Invariant natural killer T cells direct B cell responses to cognate lipid antigen in an IL-21-dependent manner.

Mouse invariant natural killer T cells (iNKT cells) provide cognate and noncognate help for lipid and protein-specific B cells, respectively. However, the long-term outcome for B cells after cognate help is provided by iNKT cells is unknown at present. Here we found that cognate iNKT cell help resulted in a B cell differentiation program characterized by extrafollicular plasmablasts, germinal-center formation, affinity maturation and a robust primary immunoglobulin G (IgG) antibody response that was uniquely dependent on iNKT cell-derived interleukin 21 (IL-21). However, cognate help from iNKT cells did not generate an enhanced humoral memory response. Thus, cognate iNKT cell help for lipid-specific B cells induces a unique signature that is a hybrid of classic T cell-dependent and T cell-independent type 2 B cell responses.

TLR-activated dendritic cells enhance the response of aged naive CD4 T cells via an IL-6-dependent mechanism.

The most effective immunological adjuvants contain microbial products, such as TLR agonists, which bind to conserved pathogen recognition receptors. These activate dendritic cells (DCs) to become highly effective APCs. We assessed whether TLR ligand-treated DCs can enhance the otherwise defective response of aged naive CD4 T cells. In vivo administration of CpG, polyinosinic-polycytidylic acid, and Pam(3)CSK(4) in combination with Ag resulted in the increased expression of costimulatory molecules and MHC class II by DCs, increased serum levels of the inflammatory cytokines IL-6 and RANTES, and increased cognate CD4 T cell responses in young and aged mice. We show that, in vitro, preactivation of DCs by TLR ligands makes them more efficient APCs for aged naive CD4 T cells. After T-DC interaction, there are enhanced production of inflammatory cytokines, particularly IL-6, and greater expansion of the aged T cells, resulting from increased proliferation and greater effector survival with increased levels of Bcl-2. TLR preactivation of both bone marrow-derived and ex vivo DCs improved responses. IL-6 produced by the activated DCs during cognate T cell interaction was necessary for enhanced aged CD4 T cell expansion and survival. These studies suggest that some age-associated immune defects may be overcome by targeted activation of APCs by TLR ligands.

Bim dictates naive CD4 T cell lifespan and the development of age-associated functional defects.

With age, peripheral naive CD4 T cells become both longer lived and functionally impaired and they express reduced levels of Bim, a proapoptotic Bcl family member. In this study, we show that reduced Bim expression by naive CD4 T cells intrinsically mediates their longer lifespan in the periphery. Moreover, using mixed bone marrow chimeras reconstituted with Bim(+/+) and Bim(+/-) bone marrow cells, Bim(+/-) naive CD4 T cells exhibit accelerated development of age-associated dysfunctions, including reduced proliferation and IL-2 production and defective helper function for B cells, without any increase in their turnover. However, newly generated Bim(+/-) naive CD4 T cells in middle-aged mice are not defective, indicating an additional requirement for their persistence in the periphery. These age-associated immune defects develop independently of the "aged" host environment and without extensive division, distinguishing them from classic "senescence." We suggest that the reduction of Bim levels with age in naive CD4 T cell is the initiating step that leads to increased cellular lifespan and development of age-associated functional defects.

Memory CD4+ T cells induce innate responses independently of pathogen.

Inflammation induced by recognition of pathogen-associated molecular patterns markedly affects subsequent adaptive responses. We asked whether the adaptive immune system can also affect the character and magnitude of innate inflammatory responses. We found that the response of memory, but not naive, CD4(+) T cells enhances production of multiple innate inflammatory cytokines and chemokines (IICs) in the lung and that, during influenza infection, this leads to early control of virus. Memory CD4(+) T cell-induced IICs and viral control require cognate antigen recognition and are optimal when memory cells are either T helper type 1 (T(H)1) or T(H)17 polarized but are independent of interferon-gamma (IFN-gamma) and tumor necrosis factor-alpha (TNF-alpha) production and do not require activation of conserved pathogen recognition pathways. This represents a previously undescribed mechanism by which memory CD4(+) T cells induce an early innate response that enhances immune protection against pathogens.

IL-10 deficiency unleashes an influenza-specific Th17 response and enhances survival against high-dose challenge.

We examined the expression and influence of IL-10 during influenza infection. We found that IL-10 does not impact sublethal infection, heterosubtypic immunity, or the maintenance of long-lived influenza Ag depots. However, IL-10-deficient mice display dramatically increased survival compared with wild-type mice when challenged with lethal doses of virus, correlating with increased expression of several Th17-associated cytokines in the lungs of IL-10-deficient mice during the peak of infection, but not with unchecked inflammation or with increased cellular responses. Foxp3(-) CD4 T cell effectors at the site of infection represent the most abundant source of IL-10 in wild-type mice during high-dose influenza infection, and the majority of these cells coproduce IFN-gamma. Finally, compared with predominant Th1 responses in wild-type mice, virus-specific T cell responses in the absence of IL-10 display a strong Th17 component in addition to a strong Th1 response and we show that Th17-polarized CD4 T cell effectors can protect naive mice against an otherwise lethal influenza challenge and utilize unique mechanisms to do so. Our results show that IL-10 expression inhibits development of Th17 responses during influenza infection and that this is correlated with compromised protection during high-dose primary, but not secondary, challenge.

Impact of post-thymic cellular longevity on the development of age-associated CD4+ T cell defects.

Elderly people are at higher risk for infections due to declining cellular and humoral immune responses. Central to this dysfunction is the reduced responsiveness of the naive CD4(+) T cell compartment. Previous data from our laboratory suggest that although defects in the aged naive CD4(+) T cell response are apparent in recent thymic emigrant populations, additional defects develop during extended post-thymic longevity in the periphery. To further investigate the factors that lead to aging defects, we took advantage of the OT-II TCR-transgenic (Tg) mouse model. We show that because of an apparent superantigen-mediated loss of naive Vbeta5(+) Tg CD4(+) T cells from the periphery of aging OT-II mice, this compartment becomes enriched for cells of reduced post-thymic longevity, resulting in a frequency of recent thymic emigrants in aged mice that is similar to that of young mice. Purification and functional analysis of aged OT-II cells with reduced post-thymic longevity reveal that they have an age-associated decrease in expansion and IL-2 production in response to Ag in vitro. However, the in vivo expansion, IL-2 production, and cognate B cell helper ability of these cells are similar to those of cells from young mice. In contrast, T cells from aged HNT Tg mice demonstrate extended post-thymic longevity and exhibit severe defects in the same in vitro and in vivo models. These data support a correlation between the requirement for increased post-thymic longevity and the development of the most severe naive CD4(+) T cell-aging defects.

Persistent depots of influenza antigen fail to induce a cytotoxic CD8 T cell response.

Encounter with Ag during chronic infections results in the generation of phenotypically and functionally heterogeneous subsets of Ag-specific CD8 T cells. Influenza, an acute infection, results in the generation of similar CD8 T cell heterogeneity, which may be attributed to long-lived depots of flu Ags that stimulate T cell proliferation well after virus clearance. We hypothesized that the heterogeneity of flu-specific CD8 T cells and maintenance of T cell memory required the recruitment of new CD8 T cells to persistent depots of flu Ag, as was the case for flu-specific CD4 T cell responses. However, robust expansion and generation of highly differentiated cytolytic effectors and memory T cells only occurred when naive CD8 T cells were primed during the first week of flu infection. Priming of new naive CD8 T cells after the first week of infection resulted in low numbers of poorly functional effectors, with little to no cytolytic activity, and a negligible contribution to the memory pool. Therefore, although the presentation of flu Ag during the late stages of infection may provide a mechanism for maintaining an activated population of CD8 T cells in the lung, few latecomer CD8 T cells are recruited into the functional memory T cell pool.

SAP is required for Th cell function and for immunity to influenza.

Ab is a crucial component of protective immunity to infection, but Ab responses do not proceed normally when defects occur in a protein called signaling lymphocytic activation molecule-associated protein (SAP). To explain this Ab defect, we analyzed B cell and plasma cell responses under conditions of SAP deficiency. Our results demonstrate that SAP-deficient (SAP knockout (KO)) mice have a profound CD4 T cell-intrinsic defect in generating Ag-specific plasma cells following challenge with model Ags or influenza virus, resulting in low Ag-specific Ab titers. We also show that SAP is required in CD4 T cells for normal division and expansion of B cells. These B cell and plasma cell defects were observed during the expansion phase of the primary immune response, indicating early defects in Th cell activity. In fact, additional experiments revealed a nearly complete lack of T cell help for B cells in SAP KO mice. Our work suggests that the ability of SAP to promote T-dependent humoral immune responses is important for antiviral immunity because mice lacking SAP are unable to prevent high dose secondary influenza infection, and because passive transfer of IgG in immune serum from wild-type, but not SAP KO mice can protect mice from an otherwise lethal influenza infection. Overall, our results demonstrate that SAP is required in CD4 T cells for their ability to help B cell responses and promote influenza-specific immunity.

Unexpected prolonged presentation of influenza antigens promotes CD4 T cell memory generation.

The kinetics of presentation of influenza virus-derived antigens (Ags), resulting in CD4 T cell effector and memory generation, remains undefined. Naive influenza-specific CD4 T cells were transferred into mice at various times after influenza infection to determine the duration and impact of virus-derived Ag presentation. Ag-specific T cell responses were generated even when the donor T cells were transferred 3-4 wk after viral clearance. Transfer of naive CD4 T cells during early phases of infection resulted in a robust expansion of highly differentiated effectors, which then contracted to a small number of memory T cells. Importantly, T cell transfer during later phases of infection resulted in a modest expansion of effectors with intermediate phenotypes, which were capable of persisting as memory with high efficiency. Thus, distinct stages of pathogen-derived Ag presentation may provide a mechanism by which T cell heterogeneity is generated and diverse memory subsets are maintained.

Repeated stimulation of CD4 effector T cells can limit their protective function.

Chronic infections often result in CD8 T-cell deletion or functional nonresponsiveness. However, to date no definitive studies have attempted to determine the impact of repeated T cell receptor stimulation on CD4 effector T cell generation. We have determined that when antigen presentation is limited to 2 d, optimum in vitro CD4 effector generation is achieved. Alternatively, repeated stimulation results in decreased CD4 effector expansion, decreased cytokine production, and altered migration. Similarly, functionally impaired effectors develop in vivo when antigen-pulsed antigen-presenting cells are replenished every 24 h during a primary immune response. CD4 effectors that are generated with repeated stimulation provide no protection during influenza infection, and have an impaired ability to provide cognate help to B cells. These results suggest that duration of antigen presentation dictates CD4 effector function, and repeated T cell receptor stimulation in vitro and in vivo that exceeds an optimal threshold results in effectors with impaired function.