Gail E. Huston

CD4 Effector T Cell Subsets in the Response to Influenza Heterogeneity, Migration, and Function

The immune response of naive CD4 T cells to influenza virus is initiated in the draining lymph nodes and spleen, and only after effectors are generated do antigen-specific cells migrate to the lung which is the site of infection. The effector cells generated in secondary organs appear as multiple subsets which are a heterogeneous continuum of cells in terms of number of cell divisions, phenotype and function. The effector cells that migrate to the lung constitute the more differentiated of the total responding population, characterized by many cell divisions, loss of CD62L, down-regulation of CCR7, stable expression of CD44 and CD49d, and transient expression of CCR5 and CD25. These cells also secrete high levels of interferon γ and reduced levels of interleukin 2 relative to those in the secondary lymphoid organs. The response declines rapidly in parallel with viral clearance, but a spectrum of resting cell subsets reflecting the pattern at the peak of response is retained, suggesting that heterogeneous effector populations may give rise to corresponding memory populations. These results reveal a complex response, not an all-or-none one, which results in multiple effector phenotypes and implies that effector cells and the memory cells derived from them can display a broad spectrum of functional potentials.

IL-7 Promotes the Transition of CD4 Effectors to Persistent Memory Cells

After transfer to adoptive hosts, in vitro–generated CD4 effectors can become long-lived memory cells, but the factors regulating this transition are unknown. We find that low doses of interleukin (IL) 7 enhance survival of effectors in vitro without driving their division. When in vitro–generated effectors are transferred to normal intact adoptive hosts, they survive and rapidly become small resting cells with a memory phenotype. CD4 effectors generated from wild-type versus IL-7 receptor−/− mice were transferred to adoptive hosts, including intact mice and those deficient in IL-7. In each case, the response to IL-7 was critical for good recovery of donor cells after 5–7 d. Recovery was also IL-7–dependent in Class II hosts where division was minimal. Blocking antibodies to IL-7 dramatically decreased short-term recovery of transferred effectors in vivo without affecting their division. These data indicate that IL-7 plays a critical role in promoting memory CD4 T cell generation by providing survival signals, which allow effectors to successfully become resting memory cells.

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-γ. 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.

CD4(+) T cell effectors can become memory cells with high efficiency and without further division.

Whether memory T lymphocytes are derived directly from effector T cells or via a separately controlled pathway has long been debated. Here we present evidence that, after adoptive transfer, a large fraction of in vitro–derived effector CD4+ T cells have the potential to become memory T cells and that this transition can occur without further division. This data supports a linear pathway from effector to memory cells and suggests that most properties of memory cells are predetermined during effector generation. We suggest, therefore, that evaluation of vaccine efficacy in the induction of memory CD4+ T cells should focus on the effector stage.

CD4+ T-cell memory: generation and multi-faceted roles for CD4+ T cells in protective immunity to influenza

Summary:  We have outlined the carefully orchestrated process of CD4+ T‐cell differentiation from naïve to effector and from effector to memory cells with a focus on how these processes can be studied in vivo in responses to pathogen infection. We emphasize that the regulatory factors that determine the quality and quantity of the effector and memory cells generated include (i) the antigen dose during the initial T‐cell interaction with antigen‐presenting cells; (ii) the dose and duration of repeated interactions; and (iii) the milieu of inflammatory and growth cytokines that responding CD4+ T cells encounter. We suggest that heterogeneity in these regulatory factors leads to the generation of a spectrum of effectors with different functional attributes. Furthermore, we suggest that it is the presence of effectors at different stages along a pathway of progressive linear differentiation that leads to a related spectrum of memory cells. Our studies particularly highlight the multifaceted roles of CD4+ effector and memory T cells in protective responses to influenza infection and support the concept that efficient priming of CD4+ T cells that react to shared influenza proteins could contribute greatly to vaccine strategies for influenza.

A method for isolation of milk fat globules

The traditional procedure for isolating milk fat globules involves repeated cycles of centrifuging to obtain globules and redispersion of them in fresh buffer to eliminate other milk components. We have evaluated a simpler, less manipulative method whereby globules are centrifuged out of the milk and through an overlying buffer layer. Human milk samples ranging from 0.1 to 35 ml were centrifuged at 1500×g for 20 min after deposition under a suitable quantity of buffer. This yielded purified globules, in less time, which could be dispersed more satisfactorily than those by the traditional procedure. Protein, phospholipid and cholesterol contents of globules by the two methods were quite similar. A lower protein content (10.4 vs 13.2 mg/g of lipid) was characteristic of globules prepared by the multiple wash method. However, large differences could not be seen in gel electrophoresis patterns of the proteins. By using plastic centrifuge tubes, tube freezing and cleavage just below the globule layer enables clean separation of globule and nonglobule phases for analysis of milk component distributions. Macro (5 to 35 ml of sample) and micro (200 μl or less) versions of the method are described. Limited trials showed that the method can be applied satisfactorily to cows and goats milks, but for highly pure globules a deeper buffer column than that used with human milk is required because of their much higher casein content.

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 (TH1) or TH17 polarized but are independent of interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α) 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.

Age-associated increase in lifespan of naïve CD4 T cells contributes to T-cell homeostasis but facilitates development of functional defects

With age, T-cell generation from the thymus is much reduced, yet a substantial naïve T-cell pool is maintained even in aged animals, suggesting that naïve T cells either persist longer or turn over faster to maintain T-cell homeostasis. We found that with age, naïve CD4 T cells became progressively longer-lived. Their longer lifespan did not depend on recognition of self-peptide/class II. Newly generated naïve T cells derived from aged stem cells had a shorter lifespan, like that of young naïve T cells. Conversely, naïve CD4 T cells derived from middle-aged thymectomized mice were longer-lived in vivo, and their development of functional defects was accelerated. These observations suggest that naïve T cells develop their longer lifespan during their sojourn in the periphery. Increased longevity of naïve CD4 T cells correlated well with reduced expression of proapoptotic molecule Bim. We suggest that the intrinsic increase in longevity helps maintain naïve T-cell homeostasis but facilitates the development of functional defects in mice.

Rapid default transition of CD4 T cell effectors to functional memory cells

The majority of highly activated CD4 T cell effectors die after antigen clearance, but a small number revert to a resting state, becoming memory cells with unique functional attributes. It is currently unclear when after antigen clearance effectors return to rest and acquire important memory properties. We follow well-defined cohorts of CD4 T cells through the effector-to-memory transition by analyzing phenotype, important functional properties, and gene expression profiles. We find that the transition from effector to memory is rapid in that effectors rested for only 3 d closely resemble canonical memory cells rested for 60 d or longer in the absence of antigen. This is true for both Th1 and Th2 lineages, and occurs whether CD4 T cell effectors rest in vivo or in vitro, suggesting a default pathway. We find that the effector–memory transition at the level of gene expression occurs in two stages: a rapid loss of expression of a myriad of effector-associated genes, and a more gradual gain of expression of a cohort of genes uniquely associated with memory cells rested for extended periods.

Environmental and intrinsic factors lead to antigen unresponsiveness in CD4+ recent thymic emigrants from aged mice

Naive CD4 cells from aged mice respond inefficiently to Ag, but the factors that underlie the age-associated defects remain unclear. We have used two approaches to isolate recent thymic emigrants (RTE) in young and aged mice and have compared their capacity to respond to antigenic stimulation ex vivo. An in situ intrathymic CFSE injection labeled developing thymocytes and allowed the identification of RTE in secondary lymphoid tissues. Analysis of CFSE-labeled RTE and control unlabeled naive CD4 cells indicated that cells from aged mice were defective in their ability to increase intracellular Ca2+ concentration following TCR cross-linking. Aged naive and RTE CD4 also secreted less IL-2 and proliferated less than that of comparable young CD4 populations. Defects in effector generation in aged RTE were overcome by the addition of IL-2 to cultures. RTE from both polyclonal and TCR transgenic mice were compromised, indicating that defects were independent of TCR specificity. In the second model, the cotransfer of congenic marker-labeled young and aged BM cells into young and aged syngeneic hosts revealed that hyporesponsiveness in aged RTE was caused by a combination of defects intrinsic to CD4 progenitors and defects induced by the aged environment. Depletion of peripheral CD4 cells in aged mice led to production of new RTE that were not defective. The results of this study suggest that defects induced by environmental and lineage intrinsic factors act together to reduce responses to Ag in aged naive CD4 cells and that these defects can be overcome in aged CD4 cells produced during recovery from lymphopenia.