Differential survival of naive CD4 and CD8 T cells

In this paper we compare survival characteristics of transgenic and polyclonal CD4 and CD8 T cells. Transgenic CD4 T cells have an intrinsically lower capacity for survival, reflected in their gradual disappearance in thymectomized hosts, their increased sensitivity to apoptosis in vitro, and fewer divisions during homeostatic proliferation upon transfer into syngeneic lymphopenic hosts compared with CD8 T cells. Homeostatic proliferation, however, does not generally result in phenotypic conversion of activation markers unless cognate or cross-reactive Ag is present. T cells from the A18 TCR transgenic strain normally selected into the CD4 lineage are fragile as CD4 T cells, yet display the typical robust survival pattern of CD8 T cells when diverted into the CD8 lineage in a CD4-deficient host. Polyclonal CD4 and CD8 T cells also show distinctive patterns of survival, emphasizing that survival signals are relayed differently in the two lymphocyte subpopulations. However, expression levels of Bcl-2 in either transgenic or polyclonal naive CD4 and CD8 T cells are similar, excluding a role for this molecule as a key factor in differential survival of CD4 vs CD8 T cells.     

IL-7 enhances the survival and maintains the size of naive T cells.

T cells require continual presence of extrinsic signals from their in vivo microenvironment to maintain viability. T cells removed from these signals and placed in tissue culture atrophied and died in a caspase-independent manner. Atrophy was characterized by smaller cell sizes, delayed mitogenic responses, and decreased glycolytic rate. Bcl-2 expression remained constant in vitro despite ongoing cell death, indicating that endogenous Bcl-2 expression is insufficient to explain the life span and size control of lymphocytes in vivo and that cell-extrinsic signals provided may be required to maintain both cell viability and size in vivo. One such signal, IL-7, was found to maintain both the size and survival of neglected T cells in vitro. IL-7 was not unique, because the common gamma-chain cytokines IL-2, IL-4, and IL-15, as well as the gp130 cytokine IL-6, also promoted both T cell survival and size maintenance. IL-7 did not induce resting T cells to proliferate. Instead, IL-7 stimulated neglected T cells to maintain their metabolic rate at levels comparable to freshly isolated cells. The survival and trophic effects of IL-7 could be separated because IL-7 was able to promote up-regulation of Bcl-2 and maintain cell viability independent of phosphatidylinositol 3-kinase and mammalian target of rapamycin activity but was unable to prevent cellular atrophy when phosphatidylinositol 3-kinase and mammalian target of rapamycin were inhibited. These data demonstrate that T cells require the continuous presence of extrinsic signals not only to survive but also to maintain their size, metabolic activity, and the ability to respond rapidly to mitogenic signals.     

TCR signals mediated by Src family kinases are essential for the survival of naive T cells

The role of TCR signals triggered by recognition of self MHCs in maintaining the survival of naive peripheral T cells remains controversial. Here we examine the role of the Src family kinases, p56(lck) (Lck) and p59(fyn) (Fyn), in the survival of naive T cells. We show that long term survival requires a combination of signals transduced by Src family kinases and signals through the IL-7R. In the absence of either one, naive T cells die slowly, but if both signals are removed, cell loss is greatly accelerated. The TCR signal can be mediated by either Fyn or Lck at wild-type levels of expression, but not by Lck alone if expressed suboptimally. The disappearance of T cells in the absence of Fyn and Lck was associated with a complete loss of TCRzeta-chain phosphorylation and down-regulation of CD5, both of which are also MHC contact dependent, indicating that the Src family kinases are critical for transducing a TCR-MHC survival signal.     

IL-15 promotes the survival of naive and memory phenotype CD8+ T cells

IL-15 stimulates the proliferation of memory phenotype CD44(high)CD8(+) T cells and is thought to play a key role in regulating the turnover of these cells in vivo. We have investigated whether IL-15 also has the capacity to affect the life span of naive phenotype (CD44(low)) CD8(+) T cells. We report that IL-15 promotes the survival of both CD44(low) and CD44(high) CD8(+) T cells, doing so at much lower concentrations than required to induce proliferation of CD44(high) cells. Rescue from apoptosis was associated with the up-regulation of Bcl-2 in both cell types, whereas elevated expression of Bcl-x(L) was observed among CD44(high) but not CD44(low) CD8(+) cells. An investigation into the role of IL-15R subunits in mediating the effects of IL-15 revealed distinct contributions of the alpha- and beta- and gamma-chains. Most strikingly, IL-15R alpha was not essential for either induction of proliferation or promotion of survival by IL-15, but did greatly enhance the sensitivity of cells to low concentrations of IL-15. By contrast, the beta- and gamma-chains of the IL-15R were absolutely required for the proliferative and pro-survival effects of IL-15, although it was not necessary for CD44(high)CD8(+) cells to express higher levels of IL-15R beta than CD44(low) cells to proliferate in response to IL-15. These results show that IL-15 has multiple effects on CD8 T cells and possesses the potential to regulate the life span of naive as well as memory CD8(+) T cells.     

A role for p120 RasGAP in thymocyte positive selection and survival of naive T cells

Activation of the Ras small GTP-binding protein is necessary for normal T cell development and function. However, it is unknown which Ras GTPase-activating proteins (RasGAPs) inactivate Ras in T cells. We used a T cell-specific RASA1-deficient mouse model to investigate the role of the p120 RasGAP (RASA1) in T cells. Death of CD4(+)CD8(+) double-positive thymocytes was increased in RASA1-deficient mice. Despite this finding, on an MHC class II-restricted TCR transgenic background, evidence was obtained for increased positive selection of thymocytes associated with augmented activation of the Ras-MAPK pathway. In the periphery, RASA1 was found to be dispensable as a regulator of Ras-MAPK activation and T cell functional responses induced by full agonist peptides. However, numbers of naive T cells were substantially reduced in RASA1-deficient mice. Loss of naive T cells in the absence of RASA1 could be attributed in part to impaired responsiveness to the IL-7 prosurvival cytokine. These findings reveal an important role for RASA1 as a regulator of double-positive survival and positive selection in the thymus as well as naive T cell survival in the periphery.     

The rapid induction of HLA-E is essential for the survival of antigen-activated naive CD4 T cells from attack by NK cells

Increasing evidence shows that NK cells regulate adaptive immunity, but the underlying mechanisms are not well understood. In this study, we show that activated human NK cells suppress autologous naive CD4 T cell proliferation in response to allogeneic dendritic cells (DCs) by selectively killing Ag-activated T cells. Naive CD4 T cells, which were initially resistant to NK cell-mediated cytotoxicity, became substantially susceptible to NK cells within a day after priming with DCs. Ag-activated T cells showed various degrees of susceptibility to NK cells. After 1 d of priming with LPS-matured DCs, T cells were less susceptible to NK cells than were T cells primed with TNF-α-matured DCs. Subsequently at day 3, Ag-activated T cells regained resistance to NK cells. The level of HLA-E expression on Ag-activated T cells was closely correlated with resistance to NK cells. HLA-E was highly expressed at day 1 by T cells primed with LPS-matured DCs but not by T cells primed with TNF-α-matured DCs. An Ab blockade revealed a critical role for the HLA-E-NKG2A interaction in the protection of Ag-activated T cells from NK cells. Collectively, this study demonstrates that NK cells impact adaptive immunity through the finely controlled kinetics of HLA-E expression on T cells. Thus, HLA-E may be a new target for immunoregulation.     

The tuberous sclerosis complex-mammalian target of rapamycin pathway maintains the quiescence and survival of naive T cells

Naive T cells receive stimulation from the positive selecting ligand in the periphery for their survival. This stimulation does not normally lead to overt activation of T cells, as the T cells remain largely quiescent until they receive either antigenic or lymphopenic stimuli. The underlying mechanism responsible for survival and quiescence of the naive T cells remains largely unknown. In this study, we report that T cell-specific deletion of Tsc1, a negative regulator of mammalian target of rapamycin, resulted in both spontaneous losses of quiescence and cellularity, especially within the CD8 subset. The Tsc1-deficient T cells have increased cell proliferation and apoptosis. Tsc1 deletion affects the survival and quiescence of T cells in the absence of antigenic stimulation. Loss of quiescence but not cellularity was inhibited by rapamycin. Our data demonstrate that tuberous sclerosis complex-mammalian target of rapamycin maintains quiescence and survival of T cells.     

Maturation-dependent licensing of naive T cells for rapid TNF production

The peripheral naïve T cell pool is comprised of a heterogeneous population of cells at various stages of development, which is a process that begins in the thymus and is completed after a post-thymic maturation phase in the periphery. One hallmark of naïve T cells in secondary lymphoid organs is their unique ability to produce TNF rapidly after activation and prior to acquiring other effector functions. To determine how maturation influences the licensing of naïve T cells to produce TNF, we compared cytokine profiles of CD4⁺ and CD8⁺ single positive (SP) thymocytes, recent thymic emigrants (RTEs) and mature-naïve (MN) T cells during TCR activation. SP thymocytes exhibited a poor ability to produce TNF when compared to splenic T cells despite expressing similar TCR levels and possessing comparable activation kinetics (upregulation of CD25 and CD69). Provision of optimal antigen presenting cells from the spleen did not fully enable SP thymocytes to produce TNF, suggesting an intrinsic defect in their ability to produce TNF efficiently. Using a thymocyte adoptive transfer model, we demonstrate that the ability of T cells to produce TNF increases progressively with time in the periphery as a function of their maturation state. RTEs that were identified in NG-BAC transgenic mice by the expression of GFP showed a significantly enhanced ability to express TNF relative to SP thymocytes but not to the extent of fully MN T cells. Together, these findings suggest that TNF expression by naïve T cells is regulated via a gradual licensing process that requires functional maturation in peripheral lymphoid organs.     

Memory T cells enhance the expression of high-avidity naive B cells

The secondary immune response classically differs from the primary response in magnitude, avidity, and isotype of the antibodies produced. Cell transfer studies to assess the contribution of memory B and memory T cells to each of these parameters are described. Avidities of the anti-DNP plaque-forming cells (PFC) generated in lethally irradiated recipients of naive B cells and keyhole limpet hemocyanin (KLH)-primed T cells, followed by immunization with soluble DNP-KLH, are medium to high, and do not differ significantly from the avidities of anti-DNP PFC in recipients of DNP-primed B cells and KLH-primed T cells. However, the number of indirect (I)-PFC and the ratio of I-PFC to direct (D)-PFC are significantly greater in the recipients of primed B and primed T cells. The results suggest that carrier primed T cells can selectively activate virgin B cells which are committed to produce medium- and high-avidity antibodies, and/or enhance the generation of somatic mutation which leads to antibodies of higher avidity. Priming of B cells is necessary for the increased magnitude of the I-PFC.     

Adult human liver contains CD8pos T cells with naive phenotype, but is not a site for conventional alpha beta T cell development

Normal adult human liver (AHL) contains populations of unconventional lymphocytes that have been shown in the mouse to mature locally. The presence of lymphoid progenitors together with IL-7, recombinase-activating gene, and pre-TCR-alpha expression in AHL suggests similar local T cell development activity in humans. Flow cytometry was used to characterize potentially naive hepatic alphabeta-T cells. We looked for evidence of TCR-alphabeta cell development in AHL by quantifying delta deletion TCR excision circles (TRECs) in CD3(pos) populations isolated from the liver and matched blood of eight individuals. Phenotypic analysis of hepatic T cells suggests the presence of Ag-inexperienced populations. TRECs were detected in all blood samples (mean, 164.10 TRECs/ micro g DNA), whereas only two hepatic samples were positive at low levels (59.40 and 1.92). The relatively high level of CD8(pos) T cells in these livers with a naive phenotype suggests that in addition to its role as a graveyard for Ag-specific activated CD8(pos) T cells, naive CD8(pos) T cells may enter the liver without prior activation. The almost complete absence of TRECs suggests that normal AHL is not a site for the development of conventional alphabeta T cells.     

Coronin 1-mediated naive T cell survival is essential for the development of autoimmune encephalomyelitis

Autoimmune encephalomyelitis is a disease of the CNS that can develop when an initial peripheral inflammatory stimulus is followed by infiltration and reactivation of T lymphocytes in the CNS. We report a crucial role for coronin 1, which is essential for maintenance of the naive T cell pool, for the development of murine experimental autoimmune encephalomyelitis (EAE), a model for multiple sclerosis. In the absence of coronin 1, immunization with myelin oligoglycoprotein (MOG(35-55)) peptide largely failed to induce EAE symptoms, despite normal mobilization of leukocyte subsets in the blood, as well as effector cytokine expression comparable with wild-type T cells on polyclonal stimulation. Susceptibility of coronin 1-deficient mice to EAE induction was restored by transfer of wild-type CD4(+) T cells, suggesting that the observed resistance of coronin 1-deficient mice to EAE development is T cell intrinsic. Importantly, although coronin 1-deficient regulatory T cells (Tregs) showed a suppressor activity comparable with wild-type Tregs, Treg depletion failed to restore EAE development in coronin 1-deficient animals. These results suggest a hitherto unrecognized role of naive T cells in the development of autoimmune encephalomyelitis and reveal coronin 1 as a crucial modulator of EAE induction.     

Requirement for protein synthesis for survival of unmodified bacteriophage T1 in a restricting host.

At high multiplication of infection, a substantial fraction of restricting cells (P1 lysogens) could be productively infected by unmodified coliphage T1 (T1.0) provided that protein synthesis was uninhibited during the first 5 min of infection. Successful infection under restricting conditions was accompanied by more genetic recombination than was seen under nonrestricting host, the recombination frequency declined for markers on T1.0 genomes; no effect was seen on recombination between markers on modified (T1.P) genomes. This suggested that recombination between unmodified genomes may be essential for their survival under conditions of host restriction. In a restricting host, genetic markers on T1.0 could recombine with T1.P even when the rescuing phage was added 6 min after T1.0 infection. However, even marker rescue recombination was diminished when protein synthesis was inhibited during early infection. Since DNA restriction is an early event, protein synthesis may be required soon after infection of a restricting host by T1.0 in order to preserve restriction-damaged DNA in a form that can participate in recombination. Experiments are also described that rule out some possibilities for the role of such a protein(s).     

Antigen-experienced T cells limit the priming of naive T cells during infection with Leishmania major

One mechanism to control immune responses following infection is to rapidly down-regulate Ag presentation, which has been observed in acute viral and bacterial infections. In this study, we describe experiments designed to address whether Ag presentation is decreased after an initial response to Leishmania major. Naive alphabeta-Leishmania-specific (ABLE) TCR transgenic T cells were adoptively transferred into mice at various times after L. major infection to determine the duration of presentation of parasite-derived Ags. ABLE T cells responded vigorously at the initiation of infection, but the ability to prime these cells quickly diminished, independent of IL-10, regulatory T cells, or Ag load. However, Ag-experienced clonal and polyclonal T cell populations could respond, indicating that the diminution in naive ABLE cell responses was not due to lack of Ag presentation. Because naive T cell priming could be restored by removal of the endogenous T cell population, or adoptive transfer of Ag-pulsed dendritic cells, it appears that T cells that have previously encountered Ag during infection compete with naive Ag-specific T cells. These results suggest that during L. major infection Ag-experienced T cells, rather than naive T cells, may be primarily responsible for sustaining the immune response.     

A comparative study of the calcium system in memory T cells and naive T cells

The comparative analysis of responses of memory and naive T lymphocytes to Ca2+-mobilizing agents, namely Con A, thimerosal, thapsigargin and ionomycin, was carried out. The effect of these agents on both types of T cells differed qualitatively and quantitatively. The lack of intracellular Ca2+ stores in memory T cells was shown. Ca2+-mobilizing agents did not induce influx of Ca2+ in memory T cells from outside and this was the reason for their stability to Ca2+ ionophores. It was also shown that memory T cells were resistant to the 'Ca2+ paradox'.     

Differential expression of a 70 kDa O-glycoprotein on T cells: a possible marker for naive and early activated murine T cells

We purified a 70 kDa O-glycoprotein that binds to the GalNAc specific lectin from Amaranthus leucocarpus (ALLr) and determined its expression pattern on T lymphocytes from different murine lymphoid organs. High level of ALLr expression was demonstrated in 95-98% of both CD4(+)8(+) and CD4(-)8(+) thymocytes, and in 80-95% of CD8(+) T cells from peripheral blood, lymph nodes, and spleen, whereas a minor fraction of CD4(+)8(-) thymocytes (46-67%) and peripheral CD4(+) T cells (9-40%) showed low ALLr expression. Peripheral CD19(+) B cells were ALLr negative and most of the peripheral ALL(+) T cells showed a CD62L(hi)CD45RB(hi)CD44(lo/-) phenotype, indicating features of naive cells. Mitogenic activation of peripheral T cells increased 3-fold the number of ALL(+)CD4(+) T cells 24 h after stimulation, as opposed to a >80% decrease in CD8(+) T cells 72 h after stimulation. Our results suggest that ALL detects a non-described surface O-glycoprotein selectively expressed by naive CD8(+) T cells and by early activated CD4(+) T cells.     

Cutting edge: naive T cells masquerading as memory cells

This study shows that naive CD8 T cells can acquire characteristics of memory T cells in the absence of stimulation with specific Ag simply by the process of homeostatic proliferation under lymphopenic conditions. This Ag-independent T cell differentiation pathway did not result in up-regulation of early activation markers (CD69, CD25, CD71), but expression of several memory markers (CD44, CD122, Ly6C) increased progressively with successive divisions. These markers were then stably expressed, and these cells also became more responsive functionally to specific Ag. Thus, all "memory" phenotype T cells in an individual may not be true Ag-experienced cells and may include naive cells masquerading as memory cells. These findings are specially relevant in cases of disease or treatment-induced lymphopenia such as in HIV-infected individuals or transplant recipients. In addition, this study may have implications for autoimmunity because homeostatic proliferation of naive T cells requires interaction with self peptide plus MHC molecules.     

Conversion of naive T cells to a memory-like phenotype in lymphopenic hosts is not related to a homeostatic mechanism that fills the peripheral naive T cell pool

To examine directly whether a limited number of naive T cells transferred to lymphopenic hosts can truly fill the peripheral naive T cell pool, we compared the expansion and phenotype of naive T cells transferred to three different hosts, namely recombination-activating gene-deficient mice, CD3epsilon-deficient mice, and irradiated normal mice. In all three recipients, the absolute number of recovered cells was much smaller than in normal mice. In addition, transferred naive T cells acquired a memory-like phenotype that remained stable with time. Finally, injected cells were rapidly replaced by host thymic migrants in irradiated normal mice. Only continuous output of naive T cells by the thymus can generate a full compartment of truly naive T cells. Thus, conversion of naive T cells to a memory-like phenotype in lymphopenic hosts is not related to a homeostatic mechanism that fills the peripheral naive T cell pool.     

On the self-referential nature of naive MHC class II-restricted T cells

The use of mutant mice expressing a normal MHC class II molecule surface level but a severely restricted self-peptide diversity (H-2Malpha(-/-)) previously revealed that T cells carrying the Ealpha(52-68)-I-A(b) complex-specific 1H3.1 TCR rely on self-peptide(s) recognition for both their peripheral persistence in irradiated hosts and their intrathymic positive selection. Here, we identify Ealpha(52-68) structurally related self-peptide(s) as a major contributor to in vivo positive selection of 1H3.1 TCR-transgenic thymocytes in I-A(b+)/I-Ealpha(-) mice. This is demonstrated by the drastic and specific reduction of the TCR high thymocyte population in 1H3.1 TCR-transgenic (Tg) mice treated with the Ealpha(52-68)-I-A(b) complex-specific Y-Ae mAb. Self-peptide(s) recognition is also driving the maturation of T cells carrying a distinct MHC class II-restricted specificity (the Ealpha(6) alphass TCR), since positive selection was also deficient in Ealpha(6) TCR Tg H-2Malpha(-/-) thymi. Such a requirement for recognition of self-determinants was mirrored in the periphery; Ealpha(6) TCR Tg naive T cells showed an impaired persistence in both H-2Malpha(-/-) and I-A(b)ss(-/-) irradiated hosts, whereas they persisted and slowly cycled in wild-type recipients. This moderate self-peptide(s)-dependent proliferation was associated with a surface phenotype intermediate between those of naive and activated/memory T cells; CD44 expression was up-regulated, but surface expression of other markers such as CD62L remained unaltered. Collectively, these observations indicate that maturation and maintenance of naive MHC class II-restricted T cells are self-oriented processes.