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.

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.

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.

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.

New Insights into the Generation of CD4 Memory May Shape Future Vaccine Strategies for Influenza.

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.

Multipronged CD4(+) T-cell effector and memory responses cooperate to provide potent immunity against respiratory virus.

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.

Spectratyping analysis of the islet-reactive T cell repertoire in diabetic NOD Igµ(null) mice after polyclonal B cell reconstitution.

BACKGROUND: Non Obese Diabetic mice lacking B cells (NOD.Igµ(null) mice) do not develop diabetes despite their susceptible background. Upon reconstitution of B cells using a chimera approach, animals start developing diabetes at 20 weeks of age. METHODS: We have used the spectratyping technique to follow the T cell receptor (TCR) V beta repertoire of NOD.Igµ(null) mice following B cell reconstitution. This technique provides an unbiased approach to understand the kinetics of TCR expansion. We have also analyzed the TCR repertoire of reconstituted animals receiving cyclophosphamide treatment and following tissue transplants to identify common aggressive clonotypes. RESULTS: We found that B cell reconstitution of NOD.Igµ(null) mice induces a polyclonal TCR repertoire in the pancreas 10 weeks later, gradually diversifying to encompass most BV families. Interestingly, these clonotypic BV expansions are mainly confined to the pancreas and are absent from pancreatic lymph nodes or spleens. Cyclophosphamide-induced diabetes at 10 weeks post-B cell reconstitution reorganized the predominant TCR repertoires by removing potential regulatory clonotypes (BV1, BV8 and BV11) and increasing the frequency of others (BV4, BV5S2, BV9, BV16-20). These same clonotypes are more frequently present in neonatal pancreatic transplants under the kidney capsule of B-cell reconstituted diabetic NOD.Igµ(null) mice, suggesting their higher invasiveness. Phenotypic analysis of the pancreas-infiltrating lymphocytes during diabetes onset in B cell reconstituted animals show a predominance of CD19+ B cells with a B:T lymphocyte ratio of 4:1. In contrast, in other lymphoid organs (pancreatic lymph nodes and spleens) analyzed by FACS, the B:T ratio was 1:1. Lymphocytes infiltrating the pancreas secrete large amounts of IL-6 and are of Th1 phenotype after CD3-CD28 stimulation in vitro. CONCLUSIONS: Diabetes in NOD.Igµ(null) mice appears to be caused by a polyclonal repertoire of T cell accumulation in pancreas without much lymphoid organ involvement and is dependent on the help by B cells.