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Interferon regulatory factor 4 (IRF4) regulates the clonal expansion and metabolic activity of activated T cells, but the precise context and mechanisms of its function in these processes are unclear. In this issue of the European Journal of Immunology, Miyakoda et al. [Eur. J. Immunol. 2018. 48: 1319-1328] show that IRF4 is required for activation and expansion of naive and memory CD8+ T cells driven by T-cell receptor (TCR) signaling, but dispensable for memory CD8+ T-cell maintenance and homeostatic proliferation driven by homeostatic cytokines. The authors show that the function of IRF4 in CD8+ T-cell expansion is partially dependent upon activation of the PI3K/AKT pathway through direct or indirect attenuation of PTEN expression. These data shed light upon the differential intracellular pathways required for naive and memory T cells to respond to self-antigens and/or homeostatic cytokines, and highlight the potential translational relevance of these findings in the context of immune reconstitution such as following allogeneic stem cell transplantation.

Following antigen encounter and subsequent resolution of the immune response, a single naive T cell is able to generate multiple subsets of memory T cells with different phenotypic and functional properties and gene expression profiles. Single-cell technologies, first and foremost flow cytometry, have revealed the complex heterogeneity of the memory T-cell compartment and its organization into subsets. However, a consensus has still to be reached, both at the semantic (nomenclature) and phenotypic level, regarding the identification of these subsets. Here, we review recent developments in the characterization of the heterogeneity of the memory T-cell compartment, and propose a unified classification of both human and nonhuman primate T cells on the basis of phenotypic traits and in vivo properties. Given that vaccine studies and adoptive cell transfer immunotherapy protocols are influenced by these recent findings, it is important to use uniform methods for identifying and discussing functionally distinct subsets of T cells.

Mutations in PTEN-induced kinase 1 (PINK1), a serine/threonine kinase linked to familial early-onset Parkinsonism, compromise mitochondrial integrity and metabolism and impair AKT signaling. As the activation of a naive T cell requires an AKT-dependent reorganization of a cell's metabolic machinery, we sought to determine if PINK1-deficient T cells lack the ability to undergo activation and differentiation. We show that CD4(+) T cells from PINK1 knockout mice fail to properly phosphorylate AKT upon activation, resulting in reduced expression of the IL-2 receptor subunit CD25. Following, deficient IL-2 signaling mutes the activation-induced increase in respiratory capacity and mitochondrial membrane potential. Under polarization conditions favoring the development of induced regulatory T cells, PINK1(-/-) T cells exhibit a reduced ability to suppress bystander T-cell proliferation despite normal FoxP3 expression kinetics. Our results describe a critical role for PINK1 in integrating extracellular signals with metabolic state during T-cell fate determination, and may have implications for the understanding of altered T-cell populations and immunity during the progression of active Parkinson's disease or other immunopathologies.

The immune system relies on homeostatic mechanisms in order to adapt to the changing requirements encountered during steady-state existence and activation by antigen. For T cells, this involves maintenance of a diverse repertoire of naive cells, rapid elimination of effector cells after pathogen clearance, and long-term survival of memory cells. The reduction of T-cell counts by either cytotoxic drugs, irradiation, or certain viruses is known to lead to lymphopenia-induced proliferation and restoration of normal T-cell levels. Such expansion is governed by the interaction of TCR with self-peptide/MHC (p/MHC) molecules plus contact with cytokines, especially IL-7. These same ligands, i.e. p/MHC molecules and IL-7, maintain naive T lymphocytes as resting cells under steady-state T-cell-sufficient conditions. Unlike naive cells, typical "central" memory T cells rely on a combination of IL-7 and IL-15 for their survival in interphase and for occasional cell division without requiring signals from p/MHC molecules. Other memory T-cell subsets are less quiescent and include naturally occurring activated memory-phenotype cells, memory cells generated during chronic viral infections, and effector memory cells. These subsets of activated memory cells differ from central memory T cells in their requirements for homeostatic proliferation and survival. Thus, the factors controlling T-cell homeostasis can be seen to vary considerably from one subset to another as described in detail in this review.

ERK5 has been implicated in regulating the MEF2-dependent genes Klf2 and nur77 downstream of the TCR and the maintenance of expression of CD62L on peripheral T cells. Based on this data, knockout of ERK5 would be predicted to compromise T-cell development and the maintenance of T cells in the periphery. Using an ERK5 conditional knockout, driven by CD4-CRE or Vav-CRE transgenes resulting in the loss of ERK5 in T cells, we have found that ERK5 is not required for T-cell development. In addition, normal numbers of T cells were found in the spleens and lymph nodes of these mice. We also find that TCR stimulation is not a strong signal for ERK5 activation in primary murine T cells. ERK5 was found to contribute to the induction of Klf2 but not nur77 mRNA following TCR activation. Despite the reduction in Klf2 mRNA, no effect was seen in ERK5 knockouts on either the mRNA levels for the Klf2 target genes CD62L, CCR7 and S1P, or the cell surface expression of CD62L. These results suggest that while ERK5 does contribute to Klf2 regulation in T cells, it is not essential for the expression of CD62L or T-cell survival.

The ability of IL-21 to promote in vitro T cell survival led us to investigate its biological activity in vivo. We report that overexpression of IL-21 in transgenic mice drives CD8(+) memory T cell accumulation with a concomitant reduction in naive T cell numbers. These memory T cells are functional, given their ability to rapidly produce IFN-gamma and proliferate following stimulation. Since the homeostasis of naive and memory T cells is controlled by cytokines, we evaluated whether IL-21 influences cytokine receptor expression. We show that IL-21 inhibits IL-7R expression on naive T cells in vitro, suggesting impaired IL-7-mediated naive T cell survival in IL-21-transgenic mice. In contrast, IL-7R expression on CD4(+) memory T cells is not affected, allowing their IL-7-dependent survival in IL-21-transgenic mice. Although IL-21 decreases IL-7R expression on CD8(+) memory T cells, this has no impact on their survival since their maintenance in the T cell pool is IL-7-independent. Rather, we demonstrate that CD8(+) memory T cells are receptive to IL-21 survival signals allowing for their accumulation in IL-21-transgenic mice. This study identifies new roles for IL-21 in T cell homeostasis and in the regulation of T cell responses to cytokines.

Only recently have natural antigens for CD1d-dependent, invariant Valpha14+ natural killer T (iNKT) cells been identified. Similar data for CD1d-independent and CD8+ NKT cell populations are still missing. Here, we show that the MHC class I-restricted CD8+ TCR-transgenic mouse lines OT-I, P14 and H-Y contain a significant proportion of transgenic CD8+ NK1.1+ T cells. In liver, most of NK1.1+ T cells express CD8alphaalpha homodimers. Transgenic NKT cells did not bind invariant Valpha14-to-Jalpha18 TCR rearrangement (Valpha14i)-specific CD1d/alpha-galactosylceramide tetramers and the frequency of iNKT cells was severely reduced. The activated cell surface phenotype and the distribution of transgenic NKT cells in vivo were similar to that reported for iNKT cells. The OT-I and P14 CD8+ NKT cells recognized their cognate antigen in the context of H2-Kb and produced cytokines shortly after TCR stimulation. Importantly, transgenic NKT cells exerted immediate antigen-specific cytotoxicity in vitro and in vivo. Our results demonstrate the presence of transgenic CD8+ NKT cells in MHC class I-restricted TCR-transgenic animals, which are endowed with rapid antigen-specific effector functions. These data imply that experiments studying naive T cell function in TCR-transgenic animals should be interpreted with caution, and that such animals could be utilized for studying CD8+ NKT cell function in an antigen-specific manner.

The molecular mechanisms by which dendritic cells (DC) favor naive T cell survival in mice have been examined in co-cultures of DC and naive CD4+ T cells. Naive T cells can survive in the presence of IL-4 or IL-7, but DC-induced T cell survival requires direct cell-cell interactions and does not seem to be mediated by these or other soluble factors. Classical MHC II molecules on DC are not necessary for T cell survival as long as hybrid AalphaEbeta MHC class II molecules are present. In the total absence of MHC II molecules on DC, T cell survival is reduced by half, and CD3zeta phosphorylation fully disappears. These results contrast with the classical view that naive T cell survival is associated with CD3zeta phosphorylation and depends mostly on IL-7 and MHC-TCR interactions. We demonstrate that DC-induced T cell survival is a multi-factorial process that also involves CD28, LFA-1 and another (as yet undefined) surface molecule that requires the activity of src (but not phosphatidylinositol-3-) kinase.

Tissue-selective homing is established during naive T cell activation by the tissue microenvironment and tissue-specific dendritic cells (DC). The factors driving induction and maintenance of T cell homing patterns are still largely unknown. Here we show that soluble factors produced during the interaction of T cells with CD11c + DC isolated from skin- or small intestine-associated tissues differentially modulate expression of the corresponding tissue-selective homing receptors (E-selectin ligands and α4β7 integrin/CCR9, respectively) on murine CD8 + T cells. Injection of tissue-specific DC via different routes induces T cells with homing receptors characteristic of the corresponding local tissue microenvironment, independent of the origin of the DC. These data indicate an important role for signals delivered in trans. Moreover, DC can reprogram the homing receptor expression on T cells previously polarized in vitro for homing to skin or small intestine. Importantly, skin-homing memory T cells stimulated directly ex vivo can also be reprogrammed by intestinal DC to a gut-homing phenotype. Our results show that tissue-selective homing receptor expression on effector and memory T cells is governed by inductive as well as suppressive signals from both DC and tissue microenvironments. © 2005 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

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.

Aging of the murine thymus is accompanied by a measurable loss of weight and cellularity and a marked reduction in its output of T lymphocytes. This study employs a molecular approach to determine changes in the output of the murine thymus using a novel assay system based on the detection and quantitation of the excised TCRdelta DNA locus from within the TCRalpha chain genes. In alpha beta(+) T cells such delta excision circles (deltaEC) are present at higher levels in naive T cells as compared with memory T cell populations, are non-replicating, and diluted within the total peripheral alpha beta(+) T cell pool with advancing age. This assay permits the assessment of thymic output in older animals where previous analysis was hampered by the transient nature of the naive T cell surface phenotype, and so allows the assessment of the efficacy of IL-7 as an agent to reverse thymic atrophy. Treatment of old mice with IL-7 although producing no overall change in the total number of alpha beta(+) T cells in the peripheral T cell pool altered the component subsets. Mice treated with IL-7 showed increases in the number of alpha beta(+)TCR cells possessing deltaEC commensurate with improved thymic output, and the splenic T cells from IL-7-treated mice performed significantly better in in vitro functional assays compared to those from age-matched saline-treated controls.

The involvement of Fas and caspase activation during naive T cell proliferation is still controversial. To explore this paradox, we used antigenic variation of PCC 88-104 peptide to dissect pathways of TCR signaling leading to cell proliferation. We demonstrated that strong TCR stimulation but not weak TCR stimulation induced T cell proliferation and was dependent on IETD- but independent of DEVD-specific caspases. In addition, altered TCR ligand induced T cell proliferation in the absence of IETD-, DEVD-specific caspase activities and Fas ligand expression. However, AND-TCR-transgenic mice in lpr/lpr background generated recently have no defect T cell proliferation after stimulation by the agonist peptide, demonstrating that antigen-induced T cell proliferation is independent of Fas-activation pathways. Thus, Fas-independent caspase activation is tightly regulated by the strength of antigenic stimulation during T cell proliferation. These data reveal a novel facet of antigenic caspase regulation during naive CD4 T cell proliferation and provide insights into the function of caspases during T cell homeostasis.

Endogenous superantigens (SAg) presented by MHC class II IA molecules induce slow-evolving negative selection of alpha beta T cells. The role of both B and gamma delta T cells on the regulation of these SAg-specific alpha beta T cell responses was addressed in IA(b+)IE(b-) C57BL/6 mice bearing genetically induced B cell and gamma delta T cell deficiencies. B lymphocytes were required in the negative selection of Vbeta5(+)/Vbeta12(+) CD4(+) T cells. In contrast, gamma delta T cells positively stimulated the utilization of the same SAg-responsive alpha beta T cell subsets. These differences started in mature CD4(+) thymocytes and extended to naive T cell pools for B cell negative selection, and up to memory T cells for gamma deltaT cell influences. The levels of SAg-responsive T cells did not vary between C57BL/6 and double deficient (B cell and gamma delta T cell-deficient) congenic mice, implying that both B and gamma delta T cells acted through independent mechanisms.

A diverse repertoire among peripheral T cells is established in early life by thymic export when the naive T cell pool is first formed. In contrast, during adult life the thymus has been thought to play only a minor role in T cell homeostasis. As individuals age there is an increasing proportion of peripheral T cells bearing a memory phenotype, as well as a corresponding decrease in the number of naive T cells. The change in the composition of the peripheral T cell pool with age is thought to occur as a result of reduced or completely curtailed thymic export following thymic involution at puberty together with the antigen-driven expansion of naive T cells in the periphery. We examined thymic export throughout life in fetal, neonatal and aged sheep. We found that the thymus in adult animals showed an efficiency of production and export on a per gram basis equivalent to that observed for much younger animals, and continued to export substantial numbers of T cells long after puberty. The data demonstrate that naive T cells constantly enter the peripheral T cell pool at the same rate throughout fetal, neonatal and adult life, and that one in every 50 T cells in the peripheral lymphoid tissues of aged sheep had emigrated from the thymus in the previous 24 h. The data suggest that restoration by the thymus of a normal peripheral T cell repertoire in chronic T cell-depleting conditions should be possible in adult patients, provided the thymus is not damaged by disease or therapy.

Naive CD4(+) T cells differ from memory cells by their heightened expression of the disialoceramide recognized by antibody 3G11. 3G11(bright) cells respond well to immobilized anti-CD3 / anti-CD28 and to their cognate antigens but produce little or no IFN-gamma or IL-4 "acutely" and undergo cell death even in the presence of IL-2. They can be rescued by IL-4, IL-6 or IL-12. IL-6 is particularly notable since it is neutral in regard to Th1 / Th2 priming, allowing an assessment of the role of endogenous IL-4 in priming for IL-4 production. Naive TCR-transgenic BALB / c scid T cells cultured with an ovalbumin peptide and IL-4(- / -) antigen-presenting cells in the presence of IL-6 showed a modest degree of priming for IL-4 production if both IFN-gamma and IL-12 were neutralized. This priming is far less than that observed if IL-4 is added to the priming culture. These results indicate that IL-4 production as a result of TCR engagement is sufficient for only a minor component of the polarization observed when unseparated BALB / c CD4 T cell populations are primed or when IL-4 is intentionally added to the priming culture.

We have studied the kinetics of the antigen induced response of naive and memory CD8 T cells expressing a transgenic T cell receptor (TCR) specific for the glycoprotein peptide amino acid 33-41 (GP33) of the lymphocytic choriomeningitis virus (LCMV). Memory T cells were generated in vivo by adoptive transfer of LCMV TCR transgenic T cells into normal recipient mice, followed by LCMV infection. The results demonstrated that the cell cycle progression and kinetics of TCR down-modulation, CD25 and CD69 up-regulation were identical in naive and memory T cells after antigen recognition. Moreover, the two T cell populations did not differ in respect of activation thresholds and in their proliferative capacities neither in vitro nor in vivo. However, memory CD8 T cells could be more rapidly induced to become cytolytic and to secrete high levels of interleukin-2 and interferon-gamma than naive T cells. LCMV GP33-specific CD8 memory T cells were only slightly more efficient in reducing LCMV titers in the spleen but were far more effective than naive LCMV GP33-specific T cells in controlling subcutaneous tumor growth of B16.F10 melanoma cells which expressed the LCMV GP33 epitope as tumor-associated antigen. Thus, in our experiments the main difference between CD8 memory T cells and naive cells is the ability of the former to rapidly acquire effector cell functions.

This study examines the influence of IL-7 on post-thymic CD4+ T cells using cord blood as a model system. Survival of naive cord blood T cells in the presence of IL-7 alone was significantly prolonged by up-regulating bcl-2, thereby preventing apoptosis while maintaining maximal cell viability. Cultures without IL-7 showed high rates of apoptosis resulting in 50% cell death by day 5 of culture. Upon phorbol 12-myristate 13-acetate + ionomycin stimulation, accumulation of cytoplasmic IL-2 was similar to that observed in freshly isolated cells, but no IL-4- or IFN-gamma-positive cells were detected. IL-7 maintained the naive T cells in a quiescent state expressing the CD45RA antigen. A significant finding was the loss of CD38 antigen expression on the naive cord blood T cells to levels similar to that observed on adult naive T cells. In contrast to the reduced proliferative response of fresh cord blood T cells to anti-CD2 + CD28 stimulation, the proliferative response of IL-7-treated cells was similar to that of adult naive T cells. This study shows that as well as maintaining the naive T cell pool by enhancing cell survival and up-regulating bcl-2 expression, IL-7 also functions as a maturation factor for post-thymic naive T cells.

Bacterial DNA and immunostimulatory (i.s.) synthetic CpG-oligodeoxynucleotides (ODN) act as adjuvants for Th1 responses and cytotoxic T cell responses to proteinaceous antigens. Dendritic cells (DC) can be referred to as "nature's adjuvant" since they display the unique capacity to sensitize naive T cells. Here, we demonstrate that bacterial DNA or i.s. CpG-ODN cause simultaneous maturation of immature DC and activation of mature DC to produce cytokines. These events are associated with the acquisition of professional antigen-presenting cell (APC) function. Unfractionated murine bone marrow-derived DC and FACS-fractionated MHC class IIlow (termed immature DC) or MHC class IIhigh populations (termed mature DC) were stimulated with bacterial DNA or i.s. CpG-ODN. Similar to lipopolysaccharide, i.s. CpG-ODN caused up-regulation of MHC class II, CD40 and CD86, but not CD80 on immature and mature DC. In parallel both DC subsets were activated to produce large amounts of IL-12, IL-6 and TNF-alpha. CpG-ODN-activated DC displayed professional APC function in allogeneic mixed lymphocyte reaction and in staphylococcal enterotoxin B-driven naive T cell responses. We interpret these findings to mean that bacterial DNA and i.s. CpG-ODN cause maturation (first step) and activation (second step) of DC to bring about conversion of immature DC into professional APC.

The role of antigen-presenting cell (APC)-derived cytokines in T cell activation is still controversial. Highly purified CD4 T cell populations of the naive and short-term Th1 and Th2 effector subsets were examined. Stimulation from anti-CD3 in the absence of APC was used to analyze directly T occurring cell-mediated effects, and the requirement for co-signaling was addressed using anti-CD28. Exogenous IL-6, IL-1 and TNF each enhanced proliferation and IL-2 secretion from naive cells, although IL-6 was most active in this regard. Peak responses, however, were obtained with IL-1 or TNF in combination with IL-6 resulting in up to 11-fold increases in IL-2 secretion. Enhanced naive T cell responses were only observed with anti-CD3 and anti-CD28, suggesting that co-signaling through surface-bound receptors was required to initiate IL-2 production. Although the cytokines enhanced naive activation, little effect was seen on differentiation into effector populations. IL-6 alone, or in combination, partially suppressed effectors secreting IFN-gamma, but did not promote generation of effectors secreting IL-4. In contrast to reports on cloned cell lines, IL-6, TNF and IL-1 had enhancing activities on all cytokines elicited from already generated Th1 and Th2 effector populations. Again combinations of IL-6, TNF and IL-1 were most effective and generally required CD28 signaling. Induced responses with preexisting effector cells were far less than with naive cells and predominantly directed at augmenting IFN-gamma and IL-5 secretion rather than IL-2 and IL-4. These studies show that APC-derived cytokines can promote T cell responses directly but largely after co-stimulation from accessory molecule co-receptors, that the effect is not specific for one T cell subset or cytokine, and that the naive T cell is the main target of action.

Stimulation of T cells via the T cell receptor (TCR) leads to an increase intracellular in free Ca2+ levels ([Ca2+]i) and the activation of the MAP kinase signaling pathway. This study analyzes for the first time Ca2+ fluxes in naive cytotoxic T cells stimulated with full agonists, partial agonists, or antagonists. Four different types of Ca2+ responses could be observed. Full agonists triggered a strong and sustained increase in [Ca2+]i. In contrast, partial T cell agonists induced either a strong but transient Ca2+ flux or very low to no increases in [Ca2+]i, while T cell antagonists failed to induce any measurable Ca2+ flux. The ability of peptides to induce elevated [Ca2+]i perfectly paralleled their ability to trigger TCR internalization and T cell activation. Thus, stimulation of naive cytotoxic T cells with a panel of defined altered peptide ligands reveals a consistent picture, where Ca2+ fluxes predict agonist, partial agonist and antagonist properties of peptides.

B cells activated with anti-mu antibody plus interferon (IFN)-gamma exerted strong antigen presentation activity for T cell proliferation. The enhanced antigen presentation function was shown to be due to the increase in B7-2 expression. When B cells were stimulated with anti-mu, expression of MHC major histocompatibility complex class II, heat-stable antigen (HSA), ICAM-1 and B7-2 was increased. The presence of IFN-gamma further augmented the expression of B7-2 on anti-mu-stimulated B cells. B7-1 was not expressed on B cells under these conditions. The participation of B7-2 in the elicitation of the proliferative response of T cells was confirmed by the inclusion of anti-B7-2 antibody in cultures. The enhanced expression of either HSA or ICAM-1 was shown not to play a major role in the increased B cell antigen presentation capacity. The major T cell population responding to this activated B cell antigen presentation was shown to be CD44low naive CD4+ T cells, whereas CD45RBlow memory CD4+ T cells responded only weakly. The difference in proliferative responses between naive and memory CD4+ T cells was explained by the different efficiency in IL-2 production of these cell populations in response to antigen presentation by B cells activated by anti-mu plus IFN-gamma. These results suggest that IFN-gamma plays an important role in recruitment of naive T cells for an immune response.

The Notch pathway is involved in cell differentiation processes in various organs and at several developmental stages. The importance of Notch for early T lymphocyte development is well established. Recently, Notch has been implicated in directing naive T helper cell differentiation towards the Th1, Th2 or regulatory T cell lineages. However, the molecular events underlying these processes are poorly understood. We show that the Notch ligands Delta-like1, Delta-like4 and Jagged1 differentially affect early T cell activation and proliferation following T cell receptor cross-linking. Delta-like1 and Jagged1 induce a dose-dependent inhibition of early activation markers CD69 and CD25, as well as inhibition of proliferation after anti-CD3 stimulation of purified CD4+ T cells. Similarly, the rapid activation of transcription factors NF-AT, AP-1 and NF-kappaB is suppressed. In contrast, triggering of Notch by Delta-like4 enhances T cell activation and proliferation. The observed effects are dependent on simultaneous cross-linking of TCR and Notch but independent of gamma-secretase-mediated cleavage of Notch. These data suggest direct interference between Notch and early TCR signal transduction events, independent of the classical Notch pathway via release of the Notch intracellular domain. A Notch-mediated alteration of TCR signaling strength may contribute to the recently described modulation of naïve T cell differentiation by Notch ligands.

We show here that T cell differentiation can be altered by exposing naive mouse CD4 T cells to altered T cell receptor signaling, achieved by transducing them with a fusion protein consisting of a modified Lck protein lacking the kinase domain and the HIV-Tat protein transduction domain. The Lck-HIV-Tat fusion protein is internalized into naive mouse T cells within 30 min after application to the medium. Activation of transduced cells in vitro resulted in strongly reduced intracellular calcium mobilization, alterations in cytokine profile, and sustained up-regulation of CD25. The cells had suppressive activity in vitro, but no Foxp3 expression. Our data indicate that signals encountered by a naive T cell during its initial activation can profoundly influence its subsequent functional behavior and elicit T cells, which can have regulatory activity.

Recent demonstrations that naive T cells proliferate after transfer to lymphopenic hosts have led to the theory that active homeostatic mechanisms fill the peripheral pool of naive T cells. To extend these data, we injected naive CD4(+) T cells from AND TCR transgenic mice (H-2(b/b) or H-2(k/k)) into CD3 epsilon-deficient mice, and studied the absolute number, phenotype and functional capacities of the transferred lymphocytes, from the first days to a few months after transfer. Proliferation of naive CD4(+) T cells did not fill the peripheral naive T cell pool. Injected naive T cells acquired a memory-like phenotype that was stable with time, despite the absence of foreign antigenic stimulation. Their functional capacities were modified, enhanced or abolished depending on the MHC haplotype. Thus, "homeostatic" proliferation of naive CD4(+) T cells in T cell-deprived recipients does not regenerate the naive CD4(+) T cell pool.

We have compared the efficiency of central nervous system and peripheral antigen-presenting cells (APC) in T cell priming and restimulation. OVA peptide 323 - 339-dependent activation of DO11.10 TCR-transgenic naive CD4+ and polarized Th1 or Th2 cells was assessed in the presence of microglia and astrocytes from the neonatal mouse brain as well as dendritic cells (DC) and B cells purified from adult mouse lymph nodes. DC were the most efficient in inducing naive T cell proliferation, IL-2 secretion and differentiation into Th1 cells, followed by IFN-gamma-preactivated microglia, large and small B cells. Astrocytes failed to activate naive T cells. IFN-gamma-pretreated microglia were as efficient as DC in the restimulation of Th1 cells, whereas IFN-gamma-pretreated astrocytes, large and small B cells were much less efficient. Conversely, Th2 cells were efficiently restimulated by all the APC types examined. During T cell priming, DC secreted more IL-12 than microglia but similar amounts of IL-12 were secreted by the two cell types upon interaction with Th1 cells. The hierarchy of APC established in this study indicates that DC and microglia are the most efficient in the stimulation of naive CD4(+) T cells and in the restimulation of Th1 cells, suggesting that activated microglia may effectively contribute to Th1 responses leading to central nervous system inflammation and tissue damage. These potentially pathogenic responses could be counteracted by the high efficiency of astrocytes as well as microglia in restimulating Th2 cells.

Whether memory T cells require persisting antigen for their survival has been a matter of debate. One prominent view that memory T cells do not require persisting antigen is based in part on studies in which T cell populations have been transferred into antigen-free mice. To generate "space" recipients were often irradiated; the functional properties of the transfused T cells were then evaluated after prolonged periods. In this report we show that transferring cytotoxic T lymphocytes (CTL) into irradiated or T and B cell-deficient hosts results in their proliferation and a change of their activation state. Moreover, naive T cell receptor-transgenic CTL specific for the lymphocytic choriomeningitis virus glycoprotein spontaneously developed cytotoxic effector function under such conditions. Therefore, some of the conclusions based on transfer of T cell populations into irradiated recipients to investigate T cell memory may have to be reevaluated.

In addition to their capacity to induce pain, vasodilatation and fever, prostaglandins E (PGE) exert anti-inflammatory activities by inhibiting the release of pro-inflammatory cytokines by macrophages and T cells, and by increasing interleukin (IL)-10 production by macrophages. We here report that PGE2, the major arachidonic acid metabolite released by antigen-presenting cells (APC), primes naive human T cells for enhanced production of anti-inflammatory cytokines and inhibition of pro-inflammatory cytokines. Unfractionated as well as CD45RO- CD31+ sort-purified neonatal CD4 T cells acquire the capacity to produce a large spectrum of cytokines after priming with anti-CD3 and anti-CD28 monoclonal antibodies (mAb), in the absence of both APC and exogenous cytokines. PGE2 primes naive T cells in a dose-dependent fashion for production of high levels of IL-4, IL-10 and IL-13, and very low levels of IL-2, interferon (IFN)-gamma, tumor necrosis factor (TNF)-alpha, and TNF-beta. PGE2 does not significantly increase IL-4 production in priming cultures, whereas it suppresses IL-2 and IFN-gamma. Addition of a neutralizing mAb to IL-4 receptor in primary cultures, supplemented or not with PGE2, prevents the development of IL-4-producing cells but does not abolish the effects of PGE2 on IL-10 and IL-13 as well as T helper (Th)1-associated cytokines. Addition of exogenous IL-2 in primary cultures does not alter the effects of PGE2 on naive T cell maturation. Thus PGE2 does not act by increasing IL-4 production in priming cultures, and its effects are partly IL-4 independent and largely IL-2 independent. Together with the recent demonstration that PGE2 suppresses IL-12 production, our results strongly suggest that this endogenously produced molecule may play a significant role in Th subset development and that its stable analogs may be considered for the treatment of Th1-mediated inflammatory diseases.

Nonobese diabetic (NOD) mice develop spontaneous insulin-dependent diabetes mellitus (IDDM), and the pancreas-infiltrating T cells invariably show a Th1 phenotype. We demonstrated here that the interleukin (IL)-12 antagonist (p40)2 can deviate the default Th1 development of naive T cell receptor (TCR)-transgenic CD4+ cells to the Th2 pathway in vitro. Although (p40)2 does not modify the cytokine profile of polarized Th1 cells, it prevents further recruitment of CD4- cells into the Th1 subset. To study the involvement of Th1 and Th2 cells in the initiation and progression of IDDM, we targeted endogenous IL-12 by administration of (p40)2 in NOD mice. (p40)2 administration to NOD mice inhibits interferon-gamma but not IL-10 production in response to lipopolysaccharide (LPS) or to the putative autoantigen IA-2. Serum immunoglobulin isotypes determined after (p40)2 treatment indicate an increase in Th2 and a decrease in Th1 helper activity. Administration of (p40)2 from 3 weeks of age onwards, before the onset of insulitis, results in the deviation of pancreas-infiltrating CD4+ but not CD8+ cells to the Th2 phenotype as well as in the reduction of spontaneous and cyclophosphamide-accelerated IDDM. After treating NOD mice with (p40)2 from 9 weeks of age, when insulitis is well established, few Th2 and a reduced percentage of Th1 cells are found in the pancreas. This is associated with a slightly decreased incidence of spontaneous IDDM, but no protection from cyclophosphamide-accelerated IDDM. In conclusion, deviation of pancreas-infiltrating CD4+ cells to Th2 is associated with protection from IDDM. However, targeting IL-12 after the onset of insulitis, when the pancreas contains polarized Th1 cells, is not sufficient to induce an effective immune deviation able to significantly modify the course of disease.

Inoculation with replicating virus leads to an increase in T cell numbers within lymph nodes that drain the site of infection. This increase has been associated with a nonspecific proliferation of bystander cells, with only a minority thought to be directed to the infectious agent. Such an assumption is largely based on precursor cytotoxic T lymphocyte (CTL) estimations using limiting dilution analysis. Recently, studies using more advanced molecular approaches have suggested that such functionally derived precursor frequencies considerably underestimate the proportion of T cells specific for the antigen under investigation. We have defined T cell receptor sequences characteristic of CTL populations directed to a dominant determinant of the herpes simplex virus (HSV) glycoprotein B (gB). In this investigation, we used this receptor signature as a probe to directly monitor changes occurring within lymph nodes draining the sites of active infection with HSV. We found that although lymph node CD8+ T cell numbers increase as a consequence of HSV infection, the majority of these cells are small resting cells that are not enriched for gB-specific receptors. In contrast, a significant proportion of activated T cells are highly enriched for CTL bearing gB-specific receptors. Our results are therefore consistent with a nonspecific migration of CTL precursors into the lymph nodes draining the site of infection, followed by the activation and proliferation of the antigen-specific subset that normally makes up a small proportion of the naive T cell repertoire.

Expression of activation markers and proliferative status were measured in peripheral CD4+ and CD8+ T cells of various T cell receptor (TCR)-transgenic mice either before or after intentional antigenic stimulation. In the absence of intentional immunization, CD4+ T cells persisted as resting or partially activated and cycling cells depending on the specificity of their TCR. Similar results were obtained following transfer into T cell-deficient recipients, i.e. T cells that were not cycling in situ did not cycle after transfer, whereas cells that were proliferating in situ also cycled after transfer. Thus, the TCR of some cells in the absence of intentional antigenic stimulation may bind to some unidentified ligand that does not induce tolerance, but rather slow expansion. In a different sort of experiment, activated T cells that were derived from noncycling naive T cells by deliberate antigenic stimulation continued to cycle slowly even a long time after transfer into antigen-free recipients that did not induce proliferation of the naive cells. Thus, lymphokines or ligands that do not induce activation of naive T cells may be responsible for the maintenance of memory cells. Our experiments show that the latter does not depend on a second TCR expressed by the memory cells, since memory T cells from RAG-2(-/-) TCR-transgenic mice persisted to a similar extent.

We produced transgenic mice expressing T cell receptor-alpha beta chain genes, derived from the chicken ovalbumin (OVA)-specific I-Ad-restricted CD4+CD8- T helper cell clone 7-3-7. In transgenic mice with H-2d genetic background (Tg-d mice), delayed-type hypersensitivity (DTH) was induced in the hind footpad by one inoculation with OVA without any previous sensitization, suggesting that naive T cells have the potential to be involved in DTH response. Spleen cells from nonimmunized Tg-d mice showed a strong T cell proliferative response to in vitro stimulation with OVA. Furthermore, these spleen cells produce cytokines including interleukin(IL)-2, IL-3, interferon-gamma, granulocyte/macrophage colony-stimulating factor, macrophage inflammatory protein (MIP)-1 alpha and MIP-1 beta, which may play an important role in the attraction of mononuclear cells to an antigen-challenging site.

In the immune system the first activation of a naive T cell by antigen is a key step in the shaping of the peripheral T cell specificity repertoire and maintenance of self-tolerance. In the present study, analysis of the interleukin-2 (IL-2) gene activation shows that naive human helper T cells (cord blood CD4+ T cells, adult CD4+CD45RO- T cells) regulate IL-2 transcription by a mechanism involving both a silencer and an activator acting on the purine-rich IL-2 promoter elements (NF-AT binding sites). By contrast, memory cells, either in vitro activated helper T cells reverting to a resting state, or CD4+ T (memory) clones, or CD4+CD45RO+ T cells isolated ex vivo, no longer have a silencer. Their IL-2 transcription seems to be controlled solely by the transition from inactive to active functional state of a positive transcription factor binding to these promoter elements as well as its cytoplasmic or nuclear location: in resting memory T cells the activator is located in the cytoplasm and is inactive, whereas in stimulated cells it is functional in promoting transcription and now resides in the nucleus. Thus, the regulation of the gene coding for the main T cell growth factor changes irreversibly after the first encounter of T cells with antigen. It is most likely that the presence of a silencer contributes to the more stringent activation requirements of naive CD4+ T cells.

A proportion of T cells recirculate in a tissue-selective manner. Recent studies which showed that the skin-tropic subset of T cells was of memory/activated type, led us to examine whether the preferential homing of T cells to the gut also involved memory T cells, and if so whether these memory T cells were phenotypically distinct from other memory T cells. Lymphocytes migrating through the gut and the skin of sheep was collected by cannulating the lymphatic ducts draining these tissues. Both naive and memory T cells were found to recirculate through the gut, although only memory T cells migrated through the skin. However, when T cells from the gut were labeled with fluorescein isothiocyanate and assessed for their migration back to the gut, it was the memory population which showed a tropism for the gut. Gut-tropic memory T cells migrated poorly through the skin, indicating that these cells were distinct from skin-tropic memory T cells. This was confirmed by phenotypic analysis. Gut memory T cells expressed very low levels of the alpha 6 and beta 1 integrins, in contrast to skin memory T cells which expressed high levels. There was no evidence for heterogeneity within the naive T cell population, which migrated preferentially to lymph nodes. This migration pattern could be explained in part by the high expression of the L-selectin (lymph node homing receptor, LAM-1) on naive T cells, in contrast to memory T cells from gut or skin which were mostly L-selectin negative. These results in sheep indicate that subsets of alpha/beta memory T cells show tissue-selective migration patterns, which probably develop in a particular environment following encounter with antigen.

A limiting dilution assay capable of detecting interleukin 4 (IL 4) production by a single cell has been developed. This assay is based on the stimulation of T cells, in the presence of IL 2 (5 U/ml), with anti-CD3 antibody bound to the surface of Terasaki wells. Cells of the IL 4-selective indicator line, CT.4S, are added 24–36 h later and IL 4 production is determined based on their survival 24–48 h thereafter. A frequency of 0.98 was obtained for IL 4 production by T cells of the D10.G4 cell line. T cells from naive donors capable of producing IL 4 in response to anti-CD3 plus IL 2 were quite rare, with a frequency in four experiments ranging between 0.0003 and 0.0018. Treatment of mice with polyclonal activators known to increase the IL 4-producing capacity of T cells when assayed in bulk culture caused striking increases in the frequency of IL 4-producing cells. Similarly, culturing cells in vitro with anti-CD3, IL 2 and IL 4 for 5 days caused a marked increase in the frequency of cells capable of producing IL 4, to 0.031.IL 4 production by individual T cells is dependent upon IL 2. Thus, in naive T cell populations, the frequency of IL 4-producing cells in response to stimulation with anti-CD3 in the absence of IL 2 was below the limit of detection. T cells from primed donors showed a striking inhibition in the frequency of IL 4-producing cells in response to anti-CD3 when IL 2 was not present. The availability of a simple assay to measure the frequency of cells capable of producing IL 4 should have substantial utility in allowing the evaluation of conditions that regulate IL 4 production in vivo and in vitro.