Positive Selection Events Research Articles

Positive and negative selection events during B lymphopoiesis

Early in B-cell development, large numbers of cells have to be generated, each of which expresses only one type of B-cell receptor (i.e. Ig) on its surface. This is achieved by the surface expression of a pre-B cell receptor containing a μ heavy chains/surrogate light chain which differentially provides signals for two responses of precursor B cells at this stage of development. On the one hand, it signals inhibition of further rearrangements of variable heavy chain to diverse-joining heavy chain loci to achieve allelic exclusion at the heavy-chain locus. On the other hand, it signals proliferative expansion by factors between 20 and 100. Later in B-cell development, tolerance to autoantigens must be established and maintained. Tolerance is achieved by developmental arrest and induction of secondary light-chain gene rearrangements in those IgM + immature B cells that are reactive to autoantigens presented in the primary B-cell generating organs. Even later in development, when mature surface (s)IgM +/sID + B cells encounter autoantigens presented to them in the periphery, either deletion or anergy of the autoantigen-reactive cells occurs. Anergic cells have a sIg-dependent, slg-proximal defect in signaling and are short-lived. Anergy can be broken in vitro by polyclonal activation via ligation of CD40 in the presence of IL-4. A small part of the remaining immature B cells not reactive to autoantigens are selected to become mature, antigen-reactive slgM +/slgD + B cells. Molecules which might guide such positive selection of B cells still remain to be identified.

T cell receptor-mediated signaling events in CD4+CD8+ thymocytes undergoing thymic selection: requirement of calcineurin activation for thymic positive selection but not negative selection.

The goal of this study was to identify the differences of intracellular signals between the processes of thymic positive and negative selection. The activation of calcineurin, a calcium- and calmodulin-dependent phosphatase, is known to be an essential event in T cell activation via the T cell receptor (TCR). The effect of FK506, an inhibitor of calcineurin activation, on positive and negative selection in CD4+CD8+ double positive (DP) thymocytes was examined in normal mice and in a TCR transgenic mouse model. In vivo FK506 treatment blocked the generation of mature TCRhighCD4+CD8- and TCRhighCD4-CD8+ thymocytes, and the induction of CD69 expression on DP thymocytes. In addition, the shutdown of recombination activating gene 1 (RAG-1) transcription and the downregulation of CD4 and CD8 expression were inhibited by FK506 treatment suggesting that the activation of calcineurin is required for the first step (or the very early intracellular signaling events) of TCR-mediated positive selection of DP thymocytes. In contrast, FK506-sensitive calcineurin activation did not appear to be required for negative selection based on the observations that negative selection of TCR alpha beta T cells in the H-2b male thymus (a negative selecting environment) was not inhibited by in vivo treatment with FK506 and that there was no rescue of the endogenous superantigen-mediated clonal deletion of V beta 6 and V beta 11 thymocytes in FK506-treated CBA/J mice. DNA fragmentation induced by TCR activation of DP thymocytes in vitro was not affected by FK506. In addition, different effects of FK506 from Cyclosporin A on the T cell development in the thymus were demonstrated. The results of this study suggest that different signaling pathways work in positive and negative selection and that there is a differential dependence on calcineurin activation in the selection processes.

Anchored polymerase chain reaction based analysis of the V beta repertoire in the non-obese diabetic (NOD) mouse.

We have performed extensive analyses of T cell receptor V beta usage in the thymus, the spleen and the infiltrated islets of preclinical non-obese diabetic (NOD) mice. A semiquantitative anchored polymerase chain reaction (An-PCR) protocol has been developed for this purpose. The validity of the method has been first assessed by antibody staining with a panel of anti-V beta monoclonal antibodies (mAb). The results obtained by An-PCR are accurate, reproducible, and in good agreement with cell surface protein staining. A strict comparison between thymus and spleen repertoires reveals no major V beta-specific deletion except the already reported V beta 3 deletion due to Mtv-3. Certain V beta such as V beta 15, 18, 20 are found with a low frequency in the spleen, but the fact that they are also scarce in the thymus probably reflects a poor availability of these genetic elements during beta chain rearrangement rather than negative selection. Other V beta, such as V beta 2, V beta 12 and V beta 14 are significantly more abundant in the spleen than in the thymus. This finding was confirmed by mAb staining for V beta 2 and V beta 14. The expansion asymmetrically affects the CD4+ subset and can be traced back to the mature, single-positive thymocyte subset, suggesting an intrathymic positive selection event. V beta repertoires in infiltrated islets of 13- and 18-week-old, non-diabetic mice are polymorphic. Practically all the V beta found in the peripheral lymphoid tissues are present in the islets, in similar proportions. The major exception is V beta 12, one of the V beta which is subject to expansion during intrathymic differentiation and which is further augmented in the islets, both at 13 and 18 weeks. This increase probably reflects further peripheral amplification of the V beta 12-bearing subset due to encounter with the same ligand as in the thymus or with a cross-reactive motif. Finally, the nucleotide sequencing of all the V beta segments in usage in the NOD strain confirms the absence of allelic polymorphism of V beta-coding regions.

Dynamic behavior of hepatitis C virus quasispecies in patients undergoing orthotopic liver transplantation.

We have studied the distribution of viral sequences from the 5' noncoding region and from a fragment of the E2/NS2 region of the hepatitis C virus (HCV) genome in samples obtained before and after liver transplantation in two patients with HCV cirrhosis. The population of viral sequences in both regions were established by sequencing cloned PCR products. In both cases, the complexity of the viral quasispecies decreased after transplantation, although the consensus nucleotide and amino acid sequences remained unchanged. It is suggested that both positive and negative selection and random sampling events contribute substantially in shaping the genetic composition of HCV quasispecies and that recurrence of HCV infection may occur under equilibrium conditions.

Constitutive activation of integrin alpha 4 beta 1 defines a unique stage of human thymocyte development.

Our understanding of thymocyte development and of the positive and negative selection events involved in shaping the repertoire of mature T lymphocytes has been greatly facilitated by the use of transgenic and gene knockout animals. Much less is known about the factors that control the homing and population of the thymus by T cell precursors and the subsequent migration of developing thymocytes through the thymic architecture. As the integrins represent a candidate group of cell surface receptors that may regulate thymocyte development, we have analyzed the expression and function of alpha 4 beta 1 and alpha 5 beta 1 on human thymocytes. A major portion of double positive (CD4+ CD8+) human thymocytes express alpha 4 beta 1 in a constitutively active form and adhere to fibronectin and vascular cell adhesion molecule 1. alpha 4 beta 1 expression is similar on adherent and nonadherent populations, thus, activity reflects the receptor state and not simple expression. The adherent cells are immature, expressing high levels of CD4/CD8 and low levels of CD3 and CD69. In contrast, nonadherent cells possess the phenotype of thymocytes after positive selection, expressing intermediate levels of CD4 and/or CD8 and high levels of CD3 and CD69. The adherent population fails to respond to activation with anti-CD3 and fibronectin, whereas nonadherents exhibit an alpha 5 beta 1-dependent proliferation. Differential regulation of alpha 4 beta 1 and alpha 5 beta 1 receptors may provide a mechanism controlling cellular traffic, differentiation, and positive selection of thymocytes.

CD45RA and CD45RBhigh expression induced by thymic selection events.

CD45 is a protein tyrosine phosphatase involved in T and B cell signaling. While peripheral T cells switch CD45 isoforms upon activation, events leading to exon switching during T cell development in the thymus have not been determined. The expression of high molecular weight isoforms of CD45 was examined on thymocytes from nontransgenic and T cell receptor (TCR) transgenic mice. All thymocytes from nontransgenic mice were CD45RB+ as assessed by staining with MB23G2, an anti-CD45RB-specific monoclonal antibody. Interestingly, there was a small population (1-3%) of thymocytes that displayed a higher intensity of staining with MB23G2, CD45RBhigh. CD45RBhigh thymocytes were found in all subsets defined by CD4 and CD8 expression and were also present within the TCR-alpha/beta high population. To analyze whether or not CD45 expression correlated with thymic selection events, expression of CD45RBhigh and a second isoform, CD45RA, was examined on thymocytes from H-Y and 2C TCR transgenic mice and found to correlate with positive and negative selection events but did not occur in nonselecting backgrounds. CD45RA and CD45RBhigh upregulation was also not observed in transgenic mice backcrossed into CD8-deficient mice, a scenario in which there is no positive selection of transgene-expressing thymocytes. These data suggest that modulation of CD45 isoform expression may be involved in thymic selection events.

Peripheral T cell receptor gamma delta variable gene repertoire maps to the T cell receptor loci and is influenced by positive selection.

Although the mechanisms that determine TCR-alpha beta V gene repertoire are well studied, the genetic influences involved in TCR-gamma delta repertoire development are unclear. Unlike the TCR-gamma delta populations that localize in epithelial tissues, the circulating peripheral TCR-gamma delta V region repertoire is quite diverse. Previous studies have shown that three TCR-gamma chains and at least six TCR-V delta genes are expressed by splenic TCR-gamma delta cells. However, the relative frequency of individual gamma delta subsets among genetically diverse mice has not been determined. Therefore, the repertoire of TCR-gamma delta cells was examined using anti-TCR V region specific mAb against V gamma 2 and V delta 4 on TCR-gamma delta + cells from total splenocytes. We found that there was a strain-specific variation in TCR-gamma delta usage. The frequency of V gamma 2 expression in different strains varied from 54 to 12%, and the frequency of V delta 4 expression in different strains varied from 38 to 10%. However, the level of V delta 4 and V gamma 2 expression for an individual strain was highly consistent from experiment to experiment. F1 analysis between parental strains that differed in relative frequency of either V gamma 2+ or V delta 4+ cells revealed that high expression was genetically dominant, suggesting that positive selection events play a major role in the peripheral gamma delta repertoire. Variations in the levels of V gamma 2+ cells and V delta 4+ cells was not associated with Mls or MHC haplotype. Analysis of recombinant inbred strains revealed that high V delta 4 expression mapped to the TCR-gamma locus, while high V gamma 2 expression was influenced by the TCR-delta locus. Back-cross analysis confirmed that the TCR loci dominantly influenced the level of V delta 4+ cells and V gamma 2+ cells; however, there was clear evidence that multiple genes affect the TCR-gamma delta repertoire.

Distinctive selection mechanisms govern the T cell receptor repertoire of peripheral CD4-CD8- alpha/beta T cells.

The T cell receptor (TCR) repertoire of CD4+ and CD8+ alpha/beta T cells is heavily influenced by positive and negative selection events that occur during T cell development in the thymus. The coreceptors CD4 and CD8 appear to be essential for this selection to occur. To gain insight into whether T cells that express TCR alpha/beta but lack either coreceptor (CD4- CD8- TCR alpha/beta or alpha/beta double-negative [DN] cells) are also subject to positive and negative selection, and whether selection can occur in the absence of coreceptors, we have performed an extensive immunogenetic analysis of the TCR V beta repertoire of alpha/beta DN cells in lymph nodes of normal mice. Our results show that alpha/beta DN cells appear to be unaffected by clonal deletion of V beta 5 and V beta 11 in I-E-expressing mice, and do not undergo deletion of V beta 6- and V beta 8.1-expressing T cells in Mls-1a-positive mice. They are also unaffected by positive selection of V beta 17a+ T cells in the context of I-Aq. The results suggest that most selection events require the participation of CD4 and CD8, while alpha/beta DN cells are unselected. This argues that most alpha/beta DN cells probably have never expressed CD4 or CD8. However, a unique form of repertoire selection occurs: enrichment of V beta 17a+ alpha/beta DN cells in I-E+ mice. This could be an instance of coreceptor-independent selection.

Expression and combinatorial diversity of germ line-encoded T cell receptor V genes in human peripheral blood T cells

The potential diversity of the T cell receptor (TcR) is defined by the combinatorial expression of variable segments and by mechanisms that insert or delete nucleotides at the junctional regions. The available repertoire is strongly influenced by negative and positive selection events. To study whether the diversity of the human T cell receptor of peripheral T cells is further restricted by the interaction between the TcR α and β chains, we compared the level of transcription of different Va elements in human T cell blasts expressing either restricted or unrestricted sets of Vβ genes. Our data establish that in some individuals, but not in others, the transcription of a given Vα element is independent from the presence of particular Vβ transcripts. Furthermore, our data also suggest that, in contrast to mouse, major TcR V gene deletions are absent in humans. Taken collectively, these results indicate that the diversity of the peripheral human TcR repertoire can benefit from the combinatorial expression of all the V elements present in the genome.

Interleukin 3 Stimulation Enhances Tyrosine Phosphorylation and Proliferative Activity of Human Fetal Thymocytes and Leukemic T-cell Precursors at Multiple Developmental Stages of T-Cell Ontogeny

The purposes of this study were to elucidate the biologic effects of recombinant human interleukin 3 (rhIL-3) on leukemia T-cell precursors and fetal thymocytes corresponding to discrete and sequential developmental stages of human T-cell ontogeny, and to examine the biochemical nature of the IL-3 receptor linked transmembrane signal in human T-cell precursor populations. The specific binding of biosynthetically labeled 35S-rhIL-3 to leukemic T-cell precursors from T-lineage ALL patients was initially investigated. In 5 of 9 cases, the binding of 35S-rhIL-3 was significantly blocked by excess cold rhIL-3, and the percentage of inhibitable binding ranged from 40% to 64% (mean ± SE = 53 ± 4%). In these cases, 5–13 femtomols (mean + SE = 7.0 ± 1.5 fms) of 35S-rhIL-3/107 cells were specifically bound. rhIL-3 stimulated in a dose dependent fashion the in vitro clonal proliferation of leukemic T-cell precursors with composite immunophenotypes corresponding to the developmental stages of prothymocytes/double negative immature thymocytes (3 of 5 cases) and corticothymocytes/double positive CD4+CD8+ immature thymocytes (4 of 4 cases). By contrast, leukemic T-cell precursors from 2 T-lineage ALL cases with a composite immunophenotype of medullary thymocytes/single positive CD4+CD8−/CD4−CD8+ mature thymocytes did not show an enhanced proliferative activity after stimulation with increasing concentrations of rhIL-3. All of the 5 cases with significant 35S-rhIL-3 binding and none of the 4 cases with no 35S-rhIL-3 binding showed a proliferative response to rhIL-3. Thus, there was a high correlation between 35S-rhIL-3 binding and proliferative response of leukemic T-cell precursors in colony assays, indicating that functional IL-3 receptors were detected in ligand binding assays. rhIL-3 also stimulated the proliferation of immature double positive CD4+CD8+ thymocytes from 9 of 10 fetal thymuses without inducing differentiation and with no selective advantage for the development of CD4+CD8− or CD4− or CD4−CD8+ single positive thymocytes. The observed differences in IL-3 responsiveness among T-cell precursor populations at distinct developmental stages indicates that IL-3 receptors may be expressed only during the early stages of human T-cell ontogeny preceding the negative or positive selection events within the thymic microenvironment. Stimulation of fetal thymocytes with rhIL-3 resulted in enhanced tyrosine phosphorylation of 8 distinct cellular substrates with molecular weights of 44 kDa, 55 kDa, 60 kDa, 69 kDa, 98 kDa, 123 kDa, 150 kDa and 190 kDa, but it did not result in stimulation of phosphoinositide turnover and increased Ins-1,4,5-P3 production. The induction of tyrosine phosphorylation by rhIL-3 was augmented by further crosslinking surface bound IL-3 molecules with an anti-IL-3 antibody and it was abrogated by the tyrosine kinase inhibitor genistein. The ligation of the CD45 protein tyrosine phosphatase markedly suppressed the tyrosine phosphorylation of specific substrates induced by IL-3 stimulation. Thus, the mitogenic transmembrane signal triggered by the engagement of the IL-3 R on human T-cell precursors is linked to a functional protein tyrosine kinase (PTK)/protein tyrosine phosphatase (PTP) regulatory pathway. Taken together, these results indicate that IL-3 may have an important growth regulatory role in early stages of human T-cell ontogeny.

Alteration of T cell lineage commitment by expression of a hybrid CD8/CD4 transgene.

Thymocyte differentiation and the subsequent production of peripheral functional T cells involve positive and negative selection events on developing thymocytes expressing a number of specific surface molecules, including the T cell receptor (TCR) and the coreceptor molecules CD4 and CDS. At least two different mechanisms may be responsible for the effects of CD4 and CDS on T cell development. CD4 binding to major histocompatibility complex (MHC) class II proteins1 and CDS binding to MHC class I proteins2 may increase the strength of interaction between the cells involved in the selection processes.3 Signal transduction through these coreceptors may also play an important role during thymocyte development. Recent evidence indicates that CD4 and CDS play important roles in signal transduction during T cell activation.4-9

"Increased frequency of T cell receptor V alpha 12.1 expression on CD8+ T cells: evidence that V alpha participates in shaping the peripheral T cell repertoire".

The T cell receptor repertoire has a potential for vast diversity. However, this diversity is limited by the fact that the majority of thymocytes die as the repertoire is shaped by positive and negative selection events during development. Such thymic selection affecting TCR V beta gene segment usage has been demonstrated in the mouse. However, similar data has not been forthcoming in man, and little is known about the role of the TCR alpha chain in antigen/major histocompatibility complex (MHC) recognition in any species. Here, we used a monoclonal antibody recognizing the TCR V alpha 12.1 gene product to assess the expression of this gene in the peripheral blood of man. In most individuals tested, the percentage of cells expressing V alpha 12.1 was significantly higher in CD8+ T cells than in CD4+ T cells. That the V alpha gene product itself was responsible for this increased expression in CD8+ T cells was underscored by the lack of substantial skewing of V beta usage in the V alpha 12.1-bearing T cells. Moreover, the skewed expression of V alpha 12.1 was already present at birth, indicating that it was likely to be due to a developmental process rather than the result of exposure to environmental antigens. Based on the established role for CD8 in binding to class I MHC molecules, we suggest that increased expression of V alpha 12.1 on CD8+ T cells points to a role for TCR's using V alpha 12.1 in class I MHC/Ag recognition. These results indicate that V alpha gene usage in the peripheral blood of man is not random, and they support a role for V alpha as a participant in the self-MHC recognition process that shapes the TCR repertoire.

Increased frequency of T cell receptor V alpha 12.1 expression on CD8+ T cells: evidence that V alpha participates in shaping the peripheral T cell repertoire.

The T cell receptor repertoire has a potential for vast diversity. However, this diversity is limited by the fact that the majority of thymocytes die as the repertoire is shaped by positive and negative selection events during development. Such thymic selection affecting TCR V beta gene segment usage has been demonstrated in the mouse. However, similar data has not been forthcoming in man, and little is known about the role of the TCR alpha chain in antigen/major histocompatibility complex (MHC) recognition in any species. Here, we used a monoclonal antibody recognizing the TCR V alpha 12.1 gene product to assess the expression of this gene in the peripheral blood of man. In most individuals tested, the percentage of cells expressing V alpha 12.1 was significantly higher in CD8+ T cells than in CD4+ T cells. That the V alpha gene product itself was responsible for this increased expression in CD8+ T cells was underscored by the lack of substantial skewing of V beta usage in the V alpha 12.1-bearing T cells. Moreover, the skewed expression of V alpha 12.1 was already present at birth, indicating that it was likely to be due to a developmental process rather than the result of exposure to environmental antigens. Based on the established role for CD8 in binding to class I MHC molecules, we suggest that increased expression of V alpha 12.1 on CD8+ T cells points to a role for TCR's using V alpha 12.1 in class I MHC/Ag recognition. These results indicate that V alpha gene usage in the peripheral blood of man is not random, and they support a role for V alpha as a participant in the self-MHC recognition process that shapes the TCR repertoire.

Rescue of thymocytes and T cell hybridomas from glucocorticoid‐induced apoptosis by stimulation via the T cell receptor/CD3 complex: a possible in vitro model for positive selection of the T cell repertoire

Positive and negative selection events are involved in determining useful T cell clones to mature in the thymus. Accumulating evidence suggests that immature self-reactive thymocytes undergo apoptotic death (negative selection) upon stimulation via the T cell receptors (TcR). A similar phenomenon of activation-induced death has been reported in T cell hybridomas. On the other hand, little is known about the mechanism of the positive selection. Apoptosis in rodent thymocytes or T cell hybridomas is also known to be induced by glucocorticoids in vitro at concentrations within the physiologic range. We report here that the TcR/CD3-mediated stimulation and glucocorticoids mutually inhibit the apoptosis in T cell hybridomas. The production of interleukin 2 by the rescued cells indicated that the TcR/CD3-mediated signal was transduced into the cells. Thymocytes were also rescued from glucocorticoid-induced apoptosis by the stimulation with antibodies to TcR/CD3 molecules. The rescue of thymocytes, however, was observed only at a narrow concentration range of each of the antibodies, suggesting that the proper stimulation via the TcR/CD3 is required for the rescue. If thymocytes in situ are differentially stimulated according to the affinity of the TcR towards self, only the thymocytes whose TcR have proper affinity towards self may be rescued from glucocorticoid-induced apoptosis. Therefore, we propose a hypothesis that the positive selection of the T cell repertoire is based on the inhibition of glucocorticoid-induced apoptosis in immature thymocytes bearing TcR with proper affinity for self by the TcR-mediated signals in situ. Furthermore, the selection may be influenced by the peak level of glucocorticoid concentration, since the proper concentration range of the anti-TcR/CD3 antibody for the rescue was variable depending on the glucocorticoid concentration.

Deviations in thymocyte development induced by divalent and monovalent ligation of the alpha/beta T cell receptor and by its cross-linking to CD8.

Fetal thymic organ cultures (FTOC) were tested as a model system to induce, in a polyclonal fashion, negative and positive thymic selection events. By flow cytometry, thymocytes developed in FTOC differed in several parameters from their in vivo differentiated counterparts. In particular, no clear distinction was possible between CD4+CD8+ immature cells with low TCR expression and mature CD4+ or CD8+ cells with high TCR expression. Thymocyte development in FTOC was manipulated with three different antibody reagents: anti-V beta 8 (F23.1), anti-Lyt-2.2 (19/178) and the quadroma derived bifunctional antibody HPHT-2, carrying one binding site of each. This antibody served also as a monovalent anti-V beta 8 reagent in FTOC from Lyt-2.1 mouse strains. Antibody 19/178 suppressed the development of single positive CD8+ cells, but only at very high concentrations. F23.1 and HPHT-2 suppressed the development of CD4+V beta 8+ and CD8+V beta 8+ thymocytes at relatively low concentrations giving rise to V beta 8 occupancies from about 2% upwards. Suppression was equally pronounced in cells with low and high TCR densities. Moreover, V beta 8 suppression occurred upon divalent and monovalent V beta 8 binding and was not significantly influenced by V beta 8-CD8 cross-linking. This suggests that ligation of the TCR alone is sufficient for clonal deletion. The data do not exclude a role for CD8 as an accessory adhesion molecule but suggest that exogenous cross-linking of CD8 to the TCR is not essential in transmembrane signaling for clonal deletion. At lower antibody concentrations giving rise to V beta 8 occupancies below detection, V beta 8-CD8 cross-linking by HPHT-2, but no divalent and monovalent V beta 8 ligation, induced an increase of CD8+V beta 8+ cells at the expense of CD4+ V beta 8+ cells with no change in the proportion of total V beta 8+ thymocytes. The latter effect was quantitatively of borderline significance but reproducible. These latter results are compatible with the hypothesis that cross-linking of the alpha beta TCR and CD8 on the thymocyte surface provides a maturation signal resulting in loss of CD4 from CD4+ CD8+ double positive immature thymocytes.

Positive and negative selection of T cell receptor V beta domains controlled by distinct cell populations in the thymus.

The role of thymic MHC class II-bearing cells in the selection of the TCR repertoire has been investigated in allogeneic radiation bone marrow chimeras. Positive selection of mature CD4+ T lymphocytes expressing the V beta 6+ TCR domain was found to depend upon radioresistant (presumably epithelial) I-E+ thymic cells. On the other hand, negative selection of CD4+ V beta 6+ cells (which was additionally dependent upon expression of the Mls-1a gene product) was controlled by a radiosensitive I-E+ thymic component (most likely dendritic cells). These data argue in favor of a compartmentalization of positive and negative selection events during T cell development.

Intrathymic deletion of self-reactive cells prevented by neonatal anti-CD4 antibody treatment.

T-cell differentiation in the thymus involves the coordinate expression of genes encoding the alpha and beta chains of the major histocompatibility complex-restricted heterodimeric antigen receptor (TCR) complex, as well as other functionally important molecules such as CD4 and CD8. The repertoire of TCR expressed by T cells is generally thought to be influenced by positive and/or negative selection events occurring when TCRs on developing T cells interact with self-antigens and major histocompatibility complex components. Using a model system in which specific antigen-reactive cells can be monitored by virtue of their preferential expression of certain TCR beta-chain variable (V beta) domains, it has been shown that self-reactive T cells are clonally deleted during development. We report here that clonal deletion of V+ beta 6 cells in Mlsa mice can be prevented by in vivo neonatal administration of monoclonal antibodies directed against CD4. Furthermore, as anti-CD4 monoclonal antibody treatment resulted in the reappearance of V+ beta 6 cells in the mature CD8+ T-cell subset, it is likely that clonal deletion acts on the CD4+CD8+ thymocyte subset and that this subset is an intermediate stage in the differentiation pathway of both CD4+ and CD8+ T-cell lineages.