T cell receptor-mediated selection of functional rat CD8 T cells from defined immature thymocyte precursors in short-term suspension culture

Recent results have indicated that positive and negative repertoire selection act on the major population of CD4,8 double-positive (DP) thymocytes that express 5-10-fold less T cell receptor (TCR) than mature T cells (i.e., they are TCRlow). Since DP cells obtained ex vivo are heterogeneous with regard to their stage within thymic selection, a homogeneous population of virgin DP cells suitable for selection studies was generated in vitro from their immediate precursors, the CD8 single-positive (SP) immature blast cells. To mimic TCR-mediated selection signals, these virgin DP cells were then cultured for another 2 d in the presence of immobilized anti-TCR monoclonal antibodies with or without interleukin 2 (IL-2). Daily monitoring of recovery and phenotype showed that without TCR stimulation, the cells remained DP and became small, TCRlow cells that were lost with a half-life of 1 d, regardless of the presence of IL-2. TCR stimulation resulted in rapid downregulation of CD4 and CD8, maintenance of a larger cell size, and induction of the CD53 antigen that marks mature and CD4,8 double-negative rat thymocytes. In the absence of IL-2, viability decreased as rapidly as without TCR stimulation. Addition of IL-2 rescued TCR-stimulated virgin DP cells and prevented CD8 downregulation, so that 50-80% of input DP cells were recovered after 2 d as CD4-8+53+ cells. After release from modulation, these in vitro generated CD8 SP cells quantitatively upregulated the TCR to the TCRhigh phenotype and were readily induced to proliferate and exhibit cytotoxic T lymphocyte (CTL) activity in a polyclonal readout. Evidence is presented implicating an IL-2 receptor (IL-2R) not containing the p55 chain (i.e., most likely the p70 intermediate affinity IL-2R) in the TCR plus IL-2-driven in vitro differentiation of virgin DP cells towards the mature CD8 SP phenotype.     

Involvement of the interleukin 2 pathway in the rearrangement and expression of both alpha/beta and gamma/delta T cell receptor genes in human T cell precursors

In this report, we have undertaken the phenotypic, functional and molecular characterization of a minor (less than 5%) subpopulation of adult thymocytes regarded as the earliest intrathymic T-cell precursors. Pro-T cells were immunoselected and shown to express different hematopoietic cell markers (CD45, CD38, CD7, CD5) and some activation-related molecules (4F2, Tr, HLA class II), but lack conventional T cell antigens (CD2-1-3-4-8-). TCR-gamma RNA messages are already expressed at this early ontogenic stage, while alpha and beta chain TCR genes remain in germline configuration. In vitro analyses of the growth requirements of pro-T cells demonstrated the involvement of the IL-2 pathway in promoting their proliferation and differentiation into CD3+ CD4+ or CD8+ mature thymocytes. Moreover, during the IL-2-mediated maturation process rearrangements and expression of both alpha and beta chain TCR genes occurred, and resulted in the acquisition of alpha/beta as well as gamma/delta (either disulphide-linked or non-disulphide-linked) heterodimeric TCR among the pro-T cell progeny.     

In vitro evidence that cytokine receptor signals are required for differentiation of double positive thymocytes into functionally mature CD8+ T cells

CD4(+)8(+) double positive (DP) thymocytes differentiate into CD4(+) and CD8(+) mature T cells in response to TCR signals. However, TCR signals that are initiated in DP thymocytes are unlikely to persist throughout all subsequent differentiation steps, suggesting that other signals must sustain thymocyte differentiation after TCR signaling has ceased. Using an in vitro experimental system, we now demonstrate that cytokine receptor signals, such as those transduced by IL-7 receptors, are required for differentiation of signaled DP thymocytes into functionally mature CD8(+) T cells as they: (a) up-regulate Bcl-2 expression to maintain thymocyte viability; (b) enhance CD4 gene silencing; (c) promote functional maturation;and (d) up-regulate surface expression of glucose transporter molecules, which improve nutrient uptake and increase metabolic activity. IL-7Rs appear to be unique among cytokine receptors in maintaining the viability of newly generated CD4(-)8(+) thymocytes, whereas several different cytokine receptors can provide the trophic/differentiative signals for subsequent CD8(+) thymocyte differentiation and maturation. Thus, cytokine receptors provide both survival and trophic/differentiative signals with varying degrees of redundancy that are required for differentiation of signaled DP thymocytes into functionally mature CD8(+) T cells.     

Regulation of thymocyte development through CD3. I. Timepoint of ligation of CD3 epsilon determines clonal deletion or induction of developmental program

Several recent observations suggest that successful rearrangement of the T cell receptor (TCR) beta locus induces several important events in thymocyte maturation. Allelic exclusion is achieved by interruption of further rearrangement of the beta locus, and CD4-8- interleukin (IL)- 2R+ cells enter the CD4+8+IL-2R- stage. The actual molecular events regulating this important control point are unknown, but may be related to the expression of the TCR-beta locus in immature CD4-8- thymocytes. It is not clear whether maturation is induced by intracellular appearance of TCR-beta chain or by signal transduction through an immature TCR complex on the thymocyte membrane, possibly involving TCR- beta chain homodimers and CD3. Here we show that early addition of anti- CD3 mAb to fetal thymic organ cultures induces all known events associated with the acquisition of the CD4+8+ stage. Expression of CD4 and CD8 is accelerated, IL-2R alpha is downregulated, and the cells fail to produce TCR-beta, possibly based on premature cessation of beta gene rearrangement. Upon stimulation with anti-CD3 antibodies, we see calcium mobilization in 15% of all CD4-8- thymocytes with no detectable surface TCR expression. These results suggest that functional CD3 is expressed on immature thymocytes at very low concentrations before the appearance of a complete TCR-beta chain. Ligation of CD3 at this stage may mimic the maturation signal normally generated by the immature TCR- beta homodimer-CD3 complex. The results are consistent with the notion that acquisition of the CD4+8+ stage involves signal transduction through an immature TCR complex. Later in thymocyte development, ligation of CD3 results in deletion of CD4+8+ cells. Thus, signal transduction through CD3 may result in entirely different cellular responses, depending on the stage of thymocyte differentiation. These results suggest an involvement of CD3 as a link in signal transduction for at least two different decision points in the development of a thymocyte.     

Differential effects of interleukin-15 and interleukin-2 on differentiation of bipotential T/natural killer progenitor cells

Bipotential T/natural killer (NK) progenitor cells are destined to differentiate mainly into T cell receptor (TCR) alpha beta and TCR gamma delta cells in a thymic microenvironment, whereas extrathymically they selectively develop into NK cells. The exact environmental conditions that are required for differentiation into these three leukocyte populations are largely unknown. In this report, we have investigated and compared the effect of interleukin (IL)-15 and IL-2 in this process. The IL-15 receptor is composed of the gamma and beta chains of the IL-2 receptor (IL-2R gamma and IL-2R beta) and of a specific alpha chain (IL-15R alpha). Here, it is shown that IL-15 mRNA is mainly expressed in thymic epithelial stromal cells, whereas IL-2 mRNA is exclusively expressed in thymocytes. IL-2R beta-expressing cells were present in the fetal thymus with a CD25-CD44+Fc gamma R+HSA- /low TCR- phenotype, which is characteristic of progenitor cells. These cells also expressed IL-15R alpha messenger RNA. Sorted IL-2R beta + TCR- cells differentiated into TCR alpha beta and TCR gamma delta cells after transfer to alymphoid thymic lobes, whereas culture of the same sorted cells in cell suspension in the presence of IL-15 resulted in the generation of functional NK cells. This shows that IL-2R beta +TCR- cells of the fetal thymus contain bipotential T/NK progenitors. Addition of low concentrations of IL-15 to fetal thymic organ culture (FTOC) resulted in an increase of all T cell subpopulations. The largest expansion occurred in the TCR gamma delta compartment. In contrast, low concentrations of IL-2 did not result in a higher total cell number and did not induce outgrowth of TCR gamma delta cells. High concentrations of IL-15 blocked TCR alpha beta development and shifted differentiation towards NK cells. Differentiation towards TCR gamma delta cells still proceeded. High concentrations of IL-2 similarly induced development into NK cells, but the cell number was fourfold lower than in IL-15 cultures. Importantly, blocking of IL-2R alpha in IL-2-treated FTOC resulted in a drastic increase in cell number, indicating that IL-2R alpha negatively regulates cell expansion. Collectively, these experiments provide direct evidence that IL-15 and IL-2 differentially affect the differentiation of bipotential T/NK progenitors.     

Different use of T cell receptor transducing modules in two populations of gut intraepithelial lymphocytes are related to distinct pathways of T cell differentiation

Most gut intraepithelial cells (IEL) of the mouse are T cells that bear CD8 molecules, present either as alpha-beta chain heterodimers (CD8 beta+) or as alpha chain homodimers (CD8 beta-). All CD8 beta+ IEL bear alpha/beta T cell receptors (TCR); CD8 beta- IEL bear either alpha/beta or gamma/delta TCR and are considered to be a thymus-independent (TI) population, probably arising locally from a small fraction of CD3- IEL containing the recombinant activating gene RAG proteins. Here we report that TI CD8 beta- IEL, whether bearing alpha/beta or gamma/delta TCR, contain, in normal mice, mRNAs for both zeta and Fc epsilon RI gamma chains. These chains are present in their CD3-TCR complexes as homo- or heterodimers. In contrast, only zeta chain mRNA and homodimers are found in gut CD8 alpha/beta+ IEL and in peripheral T lymphocytes. Intestinal CD3- precursor cells contain only gamma chain, and CD3- IL- 2R+ thymocyte precursors only zeta chain mRNAs. Only very primitive thymocyte precursors contain detectable gamma chain mRNA, and it thus appears that Fc epsilon RI gamma chain use is switched off at a very early stage during thymocyte differentiation. Thus, T cell differentiation in the gut epithelium differs from that occurring in the thymus, from which CD8 beta+ IEL appear to derive. Use of different TCR transducing modules and CD8 accessory molecules between the TI and the thymus-derived T cell populations provides an explanation for their difference in reactivity to antigenic stimulations and thus in selection of repertoires.     

Development of CD4+CD8+ thymocytes in RAG-deficient mice through a T cell receptor beta chain-independent pathway

Antigen-binding diversity is generated by site-specific V(D)J recombination of the T cell receptor (TCR) and immunoglobulin loci in lymphocyte precursors. Coordinate expression of two structurally distinct recombinase activating genes, RAG-1 and RAG-2, is necessary for activation of site-specific V(D)J recombination. In mice bearing targeted disruptions of either the RAG-1 or RAG-2 genes, T and B lymphocyte development is arrested at the CD4-8- double negative (DN) thymocyte or B220+/CD43+ pro-B cell stage. Development of CD4+CD8+ double positive (DP) thymocytes is restored by expression of a functionally rearranged TCR beta transgene, suggesting that TCR beta expression is critical for this developmental transition. We have found that treatment of adult or newborn RAG-deficient mice with a single sublethal dose of gamma-irradiation rescues the DN to DP transition in early thymocytes, and this is accompanied by a dramatic increase in thymus cellularity. In contrast to the observed induction of thymocyte maturation, there was no phenotypic or functional evidence of coincident B lymphocyte development in irradiated RAG-deficient mice. Interestingly, maturation of DP thymocytes occurred without expression of TCR beta protein in the cytoplasm or on the cell surface. These results suggest an in vivo pathway for DP thymocyte development which is TCR beta chain independent.     

Two gut intraepithelial CD8+ lymphocyte populations with different T cell receptors: a role for the gut epithelium in T cell differentiation

Mouse gut intraepithelial lymphocytes (IEL) consist mainly (90%) of two populations of CD8+ T cells. One bears heterodimeric alpha/beta CD8 chains (Lyt-2+, Lyt-3+), a T cell receptor (TCR) made of alpha/beta chains, and is Thy-1+; it represents the progeny of T blasts elicited in Peyer's patches by antigenic stimulation. The other bears homodimeric alpha/alpha CD8+ chains, contains no beta chain mRNA, and is mostly Thy-1- and TCR-gamma/delta + or -alpha/beta +; it is thymo-independent and does not require antigenic stimulation, as shown by its presence: (a) in nude and scid mice; (b) in irradiated and thymectomized mice repopulated by T-depleted bone marrow cells bearing an identifiable marker; (c) in thymectomized mice treated by injections of monoclonal anti-CD8 antibody, which lead to total depletion of peripheral CD8+ T lymphocytes; and (d) in germ-free mice and in suckling mice. In young nude mice, alpha/alpha CD8 chains, CD3-TCR complexes, and TCR mRNAs (first gamma/delta) are found on IEL, while they are not detectable on or in peripheral or circulating lymphocytes or bone marrow cells. IEL, in contrast to mature T cells, contain mRNA for the RAG protein, which is required for the rearrangement of TCR and Ig genes. We propose that the gut epithelium (an endoderm derivative, as the thymic epithelium) has an inductive property, attracting progenitors of bone marrow origin, and triggering their TCR rearrangement and alpha/alpha CD8 chains expression, thus giving rise to a T cell population that appears to belong to the same lineage as gamma/delta thymocytes and to recognize an antigenic repertoire different from that of alpha/beta CD8+ IEL.     

Fc gamma RII/III and CD2 expression mark distinct subpopulations of immature CD4-CD8- murine thymocytes: in vivo developmental kinetics and T cell receptor beta chain rearrangement status

We have recently identified a dominant wave of CD4-CD8- (double- negative [DN]) thymocytes in early murine fetal development that express low affinity Fc gamma receptors (Fc gamma RII/III) and contain precursors for Ti alpha/beta lineage T cells. Here we show that Fc gamma RII/III is expressed in very immature CD4low single-positive (SP) thymocytes and that Fc gamma RII/III expression is downregulated within the DN subpopulation and before the CD3-CD8low SP stage in T cell receptor (TCR)-alpha/beta lineage-committed thymocytes. DN Fc gamma RII/III+ thymocytes also contain a small fraction of TCR-gamma/delta lineage cells in addition to TCR-alpha/beta progenitors. Fetal day 15.5 DN TCR-alpha/beta lineage progenitors can be subdivided into three major subpopulations as characterized by cell surface expression of Fc gamma RII/III vs. CD2 (Fc gamma RII/III+CD2-, Fc gamma RII/III+CD2+, Fc gamma RII/III-CD2+). Phenotypic analysis during fetal development as well as adoptive transfer of isolated fetal thymocyte subpopulations derived from C57B1/6 (Ly5.1) mice into normal, nonirradiated Ly5.2 congenic recipient mice identifies one early differentiation sequence (Fc gamma RII/III+CD2(-)-->Fc gamma RII/III+CD2(+)-->Fc gamma RII/III- CD2+) that precedes the entry of DN thymocytes into the CD4+CD8+ double- positive (DP) TCRlow/- stage. Unseparated day 15.5 fetal thymocytes develop into DP thymocytes within 2.5 d and remain at the DP stage for > 48 h before being selected into either CD4+ or CD8+ SP thymocytes. In contrast, Fc gamma RII/III+CD2- DN thymocytes follow this same developmental pathway but are delayed by approximately 24 h before entering the DP compartment, while Fc gamma RII/III-CD2+ display accelerated development by approximately 24 h compared with total day 15.5 thymocytes. Fc gamma RII/III-CD2+ are also more developmentally advanced than Fc gamma RII/III+CD2- fetal thymocytes with respect to their TCR beta chain V(D)J rearrangement. At day 15.5 in gestation, beta chain V(D)J rearrangement is mostly, if not entirely, restricted to the Fc gamma RII/III-CD2+ subset of DN fetal thymocytes. Consistent with this analysis in fetal thymocytes, > 90% of adult thymocytes derived from mice carrying a disrupting mutation at the recombination- activating gene 2 locus (RAG-2-/-) on both alleles are developmentally arrested at the DN CD2- stage. In addition, there is a fivefold increase in the relative percentage of thymocytes expressing Fc gamma RII/III in TCR and immunoglobulin gene rearrangement-incompetent homozygous RAG-2-/- mice (15% Fc gamma RII/III+) versus rearrangement- competent heterozygous RAG-2+/- mice (< 3% Fc gamma RII/III+). Thus, Fc gamma RII/III expression defines an early DN stage preceding V beta(D beta)I beta rearrangement, which in turn is followed by surface expression of CD2. Loss of Fc gamma RII/III and acquisition of CD2 expression characterize a late DN stage immediately before the conversion into DP thymocytes.     

Induction of interleukin 2 receptor beta chain expression by self- recognition in the thymus

1-2% of adult mouse thymocytes express the T cell receptor alpha/beta (TCR-alpha/beta) together with the interleukin (IL) 2R beta (p70), but not the alpha (p 55) chain. We show that the previously described alpha/beta-TCR +CD4-8- and the partially overlapping Ly6C+ thymocytes are contained within this subset. Most IL-2R beta+ alpha/beta-TCR+ cells have a mature and activated (heat stable antigen [HSA]-, thymic shared antigen 1 [TSA-1]-, CD44high, CD69+) phenotype. Overrepresentation of V beta 8.2 in both CD4-8- and CD4 and/or CD8+ IL- 2R beta+ thymocytes suggests that IL-2R beta expression is induced by a TCR-mediated activation event. In mice transgenic for an H-2Kb-specific TCR, IL-2R beta+ cells were abundant under conditions of mainstream negative selection, i.e., in the presence of Kb, but absent under conditions of mainstream positive selection or in a nonselecting environment. Together, these results show that in addition to clonal deletion, self-recognition by immature thymocytes leads to phenotypic maturation of a small subset of thymocytes expressing IL-2R beta. IL-2- deficient mice contain normal numbers of IL-2R beta+ alpha/beta-TCR+ thymocytes, indicating that like mainstream T cell development, this minor pathway of positive selection does not depend on IL-2. However, in the absence of IL-2, the CD4/CD8 subset composition of IL-2R beta+ thymocytes is skewed towards CD4-8+, mostly at the expense of CD4-8-. A possible relevance of this finding for the development of the immune pathology of IL-2-deficient mice is discussed.     

Stoichiometry of the T cell antigen receptor (TCR) complex: each TCR/CD3 complex contains one TCR alpha, one TCR beta, and two CD3 epsilon chains

The stoichiometry of the subunits that comprise the T cell antigen receptor (TCR) complex is not completely known. In particular, it is uncertain whether TCR alpha and TCR beta proteins are present in the TCR complex as one or multiple heterodimeric pairs. In this study we have used mice transgenic for two different TCR alpha and two different TCR beta proteins to determine the number of TCR alpha and TCR beta chains in a single TCR complex. Individual thymocytes and splenic T cells from double TCR transgenic mice simultaneously expressed all four transgenic TCR proteins on their surfaces. Because the individual TCR alpha and individual TCR beta proteins were biochemically distinguishable, we were able to examine association among the transgenic TCR products. We found that each TCR alpha chain paired with each TCR beta chain, but that each TCR complex contained only one TCR alpha and one TCR beta protein. Furthermore, quantitative immunofluorescence revealed that T cells expressed twice as many CD3 epsilon as TCR beta proteins. These findings demonstrate that there are precisely one TCR alpha, one TCR beta, and two CD3 epsilon chains in each TCR/CD3 complex expressed on the surfaces of both thymocytes and mature T cells.     

Evidence that gammadelta versus alphabeta T cell fate determination is initiated independently of T cell receptor signaling

Two types of T cells, alphabeta and gammadelta, develop in vertebrates. How these two T cell lineages arise from a common thymic T progenitor is poorly understood. Differentiation of alphabeta lineage T cells requires the surrogate alpha chain (pTalpha), which associates with the T cell receptor (TCR) beta chain to form the pre-TCR. gammadelta lineage development does not appear to involve an obligatory surrogate chain, but instead requires productive rearrangement and expression of both TCR gamma and delta genes. It has been proposed that the quality of signals transmitted by the pre-TCR and gammadelta TCR are distinct and that these "instructive" signals determine the lineage fate of an uncommitted progenitor cell. Here we show that the thymic T progenitor cells (CD25(+)CD44(+)c-kit(+)CD3(-)CD4(-)CD8(-) thymocytes, termed pro-T cells) from young adult mice that have yet to express TCRs can be subdivided based on interleukin 7 receptor (IL-7R) expression. These subsets exhibit differential potential to develop into gammadelta versus alphabeta lineage (CD4+CD8+ cells) in the thymus. Upon intrathymic injection, IL-7R(neg-lo) pro-T cells generated a 13-fold higher ratio of alphabeta lineage to gammadelta lineage cells than did IL-7R(+) pro-T cells. Much of this difference was due to a fivefold greater potential of IL-7R(+) pro-T cells to develop into TCR-gammadelta T cells. Evidence indicates that this biased developmental potential is not a result of enhanced TCR-gamma gene rearrangement/expression in IL-7R(+) pro-T cells. These results indicate that the pro-T cells are heterogeneous in developmental potential before TCR gene rearrangement and suggest that in some precursor cells the initial lineage commitment is independent of TCR-mediated signals.     

Early molecular events induced by T cell receptor (TCR) signaling in immature CD4+ CD8+ thymocytes: increased synthesis of TCR-alpha protein is an early response to TCR signaling that compensates for TCR-alpha instability, improves TCR assembly, and parallels other indicators of positive selection

Differentiation of immature CD4+ CD8+ thymocytes into mature CD4+ or CD8+ T cells occurs within the thymus and is dependent upon expression of antigen receptor complexes (T cell receptor [TCR]) containing clonotypic alpha/beta proteins. We have recently found that CD4+ CD8+ thymocytes express low levels of surface TCR because of limitations placed on TCR assembly by the instability of nascent TCR-alpha proteins within the endoplasmic reticulum (ER) of immature thymocytes. Because TCR-alpha/beta expression increases during development, a molecular mechanism must exist for increasing the number of assembled TCR complexes present in immature CD4+ CD8+ thymocytes that have been signaled to differentiate into mature T cells, although no such mechanism has yet been described. In the current report we have examined the molecular consequences of intracellular signals generated by engagement of surface TCR complexes on immature CD4+ CD8+ thymocytes. Isolated TCR engagement generated signals that increased TCR-alpha RNA levels and increased synthesis of TCR-alpha proteins, which, in turn, significantly increased assembly of complete TCR- alpha/beta complexes in CD4+ CD8+ thymocytes. Increased TCR-alpha protein levels in TCR-signaled CD4+ CD8+ thymocytes was the result of increased synthesis and not increased stability of TCR-alpha proteins, indicating that TCR engagement compensates for, but does not correct, the inherent instability of TCR-alpha proteins in the ER of immature thymocytes. Consistent with the delivery by TCR engagement of a positive selection signal, TCR engagement also increased CD5 expression, decreased RAG-1 expression, and decreased CD4/CD8 coreceptor expression in immature CD4+ CD8+ thymocytes. These data identify amplified TCR-alpha expression as an initial response of immature CD4+ CD8+ thymocytes to TCR-mediated positive selection signals and provide a molecular basis for increased surface TCR density on developing thymocytes undergoing selection events within the thymus.     

A novel subset of CD2-, CD3/T cell receptor alpha/beta+ human peripheral blood T cells. Phenotypic and functional characterization of interleukin 2-dependent CD2-CD3+ T cell clones

It is generally believed that CD2 (T11, sheep erythrocyte receptor) is expressed on all human T cells. In the present study we have identified and characterized a minor subset of CD2- CD3/TCR alpha/beta+ T cells in the peripheral blood of healthy individuals. CD2-CD3+ T cells were enriched in PBMC depleted of plastic-adherent macrophages, E-rosetting (i.e., CD2+) T cells and surface Ig+ B cells. CD2-CD3+ T cells accounted for 0.1-0.8% of PBMC in six individuals. IL-2-dependent long-term clones of CD2-CD3+ T cells neither reacted with a panel of anti-CD2 mAbs nor expressed detectable levels of CD2 mRNA by Northern blot analysis. These clones, however, expressed a full-length TCR C beta mRNA and reacted with mAbs against TCR-alpha/beta, CD3, and CD4, and thus were mature T cells. CD2-CD3/TCR+ T cell clones could be triggered into proliferation, IL-2 production, and cytotoxic effector activity by anti-CD3 and anti-TCR mAbs. We conclude that (a) a minor subset of CD2-, CD3/TCR-alpha/beta+ T cells is present in normal peripheral blood; and (b) expression of CD2 at the level of protein and/or mRNA is not required for T cell signaling via the CD3/TCR molecular complex.     

Beta 2-microglobulin-dependent T cells are dispensable for allergen- induced T helper 2 responses

CD4+ and CD8+ alpha/beta+ T cells of the T helper cell (Th)2 phenotype produce the cytokines IL-4, IL-5, and IL-13 that promote IgE production and eosinophilic inflammation. IL-4 may play an important role in mediating the differentiation of antigenically naive alpha/beta+ T cells into Th2 cells. Murine NK1.1+ (CD4+ or CD4-CD8-) alpha/beta+ T cells comprise a beta 2-microglobulin (beta 2m)-dependent cell population that rapidly produces IL-4 after cell activation in vitro and in vivo and has been proposed as a source of IL-4 for Th2 cell differentiation. alpha/beta+ CD8+ T cells, most of which require beta 2m for their development, have also been proposed as positive regulators of allergen-induced Th2 responses. We tested whether beta 2m- dependent T cells were essential for Th2 cell-mediated allergic reactions by treating wild-type, beta 2m-deficient (beta 2m -/-), and IL-4-deficient (IL-4 -/-) mice of the C57BL/6 genetic background with ovalbumin (OVA), using a protocol that induces robust allergic pulmonary disease in wild-type mice. OVA-treated beta 2m -/- mice had circulating levels of total and OVA-specific IgE, pulmonary eosinophilia, and expression of IL-4, IL-5, and IL-13 mRNA in bronchial lymph node tissue similar to that of OVA-treated wild-type mice. In contrast, these responses in OVA-treated IL-4 -/- mice were all either undetectable or markedly reduced compared with wild-type mice, confirming that IL-4 was required in this allergic model. These results indicate that the NK1.1+ alpha/beta+ T cell population, as well as other beta 2m-dependent populations, such as most peripheral alpha/beta+ CD8+ T cells, are dispensable for the Th2 pulmonary response to protein allergens.     

CD8 beta chain influences CD8 alpha chain-associated Lck kinase activity

The CD8 molecule plays an important role in the differentiation of CD8+ T cells in the thymus and in their normal function in the periphery. CD8 exists on the cell surface in two forms, the alpha alpha homodimer and the alpha beta heterodimer. Recent studies indicate an important role for the CD8 beta chain in thymic development of CD8+ T cells and suggest that signaling via CD8 alpha beta may be distinct from CD8 alpha alpha. To better understand these differences, we introduced the CD8 beta gene into a T cell hybridoma which only expressed the CD8 alpha alpha homodimer. In the parent hybridoma, cross-linking of the CD8 alpha chain led to minimal enhancement of CD8-associated Lck tyrosine kinase activity. In the CD8 beta+ transfectants, several observations suggested that CD8 beta modifies CD8 alpha-associated Lck tyrosine kinase activity: (a) in in vitro kinase assays, antibody- mediated crosslinking of CD8 alone, or CD8 cross-linking with the TCR, resulted in 10-fold greater activation of Lck kinase activity, compared to cells expressing CD8 alpha alpha alone; (b) in vivo, markedly enhanced tyrosine phosphorylation of several intracellular proteins was observed upon CD8 cross-linking with the TCR in CD8 alpha beta- expressing cells, compared to cells expressing CD8 alpha alpha alone; and (c) Lck association with CD8 alpha was stabilized by the coexpression of CD8 beta. Thus, the differential Lck kinase activation and tyrosine phosphorylation seen with CD8 alpha alpha vs. CD8 alpha beta may reflect the unique signaling capabilities of the CD8 beta molecule. These differences in signaling may, in part, account for the diminished ability to generate CD8 single positive thymocytes in mice bearing a homozygous disruption of the CD8 beta gene.     

Th2-like CD8+ T cells showing B cell helper function and reduced cytolytic activity in human immunodeficiency virus type 1 infection

We analyzed at clonal level the functional profile of circulating or skin-infiltrating T lymphocytes from two individuals infected with the human immunodeficiency virus type 1 (HIV-1), suffering from a Job's- like syndrome (eczematous dermatitis, recurrent skin and sinopulmonary infections, and hypergammaglobulinemia E) and showing virtually no circulating CD4+ T cells. Most of the CD3+ T cell clones generated from both patients were CD4- CD8+ TCR alpha beta +. The others were CD4- CD8- TCR alpha beta + which exhibited reduced mRNA expression for the CD8 molecule or no mRNA expression for either CD4 or CD8 molecules. The great majority of both CD4- CD8+ and CD4- CD8- did not produce interferon (IFN) gamma and exhibited reduced cytolytic activity. Rather, most of them produced large amounts of both interleukin (IL) 4 and IL-5 and provided B cell helper function for IgE synthesis. These data suggest that a switch of cytolytic CD8+ T cells showing a Th1-like cytokine secretion profile to cells that make Th2-type cytokines, exhibit reduced cytolytic potential, and provide B cell helper function can occur in the course of HIV-1 infection. These cells may contribute to the reduced defense against viral infections and intracellular parasites and account for the elevated IgE serum levels, eosinophilia, and the allergic-like clinical manifestations seen in a proportion of HIV-1-infected individuals.     

Restricting Zap70 expression to CD4+CD8+ thymocytes reveals a T cell receptor-dependent proofreading mechanism controlling the completion of positive selection

Although T cell receptor (TCR) signals are essential for intrathymic T cell-positive selection, it remains controversial whether they only serve to initiate this process, or whether they are required throughout to promote thymocyte differentiation and survival. To address this issue, we have devised a novel approach to interfere with thymocyte TCR signaling in a developmental stage-specific manner in vivo. We have reconstituted mice deficient for Zap70, a tyrosine kinase required for TCR signaling and normally expressed throughout T cell development, with a Zap70 transgene driven by the adenosine deaminase (ADA) gene enhancer, which is active in CD4(+)CD8(+) thymocytes but inactive in CD4(+) or CD8(+) single-positive (SP) thymocytes. In such mice, termination of Zap70 expression impaired TCR signal transduction and arrested thymocyte development after the initiation, but before the completion, of positive selection. Arrested thymocytes had terminated Rag gene expression and up-regulated TCR and Bcl-2 expression, but failed to differentiate into mature CD4 or CD8 SP thymocytes, to be rescued from death by neglect or to sustain interleukin 7R alpha expression. These observations identify a TCR-dependent proofreading mechanism that verifies thymocyte TCR specificity and differentiation choices before the completion of positive selection.