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. 2021 Nov;13(11):a038182. doi: 10.1101/cshperspect.a038182

Durable CD4 T-Cell Memory Generation Depends on Persistence of High Levels of Infection at an Effector Checkpoint that Determines Multiple Fates

Susan L Swain 1, Michael C Jones 1, Priyadharshini Devarajan 1, Jingya Xia 1, Richard W Dutton 1, Tara M Strutt 2, K Kai McKinstry 2
PMCID: PMC8559547  PMID: 33903157

Abstract

We have discovered that the determination of CD4 effector and memory fates after infection is regulated not only by initial signals from antigen and pathogen recognition, but also by a second round of such signals at a checkpoint during the effector response. Signals to effectors determine their subsequent fate, inducing further progression to tissue-restricted follicular helpers, cytotoxic CD4 effectors, and long-lived memory cells. The follicular helpers help the germinal center B-cell responses that give rise to high-affinity long-lived antibody responses and memory B cells that synergize with T-cell memory to provide robust long-lived protection. We postulate that inactivated vaccines do not provide extended signals from antigen and pathogen beyond a few days, and thus elicit ineffective CD4 T- and B-cell effector responses and memory. Defining the mechanisms that underlie effective responses should provide insights necessary to develop vaccine strategies that induce more effective and durable immunity.

It has been long appreciated that the response of resting naive T cells requires multiple signals. First and foremost are signals from T-cell receptors that must recognize peptides derived from antigens (Ags) presented by antigen-presenting cells (APCs) during their cognate interactions. Naive and even resting memory cells require additional “costimulatory” signals that are received when cell surface molecules interact with their ligands expressed on activated APCs (e.g., CD28 on the T-cell binding CD80/CD86 on the APCs). Inflammatory cytokines made by APCs (e.g., IL-6, IL-12/IL-18), or produced systemically (e.g., IFN-γ and type I IFN) in response to infection or other inflammation, deliver additional “third” signals that synergize to drive activation of naive cells, expansion of the responding population, and the generation of multiple effector subsets at the height of the response. Optimal responses of naive T cells following infection require both T-cell recognition of abundant Ag on APCs and potent signals generated by pathogen-associated molecular patterns (PAMPs) that act to both activate the APCs, so that they provide costimulatory signals to the T cells and to induce inflammatory cytokines (Thompson et al. 2006). Additional signals likely prolong T-cell division and result in greater effector responses (Zehn et al. 2012; León et al. 2014), although others argue these steps are driven sufficiently by a brief early encounter (van Stipdonk et al. 2001).

In contrast to naive T-cell activation and effector generation, the signals that support the transition to and maintenance of memory are not well defined. Many of the heterogenous effectors generated by infection or high-dose antigen with adjuvants supplying PAMPs undergo apoptosis by programmed cell death and/or survival factor starvation. Only a small cohort survives to become resting and transitions to what we call “memory” cells (McKinstry et al. 2007). Memory T cells, in general, survive much longer than their effector precursors or naive cells and are also distributed both centrally, in secondary lymphoid organs (SLOs), and in peripheral tissues.

The inflammatory environment generated by infection via PAMP signaling impacts naive CD4 T-cell activation and polarization repeatedly. Initial exposure to polarizing cytokines drives a spectrum of T-cell subsets (e.g., for CD4 T cells: Th1, Th2, Th17), which themselves make “signature” patterns of cytokines, resulting in multiple functionally distinct subsets that can provide effective protection against multiple kinds of pathogens. CD4 T-cell subsets are better studied, but CD8 T cells also respond to the polarizing cytokines and can yield similar cytokine-defined subsets (e.g., Tc1, Tc2, Tc17) (Hamada et al. 2013). Many CD8 T-cell effectors are cytotoxic T lymphocytes (known as CTLs) and are able to kill Ag-expressing targets. We and others find some CD4 T cells also become cytolytic following influenza A virus (IAV) infection and we call these ThCTLs (Brown et al. 2012; Marshall et al. 2017). Another cohort of CD4 effector T cells interacts with responding B cells and become T follicular helper (Tfh) cells, which are responsible for helping B cells initiate germinal centers (GCs), and for driving the GC B cells to undergo isotype switch and further differentiate to Ab-secreting cells and long-lived plasma cells (LLPCs) as well as memory B cells. This puts the generation of Tfh at the heart of the protective humoral immune response.

It is clear that if we are to understand how to generate strong protective immunity, we must identify what mechanisms drive CD4 T cells to more specialized effectors such as Tfh and ThCTL, as well as to long-lived central memory (Tcm) and tissue-resident memory (Trm) cells, so that strategies to provide the necessary signals during vaccination can be developed.

Few mechanisms that act at the effector phase have been well defined. It is clear that both CD4 (Li et al. 2003; Bradley et al. 2005) and CD8 (Hashimoto et al. 2019) memory generation requires IL-7 or other common γ-chain cytokines such as IL-15, which keep cells alive as they become resting and thereafter. A large number of studies have supported a need for costimulatory interactions in the development of CD4 and CD8 memory, including CD28 with CD80:CD86 (Harding et al. 1992), CD154:CD40 (Fähnrich et al. 2018), 41BB:41BB ligand (Zhou et al. 2017), and OX40:OX40L (Gramaglia et al. 2000). Some of these are thought to occur after initial steps (Gramaglia et al. 2000; Zhou et al. 2017), but the time and context in which these interactions occur is for the most part unclear. In contrast, multiple studies support a requirement for effector CD4 T cells to interact with B cells during GC responses and implicate repeated ICOS:ICOSL interactions as critical to Tfh and more differentiated GC-Tfh formation (Wan et al. 2019). The generation of Tfh cells but not other CD4 subsets, requires the SLAM-associated protein (SAP) (Crotty et al. 2003; Kamperschroer et al. 2006; Qi 2016). The peak of the Tfh response occurs after the peak of total CD4 effector cells and then GC-Tfh cells persist until the GC reaction wanes. This suggests a distinct program for Tfh versus conventional Th1, Th2, and Th17 effectors, which respond to APCs and then rapidly contract. Collectively, these observations raise the possibility that effector CD4 T-cell recognition of cognate antigen on APCs could drive multiple effector fates as well as memory generation.

IDENTIFYING AND CHARACTERIZING THE CD4 EFFECTOR FATE-DETERMINING CHECKPOINT THAT REGULATES MEMORY CD4 T CELLS, Tfh, AND ThCTL

Here we will summarize our studies identifying characteristics of, and mechanisms required for, generation of strong CD4 memory to IAV and discuss their relevance to the induction of the most effective protective immunity. In short, we find that strong signals from Ag and pathogen recognition are required by CD4 T cells not only initially, but also at the effector stage, to drive both memory and specialized CD4 effector generation. We suggest it is a general feature of CD4 responses to most acute infections and that the lack of continuing Ag and pathogen signals at the effector time point is at least partly responsible for the suboptimal responses to nonliving vaccines that contain limited and transient levels of Ag and PAMP.

Multiple Memory CD4 T-Cell Subsets Mediate Optimal CD4 Memory-Mediated Protection

A key question is what set of functions constitute optimally primed CD4 T cells. For CD8 T cells, which are thought to convey protection largely by killing infected cells, cytotoxic function against infected cells is an obvious functional correlate. However, we found impressive viral control by CD8 T cells even when they were deficient for the cellular machinery required for cytotoxicity including perforin and FAS ligand (Hamada et al. 2013), so other CD8 effector mechanisms also must play key roles. For CD4 T cells, the situation is more complex given that effectors include multiple functionally specialized subsets. Reflecting this complexity, Th1, Th17, ThCTL, and Tfh functions have all been shown to contribute to viral clearance (Brown et al. 2006; Kamperschroer et al. 2006; McKinstry et al. 2009, 2013).

A striking feature of immune responses to viral and other infections is that the memory CD4 T-cell populations that form largely reflect the heterogeneity observed within the effector response (Strutt et al. 2013). The ability of T-cell memory to mediate strong protection probably stems both from their increased numbers and rapid restimulation, and from their ability to recapitulate key aspects of the primary (1°) response and again develop into multiple secondary (2°) effector subsets (Strutt et al. 2012). The multiple functional subsets generated in 2° responses carry out direct antiviral effects, “help” B-cell responses, and maximize the efficacy of CD8 responses. These activities synergize to provide extraordinarily strong protection that can combat supralethal viral challenge (McKinstry et al. 2012). Furthermore, CD4 T-cell “help” also is needed for generation of CD8 memory T cells (Cullen et al. 2019) as well as LLPC and memory B cells (Gage et al. 2019). The diverse protective actions of memory CD4 T cells are redundant in many cases, such that complete clearance of IAV can still be observed when any given pathway is disrupted (McKinstry et al. 2012).

The location of memory CD4 T cells represents another axis of diversity. Trm cells have emerged as key players in coordinating successful recall against pathogens, and there is much evidence that lung-resident Trm cells confer potent immunity against IAV (Teijaro et al. 2011; Strutt et al. 2018). The extent of functional diversity that develops in 2° effectors derived from Trm cells versus conventional memory cells is unclear.

We would argue that the most effective and durable immunity requires diverse, long-lived memory CD4 and CD8 T cells, working by multiple functional pathways, as well as long-lived production of antibodies (Abs) of multiple isotypes and long-lived memory B cells. Thus, although there is no clear-cut answer as to the “best” type(s) of memory CD4 cell that vaccines should aim to prime, the most logical approach to optimize protection is for vaccines to induce comparable diversity of memory subsets to the highly protective immunity formed after natural infection.

For the remainder of this perspective, we will discuss newly defined mechanisms that are required for optimal memory formation following infection. Our studies make use of reductionist in vivo models that allow us to pinpoint when different signals from viral infection are needed and what roles they play. We have found that the fate of the CD4 effector population is determined by a distinct, second round of signals some days after the initiation of the response.

The Generation of Memory CD4 T Cells Requires Autocrine IL-2 at a Defined Effector Checkpoint

While initially identified as an obligate autocrine T-cell growth factor, IL-2 is now known to have diverse roles in both promoting and restraining immune responses (Liao et al. 2013). IL-2 also delivers signals to CD4 T cells that enhance the ability of effectors to survive long term after the resolution of an immune response. Abbas and colleagues found IL-2 signaling for the few days following CD4 T-cell activation is necessary to enable cells to compete for survival in the memory niche (Dooms et al. 2004). In this context, IL-2 acts by up-regulating surface expression of the IL-7 receptor α chain (CD127) (Dooms et al. 2007), which ensures better access to limiting amounts of IL-7 presented on stromal cells that support homeostatic memory cell survival. However, these studies were conducted with nonreplicating antigens, limiting the span of TCR-mediated signaling to only a few days poststimulation.

Using in vitro–generated effectors, we found that IL-2 and TGF-β together prevent otherwise default apoptosis of CD4 effectors (Zhang et al. 1995), so we investigated whether IL-2 is needed at the effector stage in vivo to block apoptosis and promote memory. We tracked memory formation induced by IAV, from IL-2-deficient or wild-type (WT) DO11.10 transgenic CD4 T cells specific for ovalbumin expressed by a recombinant IAV (PR8-OVAII). Both donor CD4 T cells formed similar effector populations 7 d postinfection (dpi), but only WT, not IL-2-deficient cells, were detected at a memory time point 3 wk later (McKinstry et al. 2014). To determine the precise timing of the IL-2 signals needed to promote memory formation, we provided exogenous IL-2 or blocked endogenous signals in recipient mice of WT and IL-2-deficient donor cells at different time points and assessed donor memory recovery. IL-2 given during the priming phase of the immune response (day 1–3) did not rescue donor memory formation, but giving IL-2 during priming (in vitro) as well a second hit at 5–7 d posttransfer (dpt), when they were highly activated effector cells, but not other 3 d intervals, restored memory formation to WT levels (Fig. 1). Separately, mice treated at 5–7 dpt with anti-IL-2 or anti-CD70 Ab (CD70-CD27 signaling is a key regulator of effector IL-2 production) showed strikingly reduced memory formation from WT donor-naive CD4 T cells (Fig. 2). Because IL-2 is produced by T cells only after TCR stimulation (Sojka et al. 2004), these results suggest memory formation induced by IAV infection requires CD4 T cells to recognize cognate Ag at an effector checkpoint at 5–7 dpt.

Figure 1.

Figure 1.

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IL-2 signals rescue memory formation from IL-2−/− effector CD4 T-cell responses only at 5–7 dpi, defining an effector “checkpoint” for memory generation. Equal numbers of wild-type (WT) or IL-2-deficient (IL-2−/−) DO11.10 TCR Tg CD4 T cells were transferred to congenic BALB/c hosts that were then challenged with PR8-OVAII. The donor CD4 T-cell populations were primed in vitro in the presence of IL-2 to ensure delivery of early IL-2 signals that are needed for memory development. At various time points postchallenge, groups of three to five mice were treated with intraperitoneal injections of IL-2 complexes formed by incubating 2 μg of recombinant IL-2 with the anti-IL-2 antibody clone S4B6 for three consecutive days. At 30 dpi, donor cells were enumerated in the spleen. Only IL-2 complex treatment between 5 and 7 dpi restored IL-2−/− donor memory formation to WT levels (K McKinstry and T Strutt, unpubl.). (ns) Not significant.

Figure 2.

Figure 2.

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Signals needed for CD4 effector to memory transition. This figure summarizes our published work indicating that three major classes of stimulation (IL-2, Ag displayed by major histocompatibility complex [MHC] class II, and costimulation) can be modulated to either increase or decrease memory formation from wild-type (WT) CD4 effector cells responding to influenza A virus (IAV). We added treatments to block pathways only at 5–7 d postinfection, restricting its impact to a window well into the effector stage of the CD4 T-cell response against IAV and determined memory CD4 recovery at 28 dpi (McKinstry et al. 2014). The picture represents all the pathways that were identified (McKinstry et al. 2014; Bautista et al. 2016).

Additional studies in this model indicated that the IL-2 signals to effector cells are required to up-regulate the IL-7Rα receptor to promote prolonged survival (Kondrack et al. 2003; Li et al. 2003) and to down-regulate proapoptotic protein Bim to reduce apoptosis (McKinstry et al. 2014). These mechanisms, portrayed in Figure 2, suggest autocrine IL-2 is pivotal to rescue IAV-induced effectors from default apoptosis and support their transition to long-lived resting memory.

While autocrine IL-2 signaling at the effector phase of the response is critical for establishing all subsets of memory, Trm generation is controlled, in part, by another mechanism. The only long-lived memory CD4 T cells we found in recipients of IL-2-deficient donor cells were in the lung (McKinstry et al. 2014). We found the IL-2-independent memory cells were highly functional and expressed the reported phenotypic signatures of “tissue-resident” memory cells (Strutt et al. 2018). Furthermore, they shared a gene expression signature, which partially overlapped with CD8 tissue-resident memory cells isolated from multiple organs. We propose that IL-15 acts as a common “alarm” signal during infections to instruct formation of this IL-2-independent population of Trm cells (Jabri and Abadie 2015; Dhume and McKinstry 2018). Following IAV infection, IL-15 levels peak in the lung during the same time frame as when IL-2 signals are needed to instruct memory fate (Strutt et al. 2018). In IL-15-deficient mice, or in mice treated with Ab to block both IL-2 and IL-15 signaling, generation of IAV-specific CD4 effectors cells was not impacted, but the formation of lung Trm cells was abolished (Strutt et al. 2018).

The Generation of Memory CD4 T Cells Requires that Effector Cells Recognize APCs and Receive Costimulation

The clear-cut need for autocrine IL-2 production at the peak of the CD4 effector phase to support intrinsic CD4 memory generation described above, suggests that effectors need to again recognize Ag on costimulatory APCs to secrete sufficient IL-2 to support memory formation. Our studies also define a short window during the effector response at which IL-2, which is itself very short-lived, must be produced, supporting the idea that this is a discrete fate decision checkpoint. This hypothesis is highly attractive since such a requirement for continuing signs of infection would restrict memory generation to those situations in which a pathogen has persisted throughout the effector phase, preventing unnecessary memory cells that occupy space and consume resources, when infections are short term or readily cleared.

To examine whether cognate Ag recognition is indeed required for effectors at the identified checkpoint to differentiate into memory cells, we designed a new CD4 effector transfer approach to define the signals and mechanisms that drive them to memory (Bautista et al. 2016). We generated a marked cohort of Ag-specific CD4 effectors by transferring naive OT-II CD4 (Thy1.1 or CD45.1) cells into WT host B6 mice and infected with PR8-OVAII virus recognized by the OT-II TCR. At 6 dpi, we re-isolated the effectors, which had all responded in the first hosts, and transferred them to second hosts that were either infected with IAV with cognate Ag (PR8-OVAII) or not (PR8) 6 d prior (Fig. 3). The donor effectors persisted and went on to form protective memory in the second hosts infected with PR8-OVAII. The difference between donor memory recovery when Ag was introduced by infection or as Ag/APC versus no Ag was impressive, ranging from 40- to 250-fold in spleen, draining lymph node (DLN), and lung, and persisted for at least 7 wk (Bautista et al. 2016). Moreover, in over a dozen studies we found equal levels of memory when we introduced Toll-like receptor (TLR)-ligand-activated APCs incubated with OT-II peptide (Ag/APC) in the absence of any infection in the second host, indicating definitively that cognate Ag but not infection was needed at the effector checkpoint to drive memory formation and persistence. This set of results is shown in Figure 3.

Figure 3.

Figure 3.

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CD4 effectors require cognate Ag recognition but not infection to become memory, but require both to become Tfh and ThCTL cells. Critical protective CD4 subsets, Tfh, ThCTL, and memory, require a second round of antigen encounter during the effector checkpoint. While we find that the tissue-restricted effectors, Tfh and ThCTL, additionally require signals from infection, the formation of long-lasting memory is independent of this signal (Bautista et al. 2016; Devarajan et al. 2020). (BMDC) Bone marrow–derived dendritic cell, (DC) dendritic cell.

In vitro stimulation of the same donor effectors with Ag/APC, induced a shift in phenotype after 2–3 d, including expression of Stat3, CXCR3, Bcl2, and CD25 and down-regulation of Bim and apoptosis, as well as several rounds of division and progression to a more resting state. The shifts required CD80/86 on the APC and production of IL-2. Optimum transition of effectors to a memory phenotype in vitro occurred when effectors were harvested and restimulated at 6 dpi, but was progressively lost if delayed over the next 3–7 d. We conclude that the two studies identify a single effector checkpoint at which almost all the IAV-induced effectors that do not recognize Ag die of apoptosis, while a cohort of those that do see cognate Ag on activated APC make autocrine IL-2 survive and become memory-like.

Importantly, when the IAV-generated effector population in the spleen is transferred to hosts with either PR8-OVAII infection or with peptide-pulsed Ag/APC, persisting memory is generated in spleen, lymph node, and in the lung, and this CD4 memory is able to provide CD4-mediated protection from a higher dose of virus that is lethal in unimmunized mice (Bautista et al. 2016).

The Generation of CD4 Memory Is Favored by Strong TCR Signals at the Effector Checkpoint

During live sublethal IAV infection of WT-naive animals, virus titers remain high for at least 7 dpi, until CD8 and CD4 cytolytic cells clear virus-infected cells by about 10 dpi or so. Thus, high levels of virus- and pathogen-mediated inflammation are present at the effector checkpoint we have defined. This should generate activated APC bearing a high level of IAV Ag and we see strong Ag presentation at 7 dpi (Jelley-Gibbs et al. 2005).

Studies that evaluate the impact of Ag dose and affinity, when Ag is introduced only at day 0, have demonstrated that weaker interactions generally favor a Th2 response, while stronger interactions promote Th1 skewing (Brogdon et al. 2002; Corse et al. 2011). Increased peptide MHC (pMHC)-TCR signal strength during CD4 T-cell priming, through combinations of higher affinity interactions, increased Ag density, and/or increased dwell time, generates a more potent effector population that is more likely to become a specialized effector (Tfh > Th1) (Fazilleau et al. 2009; Tubo et al. 2013; Vanguri et al. 2013).

CD4 effector cells are likely to behave differently from naive resting cells and it is important from a vaccine perspective to know what dose and affinity are necessary to drive memory fate. We thus wanted to establish the extent to which high affinity and density of Ag presentation are needed at the effector checkpoint to drive optimal memory. Our collaborator, Eric Huseby, developed a TCR transgenic mouse strain on a C57BL6 background recognizing a CD4 immunodominant IAb-binding epitope in nucleoprotein (NP) (residues 311–325), which we call FluNP (ES Huseby and SL Swain, unpubl.). The naive TCR transgenic CD4 T cells of the FluNP strain respond well to IAV infection, generating a robust Th1 effector response and long-term memory after viral clearance. Larry Stern's group developed a truncation in the NP311–325 epitope peptide as well as a series of single amino acid peptide variants that still bound class II (IAb) very well (LJ Stern, unpubl.), but which showed a range of dose-dependent abilities to induce naive TCR-transgenic CD4 T-cell activation (MC Jones, unpubl.) when bound to activated APC, including CD25 induction, IL-2 production, CD69 expression, and up-regulation of Nur77, indicating recent TCR triggering (Moran et al. 2011; Bautista et al. 2016). We used APC pulsed with the different peptides to present Ag in vitro and found IL-2 production and CD25 up-regulation were most dependent on the functionally defined affinity of the peptide.

To evaluate the impact of TCR signal strength on memory generation, we pulsed bone marrow–derived dendritic cells (BMDCs), activated by virus-associated TLR agonists (polyI:C, CpG), with the different NP epitopes at a concentration of 10 µM; we used these as Ag/APC to support the transition of 6 dpi spleen FluNP effectors transferred to unimmunized hosts to memory cells. We found that relatively high doses of peptide were required to pulse APCs (Xia et al. 2020), supporting the need for high signal strength. Recovery of donor CD4 memory cells correlated strongly with functional avidity and there was negligible recovery with low-avidity peptides as depicted in Figure 4. Thus, our preliminary data suggest that very strong TCR stimulation is needed at the effector stage to drive optimum numbers of CD4 memory cells as shown in Figure 4. Recent results provide support that the higher levels of memory correlate to protection to lethal rechallenge (M Jones, unpubl.). This requirement for both high-dose and high functional avidity to generate memory CD4 T cells reinforces the concept that the strong generation of CD4 memory is strictly regulated at the effector stage, precluding significant memory formation unless abundant antigen such as that provided by infection is present for a week or so into the response. This requirement would imply that much of the persisting memory pool is directed to highly expressed epitopes for which there are CD4 T cells with high affinity, possibly limiting the breadth of the memory pool.

Figure 4.

Figure 4.

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Strong T-cell receptor (TCR) signals are needed at the checkpoint to drive protective CD4 memory. Our preliminary data suggest that over a broad range of dose (density of peptide on antigen-presenting cells [APCs]) and affinity of interaction with peptide major histocompatibility complex (pMHC), the strength of signal CD4 effector T cells receive correlates with size of the recovered CD4 memory population and with protection of the host from lethal influenza A virus (IAV) challenge (MC Jones et al., unpubl.).

The Generation of Tfh and ThCTL from Effectors, Also Requires Ag Presentation and Other Signals from Continuing IAV Infection

The donor effectors present at 6 dpi with IAV are all well divided, activated cells that can make IFN-γ, TNF, and in some cases, IL-2. A minor fraction of effectors at this stage have begun to express moderate levels of markers for Tfh cells, but not of the signature markers of ThCTL, which become lung restricted after IAV infection (Marshall et al. 2017). Tfh cells are “restricted” to spleen and LN follicular regions. Both Tfh and ThCTL effectors peak at days 8–10. Both Tfh and ThCTL are tissue-restricted (TR) effectors that acquire key properties associated with tissue residence including down-regulation of KLF2 and S1pr1 (Lee et al. 2015; Marshall et al. 2016) and expression of CD69 (Devarajan et al. 2020).

Previous studies indicated that prolonged Ag exposure (Benson et al. 2015) or repeated immunization (Deenick et al. 2010; Baumjohann et al. 2013; Tam et al. 2016) was required to support Tfh generation. Depletion of germinal center B (GCB) cells 6–8 d after immunization also reduced Tfh generation on day 9 (Baumjohann et al. 2013). However, it is unclear whether these results are driven by mechanisms acting during priming or at the CD4 effector stage and whether these reductions reflect a requirement for cognate Ag, for costimulation (e.g., through CD28, ICOS, 4-1BB, or CD40L), and/or for inflammatory factors induced by infection. Therefore, we asked whether effectors at the 6 dpi effector checkpoint might also need signals that drive them further down the pathways to Tfh and ThCTL. It would make sense, since it would arguably be a poor strategy to generate these potent effectors if an infection was no longer present.

It is potentially challenging to distinguish the need for Ag recognition and infection-mediated signals during initial priming from those needed at the effector stage. To analyze this, we used the sequential transfer model described above, with 6 dpi donor CD4 effectors from spleen transferred to hosts with or without Ag and/or infection. We examined the appearance of Tfh (expressing CXCR5, PD-1, ICOS, and high BCL6) (Qi 2016) and ThCTL (expressing NKG2A/C/E, CD94, and high GrzB) (Marshall et al. 2017) 2–3 dpt. Robust Tfh and ThCTL cells developed only when Ag was available (e.g., in PR8-OVAII, but not PR8 infection–matched hosts as summarized in Figure 3 (Devarajan et al. 2020). However, unlike the generation of memory, Ag/APC alone in the second host were not able to provide the signals necessary to efficiently drive either Tfh or ThCTL cells, and instead both required infection as indicated in Figure 3. To separate the requirement for infection from that for infection-mediated activation of APCs, we tested whether Ag/APC and PR8 (not expressing OVAII Ag) seen by the donor effectors would synergize to enhance the generation of TR effectors. Indeed, separate Ag/APC and PR8 infection were as effective as PR8-OVAII infection. This implies that two nonlinked events were needed, with infection-mediated signals acting independently of activation of APCs (Devarajan et al. 2020). Figure 5 summarizes the requirements for generating CD4 memory as well as Tfh and ThCTL effectors.

Figure 5.

Figure 5.

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Distinct signals determine Tfh and ThCTL effector fate and CD4 memory development. This cartoon summarizes our studies that have defined the reguirements for the signals that are needed to drive distinct effector fates at the effector checkpoint. Without Ag/antigen-presenting cell (APC) signals, most effectors die of apoptosis. When Ag/APC are present, a sufficiently strong T-cell receptor (TCR) signal, coupled with CD28 and CD27 costimulation and resulting in IL-2 production, leads to generation of long-lived memory cells. The memory cells also depend on IL-7 (or IL-15 for some Trm cells in lung). On the other hand, ThCTLs need Ag/APCs but do not depend on IL-2 or CD28 or CD27 costimulation, but unlike memory cell generation, their generation is dependent on infection in the host. Tfh cells need Ag/APCs plus infection and costimulation through CD28, but not IL-2 (McKinstry et al. 2014; Bautista et al. 2016; Devarajan et al. 2016, 2018, 2020; Marshall et al. 2017). (TR) Tissue-restricted, (SLO) secondary lymphoid organ.

We found further support for the concept that there are distinct pathways for memory versus Tfh and ThCTL effectors and they also differed between the TR effectors (Fig. 5). First, location of the Ag delivered at the effector stage influenced how many effectors developed in each site. Local Ag presentation in the tissue niche was required for Tfh and ThCTL formation even if effectors encountered antigen in the periphery before migrating to the tissue. Neither Tfh nor ThCTL cells required IL-2 at the effector checkpoint; however, Tfh but not ThCTL cells required CD28:CD80/86 interaction. Thus, both development of memory and robust generation of Tfh and ThCTL effectors require that effectors recognize Ag. Moreover, the TR effectors also require local Ag in their tissue site and likely require unidentified infection-mediated signals at, or coming from, the site of infection. Our studies suggest these signals are induced by PAMP-stimulating extrinsic pathogen-recognition pathways (P Devarajan, unpubl.). These results, summarized in the cartoon in Figure 5, indicate that effectors make multiple critical fate decisions at the effector stage driven by the presence and location of Ag presentation, and by the presence of viral PAMP.

CAN THE POOR EFFICACY OF INFLUENZA VACCINES BE ATTRIBUTED TO THE ABSENCE OF SUFFICIENT Ag PRESENTATION AND PAMP AT THE EFFECTOR CHECKPOINT?

Given that Tfh cells develop later in the effector stage and are required for effective B-cell responses, including GC formation, isotype switching, somatic hypermutation, and generation of LLPC and memory B cells, strategies to maximize Tfh cells are critical for optimal long-term protection.

Assuming we can apply the paradigm of the need for strong TCR signals to effectors and for PAMP signals at the effector checkpoint broadly, then responses to replicating pathogens that persist for a little more than a week will generate durable memory CD4 T cells of all key protective subsets and, indirectly, CD8 and B-cell protective memory. On the other hand, current nonliving vaccines composed of proteins likely cannot provide prolonged high doses of Ag and or strong prolonged PAMP-induced responses, and are liable to be relatively poor at inducing CD4 T-cell differentiation and Tfh-dependent B-cell responses, other than through some level of T-independent Ab induction (Devarajan et al. 2018). This is indeed the pattern seen when live infection with IAV is compared to immunization with inactivated or component vaccines (Devarajan et al. 2016; Miyauchi et al. 2016; Gaya et al. 2018).

To evaluate whether the ineffective responses to inactivated and subunit vaccines are caused by weak induction of prolonged Ag presentation and inflammation at the effector checkpoint, we compared Ag presentation by whole inactivated influenza vaccine (WIV) to live infection. We used several approaches to evaluate functional Ag presentation, including the ability to stimulate the expansion of TCR transgenic-naive CD4 T cells, as indicated by loss of vital dye as cells divide, the ability to induce expression of Nur77, and an examination of expression of activation markers by responding CD4 T cells. Results indicated WIV Ag presentation rose soon after immunization, continued for 2 days or so, and then was largely lost by day 4–5. In synchrony, the naive CD4 T-cell response peaked around day 3–4 and then began contracting. In contrast, Ag presentation and CD4 T cells responding to live IAV infection took 2–3 d to be detectable, then, as expected from earlier analyses, rose and stayed high for more than 8 d. Phenotypic comparison of the peak effectors generated by WIV versus live IAV indicated both populations had differentiated into effectors with an up-regulated expression of CD44 and down-regulated CD62L. However, only the live virus effectors went on to express more IRF4, Eomes, IFN-γ, CXCR5, and T-bet. And only the live virus induced effectors that migrated efficiently to the lung (Xia et al. 2020).

The transient Ag presentation is likely to be accompanied by short-term PAMP expression and induction of inflammation for a limited time. We have not directly measured this yet, but plan to in the future. Given the lack of late Ag presentation and by inference PAMP stimulation with WIV vaccination, the kinetics of the CD4 effector response was predictable. The infection generated more CD4 effectors especially in the lung, more GCB cells, and more LLPCs. These observations support the hypothesis that short presentation of Ag with nonliving vaccines may be a critical factor that is responsible for causing inactivated vaccines to induce low levels of CD4 memory and ineffective protection. It is now important to examine later time points and function of the memory produced, and define optimal strategies to provide Ag and PAMP at the effector stage to mimic the pattern of live infection. We suggest that the initial vaccine should include high doses of as many viral antigens as possible to ensure high-affinity interactions and a source of PAMP that activates APCs for CD4 T cells (Brahmakshatriya et al. 2017) plus a subsequent regimen of high dose Ag and PAMP that activates APCs and provides infection signals in key tissue sites as well as systemically (Fig. 6; Devarajan et al. 2020; Xia et al. 2020).

Figure 6.

Figure 6.

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Improving vaccine-induced CD4 immunity. This cartoon summarizes our predictions about some strategies that we predict will improve efficacy of noninfectious vaccines. We postulate that nonliving vaccines present Ag and provide pathogen-associated molecular pattern (PAMP) signals over too short a time to generate a robust Tfh or ThCTL effector response or abundant long-term CD4 memory. We suggest vaccines should present high doses of antigens from pathogens to maximize potential for high-affinity epitopes, and that these need to be associated with sources of PAMPs or the infection-mediated signal, which are not yet identified. These need to be administered both initially and again when vaccine presentation of Ag and PAMPs starts to fade. The antigen-presenting cells (APCs) induced by vaccines need to express costimulatory ligands indicated in Figure 5. We predict that vaccine-induced response can be augmented by providing more optimum Ag presentation for CD4 T cells at day 0 and again as soon as vaccine Ag and PAMP signals wane. We predict this general strategy will drive more Tfh and ThCTL effectors, more germinal center B (GCB) cells, and more long-lived CD4 and B-cell memory and sustained protective Ab and thus achieve much more durable immunity (Devarajan et al. 2016, 2018, 2020; Brahmakshatriya et al. 2017; Xia et al. 2020). (DC) Dendritic cell.

CONCLUDING REMARKS

The studies discussed here support the hypothesis that the immune response has evolved to require indications of continuing infection at the effector phase to fully to commit to generating more specialized CD4 effectors and memory induction. A key signal regulating the decision appears to be the continued presence of high levels of Ag beyond the first few days of infection, with cognate interactions and costimulatory pathways needed to induce IL-2 and provide other signals, which rescue effector CD4 T cells from apoptosis and drive memory fate. Both Ag- and pathogen-provided “infection signals” are also required for the generation of TR Tfh and ThCTL cells (Fig. 6). If these are not present following IAV infection, only the non-tissue-resident effectors and short-lived CD4 memory cells develop. We suggest this need for CD4 T cells for Ag recognition and PAMP-induced signals at the effector phase is a general paradigm applying to immune response of all or most kinds. We stress that further definition of exactly what signals are needed, when they are needed, and what mechanisms they mediate will support the design of more effective vaccines that provide such signals. Then it will be possible to induce much more effective “durable” memory and much more effective and long-lasting protection.

ACKNOWLEDGMENTS

We want to thank colleagues who contributed to these studies including Lawrence Stern, Eric Huseby, Vinayak Brahmakshatriya, Bianca Bautista, Allen Vong, and Nikki Marshall. We also thank the Trudeau Institute, University of Massachusetts Medical School, and the National Institutes of Health (NIH) for financial support.

Footnotes

Editors: David Masopust and Rafi Ahmed

Additional Perspectives on T-Cell Memory available at www.cshperspectives.org

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