Molecular and cellular mechanisms of T cell development.

The thymus is central to the establishment of a functioning immune system. Here is the place where T cells mature from hematopoietic progenitors, driven by mutual interactions of stromal cells and the developing thymocytes. As a result, different types of T cells are generated, all of which have been carefully selected for the ability to act in host defense towards non-self and against the potential to mount pathogenic self-reactive autoimmune responses. In this review we summarize our present knowledge on the lineage decisions taking place during this development, the selection processes responsible for shaping the T cell antigen-receptor repertoire, the interactions with the stromal components and the signal transduction pathways which transform the interactions with the thymic microenvironment into cellular responses of survival, proliferation, differentiation and, importantly, also of cell death.

Disruption of Akt kinase activation is important for immunosuppression induced by measles virus.

Surface-contact-mediated signaling induced by the measles virus (MV) fusion and hemagglutinin glycoproteins is necessary and sufficient to induce T-cell unresponsiveness in vitro and in vivo. To define the intracellular pathways involved, we analyzed interleukin (IL)-2R signaling in primary human T cells and in Kit-225 cells. Unlike IL-2-dependent activation of JAK/STAT pathways, activation of Akt kinase was impaired after MV contact both in vitro and in vivo. MV interference with Akt activation was important for immunosuppression, as expression of a catalytically active Akt prevented negative signaling by the MV glycoproteins. Thus, we show here that MV exploits a novel strategy to interfere with T-cell activation during immunosuppression.

MEK activity regulates negative selection of immature CD4+CD8+ thymocytes.

CD4+CD8+ thymocytes are either positively selected and subsequently mature to CD4 single positive (SP) or CD8 SP T cells, or they die by apoptosis due to neglect or negative selection. This clonal selection is essential for establishing a functional self-restricted T cell repertoire. Intracellular signals through the three known mitogen-activated protein (MAP) kinase pathways have been shown to selectively guide positive or negative selection. Whereas the c-Jun N-terminal kinase and p38 MAP kinase regulate negative selection of thymocytes, the extracellular signal-regulated kinase (ERK) pathway is required for positive selection and T cell lineage commitment. In this paper, we show that the MAP/ERK kinase (MEK)-ERK pathway is also involved in negative selection. Thymocytes from newborn TCR transgenic mice were cultured with TCR/CD3epsilon-specific Abs or TCR-specific agonist peptides to induce negative selection. In the presence of the MEK-specific pharmacological inhibitors PD98059 or UO126, cell recovery was enhanced and deletion of DP thymocytes was drastically reduced. Furthermore, development of CD4 SP T cells was blocked, but differentiation of mature CD8 SP T cells proceeded in the presence of agonist peptides when MEK activity was blocked. Thus, our data indicate that the outcome between positively and negatively selecting signals is critically dependent on MEK activity.

Activation of the extracellular signal-related kinase/mitogen-activated protein kinase pathway discriminates CD4 versus CD8 lineage commitment in the thymus.

We have investigated the role of the mitogen-activated protein kinase (MAPK) pathway in the differentiation of CD4+ and CD8+ T cells by looking specifically at the effects of inhibitors of MAPK-activating enzyme, MAPK/extracellular signal-related kinase (ERK) kinase (MEK), during the positive selection step from double-positive to single-positive (SP) thymocytes. Using a variety of transgenic/knockout mouse strain combinations that fail to differentiate individual lineages of SP thymocytes together with genetically engineered F(ab')2 reagents that induce maturation preferentially to either the CD4 or CD8 subpopulations, we show that induction of CD4 differentiation cells is highly sensitive to levels of MEK inhibition that have no effect on CD8 maturation. In addition, the presence of MEK inhibitor is able to modify signals that normally induce CD4 differentiation to instead promote CD8 differentiation. Finally, we show that continuous culture in the presence of inhibitor interferes with TCR up-regulation in SP thymocytes, suggesting that MAPK signaling may be involved in final maturation steps for both lineages. These data indicate that there is discrimination in the biochemical pathways that are necessary to specify CD4 and CD8 lineage commitment and can reconcile previously conflicting reports on the influence of MAPK activation in commitment and maturation of thymocytes.

Molecular requirements for lineage commitment in the thymus--antibody-mediated receptor engagements reveal a central role for lck in lineage decisions.

Recent experiments in our laboratory have focused on the receptor engagements required for the differentiation of fully mature, single positive thymocytes from their double positive precursors. We have used a novel approach which involves the ligation of surface receptors on immature thymocytes with genetically engineered F(ab1)2 reagents, which, unlike conventional antibodies, do not aggregate the CD3 complex to such an extent as to induce extensive deletion of these cells. The experimental data presented in this review indicate that differentiation of the two mature CD4 and CD8 lineages occurs in response to distinct intracellular signals induced by particular receptor engagements. The data suggest that the tyrosine kinase p56lck (lck) plays a crucial role in determining lineage choice, in that maturation of thymocytes into the CD4 lineage occurs upon recruitment of active lck to the T-cell receptor (TCR)/CD3 complex, whereas CD8 maturation can be induced by CD3 ligation in the absence of co-receptor-mediated lck recruitment. A central role for lck activity in determining the threshold for differentiation of the CD4 lineage is revealed in experiments with thymi deficient for a regulator of lck activity, CD45. A model of thymocyte differentiation is presented in which we propose that the relative balance of signals delivered by TCR engagement and lck activation determines lineage choice.

Retarded thymic involution and massive germinal center formation in NF-ATp-deficient mice.

NF-ATp and NF-ATc are the most prominent nuclear NF-AT transcription factors in peripheral T lymphocytes. After T cell activation both factors bind to and control the promoters and enhancers of numerous lymphokine and receptor ligand genes. In order to define a specific role for NF-ATp in vivo we have inactivated the NF-ATp gene by gene targeting in mice. We show that NF-ATp deficiency leads to the accumulation of peripheral T cells with a "preactivated" phenotype, enhanced immune responses of T cells after secondary stimulation in vitro and severe defects in the proper termination of antigen responses, as shown by a reduced deletion of superantigen-reactive CD4+ T cells. These alterations in the function of the immune system are correlated with drastic changes in the morphology of lymphoid organs. Approximately 25 % of NF-ATp-deficient mice older than 6 months develop large germinal centers in the spleen and peripheral lymph nodes. In addition, they exhibit a pronounced retardation in the involution of the thymus. The thymus of these NF-ATp-deficient mice exhibits large cortical areas typical for newborn mice and a massive infiltration of IgM+/ IgD+ B lymphocytes. Contrary to the T lymphocytes from IL-2-deficient mice which develop a phenotype similar to the NF-ATp-/- mice, NF-ATp-/- T cells do not show obvious defects in Fas-mediated apoptosis. This might indicate defects in other types of programmed cell death which are controlled by the activity of NF-ATp.

CD3 ligation on immature thymocytes generates antagonist-like signals appropriate for CD8 lineage commitment, independently of T cell receptor specificity.

The signals that direct differentiation of T cells to the CD4 or CD8 lineages in the thymus remain poorly understood. Although it has been relatively easy to direct differentiation of CD4 single positive (CD4+) cells using combinations of antibodies and pharmacological agents that mimic receptor engagements, equivalent stimuli do not induce efficient maturation of CD8+ cells. Here we report that, irrespective of the MHC-restriction specificity of the TCR, differentiation of mature CD8+ thymocytes can be induced by ligation of CD3 polypeptides on immature thymocytes with a F(ab')2 reagent (CD3fos-F(ab')2). The tyrosine phosphorylation patterns stimulated by CD3fos-F(ab')2 have been shown to resemble those delivered to mature T cells by antagonist peptides, which are known to direct positive selection of CD8+ cells, and we can show that this reagent exhibits potent antagonistic-like activity for primary T cell responses. Our results suggest a distinction in the signals that specify lineage commitment in the thymus. We present a model of thymocyte differentiation that proposes that the relative balance of signals delivered by TCR engagement and by p56lck activation is responsible for directing commitment to the CD8 or CD4 lineages.

How many thymocytes audition for selection?

T cell maturation requires the rearrangement of clonotypic T cell receptors (TCR) capable of interacting with major histocompatibility complex (MHC) ligands to initiate positive and negative selection. Only 3-5% of thymocytes mature to join the peripheral T cell pool. To investigate the basis for this low success rate, we have measured the frequency of preselection thymocytes capable of responding to MHC. As many as one in five MHC-naive thymocytes show upregulation of activation markers on exposure to MHC-expressing thymic stroma in short-term reaggregate culture. The majority of these cells display physiological changes consistent with entry into the selection process within 24 h. By exposing TCR transgenic thymocytes to a range of MHC-peptide complexes, we show that CD69 induction is indicative of thymocyte selection, positive or negative. Our data provide evidence that the fraction of thymocytes that qualify to enter the thymic selection process far exceeds the fraction that successfully complete it, and suggest that most MHC-reactive thymocytes are actively eliminated in the course of selection.

Signals through CD8 or CD4 can induce commitment to the CD4 lineage in the thymus.

Differentiation of thymocytes into mature single-positive T cells is an ordered process involving sequential interactions between T cell receptor (TCR), co-receptors (CD4 or CD8) and their appropriate major histocompatibility complex-encoded ligands. Precisely how these receptor/co-receptor engagements determine lineage commitment is still controversial, but recently it has been suggested that quantitative differences in the signal transmitted by co-ligation of CD4 versus CD8 with TCR might provide the discriminating signal. We examine this hypothesis, using bispecific F(ab')2 antibodies to mimic TCR/ co-receptor engagement during thymocyte differentiation. These bispecific antibodies lack Fc and can engage surface molecules without extensive cross-linking or targeting to Fc receptor-bearing cells. We show that TCR/CD3 co-ligation with CD4 induces efficient differentiation of mature CD4 lineage cells, irrespective of their TCR specificity. Interestingly, TCR/CD3 co-ligation with CD8 also induces maturation of CD4 T cells, although less efficiently, but not of CD8 T cells. Thus, although the signals delivered by co-ligation of TCR and CD8 appear weaker than from co-ligation of TCR and CD4, the outcome from either engagement is the same. These data suggest that differences in signal intensity alone do not determine lineage commitment in the thymus, but that distinct signals are required for CD4 and CD8 single-positive cell differentiation.

Developmentally regulated expression of P-glycoprotein (multidrug resistance) activity in mouse thymocytes.

P-glycoprotein (P-gly) is the transmembrane efflux pump responsible for multidrug resistance in tumor cells. The activity of P-gly in mature peripheral lymphocytes is lineage specific, with CD8+ T cells and natural killer (NK) cells expressing high levels as compared to CD4+ T cells and B cells. We have now investigated P-gly activity in immature and mature subsets of mouse thymocytes. Our data indicate that P-gly activity is undetectable in immature CD4-8- and CD4+8+ thymocyte subsets. Among mature thymocytes, P-gly activity is absent in the CD4+ subset but present in the more mature (HSAlow) fraction of CD8+ cells. Furthermore, while thymic CD4-8- T cell receptor (TCR) gamma delta cells have little P-gly activity, a minor subset of CD4-8- or CD4+ TCR alpha beta + thymocytes bearing the NK1.1 surface marker expresses high levels of P-gly activity. Collectively, our results indicate that P-gly activity arises late during thymus development and is expressed in a lineage-specific fashion.

Heterogeneity in P-glycoprotein (multidrug resistance) activity among murine peripheral T cells: correlation with surface phenotype and effector function.

P-glycoprotein (P-gly) is the transmembrane efflux pump responsible for multidrug resistance in tumor cells. Functional P-gly activity can be conveniently assessed microfluorometrically using the fluorescent dye rhodamine 123 (Rh123), which is an artificial substrate for the P-gly transporter. Here we assess P-gly activity in subsets of mouse peripheral T lymphocytes using the Rh123 efflux assay. Our data indicate that virtually all CD8+ cells extrude Rh123 efficiently, whereas only a subset of CD4+ cells exhibit P-gly activity. Correlation of P-gly activity in CD4+ cells with the expression of a panel of surface markers revealed that cells bearing an "activated/memory" phenotype (CD45RB-, CD44hi, CD62L-, CD25+, CD69+) were exclusively found in the fraction that can extrude Rh123. In contrast "naive" phenotype CD4+ cells (CD45RB+, CD44lo, CD62L+, CD25-, CD69-) could be further subdivided into two major subsets based on P-gly activity. In functional studies of sorted cell populations the Rh123-extruding subset of "naive" CD4+ cells proliferated more strongly and secreted higher levels of interleukin (IL)-2 than its Rh123-retaining counterpart when activated by a variety of polyclonal stimuli. Furthermore, this subset produced detectable levels of interferon (IFN)-gamma upon stimulation but no IL-4 or IL-10. As expected, the Rh123-retaining "naive" subset produced only IL-2 after stimulation, whereas the "memory" subset produced IFN-gamma, IL-4 and IL-10 in addition to low levels of IL-2. Collectively, our data indicate that P-gly activity is a novel parameter that can be used to distinguish a subset of "preactivated" CD4+ cells that would be considered as naive on the basis of their surface phenotype.

Quantitative and qualitative differences in IL-2 and IL-4 expression in primary and secondary T cell stimulation.

Lymphocytes were concanavalin A (Con A) primed and the signal was withdrawn 4-48 h post-stimulation by alpha-methyl-D-mannopyranoside (alpha MM) treatment. Upon restimulation IL-2 and IL-4 RNA expression was found to be greatly enhanced. Re-expression of lymphokine RNA was dependent on signals delivered by Con A, anti-CD3 antibodies or phorbolester plus ionomycin, and could not be achieved by either IL-2, phorbolester or ionomycin alone. Increased IL-2 re-expression was only possible when alpha MM was added early after primary stimulation, while the ability for enhanced IL-4 RNA re-expression persisted. IL-2 and IL-4 RNA re-expression was characterized by increases in steady state precursor RNA levels and thus, presumably, increased rates of transcription. However, the high accumulation of IL-2 RNA observed upon restimulation was also due to greatly increased RNA stability (greater than 4 h versus 30 min after primary stimulation). Thus, secondary expression of IL-4 RNA is persistent and mostly due to quantitative changes in transcription, whereas enhanced re-expression of IL-2 RNA also results from altered post-transcriptional regulation. This phenotype, however, is only short lived.

Regulation of lymphokine expression in T cell activation. I. Rapid loss of interleukin-specific RNA after removal of the stimulating signal.

Resting T lymphocytes can be induced to express a variety of lymphokines after antigenic or mitogenic stimulation. Expression is regulated both at the transcriptional level, through induction of T cell-specific DNA-binding proteins, and at the post-transcriptional level through alteration of precursors or mRNA stability. To investigate whether lymphokine expression follows a preprogrammed course after activation of T cells or whether it is dependent on the continued monitoring of activating signals, we followed the fate of interleukin (IL) 2, IL 4 and IL 2 receptor mRNA after removal of the stimulating signal concanavalin (Con A) by alpha-methylmannoside (alpha MM). IL 2 and IL 4 needed continued stimulation of the cells for RNA expression, as shown by the rapid disappearance of IL 2 and IL 4 mRNA after addition of alpha MM to stimulated cells, while IL 2 receptor mRNA which is also induced after Con A stimulation was minimally influenced. In the case of both IL no new mature mRNA was generated after removal of the stimulating signal. The T1/2 of IL 2 mRNA remained unchanged after alpha MM treatment as compared to actinomycin D treatment, while IL 4 mRNA became labilized after withdrawal of the signal.