CD4 memory has a hierarchical structure created by requirements for infection-derived signals at an effector checkpoint.

Our recent studies reveal that the persistence, location, and amount of both antigen and signals that induce pathogen recognition responses determine the number of CD4 memory cells, the subsets that develop, their location, and hence their protective efficacy. Non-replicating vaccines provide antigen that is short-lived and generate low levels of only some memory subsets that are mostly restricted to secondary lymphoid tissue. In contrast, exposure to long-lived replicating viruses and bacteria provides high levels of diverse antigens in sites of infection and induces strong pathogen recognition signals for extended periods of time, resulting in much higher levels of memory cells of diverse subsets in both lymphoid and nonlymphoid sites. These include memory subsets with highly potent functions such as T follicular helpers and cytotoxic CD4 effectors at sites of infection, where they can most effectively combat the pathogen early after re-infection. These effectors also do not develop without antigen and pathogen recognition signals at the effector stage, and both subsets must receive these signals in the tissue sites where they will become resident. We postulate that this leads to a hierarchical structure of memory, with the strongest memory induced only by replicating pathogens. This paradigm suggests a likely roadmap for markedly improving vaccine design.

Cytotoxic CD4 development requires CD4 effectors to concurrently recognize local antigen and encounter type I IFN-induced IL-15.

Cytotoxic CD4 T cell effectors (ThCTLs) kill virus-infected major histocompatibility complex (MHC) class II+ cells, contributing to viral clearance. We identify key factors by which influenza A virus infection drives non-cytotoxic CD4 effectors to differentiate into lung tissue-resident ThCTL effectors. We find that CD4 effectors must again recognize cognate antigen on antigen-presenting cells (APCs) within the lungs. Both dendritic cells and B cells are sufficient as APCs, but CD28 co-stimulation is not needed. Optimal generation of ThCTLs requires signals induced by the ongoing infection independent of antigen presentation. Infection-elicited type I interferon (IFN) induces interleukin-15 (IL-15), which, in turn, supports CD4 effector differentiation into ThCTLs. We suggest that these multiple spatial, temporal, and cellular requirements prevent excessive lung ThCTL responses when virus is already cleared but ensure their development when infection persists. This supports a model where continuing infection drives the development of multiple, more differentiated subsets of CD4 effectors by distinct pathways.

CD4 Effector TCR Avidity for Peptide on APC Determines the Level of Memory Generated.

Initial TCR affinity for peptide Ag is known to impact the generation of memory; however, its contributions later, when effectors must again recognize Ag at 5-8 d postinfection to become memory, is unclear. We examined whether the effector TCR affinity for peptide at this "effector checkpoint" dictates the extent of memory and degree of protection against rechallenge. We made an influenza A virus nucleoprotein (NP)-specific TCR transgenic mouse strain, FluNP, and generated NP-peptide variants that are presented by MHC class II to bind to the FluNP TCR over a broad range of avidity. To evaluate the impact of avidity in vivo, we primed naive donor FluNP in influenza A virus-infected host mice, purified donor effectors at the checkpoint, and cotransferred them with the range of peptides pulsed on activated APCs into second uninfected hosts. Higher-avidity peptides yielded higher numbers of FluNP memory cells in spleen and most dramatically in lung and draining lymph nodes and induced better protection against lethal influenza infection. Avidity determined memory cell number, not cytokine profile, and already impacted donor cell number within several days of transfer. We previously found that autocrine IL-2 production at the checkpoint prevents default effector apoptosis and supports memory formation. Here, we find that peptide avidity determines the level of IL-2 produced by these effectors and that IL-2Rα expression by the APCs enhances memory formation, suggesting that transpresentation of IL-2 by APCs further amplifies IL-2 availability. Secondary memory generation was also avidity dependent. We propose that this regulatory pathway selects CD4 effectors of highest affinity to progress to memory.

IgD+ age-associated B cells are the progenitors of the main T-independent B cell response to infection that generates protective Ab and can be induced by an inactivated vaccine in the aged.

Age-associated B cells (ABC) accumulate with age and are associated with autoimmunity and chronic infection. However, their contributions to acute infection in the aged and their developmental pathways are unclear. We find that the response against influenza A virus infection in aged mice is dominated by a Fas+ GL7- effector B cell population we call infection-induced ABC (iABC). Most iABC express IgM and include antibody-secreting cells in the spleen, lung, and bone marrow. We find that in response to influenza, IgD+ CD21- CD23- ABC are the precursors of iABC and become memory B cells. These IgD+ ABC develop in germ-free mice, so are independent of foreign antigen recognition. The response of ABC to influenza infection, resulting in iABC, is T cell independent and requires both extrinsic TLR7 and TLR9 signals. In response to influenza infection, IgD+ ABC can induce a faster recovery of weight and higher total anti-influenza IgG and IgM titers that can neutralize virus. Immunization with whole inactivated virus also generates iABC in aged mice. Thus, in unimmunized aged mice, whose other B and T cell responses have waned, IgD+ ABC are likely the naive B cells with the potential to become Ab-secreting cells and to provide protection from infection in the aged.

Bona Fide Th17 Cells without Th1 Functional Plasticity Protect against Influenza.

Optimal transcriptional programming needed for CD4 T cells to protect against influenza A virus (IAV) is unclear. Most IAV-primed CD4 T cells fit Th1 criteria. However, cells deficient for the Th1 "master regulator," T-bet, although marked by reduced Th1 identity, retain robust protective capacity. In this study, we show that T-bet's paralog, Eomesodermin (Eomes), is largely redundant in the presence of T-bet but is essential for the residual Th1 attributes of T-bet-deficient cells. Cells lacking both T-bet and Eomes instead develop concurrent Th17 and Th2 responses driven by specific inflammatory signals in the infected lung. Furthermore, the transfer of T-bet- and Eomes-deficient Th17, but not Th2, effector cells protects mice from lethal IAV infection. Importantly, these polyfunctional Th17 effectors do not display functional plasticity in vivo promoting gain of Th1 attributes seen in wild-type Th17 cells, which has clouded evaluation of the protective nature of Th17 programming in many studies. Finally, we show that primary and heterosubtypic IAV challenge is efficiently cleared in T-bet- and Eomes double-deficient mice without enhanced morbidity despite a strongly Th17-biased inflammatory response. Our studies thus demonstrate unexpectedly potent antiviral capacity of unadulterated Th17 responses against IAV, with important implications for vaccine design.

Strong influenza-induced TFH generation requires CD4 effectors to recognize antigen locally and receive signals from continuing infection.

While influenza infection induces robust, long-lasting, antibody responses and protection, including the T follicular helper cells (TFH) required to drive B cell germinal center (GC) responses, most influenza vaccines do not. We investigated the mechanisms that drive strong TFH responses during infection. Infection induces viral replication and antigen (Ag) presentation lasting through the CD4 effector phase, but Ag and pathogen recognition receptor signals are short-lived after vaccination. We analyzed the need for both infection and Ag presentation at the effector phase, using an in vivo sequential transfer model to time their availability. Differentiation of CD4 effectors into TFH and GC-TFH required that they recognize Ag locally in the site of TFH development, at the effector phase, but did not depend on specific Ag-presenting cells (APCs). In addition, concurrent signals from infection were necessary even when sufficient Ag was presented. Providing these signals with a second dose of live attenuated influenza vaccine at the effector phase drove TFH and GC-TFH development equivalent to live infection. The results suggest that vaccine approaches can induce strong TFH development that supports GC responses akin to infection, if they supply these effector phase signals at the right time and site. We suggest that these requirements create a checkpoint that ensures TFH only develop fully when infection is still ongoing, thereby avoiding unnecessary, potentially autoimmune, responses.

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

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.

"An Intrinsic Program Determines Key Age-Associated Changes in Adaptive Immunity that Limit Response to Non-Pathogens."

As mice age their adaptive immune system changes dramatically, leading to weakened responses to newly encountered antigens and poor efficacy of vaccines. A shared pattern emerges in the aged, with both CD4 T and B cell responses requiring higher levels of pathogen recognition. Moreover, in aged germ-free mice we find accumulation of the same novel age-associated T and B cell subsets that we and others have previously identified using mice maintained in normal laboratory animal housing conditions, suggesting that their development follows an intrinsic program.

Influenza Vaccine-Induced CD4 Effectors Require Antigen Recognition at an Effector Checkpoint to Generate CD4 Lung Memory and Antibody Production.

Previously, we discovered that influenza-generated CD4 effectors must recognize cognate Ag 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 3 d 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 nonlive vaccines will need to provide high levels of Ag recognition throughout the effector checkpoint to optimize CD4 memory generation.

CD25-Targeted IL-2 Signals Promote Improved Outcomes of Influenza Infection and Boost Memory CD4 T Cell Formation.

IL-2 is a pleotropic cytokine with potent pro- and anti-inflammatory effects. These divergent impacts can be directed in vivo by forming complexes of IL-2 and anti-IL-2 mAbs (IL-2C) to target IL-2 to distinct subsets of cells based on their expression of subunits of the IL-2R. In this study, we show that treatment of mice with a prototypical anti-inflammatory IL-2C, JES6-1-IL-2C, best known to induce CD25+ regulatory CD4 T cell expansion, surprisingly causes robust induction of a suite of inflammatory factors. However, treating mice infected with influenza A virus with this IL-2C reduces lung immunopathology. We compare the spectrum of inflammatory proteins upregulated by pro- and anti-inflammatory IL-2C treatment and uncover a pattern of expression that reveals potentially beneficial versus detrimental aspects of the influenza-associated cytokine storm. Moreover, we show that anti-inflammatory IL-2C can deliver survival signals to CD4 T cells responding to influenza A virus that improve their memory fitness, indicating a novel application of IL-2 to boost pathogen-specific T cell memory while simultaneously reducing immunopathology.

Understanding the Heterogeneous Population of Age-Associated B Cells and Their Contributions to Autoimmunity and Immune Response to Pathogens.

In humans and mice, susceptibility to infections and autoimmunity increases with age due to age-associated changes in innate and adaptive immune responses. Aged innate cells are also less active, leading to decreased naive T- and B-cell responses. Aging innate cells contribute to an overall heightened inflammatory environment. Naive T and B cells undergo cell-intrinsic age-related changes that result in reduced effector and memory responses. However, previously established B- and T-cell memory responses persist with age. One dramatic change is the appearance of a newly recognized population of age-associated B cells (ABCs) that has a unique cluster of differentiation (CD)21-CD23- phenotype. Here, we discuss the discovery and origins of the naive phenotype immunoglobulin (Ig)D+ versus activated CD11c+T-bet+ ABCs, with a focus on protective and pathogenic properties. In humans and mice, antigen-experienced CD11c+T-bet+ ABCs increase with autoimmunity and appear in response to bacterial and viral infections. However, our analyses indicate that CD21-CD23- ABCs include a resting, naive, progenitor ABC population that expresses IgD. Similar to generation of CD11c+T-bet+ ABCs, naive ABC response to pathogens depends on toll-like receptor stimulation, making this a key feature of ABC activation. Here, we put forward a potential developmental map of distinct subsets from putative naive ABCs. We suggest that defining signals that can harness the naive ABC response may contribute to protection against pathogens in the elderly. CD11c+T-bet+ ABCs may be useful targets for therapeutic strategies to counter autoimmunity.

Virus-induced natural killer cell lysis of T cell subsets.

In addition to direct anti-viral activity, NK cells regulate viral pathogenesis by virtue of their cytolytic attack on activated CD4 and CD8 T cells. To gain insight into which differentiated T cell subsets are preferred NK targets, transgenic T cells were differentiated in vitro into Th0, Th1, Th2, Th17, Treg, Tc1, and Tc2 effector cells and then tested for lysis by enriched populations of lymphocytic choriomeningitis virus (LCMV)-induced activated NK cells. There was a distinct hierarchy of cytotoxicity in vitro and in vivo, with Treg, Th17, and Th2 cells being more sensitive and Th0 and Th1 cells more resistant. Some distinctions between in vitro vs in vivo generated T cells were explainable by type 1 interferon induction of class 1 histocompatibility antigens on the effector T cell subsets. NK receptor (NKR)-deficient mice and anti-NKR antibody studies identified no one essential NKR for killing, though there could be redundancies.

Memory CD4 T cell-derived IL-2 synergizes with viral infection to exacerbate lung inflammation.

Defining the most penetrating correlates of protective memory T cells is key for designing improved vaccines and T cell therapies. Here, we evaluate how interleukin (IL-2) production by memory CD4 T cells, a widely held indicator of their protective potential, impacts immune responses against murine influenza A virus (IAV). Unexpectedly, we show that IL-2-deficient memory CD4 T cells are more effective on a per cell basis at combating IAV than wild-type memory cells that produce IL-2. Improved outcomes orchestrated by IL-2-deficient cells include reduced weight loss and improved respiratory function that correlate with reduced levels of a broad array of inflammatory factors in the infected lung. Blocking CD70-CD27 signals to reduce CD4 T cell IL-2 production tempers the inflammation induced by wild-type memory CD4 T cells and improves the outcome of IAV infection in vaccinated mice. Finally, we show that IL-2 administration drives rapid and extremely potent lung inflammation involving NK cells, which can synergize with sublethal IAV infection to promote acute death. These results suggest that IL-2 production is not necessarily an indicator of protective CD4 T cells, and that the lung environment is particularly sensitive to IL-2-induced inflammation during viral infection.

Original Antigenic Sin: Friend or Foe in Developing a Broadly Cross-Reactive Vaccine to Influenza?

In this issue of Cell Host & Microbe, two articles (Lee et al., 2019; Henry et al., 2019) find the influenza-specific antibody repertoire in humans becomes static over time and with age, despite repeated exposures. Identified persistent dominant clones target conserved viral epitopes, supporting the feasibility of a universal influenza vaccine.

Pathogen Recognition by CD4 Effectors Drives Key Effector and Most Memory Cell Generation Against Respiratory Virus.

Although much is known about the mechanisms by which pathogen recognition drives the initiation of T cell responses, including those to respiratory viruses, the role of pathogen recognition in fate decisions of T cells once they have become effectors remains poorly defined. Here, we review our recent studies that suggest that the generation of CD4 T cell memory is determined by recognition of virus at an effector "checkpoint." We propose this is also true of more highly differentiated tissue-restricted effector cells, including cytotoxic "ThCTL" in the site of infection and TFH in secondary lymphoid organs. We point out that ThCTL are key contributors to direct viral clearance and TFH to effective Ab response, suggesting that the most protective immunity to influenza, and by analogy to other respiratory viruses, requires prolonged exposure to antigen and to infection-associated signals. We point out that many vaccines used today do not provide such prolonged signals and suggest this contributes to their limited effectiveness. We also discuss how aging impacts effective CD4 T cell responses and how new insights about the response of aged naive CD4 T cells and B cells might hold implications for effective vaccine design for both the young and aged against respiratory viruses.

The properties of the unique age-associated B cell subset reveal a shift in strategy of immune response with age.

In aged mice, conventional naive B cells decrease and a new population of age-associated B cells (ABC)3 develops. When aged unprimed mice are infected with influenza virus, there is a reduced generation of helper CD4 T cell subsets and germinal center B cells, leading to limited production of IgG Ab and less generation of conventional long-lived plasma cells, compared to young. However, we find an enhanced non-follicular (GL7-) ABC response that is helper T cell-independent, but requires high viral dose and pathogen recognition pathways. The infection-induced ABC (iABC) include IAV-specific Ab-secreting cells, some of which relocate to the bone marrow and lung, and persist for >4wk., suggesting they may provide significant protection. We also speculate there is a shift with increased age to dependence on TLR-mediated pathogen-recognition in both B and CD4 T cell responses.

IL-6 Production by TLR-Activated APC Broadly Enhances Aged Cognate CD4 Helper and B Cell Antibody Responses In Vivo.

Naive CD4 T cell responses, especially their ability to help B cell responses, become compromised with aging. We find that using APC pretreated ex vivo with TLR agonists, polyinosinic-polycytidylic acid and CpG, to prime naive CD4 T cells in vivo, restores their ability to expand and become germinal center T follicular helpers and enhances B cell IgG Ab production. Enhanced helper responses are dependent on IL-6 production by the activated APC. Aged naive CD4 T cells respond suboptimally to IL-6 compared with young cells, such that higher doses are required to induce comparable signaling. Preactivating APC overcomes this deficiency. Responses of young CD4 T cells are also enhanced by preactivating APC with similar effects but with only partial IL-6 dependency. Strikingly, introducing just the activated APC into aged mice significantly enhances otherwise compromised Ab production to inactivated influenza vaccine. These findings reveal a central role for the production of IL-6 by APC during initial cognate interactions in the generation of effective CD4 T cell help, which becomes greater with age. Without APC activation, aging CD4 T cell responses shift toward IL-6-independent Th1 and CD4 cytotoxic Th cell responses. Thus, strategies that specifically activate and provide Ag to APC could potentially enhance Ab-mediated protection in vaccine responses.

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

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

Short-Lived Antigen Recognition but Not Viral Infection at a Defined Checkpoint Programs Effector CD4 T Cells To Become Protective Memory.

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