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.

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.

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.

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.

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.

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.

"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.

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.