), and host CD4 T cells do not respond to WIV. The transfer of HNT CD4 T cells into DO11.10 hosts, allows us to evaluate not only the generation of donor CD4 memory cells but also their functional contribution (e.g. helper function) independent of host CD4 responses (23, 30). The advantage of this model is the ability to follow the response and impact of the naïve cohort of cells, unambiguously and assess the impact of providing the peptide Ag to these cells without introduction of Ag seen by B cells or host T cells.

To test the impact of Ag recognition at the effector checkpoint on memory CD4 T cell generation, we transferred Ag/*APC to the vaccine immunized hosts on 5 dpt, and compared results to hosts that did not receive Ag/*APC., We enumerated donor cells in spleen and lung on 35 dpt. Addition of Ag/*APC at 5 dpt led to a slight 1.7-fold increase in total donor memory cell number in spleen (Figure 3B) but a larger 4.5-fold donor cell increase in the lung (Figure 3C). The selective increase of donor memory cells in lung, led us as to analyze whether Ag/*APC at 5 dpt favors generation of CD62L^lo memory cells, which include both tissue-restricted memory T_RM and recirculating T_EM. Following vaccine immunization with no Ag/*APC at 5 dpi, donor cells were mostly CD62^lo in both spleen (80%) and lung (78%) (Figure 3D). When Ag/*APCs were injected on 5 dpt, there was a minor increase in percentage, and a 1.7 fold increase in number of CD62L^lo donor CD4 memory cells in the spleen and a higher increase in percentage (>90%) and a 4.2-fold increase in lung (Figure 3D-F). Thus, the addition of checkpoint Ag/*APC on 5 dpt, led to more than 4-fold higher generation of both total and CD62^lo memory and CD4 T cells in the lung with a smaller increase in the spleen.

We also evaluated if memory augmentation by “checkpoint” Ag presentation occurs after immunization with WIV alone, in a different but comparable model into which we transfer FluNP TcR CD4 T cells (as in Supplementary Fig 1A) as the donor cells whose fate we follow. The mice were immunized with WIV alone and injected with Ag/*APC or not at 5 dpv as in Supplementary Fig 1B. Similar to WIV+Ag/*APC immunization, donor memory cells in both spleen (Supplementary Fig 4B) and lung (Supplementary Fig 4C) were significantly increased in both frequency (3.9-fold for spleen and 9.4-fold for lung) and number (~2.0-fold for spleen and lung). To confirm that Ag is essential for the impact, we compared the impact of Ag/*APC with that of *APC not pulsed with peptide Ag. In the HNT model, we added *APC alone without peptide at 5 dpt. We compared the generation of memory donor cells to those in immunized mice without 5 dpt *APC addition. We found adding*APC without Ag at 5 dpt had no impact on donor memory cell recovery, which was at a similar frequency and number as in untreated immunized mice (Supplementary Fig 4E and 4F) and the same general magnitude as in Figure 4, without Ag/*APC. Thus, as expected, the impact of Ag/*APC is peptide-Ag dependent.

Addition of Ag/*APC at 5 dpt enhances the generation of T_RM in the lung

Memory CD4 T cells in detected in peripheral tissues include both recirculating T_EM and retained T_RM in the tissue. T_RM play critical roles in providing long-lived immunity locally (35). Specifically, in the lung, T_RM provide strong protection against influenza challenge (36). Both T_EM and T_RM are CD62L^lo cells. Therefore, we further analyzed what fraction of the augmented CD62L^lo memory CD4 T cells are T_RM rather than T_EM.

We examined expression of CD69, a T_RM signature marker of all T_RM at 35 dpt (36, 37). Addition of Ag/*APC at 5 dpt increased both CD69⁺ donor lung memory cell frequency (2.1-fold) and number (6.6-fold) (Figure 4A-B). Moreover, expression of CXCR6, shown to be upregulated on T_RM (38), was also significantly upregulated by the addition of Ag/*APC at 5 dpt. Addition of Ag/*APC at 5 dpt also increased the frequency (2.0-fold) and number (3.3-fold) of CXCR6⁺ donor lung memory cells (Fig 4C-D). To confirm the that donor memory cells were bona fide T_RM, we used in vivo antibody labeling in the same experiments, to identify more Ab-inaccessible donor T_RM cells from those circulating which are Ab-accessible. Consistent with expectation (33), over 99% blood CD4 T cells were labelled by i.v. injected Ab, while most of the donor memory CD4 T cells at 35 dpt in the spleen remained un-labelled, confirming the spleen T_CM share tissue-resident properties (Figure 4C). We found the addition of Ag/*APC at 5 dpt led to a significantly increased fraction of un-labelled donor CD4 memory in lung (Figure 4E) but not spleen (Figure 4D), strongly suggesting many are T_RM. The total number of donor T_RM was increased 4.7-fold in the lung, with little impact on donor T_RM in spleen. Taken together, Ag/*APC addition on 5 dpt promoted immunization-generated CD4 effectors to migrate and accumulate in the lung in larger numbers and to further differentiate into CD69⁺/CXCR6⁺ T_RM cells. Thus, recognition of checkpoint Ag selectively promoted CD4 effectors to become memory lung T_RM but not T_EM.

Addition of Ag/*APC at 5 dpt increases anti-influenza Ab titers and antibody-secreting cell recovery.

Given the increased donor memory generation, we asked if adding checkpoint Ag/*APC would lead to more helper cells and to better helper activity. We assessed expression of helper phenotypes on CD4 donor effectors, the recovery of Ab-secreting B cell (AbSC) responses (35 dpt) and influenza-specific Ab production. As above, HNT naïve CD4 donor cells were transferred to DO11.10 hosts, which were immunized with WIV+Ag/*APC, and Ag/*APC were added or not on 5dpt. To assess induction of more differentiated effectors, we measured the expression of ICOS, one signature marker of functional CD4 T helper cells (39), two days later (Figure 5A-C). Adding Ag/*APC on 5 dpt enhanced expression of ICOS significantly (Figure 5A), with higher expression per effector (2.3-fold) (Figure 5B) and increased numbers of ICOS⁺ donor effectors (2.4-fold) (Figure 5C). Thus, Ag presentation at the effector checkpoint enhanced effectors to upregulate ICOS expression which might correlate with enhanced helper activity. To analyze whether the effectors developed other indications of a more T_FH-like phenotype, we evaluated the co-expression of Bcl-6 and CXCR5 on donor cells with or without Ag/*APC treatment at 5 dpt. We found adding the checkpoint source of Ag, resulted in the generation of a modest 2-fold increase in Bcl-6⁺CXCR5⁺ and Bcl-6⁺ICOS⁺ expression on a relatively small population of cells (Figure 5E and F), which were fewer and with less intensity of staining than those generated by PR8 infection (Figure 5D). This moderate impact may be because this is a time 1–2 d before the peak of T_FH and/or it may indicate the effectors require additional signals to progress to full T_FH status.

To test if checkpoint Ag presentation resulted in generation of a prolonged AbSC response, we analyzed the B cell Ab response to PR8 IAV after 35 dpt, using the same experimental design. We measured the serum titer of anti-PR8 IgG by ELISA and determined the number of anti-PR8 IgG secreting cells (AbSC) by ELISpot. Anti-PR8 IgG titers were significantly increased (2.8 fold) in the vaccine-immunized mice treated with Ag/*APC at 5 dpt (Figure 5C). Consistent with serum titers, PR8-specific IgG AbSC in spleen (Figure 5D) were modestly increased in number (1.7 fold) with the Ag/*APC treatment at 5 dpt. Anti-PR8 IgG AbSC cells in BM (Figure 5E) were significantly increased in both frequency (2.1 fold) and number (3.4 fold). In summary, addition of the checkpoint Ag/*APCs following WIV+Ag/*APC vaccine immunization increased anti-influenza long-term (35 dpt) AbSC and level of IgG1 in the serum, presumably indicating better helper CD4 effector activity.

Quantity of memory CD4 T cells is increased by higher levels of Ag density at the checkpoint

While it was clear that adding “checkpoint” Ag/*APC was effective in enhancing lung CD4 memory and inducing T_RM generation, we were still puzzled to see only a modest increase in CD4 memory in the spleen, since in the live virus studies, almost all spleen memory generation depended on the checkpoint Ag recognition which increased memory 20–200-fold (22, 23). We reasoned that live influenza virus infection provides a very high level of Ag throughout the “checkpoint” interval. Thus, we analyzed whether increasing Ag density at checkpoint (5 dpt) would result in an additional increase in CD4 memory generation. For studies above, we used a dose of HA_126–138 peptide (12.5µM) to pulse *APC at the checkpoint (5 dpt), with is higher than doses of 1–2.5 µM per mouse routinely used in studies (40). Here we pulsed *APC with three peptide doses: 1.25, 12.5 and 125µM (Figure 6A) and added these at 5 dpt. There was a clear impact of increased peptide density, which led to an increase in memory generation. At low density of peptide (1.25 μM), Ag/*APC on 5 dpt only slightly increased the frequency of donor cells in spleen compared to no Ag/*APC addition and there was no enhancement in numbers of donor memory (Figure 6B). As Ag concentration increased, donor memory cells in spleen and lung both increased (Figure 6B and 6C). The highest dose (125µM) increased spleen donor memory cells 2–4 fold and lung donor memory over 6-fold (Figure 6B). The quantity of CD69⁺ T_RM memory donor cells in lung at 35 dpt, in the immunized mice were also highly dependent on the “checkpoint” Ag dose, with no significant increase at low dose (1.25 µM), 5.9-fold increase at medium dose (12.5 µM) and a striking, 11.6-fold increase at high dose (125 µM) (Figure 6D). Thus, we suggest that augmentation of donor CD4 memory requires an unanticipated high dose of checkpoint peptide on APC to drive vaccine-generated effectors to total and T_RM lung memory.

To determine if increasing Ag density also promoted CD4 helper activity, we measured the level of persistent anti-PR8 IgG in serum collected at 35 dpt from the mice given checkpoint Ag/*APC at 5 dpt, made with different doses of peptide. The titers of anti-PR8 IgG increased, though modestly, from low dose (1.25uM) to the high doses (12.5uM and 125uM) and were about 2-fold higher than background. Of interest, there was no change from medium dose (12.5uM) to high dose (125uM), suggesting some impacts of checkpoint Ab may require higher Ag density on APC than others.

Discussion

Viral infection, including that with PR8 influenza, usually induces a vigorous CD4 T cell response. Following IAV infection, continuing viral replication generates both high levels of virus and viral Ag presentation and continuous activation of APC and other immune cells and perhaps T and B cells, by pathogen recognition. Thus, naïve CD4 T cells have the opportunity to recognize Ag initially. Then again later at the checkpoint, CD4 effectors that develop and peak about 6 days, can potentially recognize Ag again during the defined checkpoint we have identified during which signals from Ag presentation, are required to support memory formation. The recognition of Ag on activated APC with the help of multiple costimulatory interactions (e.g. CD28:CD80/CD86 and CD27/CD70) and autocrine IL-2 all at the effector checkpoint are critical for CD4 effectors to become memory cells (22). Here, we show that unlike influenza infection, inactivated vaccine (WIV), while it provides the strong signals to naïve CD4 T cells, does not persist to provide strong Ag presentation through the effector stage. This is true even when we add additional Ag/*APC initially which provides IL-6 signals during cognate interaction, to enhance the initial CD4 effector generation (27). We suggest this is a likely reason that non-replicating vaccines, such as most of those for influenza, are often ineffective in inducing memory CD4 T cells.

Given the dynamics of Ag presentation established here, we postulated that CD4 effectors generated by vaccines, like those generated by live infection, require Ag presentation at the checkpoint to become memory cells. We tested this by adding checkpoint Ag/*APC to provide, the missing Ag presentation. Indeed, we found that while WIV vaccine (with or without Ag/*APC) presented Ag rapidly and effectively to naive Ag-specific CD4 T cells presentation was gone after the first few days and lead to the generation of effectors, but few memory cells. When we provided Ag/*APC at 5 dpt there was enhanced CD4 memory generation, especially in the lung and preferentially among CD62L^-, CD69⁺ lung T_RM. This strategy also resulted in a better CD4 helper activity, including increased AbSC in the BM and enhanced serum Ab. Thus, these results support the concepts that non-replicating vaccines fail to drive the robust development of memory from CD4 effectors in part because they do not provide Ag at the effector checkpoint, and that CD4 effectors generated by vaccines as well as infection, must go through a checkpoint where they again recognize high levels of cognate Ag in order to form the bulk of memory cells, especially those in the lung.

Lung and other T_RM have been characterized by expression of CD69, CD103 (variable), CXCR6 and lack of CCR7 and by their inaccessibility to intravenous Ab injection and finally by their lack of circulation, determined by parabiosis experiments (35–38). Recently reports have indicated T_RM can on occasion migrate and re-localize (41) and there is evidence repeated Ag exposure can drive some circulating T_EM to become T_RM (42). CD4 T_RM generated by influenza infection are CD69⁺ but often CD103^- (44), but they share many transcriptional programs with CD8 TRM and are inaccessible to i.v. Ab and these all correlate with each other (43). Lung CD4 T_RM generated by influenza play a key role in protection and also mediate inflammation (44). Here we have used CD69 and CXCR6 expression and inaccessibility to anti-CD4. Ab identify lung T_RM.

We found striking enhancement of lung T_RM generation when we added Ag/*APC at the time coinciding with the peak of the effector response, that we previously correlated with the memory checkpoint (22, 23). The low T_RM proportion without checkpoint Ag/*APC, confirms our hypothesis that the transient Ag presentation by inactivated vaccine, is unable on its own, to support T_RM differentiation. CD4 effector recognition of Ag at checkpoint (5 dpt) preferentially promoted lung T_RM. While lung T_RM are commonly thought to be generated best only when vaccines are i.n. administrated, our studies using i.v. introduction of Ag/*APC indicate, the i.v. route was also effective in generating substantial populations of lung CD4 memory and T_RM. We suggest CD4 effectors generated by inactivated vaccines, may not be as fully differentiated as those generated by infection, regardless of the immunization route and that introducing the Ag/*APC further drives their differentiation. T_RM start to accumulate from the 2nd week of response (45). The success of Ag/*APC delivered i.v. at the checkpoint in enhancing lung T_RM is compatible with the concept that Ag delivery to the tissues may be important for generating T_RM providing more effective immunity (46), since i.v. Ag/*APC present Ag in the lung (22) and see here in Fig 2 , and the i.v. route has proven to be a successful in other studies (47). The enhanced lung T_RM generation raises the likelihood that one key facet of checkpoint Ag recognition is to induce a multi-faceted program associated with effector migration including CXCR3 expression (48), tissue residency depending on CXCR6 (49) and long term survival (e.g. depending on IL-2 and IL-15-dependent manner) (44, 50). We plan to determine in future studies the more global induction of gene expression on lung T_RM, induced by addition of Ag/*APC at the checkpoint.

We found the density of Ag on Ag/*APC at checkpoint was a limiting factor for memory CD4 T cell generation. As we increased the dose of HNT peptide from 12.5µM to 125µM, a marked increase in the generation of donor memory in both the spleen and lung and of lung CD69⁺ T_RM occurred. This suggests that high levels of checkpoint Ag density may be needed to drive the vaccine-induced effectors to memory. The marked increase in donor lung memory, and significant increase in spleen as well, as the concentration of peptide used to load the APC increased, likely reveals a need for high density TcR signaling, not for longer duration of the Ag/*APC presentation, since the short lifespan of APC is not impacted. This suggests high levels of Ag, connoting continuing infection are needed. We also suggest, that besides transient Ag presentation at the standard 12.5µM dose used here, more extended stimulation could be required during the checkpoint interval if indeed the effectors generated by vaccine are less differentiated that those elicited by infection.

In addition to the enhanced CD4 memory, there was a modest, but significantly enhanced B cell response when we added Ag/*APC to the CD4 effectors at 5 dpt. There was an increase in total IgG Ab to influenza and a comparable increase in AbSC in the BM. We suggest the increased AbSC and Ab are a secondary consequence of enhanced CD4 effector helper activity. We found the checkpoint Ag/*APC strongly upregulated the expression of ICOS, a critical costimulatory molecular for helper function, on CD4 effectors. There was also a modest upregulation of CXCR5 and Bcl-6 co-expression. We have not yet determined if this enhanced B cell response was dependent on greater development of T_FH or non-T_FH mediated help. Published studies of Ab responses in human to inactivated vaccine are correlated with the induction of ICOS⁺ T helper cells (51, 52) and non-T_FH CD4 T cells can help B cells produce anti-influenza Ab generation, as we saw in earlier studies (5). Specific anti-PR8 IgG production was only modestly enhanced by checkpoint Ag/*APC resulting in a 2.8-fold increase in serum Ab. Meanwhile, while memory CD4 generation was obviously Ag density-dependent, serum IgG levels were not further increased when we increased Ag dose from medium to high. We suggest the modest effects of Ag/*APC given at 5 dpt on Ab production may indicate we have not yet fully optimized the signals we are providing, either Ag presentation itself and/or other as yet unidentified signals at checkpoint . We know the “checkpoint” Ag/*APC only provides Ag recognized by CD4 for less than 48hr (22), compared to viral infection which last much longer (here more than 11 dpt) induces a much larger Ab response. Additional signals that we postulate might be useful at the checkpoint, include longer Ag presentation to CD4 effectors, simultaneous stimulation of B cells and higher levels of PAMP (53, 54). In agreement with the possibility that some signal(s) is still lacking, we note that in the WIV vaccine model, the checkpoint Ag/*APC (even at the highest Ag density, 125 µM) resulted in a smaller increase (about 6-fold) in CD4 T cell memory, compared to the increase in the live infection model where the memory population was dramatically increased equal or >20-fold in all organs (22). We assume that, after vaccination, the total amount of Ag presentation to CD4 T cells is quite limited since we show the high level of Ag was only available for less than 3 days and we suspect the same would be true if PAMP due to recognition of viral RNA, which would present only when WIV was introduced, but would then decay quickly. In contrast, following live infection with PR8, viral replication continues until effector CD8 and CD4 T cells enter the lung and clear virus and infected cells (5). Thus, after infection, high levels of Ag presentation and other infection-induced signals must persist for an extended time, at least over 10 days. We therefore suggest that the effector population after WIV vaccine has received much less stimulation (Ag presentation, costimulatory and inflammatory signals). We plan further studies to assess the functional impact on protection of adding each of the possible limiting signals and once optimized to determine further correlates of protection in a more vaccine-like setting. While the Ag/APC addition has not fully recapitulated live infection, it is clear that adding Ag/*APC at the effector checkpoint induces multiple indications of further differentiation of the cells into more specialized effectors and substantial enhancement of lung memory, especially of the T_RM subsets known to be important for protection against influenza.

There have been a number of previous studies suggesting that continuous Ag presentation can enhance T cell response (55–57). What is clear from our studies is that for optimum CD4 responses following PR8 infection, it is not continuous Ag that is required, but rather a requirement for Ag, transiently, at a defined step in CD4 effector development (22, 23). At this checkpoint, effectors that fail to recognize Ag undergo passive cell death and those that recognize Ag can be induced to undergo multiple effector fates, one of which is transition to memory (22). The generation of memory requires autocrine IL-2 production, and induction of upregulated CD25, CD127 and downregulation of the proapoptotic protein Bim, consistent with the need for preventing default apoptosis and effector contraction by Ag-dependent survival pathways to promote memory. Most relevant here, is that vaccine induced effectors, exposed to Ag, costimulatory molecular ligation and an undetermined level of inflammation for less than 3d, also need the checkpoint Ag recognition signals and they can be supplied at least in part by short-term exposure to Ag/*APC which implies they may be able to be readily supplied by strategies that give inactivated vaccine repeatedly at this time. While infection maintains high levels of Ag, for most vaccine formulations, levels of Ag decrease with time, consistent with our hypothesis that lack of strong Ag presentation at the checkpoint is a major reason for the ineffectiveness of modern vaccines using highly purified proteins.

Multiple studies support the concept that generation of more durable and universal effective vaccines for influenza must include vigorous development of CD4 memory in SLO and tissue that can provide heterosubtypic CD4 T cell recall (3, 36, 58, 59). We suggest that strong T cell memory is critical for protecting against influenza and other ssRNA viruses, including coronaviruses such as the new SARS Cov-2, since the viruses evolve rapidly and are likely to generate new strains that evade pre-existing neutralizing Ab. But in influenza outbreaks, epitopes for T cell recognition were almost always conserved (31). We suggest that vaccine strategies that provide strong Ag recognition at the CD4 effector stage checkpoint will induce more and better CD4 T cell memory generation and that inducing CD4 memory should become an integral part of the most effective immunization strategies. Though we achieved an increased generation of lung CD4 memory and a small increase in persistent Ab by the addition here of Ag/*APC strategy, the degree of enhancement is still less than optimum compared to the memory generation in live virus infection. We suggest this implies we still need to better define what additional signals to further optimize strong T_FH differentiation, necessary to induce the generation of high affinity long-lived AbSC and B cell memory and more extensive CD4 memory. Moreover, it remains unclear what exact approaches which work best to provide Ag and ensure its presentation by activated APC in a vaccine setting in humans. It is well appreciated that repeated addition of protein Ag can improve Ab responses (55). Adjuvants that greatly prolong Ag half-life and also activate or target APC may be needed both initially and we suggest, at the checkpoint. Higher doses of vaccine may be needed. The route of Ag administration also deserves consideration to ensure Ag presentation occurs in sites of desired tissue residency, so intranasal aerosol delivery or installation now widely used for FluMist and over the counter allergy treatments, is an attractive possibility to include for respiratory viral infection. Certainly, there may be resistance to the concept of the need for two closely-spaced doses of vaccine, but if the need for yearly immunization could be reduced we suggest it would be worth the cost and inconvenience.

Taken together, the results here show clearly that the WIV vaccine (enhanced with Ag/*APC), briefly but effectively presents Ag and induces effectors at the initiation of naïve CD4 T cell response, suggesting that it is particularly the lack of Ag presentation, perhaps with other signals, at the checkpoint that is largely responsible for the poor efficacy of non-replicating inactivated and component vaccines in generating strong CD4 memory and long-term immunity. Results here provide proof-or-principle that recognition of Ag by effector CD4 T cells at their fate-determining checkpoint, even those generated by non-replicating vaccines, can promote their effective transition to memory and especially the generation of total lung memory and lung T_RM. This provides support for using prolonged provision of abundant Ag recognition, as part of a vaccine strategies that should be able to induce better CD4 immunity and hence broader, more durable protection especially to heterosubtypic strains.

Key points.

WIV immunization resulted in early but transient Ag presentation and CD4 responses.

Adding checkpoint Ag at day 5 promoted lung CD4 memory T_RM cells and helper function.

High checkpoint Ag doses were required to drive the effectors to optimal memory.

Abbreviations

AbSC

antibody secreting cell

*APC

TLR agonist-activated APC

Ag/*APC

*BMDC pulsed with HA_126–138

BMDC

bone marrow derived dendritic cells

*BMDC

TLR agonist-activated BMDC

dLN

lung draining LN

dpt

days post-treatment

HA

hemagglutinin

HNT/*BMDC

*BMDC pulsed with HA_126–138

i.n.

intranasal

mLN

mediastinal LN

NA

neuraminidase

OLAW

Office of Laboratory Animal Welfare

OVA_II

OVA_323–339

PAMP

pathogen association molecular pattern

PR8

Influenza A/PR8/34

SLO

secondary lymphoid organs

T_CM

central memory cells

T_EM

effector memory cells

T_FH

T follicular helpers

T_RM

tissue resident memory cells

ThCTL

cytotoxic CD4 T cells

ss

single stranded

WIV

whole inactivated influenza virus