Search

Memory CD4+ T cells combat viral infection and contribute to protective immune responses through multiple mechanisms, but how these pathways interact is unclear. We found that several pathways involving memory CD4+ T cells act together to effectively clear influenza A virus (IAV) in otherwise unprimed mice. Memory CD4+ T cell protection was enhanced through synergy with naive B cells or CD8+ T cells and maximized when both were present. However, memory CD4+ T cells protected against lower viral doses independently of other lymphocytes through production of IFN-γ. Moreover, memory CD4+ T cells selected for epitope-specific viral escape mutants via a perforin-dependent pathway. By deconstructing protective immunity mediated by memory CD4+ T cells, we demonstrated that this population simultaneously acts through multiple pathways to provide a high level of protection that ensures eradication of rapidly mutating pathogens such as IAV. This redundancy indicates the need for reductionist approaches for delineating the individual mechanisms of protection mediated by memory CD4+ T cells responding to pathogens. [ABSTRACT FROM AUTHOR]

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., Competing Interests: The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Swain.)

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., (Copyright © 2023 by The American Association of Immunologists, Inc.)

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., (© 2022 The Authors. Aging Cell published by Anatomical Society and John Wiley & Sons Ltd.)

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., (Copyright © 2022 by The American Association of Immunologists, Inc.)

While influenza infection induces robust, long-lasting, antibody responses and protection, including the T follicular helper cells (T FH ) required to drive B cell germinal center (GC) responses, most influenza vaccines do not. We investigated the mechanisms that drive strong T FH 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 T FH and GC-T FH required that they recognize Ag locally in the site of T FH 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 T FH and GC-T FH development equivalent to live infection. The results suggest that vaccine approaches can induce strong T FH 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 T FH only develop fully when infection is still ongoing, thereby avoiding unnecessary, potentially autoimmune, responses., Competing Interests: The authors declare no competing interest.

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., (Copyright © 2021 Cold Spring Harbor Laboratory Press; all rights reserved.)

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.

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., (Copyright © 2020 by The American Association of Immunologists, Inc.)

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., (Copyright © 2020 by The American Association of Immunologists, Inc.)

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., (Copyright © 2019 Elsevier Inc. All rights reserved.)

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.

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., Competing Interests: The authors have declared that no competing interests exist.

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., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Tissue-resident memory T cells (T RM ) provide optimal defense at the sites of infection, but signals regulating their development are unclear, especially for CD4 T cells. Here we identify two distinct pathways that lead to the generation of CD4 T RM in the lungs following influenza infection. The T RM are transcriptionally distinct from conventional memory CD4 T cells and share a gene signature with CD8 T RM . The CD4 T RM are superior cytokine producers compared with conventional memory cells, can protect otherwise naive mice against a lethal influenza challenge, and display functional specialization by inducing enhanced inflammatory responses from dendritic cells compared with conventional memory cells. Finally, we demonstrate than an interleukin (IL)-2-dependent and a novel IL-2-independent but IL-15-dependent pathway support the generation of cohorts of lung T RM .

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 T FH in secondary lymphoid organs. We point out that ThCTL are key contributors to direct viral clearance and T FH 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.

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., (Copyright © 2017 Elsevier Inc. All rights reserved.)

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., (Copyright © 2017 by The American Association of Immunologists, Inc.)

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., Competing Interests: The authors declare no conflict of interest., (Copyright © 2017 by The American Association of Immunologists, Inc.)

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., (Copyright © 2016 by The American Association of Immunologists, Inc.)

Memory T cells can often respond against pathogens that have evaded neutralizing Abs and are thus key to vaccine-induced protection, yet the signals needed to optimize their responses are unclear. In this study, we identify a dramatic and selective requirement for IL-6 to achieve optimal memory CD4 T cell recall following heterosubtypic influenza A virus (IAV) challenge of mice primed previously with wild-type or attenuated IAV strains. Through analysis of endogenous T cell responses and adoptive transfer of IAV-specific memory T cell populations, we find that without IL-6, CD4 + , but not CD8 + , secondary effector populations expand less and have blunted function and antiviral impact. Early and direct IL-6 signals to memory CD4 T cells are required to program maximal secondary effector responses at the site of infection during heterosubtypic challenge, indicating a novel role for a costimulatory cytokine in recall responses., (Copyright © 2016 by The American Association of Immunologists, Inc.)

An age-related decline in cytolytic activity has been described in CD8+ T cells and we have previously shown that the poor CD8+ effector T cell responses to influenza A/H3N2 challenge result from a decline in the proportion and function of these cytolytic T lymphocytes (CTL). Here, we describe that addition of exogenous cytokines to influenza-stimulated PBMC from both aged mice and humans, enhances the generation of influenza specific CD8 CTL by increasing their proliferation and survival. Our data show that the addition of IL-2 and IL-6 to splenocytes from mice previously infected with influenza virus restores the aged CD8+ T cell response to that observed in young mice. In humans, IL-2 plus IL-6 also reduces the proportion of apoptotic effector CD8+ T cells to levels resembling those of younger adults. In HLA-A2+ donors, MHC Class I tetramer staining showed that adding both exogenous IL-2 and IL-6 resulted in greater differentiation into influenza-specific effector CD8+ T cells. Since this effect of IL-2/IL-6 supplementation can be reproduced with the addition of Toll-like receptor agonists, it may be possible to exploit this mechanism and design new vaccines to improve the CD8 T cell response to influenza vaccination in older adults., Competing Interests: Janet McElhaney has participated on advisory boards for GlaxoSmithKline, Sanofi Pasteur, Pfizer, and Merck and on data monitoring boards for Sanofi Pasteur; and has participated in clinical trials sponsored by Merck, GlaxoSmithKline and Sanofi Pasteur, has received honoraria and travel and accommodation reimbursements for presentations sponsored by Merck, GlaxoSmithKline and Sanofi Pasteur, and travel reimbursement for participation on a publication steering committee for GlaxoSmithKline. Xin Zhou, Jacob Hopkins, Chongkai Wang, Vinayak Brahmakshatriya, George Kuchel, Laura Haynes, and Susan Swain have no conflicts to declare.

Influenza viral evolution presents a formidable challenge to vaccination due to the virus' ability to rapidly mutate to evade immune responses. Live influenza infections generate large and diverse CD4 effector T cell responses that yield highly protective, long-lasting CD4 T cell memory that can target conserved viral epitopes. We review advances in our understanding of mechanisms involved in generating CD4 T cell responses against the influenza A virus (IAV), focusing on specialized follicular helper (TFH) and CD4 cytotoxic (ThCTL) effector subsets and on CD4 T cell memory. We also discuss two recent findings in context of enhancing vaccine responses. First, helper T cells require priming with APC secreting high levels of IL-6. Second, the transition of IAV-generated effectors to memory depends on IL-2, costimulation and antigen signals, just before effectors reach peak numbers, defined as the "memory checkpoint." The need for these signals during the checkpoint could explain why many current influenza vaccines are poorly effective and elicit poor cellular immunity. We suggest that CD4 memory generation can be enhanced by re-vaccinating at this time. Our best hope lies in a universal vaccine that will not need to be formulated yearly against seasonal antigenically novel influenza strains and will also be protective against a pandemic strain. We suggest a vaccine approach that elicits a powerful T cell response, by initially inducing high levels of APC activation and later providing antigen at the memory checkpoint, may take us a step closer to such a universal influenza vaccine.

One of the most profound public health consequences of immune senescence is reflected in an increased susceptibility to influenza and other acute respiratory illnesses, as well as a loss of influenza vaccine effectiveness in older people. Common medical conditions and mental and psychosocial health issues as well as degree of frailty and functional dependence accelerate changes associated with immune senescence. All contribute to the increased risk for complications of influenza infection, including pneumonias, heart diseases, and strokes that lead to hospitalization, disability, and death in the over 65 population. Changes in mucosal barrier mechanisms and both innate and adaptive immune functions converge in the reduced response to influenza infection, and lead to a loss of antibody-mediated protection against influenza with age. The interactions of immune senescence and reduced adaptive immune responses, persistent cytomegalovirus infection, inflammaging (chronic elevation of inflammatory cytokines), and dysregulated cytokine production, pose major challenges to the development of vaccines designed to improve T-cell-mediated immunity. In older adults, the goal of vaccination is more realistically targeted to providing clinical protection against disease rather than to inducing sterilizing immunity to infection. Standard assays of antibody titers correlate with protection against influenza illness but do not detect important changes in cellular immune mechanisms that correlate with vaccine-mediated protection against influenza in older people. This article will discuss: (i) the burden of influenza in older adults and how this relates to changes in T-cell function, (ii) age-related changes in different T-cell subsets and immunologic targets for improved influenza vaccine efficacy in older, and (iii) the development of correlates of clinical protection against influenza disease to expedite the process of new vaccine development for the 65 and older population. Ultimately, these efforts will address the public health need for improved protection against influenza in older adults and "vaccine preventable disability."

Type 2 effector production of IL-13, a demonstrated requirement in models of fibrosis, is routinely ascribed to CD4(+) Th2 cells. We now demonstrate a major role for CD8(+) T cells in a murine model of sterile lung injury. These pulmonary CD8(+) T cells differentiate into IL-13-producing Tc2 cells and play a major role in a bleomycin-induced model of fibrosis. Differentiation of these Tc2 cells in the lung requires IL-21, and bleomycin treated IL-21- and IL-21R-deficient mice develop inflammation but not fibrosis. Moreover, IL-21R-expressing CD8(+) cells are sufficient to reconstitute the fibrotic response in IL-21R-deficient mice. We further show that the combination of IL-4 and IL-21 skews naive CD8(+) T cells to produce IL-21, which, in turn, acts in an autocrine manner to support robust IL-13 production. Our data reveal a novel pathway involved in the onset and regulation of pulmonary fibrosis and identify Tc2 cells as key mediators of fibrogenesis., (Copyright © 2015 by The American Association of Immunologists, Inc.)

CD4+ T cells mediate protection against Mycobacterium tuberculosis (Mtb); however, the phenotype of protective T cells is undefined, thereby confounding vaccination efforts. IL-27 is highly expressed during human tuberculosis (TB), and absence of IL-27R (Il27ra) specifically on T cells results in increased protection. IL-27R deficiency during chronic Mtb infection does not impact antigen-specific CD4+ T cell number but maintains programmed death-1 (PD-1), CD69, and CD127 expression while reducing T-bet and killer cell lectin-like receptor G1 (KLRG1) expression. Furthermore, T-bet haploinsufficiency results in failure to generate KLRG1+, antigen-specific CD4+ T cells, and in improved protection. T cells in Il27ra(-/-) mice accumulate preferentially in the lung parenchyma within close proximity to Mtb, and antigen-specific CD4+ T cells lacking IL-27R are intrinsically more fit than intact T cells and maintain IL-2 production. Improved fitness of IL-27R-deficient T cells is not associated with increased proliferation but with decreased expression of cell death-associated markers. Therefore, during Mtb infection, IL-27R acts intrinsically on T cells to limit protection and reduce fitness, whereas the IL-27R-deficient environment alters the phenotype and location of T cells. The significant expression of IL-27 in TB and the negative influence of IL-27R on T cell function demonstrate the pathway by which this cytokine/receptor pair is detrimental in TB., (© 2015 Torrado et al.)

Decline in immune function and inflammation concomitantly develop with ageing. Here we focus on the impact of this inflammatory environment on T cells, and demonstrate that in contrast to successful tumour elimination in young mice, replenishment of tumour-specific CD4(+) T cells fails to induce tumour regression in aged hosts. The impaired antitumour effect of CD4(+) T cells with their defective Th1 differentiation in an aged environment is restored by interleukin (IL)-6 blockade or IL-6 deficiency. IL-6 blockade also restores the impaired ability of CD4(+) T cells to promote CD8(+) T-cell-dependent tumour elimination in aged mice, which requires IFN-γ. Furthermore, IL-6-stimulated production of IL-4/IL-21 through c-Maf induction is responsible for impaired Th1 differentiation. IL-6 also contributes to IL-10 production from CD4(+) T cells in aged mice, causing attenuated responses of CD8(+) T cells. These findings suggest that IL-6 serves as an extrinsic factor counteracting CD4(+) T-cell-mediated immunity against tumour in old age.

Immunopathologic examination of the lungs of mice with experimental influenza virus infection reveals three prominent findings. (i) There is rapidly developing perivascular (arterial) and peribronchial infiltration with T-cells and invasion of T-cells into the bronchiolar epithelium, separation of epithelial cells from each other and from the basement membrane, leading to defoliation of the bronchial epithelium. This reaction is analogous to a viral exanthema of the skin, such as measles and smallpox. This previously described but unappreciated reaction most likely is an effective way to eliminate virus-infected cells, but may contribute to acute toxicity and mortality. (ii) After this, there is formation of dense collections of lymphocytes adjacent to bronchi consisting mainly of B-cells, with a scattering of T-cells and macrophages. This is known as induced bronchial-associated lymphoid tissue (iBALT) and correlates with increased interleukin (IL)-17 in the lung. iBALT provides sites for a local immune reaction in the lung to both the original infection and related viral infections (heterologous immunity). (iii) Within the first 2-3 weeks, there is proliferation of type II pneumocytes and/or terminal bronchial epithelial cells extending from the terminal bronchioles into the adjacent alveoli, eventually leading to large zones of the lung filled with tumor-like epithelial cells with squamous metaplasia. The proliferation correlates with IL-17 and IL-22 expression, and the extent of this reaction appears to be determined by the availability of T-regulatory cells.

It is unclear how CD4 T-cell memory formation is regulated following pathogen challenge, and when critical mechanisms act to determine effector T-cell fate. Here, we report that following influenza infection most effectors require signals from major histocompatibility complex class II molecules and CD70 during a late window well after initial priming to become memory. During this timeframe, effector cells must produce IL-2 or be exposed to high levels of paracrine or exogenously added IL-2 to survive an otherwise rapid default contraction phase. Late IL-2 promotes survival through acute downregulation of apoptotic pathways in effector T cells and by permanently upregulating their IL-7 receptor expression, enabling IL-7 to sustain them as memory T cells. This new paradigm defines a late checkpoint during the effector phase at which cognate interactions direct CD4 T-cell memory generation.

In response to acute virus infections, CD8(+) T cells differentiate to form a large population of short-lived effectors and a stable pool of long-lived memory cells. The characteristics of the CD8(+) T cell response are influenced by TCR affinity, Ag dose, and the inflammatory cytokine milieu dictated by the infection. To address the mechanism by which differences in TCR signal strength could regulate CD8(+) T cell differentiation, we investigated the transcription factor, IFN regulatory factor 4 (IRF4). We show that IRF4 is transiently upregulated to differing levels in murine CD8(+) T cells, based on the strength of TCR signaling. In turn, IRF4 controls the magnitude of the CD8(+) T cell response to acute virus infection in a dose-dependent manner. Modest differences in IRF4 expression dramatically influence the numbers of short-lived effector cells at the peak of the infection, but have no impact on the kinetics of the infection or on the rate of T cell contraction. Furthermore, the expression of key transcription factors such as T cell factor 1 and Eomesodermin are highly sensitive to graded levels of IRF4. In contrast, T-bet expression is less dependent on IRF4 levels and is influenced by the nature of the infection. These data indicate that IRF4 is a key component that translates the strength of TCR signaling into a graded response of virus-specific CD8(+) T cells., (Copyright © 2014 by The American Association of Immunologists, Inc.)

Aging leads to reduced immunity, especially adaptive responses. A key deficiency is the poor ability to mount robust antibody response. Although intrinsic alterations in B cells with age are in part responsible, impaired CD4 T cell help makes a major contribution to the poor antibody response. Other CD4 effector responses and memory generation are also impaired. We find delayed and reduced development of CD4 T follicular help (Tfh) cells in aged mice in response to influenza infection with reduction of long-lived plasma cells. When we examine CD4 subsets we also find a shift towards Th1 and cytotoxic CD4 (ThCTL) responses. We summarize strategies to circumvent the CD4 T cell defect in aged, including adjuvants and proinflammatory cytokines. We find that we can strongly enhance responses of aged naïve CD4 T cells by using Toll-like receptor (TLR) activated dendritic cells (DC) as APC in vivo and that this leads to improved germinal center B cells and IgG antibody responses. The enhanced response of aged naïve CD4 T cells is dependent on IL-6 produced by the DC., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Activation of self-reactive T cells and their trafficking to target tissues leads to autoimmune organ destruction. Mice lacking the co-inhibitory receptor cytotoxic T lymphocyte antigen-4 (CTLA-4) develop fatal autoimmunity characterized by lymphocytic infiltration into nonlymphoid tissues. Here, we demonstrate that the CD28 co-stimulatory pathway regulates the trafficking of self-reactive Ctla4(-/-) T cells to tissues. Concurrent ablation of the CD28-activated Tec family kinase ITK does not block spontaneous T cell activation but instead causes self-reactive Ctla4(-/-) T cells to accumulate in secondary lymphoid organs. Despite excessive spontaneous T cell activation and proliferation in lymphoid organs, Itk(-/-); Ctla4(-/-) mice are otherwise healthy, mount antiviral immune responses and exhibit a long lifespan. We propose that ITK specifically licenses autoreactive T cells to enter tissues to mount destructive immune responses. Notably, ITK inhibitors mimic the null mutant phenotype and also prevent pancreatic islet infiltration by diabetogenic T cells in mouse models of type 1 diabetes, highlighting their potential utility for the treatment of human autoimmune disorders.

T cell migration is crucial for an effective adaptive immune response to invading pathogens. Naive and memory T cells encounter pathogen antigens, become activated, and differentiate into effector cells in secondary lymphoid tissues, and then migrate to the site(s) of infection where they exert effector activities that control and eliminate pathogens. To achieve activation, efficient effector function, and good memory formation, T cells must traffic between lymphoid and non-lymphoid tissues within the body. This complex process is facilitated by chemokine receptors, selectins, CD44, and integrins that mediate the interactions of T cells with the environment. The expression patterns of these migration receptors (MR) dictate the tissues into which the effector T cells migrate and enable them to occupy specific niches within the tissue. While MR have been considered primarily to facilitate cell movement, we highlight how the heterogeneity of signaling through these receptors influences the function and fate of T cells in situ. We explore what drives MR expression heterogeneity, how this affects migration, and how this impacts T cell effector function and memory formation.

Recent observations have uncovered multiple pathways whereby CD4 T cells can contribute to protective immune responses against microbial threats. Incorporating the generation of memory CD4 T cells into vaccine strategies thus presents an attractive approach toward improving immunity against several important human pathogens, especially those against which antibody responses alone are inadequate to confer long-term immunity. Here, we review how memory CD4 T cells provide protection against influenza viruses. We discuss the complexities of protective memory CD4 T cell responses observed in animal models and the potential challenges of translating these observations into the clinic. Specifically, we concentrate on how better understanding of organ-specific heterogeneity of responding cells and defining multiple correlates of protection might improve vaccine-generated memory CD4 T cells to better protect against seasonal, and more importantly, pandemic influenza.

Over the last decade, the known spectrum of CD4(+) T-cell effector subsets has become much broader, and it has become clear that there are multiple dimensions by which subsets with a particular cytokine commitment can be further defined, including their stage of differentiation, their location, and, most importantly, their ability to carry out discrete functions. Here, we focus on our studies that highlight the synergy among discrete subsets, especially those defined by helper and cytotoxic function, in mediating viral protection, and on distinctions between CD4(+) T-cell effectors located in spleen, draining lymph node, and in tissue sites of infection. What emerges is a surprising multiplicity of CD4(+) T-cell functions that indicate a large arsenal of mechanisms by which CD4(+) T cells act to combat viruses., (© 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

We have previously shown that mice challenged with a lethal dose of A/Puerto Rico/8/34-OVA(I) are protected by injection of 4-8 × 10(6) in vitro-generated Tc1 or Tc17 CD8(+) effectors. Viral load, lung damage, and loss of lung function are all reduced after transfer. Weight loss is reduced and survival increased. We sought in this study to define the mechanism of this protection. CD8(+) effectors exhibit multiple effector activities, perforin-, Fas ligand-, and TRAIL-mediated cytotoxicity, and secretion of multiple cytokines (IL-2, IL-4, IL-5, IL-9, IL-10, IL-17, IL-21, IL-22, IFN-γ, and TNF) and chemokines (CCL3, CCL4, CCL5, CXCL9, and CXCL10). Transfer of CD8(+) effectors into recipients, before infection, elicits enhanced recruitment of host neutrophils, NK cells, macrophages, and B cells. All of these events have the potential to protect against viral infections. Removal of any one, however, of these potential mechanisms was without effect on protection. Even the simultaneous removal of host T cells, host B cells, and host neutrophils combined with the elimination of perforin-mediated lytic mechanisms in the donor cells failed to reduce their ability to protect. We conclude that CD8(+) effector T cells can protect against the lethal effects of viral infection by means of a large number of redundant mechanisms.

Our recent studies indicate that the longer peripheral persistence of naïve CD4 T cells that occurs with age is necessary for the development of the key aging defects that lead to compromised responses to vaccination and to new pathogens or new strains of circulating infectious agents. This longer persistence is in turn is linked to the decrease in development of new thymic emigrants and thymic involution that occur at adolescence. Therefore the process of development of naïve CD4 aging defects, is closely tied to the homeostasis of T cells and the shifts that occur in their homeostasis with age. Here we review this connection between age-related changes in T cell homeostasis and the development of T cell defects and discuss the implication for approaches to better vaccinating the elderly., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Whether differences between naive cell-derived primary (1°) and memory cell-derived secondary (2°) CD4(+) T-cell effectors contribute to protective recall responses is unclear. Here, we compare these effectors directly after influenza A virus infection. Both develop with similar kinetics, but 2° effectors accumulate in greater number in the infected lung and are the critical component of memory CD4(+) T-cell-mediated protection against influenza A virus, independent of earlier-acting memory-cell helper functions. Phenotypic, functional, and transcriptome analyses indicate that 2° effectors share organ-specific expression patterns with 1° effectors but are more multifunctional, with more multicytokine (IFN-γ(+)/IL-2(+)/TNF(+))-producing cells and contain follicular helper T-cell populations not only in the spleen and draining lymph nodes but also in the lung. In addition, they express more CD127 and NKG2A but less ICOS and Lag-3 than 1° effectors and express higher levels of several genes associated with survival and migration. Targeting two differentially expressed molecules, NKG2A and Lag-3, reveals differential regulation of 1° and 2° effector functions during pathogen challenge.

CD4 effectors generated in vitro can promote survival against a highly pathogenic influenza virus via an antibody-independent mechanism involving class II-restricted, perforin-mediated cytotoxicity. However, it is not known whether CD4 cells activated during influenza virus infection can acquire cytolytic activity that contributes to protection against lethal challenge. CD4 cells isolated from the lungs of infected mice were able to confer protection against a lethal dose of H1N1 influenza virus A/Puerto Rico 8/34 (PR8). Infection of BALB/c mice with PR8 induced a multifunctional CD4 population with proliferative capacity and ability to secrete interleukin-2 (IL-2) and tumor necrosis factor alpha (TNF-α) in the draining lymph node (DLN) and gamma interferon (IFN-γ) and IL-10 in the lung. IFN-γ-deficient CD4 cells produced larger amounts of IL-17 and similar levels of TNF-α, IL-10, and IL-2 compared to wild-type (WT) CD4 cells. Both WT and IFN-γ(-/-) CD4 cells exhibit influenza virus-specific cytotoxicity; however, IFN-γ-deficient CD4 cells did not promote recovery after lethal infection as effectively as WT CD4 cells. PR8 infection induced a population of cytolytic CD4 effectors that resided in the lung but not the DLN. These cells expressed granzyme B (GrB) and required perforin to lyse peptide-pulsed targets. Lethally infected mice given influenza virus-specific CD4 cells deficient in perforin showed greater weight loss and a slower time to recovery than mice given WT influenza virus-specific CD4 cells. Taken together, these data strengthen the concept that CD4 T cell effectors are broadly multifunctional with direct roles in promoting protection against lethal influenza virus infection.

Viral pathogens often induce strong effector CD4(+) T cell responses that are best known for their ability to help B cell and CD8(+) T cell responses. However, recent studies have uncovered additional roles for CD4(+) T cells, some of which are independent of other lymphocytes, and have described previously unappreciated functions for memory CD4(+) T cells in immunity to viruses. Here, we review the full range of antiviral functions of CD4(+) T cells, discussing the activities of these cells in helping other lymphocytes and in inducing innate immune responses, as well as their direct antiviral roles. We suggest that all of these functions of CD4(+) T cells are integrated to provide highly effective immune protection against viral pathogens.

Krüppel-like factor 4 (KLF4), a transcription factor, plays a key role in the pluripotency of stem cells. We sought to determine the function of KLF4 in T-cell development and differentiation by using T-cell-specific Klf4-knockout (KO) mice. We found that KLF4 was highly expressed in thymocytes and mature T cells and was rapidly down-regulated in mature T cells after activation. In Klf4-KO mice, we observed a modest reduction of thymocytes (27%) due to the reduced proliferation of double-negative (DN) thymocytes. We demonstrated that a direct repression of Cdkn1b by KLF4 was a cause of decreased DN proliferation. During in vitro T-cell differentiation, we observed significant reduction of IL-17-expressing CD4(+) T cells (Th17; 24%) but not in other types of Th differentiation. The reduction of Th17 cells resulted in a significant attenuation of the severity (35%) of experimental autoimmune encephalomyelitis in vivo in Klf4-KO mice as compared with the Klf4 wild-type littermates. Finally, we demonstrated that KLF4 directly binds to the promoter of Il17a and positively regulates its expression. In summary, these findings identify KLF4 as a critical regulator in T-cell development and Th17 differentiation.

The mechanisms responsible for heterosubtypic immunity to influenza virus are not well understood but might hold the key for new vaccine strategies capable of providing lasting protection against both seasonal and pandemic strains. Memory CD4 T cells are capable of providing substantial protection against influenza both through direct effector mechanisms and indirectly through regulatory and helper functions. Here, we discuss the broad impact of memory CD4 T cells on heterosubtypic immunity against influenza and the prospects of translating findings from animal models into improved human influenza vaccines., (© 2011 The Association for the Publication of the Journal of Internal Medicine.)