Fibroblastic reticular cells in lymph nodes regulate the homeostasis of naive T cells

Interleukin 7 is essential for the survival of naive T lymphocytes. Despite its importance, its cellular source in the periphery remains poorly defined. Here we report a critical function for lymph node access in T cell homeostasis and identify T zone fibroblastic reticular cells in these organs as the main source of interleukin 7. In vitro, T zone fibroblastic reticular cells were able to prevent the death of naive T lymphocytes but not of B lymphocytes by secreting interleukin 7 and the CCR7 ligand CCL19. Using gene-targeted mice, we demonstrate a nonredundant function for CCL19 in T cell homeostasis. Our data suggest that lymph nodes and T zone fibroblastic reticular cells have a key function in naive CD4(+) and CD8(+) T cell homeostasis by providing a limited reservoir of survival factors.     

Aurintricarboxylic acid promotes the conversion of naive CD4+CD25- T cells into Foxp3-expressing regulatory T cells

Naive peripheral CD4(+)CD25(-) T cells can be converted into Foxp3-expressing regulatory T cells under appropriate stimulation conditions. Considering that continuous exposure to antigens is one of the prerequisites for the differentiation and maintenance of Treg cells, we investigated whether preventing activation-induced cell death while providing continuous TCR stimulation could promote the expression of Foxp3 in murine naive CD4(+) T cells. Among the several anti-apoptotic agents tested, aurintricarboxylic acid (ATA) was found to induce the in vitro conversion of naive CD4(+) T cells into Foxp3(+) Treg cells with suppressive activity. Neutralizing studies with an antibody against transforming growth factor (TGF)-β revealed that ATA requires the presence of TGF-β to induce Foxp3 expression in naive CD4(+)CD25(-) T cells. Although ATA itself did not activate the Smad signaling pathway, it down-regulated the extracellular signal-regulated kinase and mammalian target of rapamycin signaling cascade in activated T cells. Lastly, combined exposure to ATA and TGF-β had a synergistic effect on the rate of induction and maintenance of Foxp3 expression. These results indicate that ATA could be exploited to efficiently prepare inducible regulatory T cells in vitro and may aid in more precisely identifying the specific signaling pathways that drive Foxp3 expression in T cells.     

T cell homeostasis: keeping useful T cells alive and live T cells useful

It has become clear that the regulation of T cell numbers is under peripheral homeostatic control. However, the rules for homeostasis vary with the T cells' differentiation state and the overall T cell number in the animal. Furthermore, homeostatic pressures can cause unexpected changes in T cell differentiation and function which might promote or dampen T cell reactivity. In this review, we focus on the role of peptide/MHC and cytokine interactions in regulating the size and composition of the T cell pool.     

Rapid conversion of naive to effector T cell function counteracts diminished primary human newborn T cell responses.

The reduced incidence of graft versus host disease following the use of human cord blood as a source of stem cells for bone marrow reconstitution challenges our understanding of the immunocompetence of newborn T cells. Newborn CD4+ T cells express mainly the CD45RA phenotype and have been considered to respond comparably to adult CD4+ T cells exhibiting the CD45RA phenotype. We compared the in vitro kinetics of phenotypic conversion of newborn and adult CD4+CD45RA+ T cells to CD4+CD45RO+ T cells. The cytokine profile and B cell helper activity of the converted CD4+CD45RO+ T cell population were also determined. Newborn CD4+CD45RA+ T cells were converted to CD4+CD45RO+ with significantly faster time kinetics than adult CD4+CD45RA+ T cells, following either phytohaemagglutinin (PHA) or anti-CD2 activation. Freshly purified newborn naive T cells did not produce IL-2, IL-4 or interferon-gamma (IFN-gamma) following stimulation, whereas adult naive T cells secreted IL-2 and adult-derived CD4+CD45RO+ T cells secreted all three cytokines under the same stimulatory conditions. However, newborn and adult CD4+CD45RA+ T cells, following primary stimulation and maturation in vitro, acquired the ability to secrete a Th1-type cytokine profile of IL-2 and IFN-gamma after secondary stimulation. Newborn CD4+ naive T cells that acquired the CD45RO phenotype in vitro also gained B cell helper activity equivalent to that of adult in vitro matured CD4+ naive T cells. These findings suggest that newborn and adult CD4+CD45RA+ T cell subsets are differentially responsive to various stimuli. They show that newborn CD4+CD45RA+ naive T cells can transform more quickly than their adult counterparts into functionally equivalent CD4+CD45RO+ T cells, a process that may be important to counteract the immature immune environment which exists in the newborn.     

Correlation between the numbers of naive T cells infused with blood stem cell allografts and the counts of naive T cells after transplantation.

Naive T cells after allogeneic hematopoietic cell transplantation are thought to originate from the engrafted hematopoietic cells. In this report, we show that there is a correlation between the number of naive CD4 T cells infused with peripheral blood stem cell grafts and the absolute number of peripheral naive CD4 T cells on day 30 (R = 0.65; P <.001), day 80 (R = 0.63; P <.001), and day 180 (R = 0.66; P <.001) after transplantation. These results suggest that in the first 6 months after transplantation, most naive CD4 T cells are derived from the naive T cells infused with the graft.     

Panning T cells on vascular endothelial cell monolayers: a rapid method for enriching naive T cells

A key functional/phenotypic difference between naive and memory T cells is the ability of memory and activated T cells to home to sites of inflammation by adhering to vascular endothelial cells. To determine if this trait could be used to separate naive T cells from memory T cells, CD4+ T cells were incubated with monolayers of IFN-gamma-primed vascular endothelial cells after which the phenotypic and functional characteristics of the nonadherent population were assayed. The nonadherent population 1) contained a five-fold decrease in the frequency of cells displaying the CD44(high)/CD45RB(low) "memory" phenotype and 2) responded well to allostimulation but displayed a reduced ability to respond to immobilized anti-CD3 antibody and, when isolated from ovalbumin-immunized mice, displayed a reduced recall response to ovalbumin in vitro. These studies demonstrate that rwo brief incubations of T cells with monolayers of IFN-gamma-primed endothelial cells can significantly enrich for naive T cells as determined by both phenotypic and functional analyses.     

Human naive T cells activated by cytokines differentiate into a split phenotype with functional features intermediate between naive and memory T cells

We have recently shown that CD45RA⁺CD4⁺ naive T cells can beactivated to proliferate by a combination of IL-2, TNF- andIL-6, but, at variance with TCR-medlated activation, they donot acquire the CD45RO molecule. This prompted us to investigatethe phenotype of these cells and the functional features theydisplay upon TCR stimulation. Naive T cells expanded by cytokines,though remaining CD45RA⁺ express a variety of activation andadhesion molecules which are peculiar to effector or memoryT cells. Naive cells primed by cytokines, when activated withantl-CD3 mAb, produce a broad spectrum of cytokines, expressCD40 ligand, but are unable to help B cells for Ig synthesis.A subset of CD4⁺CD45RA ⁺ RO^– T cells with a phenotype(HLA-DR^–, VLA-2⁺ or IL-2R⁺) similar to that of cells activatedby cytokines In vitro can be found In vivo. These results demonstratethat activation signals delivered by cytokines, in the absenceof TCR stimulation, can activate naive T cells to proliferateand differentiate into a ‘split phenotype’ withelements common to both naive and memory T cells. This novelantigen-Independent activation may help to maintain the naiveT cell repertoire and facilitate the antigen-responsivenessof naive T cells.     

Differential T cell receptor-mediated signaling in naive and memory CD4 T cells

Naive and memory CD4 T cells differ in cell surface phenotype, function, activation requirements, and modes of regulation. To investigate the molecular bases for the dichotomies between naive and memory CD4 T cells and to understand how the T cell receptor (TCR) directs diverse functional outcomes, we investigated proximal signaling events triggered through the TCR/CD3 complex in naive and memory CD4 T cell subsets isolated on the basis of CD45 isoform expression. Naive CD4 T cells signal through TCR/CD3 similar to unseparated CD4 T cells, producing multiple tyrosine-phosphorylated protein species overall and phosphorylating the T cell-specific ZAP-70 tyrosine kinase which is recruited to the CD3ζ subunit of the TCR. Memory CD4 T cells, however, exhibit a unique pattern of signaling through TCR/CD3. Following stimulation through TCR/CD3, memory CD4 T cells produce fewer species of tyrosine-phosphorylated substrates and fail to phosphorylate ZAP-70, yet unphosphorylated ZAP-70 can associate with the TCR/CD3 complex. Moreover, a 26/28-kDa phosphorylated doublet is associated with CD3ζ in resting and activated memory but not in naive CD4 T cells. Despite these differences in the phosphorylation of ZAP-70 and CD3-associated proteins, the ZAP-70-related kinase, p72^syk, exhibits similar phosphorylation in naive and memory T cell subsets, suggesting that this kinase could function in place of ZAP-70 in memory CD4T cells. These results indicate that proximal signals are differentially coupled to the TCR in naive versus memory CD4 T cells, potentially leading to distinct downstream signaling events and ultimately to the diverse functions elicited by these two CD4 T cell subsets.     

The Ca(v)1.4 calcium channel is a critical regulator of T cell receptor signaling and naive T cell homeostasis

The transport of calcium ions (Ca(2+)) to the cytosol is essential for immunoreceptor signaling, regulating lymphocyte differentiation, activation, and effector function. Increases in cytosolic-free Ca(2+) concentrations are thought to be mediated through two interconnected and complementary mechanisms: the release of endoplasmic reticulum Ca(2+) "stores" and "store-operated" Ca(2+) entry via plasma membrane channels. However, the identity of molecular components conducting Ca(2+) currents within developing and mature T?cells is unclear. Here, we have demonstrated that the L-type "voltage-dependent" Ca(2+) channel Ca(V)1.4 plays a cell-intrinsic role in the function, development, and survival of naive T?cells. Plasma membrane Ca(V)1.4 was found to be essential for modulation of intracellular Ca(2+) stores and T?cell receptor (TCR)-induced rises in cytosolic-free Ca(2+), impacting activation of Ras-extracellular signal-regulated kinase (ERK) and nuclear factor of activated T?cells (NFAT) pathways. Collectively, these studies revealed that Ca(V)1.4 functions in controlling naive T?cell homeostasis and antigen-driven T?cell immune responses.     

Galectin-1 supports survival of naive T cells without promoting cell proliferation

Naive T cells do not proliferate but remain alive in vivo. In contrast, naive T cells rapidly die in an in vitro culture, suggesting that some factors that are present at the sites of naive T cell circulation in vivo but missing in the bovine serum-containing culture medium, are necessary for their survival. The present study was designed to search for such factors. By functional screening of the cDNA library from murine lymph node-derived stromal cells (LNS) that effectively support the survival of naive T cells, we found that nascent polypeptide-associated complex (alpha-NAC) promoted T cell survival. A conditioned medium derived from culture supernatant of Cos7 cells transfected with alpha-NAC gene supported T cell survival, indicating that alpha-NAC induced production of soluble factor(s) that were secreted into the medium. By examining the products that were cloned from a functional screening of the cDNA library from alpha-NAC-transfected NIH3T3 cells but were not detected in that from control vector-transfected cells, galectin-1 was found as a soluble factor in the conditioned medium of the LNS. Our study demonstrates the novel role of galectin-1 as a soluble factor that functions to maintain naive T cell survival without inducing cell proliferation.     

Polarization of naive T cells into Th1 or Th2 by distinct cytokine-driven murine dendritic cell populations: implications for immunotherapy

Dendritic cells (DCs) activate T cells and regulate their differentiation into T helper cell type 1 (Th1) and/or Th2 cells. To identify DCs with differing abilities to direct Th1/Th2 cell differentiation, we cultured mouse bone marrow progenitors in granulocyte macrophage-colony stimulating factor (GM), GM + interleukin (IL)-4, or GM + IL-15 and generated three distinct DC populations. The GM + IL-4 DCs expressed high levels of CD80/CD86 and major histocompatibility complex (MHC) class II and produced low levels of IL-12p70. GM and GM + IL-15 DCs expressed low levels of CD80/CD86 and MHC class II. The GM + IL-15 DCs produced high levels of IL-12p70 and interferon (IFN)-gamma, whereas GM DCs produced only high levels of IL-12p70. Naive T cells stimulated with GM + IL-4 DCs secreted high levels of IL-4 and IL-5 in addition to IFN-gamma. In contrast, the GM + IL-15 DCs induced higher IFN-gamma production by T cells with little or no Th2 cytokines. GM DCs did not induce T cell polarization, despite producing large amounts of IL-12p70 following activation. A similar pattern of T cell activation was observed after in vivo administration of DCs. These data suggest that IL-12p70 production alone, although necessary for Th1 differentiation, is not sufficient to induce Th1 responses. These studies have implications for the use of DC-based vaccines in immunotherapy of cancer and other clinical conditions.     

Cytokines and T cell homeostasis

In recent years it has become apparent that the long-term survival of T cells requires continuous contact with external stimuli. At least two types of stimuli, namely self antigens and cytokines, are involved in maintaining T cell viability. As discussed here, the factors controlling T cell survival and turnover in vivo differ considerably from one T cell subset to another.     

Frequencies of FoxP3+ naive T cells are related to both viral load and naive T cell proliferation responses in HIV disease

HIV infection results in depletion and dysfunction of na?ve CD4(+) T cells. The mechanisms underlying these deficiencies are not understood. We investigated the frequencies of CD4(+) na?ve subsets in HIV disease as defined by expression of CD25 and/or FoxP3 and the relationship of these frequencies to na?ve T cell proliferation function. We observed increased proportions of CD25(+)FoxP3(+) and CD25(+)FoxP3(-) cells and decreased proportions of CD25(-)FoxP3(-) cells within the na?ve CD4(+) cell compartment from HIV-infected persons compared with findings in healthy donors. These perturbations were related to higher plasma HIV RNA levels but not with higher immune activation, as measured by the proportions of CD38(+) memory CD4(+) T cells. Na?ve T cell proliferation responses to mitogen stimulation were inversely related to the frequencies and absolute numbers of FoxP3(+) na?ve T cells. MDA, a marker of oxidative stress, and sCD14, a marker of monocyte activation and a surrogate for microbial translocation, were increased in serum samples from HIV(+) donors; however, neither marker was related to na?ve T cell function in HIV(+) donors. These observations suggest that alterations in na?ve T cell subset frequencies could contribute to na?ve T cell dysfunction in HIV disease, but these alterations are not necessarily the result of chronic immune activation.     

Clonal induction of helper T cells: conversion of specific signals into nonspecific signals

The mechanism by which murine helper T cells specific for sheep erythrocytes (SRC) or horse erythrocytes (HRC) exert their effect in an antibody response has been studied in a specific in vitro helper assay in which T cells specific for SRC (TSRC) do not support an antibody response against HRC and in which T cells specific for HRC (T'HRC) do not support an antibody response against SRC. Either population of helper T cells can support an antibody response to both antigens if the homologous and an unrelated antigen are present at the same time. The helper effect is mediated by stable soluble products, the induction of which is antigen specific and independent of B cells. To exclude T cell-T cell interactions, pure populations of specific helper T cells were obtained by long-term culture in vitro of in vivo primed T cells followed by single cell cloning. Clones of T'SRC or T'HRC are highly specific both in vitro and in vivo. For in vivo experiments syngeneic nude mice, selectively and specifically reconstituted with cloned helper T cells, were used. While specific helper T cells can also provide help in vitro for an unrelated antigen, in vivo only specific T cell help is revealed.     

Instruction of naive CD4+ T cells by polarized CD4+ T cells within dendritic cell clusters

Cooperation between CD4(+) T cells can enhance the response and modulate the cytokine profile, and defining these parameters has become a major issue for multivalent-vaccine strategies.We explored cooperation using adoptive transfer of two populations of TCR transgenic T cells of different specificity. One was transferred without prior activation, whereas the second was activated for five days by antigen stimulation under polarizing culture conditions. Both populations were transferred into a single adoptive host and then primed by particle-mediated DNA delivery. Polarized Th1 cells (inducers) raised the frequency of IFN-gamma(+) cells within a naive (target) population, whereas Th2 inducers raised the frequency of IL-4(+) and reduced that of IL-2(+) cells. These effects were obtained when the genes for both antigens were on the same particle, favoring presentation by the same dendritic cell, but not when on different particles delivered to different dendritic cells. Autonomy of DC clusters allows linked sets of antigens (e.g. from a single pathogen) to maintain cytokine bias, but allows other independent responses, each with their own set of autonomous clusters.