Strength of signaling by CD4 and CD8 coreceptor tails determines the number but not the lineage direction of positively selected thymocytes.

The present study has assessed the impact of the intracellular domains of CD4 and CD8 on positive selection and lineage direction of MHC class I-restricted thymocytes. Contrary to current presumption, we found that the CD4 tail promotes the generation of both CD4+ and CD8+ T cells without preference for the CD4+ T cell lineage. We also found that the identity of the coreceptor tail and hence the strength of coreceptor signaling determine the number of thymocytes undergoing positive selection but not their ultimate CD4/CD8 phenotype. These findings demonstrate that the strength of coreceptor signaling has a significant quantitative but not qualitative impact on positive selection and provide a simple explanation for the greater numbers of CD4+ than CD8+ T cells selected in the normal thymus.

Analysis of flow cytometry data.

This unit provides several approaches to flow cytometry data analysis. Frequency determinations based on analysis of single-parameter fluorescence histograms and dual-parameter contour plots are presented. Next, steps are described for calculating values for signal-to-noise ratios when logarithmic amplification is used for data collection. This calculation can be used to compare the amounts of antigenic determinants per cell between different cells stained with the same reagent. Finally, a procedure is described for constructing a calibration curve for logarithmic amplifiers. This calibration curve is required for calculation of signal-to-noise values and can also be used to determine if the amplifiers are working properly.

Synchronous deletion of Mtv-superantigen-reactive thymocytes in the CD3(medium/high) CD4(+)CD8(+) subset.

Multiple model systems have demonstrated that negatively selected thymocytes can be deleted during the immature CD4(+)CD8(+) CD3(low) stage after high affinity interaction of T-cell receptors (TCRs) with antigen:major histocompatibility complex (MHC) complexes. Superantigens (SAGs) derived from endogenous mammary tumour viruses (Mtv) induce negative selection of Mtv-SAG-reactive thymocytes regardless of which peptide antigen is presented by MHC molecules. In this study, the timing of deletion of multiple subsets of Mtv-SAG-reactive CD4(+)CD8(+) thymocytes was investigated by a 4 colour flow cytometry in SJL x CBA/J cross-bred mice. Deletion of V beta 3(+), V beta 5(+), V beta 11(+), and V beta 17(+) Mtv-SAG-reactive thymocytes was found to occur synchronously in the most mature CD3(medium) and early CD3(high) subsets of CD4(+)CD8(+) thymocytes, in contrast with reports showing that the deletion of Mtv-SAG-reactive thymocytes can occur at different stages in particular model systems.

Coreceptor reversal in the thymus: signaled CD4+8+ thymocytes initially terminate CD8 transcription even when differentiating into CD8+ T cells.

A central paradigm of T cell development is that CD4+8+ (DP) thymocytes differentiate into CD4+ or CD8+ T cells in response to intrathymic signals that extinguish transcription of the inappropriate coreceptor molecule. Contrary to this prevailing paradigm, we now demonstrate that signaled DP thymocytes initially terminate CD8 transcription even when differentiating into CD8+ T cells. Remarkably, thymocytes that have selectively terminated CD8 transcription can be signaled by IL-7 to differentiate into CD8+ T cells by silencing CD4 transcription and reinitiating CD8 transcription, events we refer to as "coreceptor reversal." These observations significantly alter our understanding of CD8+ T cell differentiation and lead to a new perspective ("kinetic signaling") on CD4/CD8 lineage determination in the thymus. These observations also suggest a novel mechanism by which bipotential cells throughout development can determine their appropriate cell fate.

CD8 coreceptor extinction in signaled CD4(+)CD8(+) thymocytes: coordinate roles for both transcriptional and posttranscriptional regulatory mechanisms in developing thymocytes.

T-cell development in the thymus is characterized by changing expression patterns of CD4 and CD8 coreceptor molecules and by changes in CD4 and CD8 gene transcription. In response to T-cell receptor (TCR) signals, thymocytes progress through developmental transitions, such as conversion of CD4(+)CD8(+) (double-positive [DP]) thymocytes into intermediate CD4(+)CD8(-) thymocytes, that appear to require more-rapid changes in coreceptor expression than can be accomplished by transcriptional regulation alone. Consequently, we considered the possibility that TCR stimulation of DP thymocytes not only affects coreceptor gene transcription but also affects coreceptor RNA stability. Indeed, we found that TCR signals in DP thymocytes rapidly destabilized preexisting CD4 and CD8 coreceptor RNAs, resulting in their rapid elimination. Destabilization of coreceptor RNA was shown for CD8alpha to be dependent on target sequences in the noncoding region of the RNA. TCR signals also differentially affected coreceptor gene transcription in DP thymocytes, terminating CD8alpha gene transcription but only transiently reducing CD4 gene transcription. Thus, posttranscriptional and transcriptional regulatory mechanisms act coordinately in signaled DP thymocytes to promote the rapid conversion of these cells into intermediate CD4(+)CD8(-) thymocytes. We suggest that destabilization of preexisting coreceptor RNAs is a mechanism by which coreceptor expression in developing thymocytes is rapidly altered at critical points in the differentiation of these cells.

Positive selection as a developmental progression initiated by alpha beta TCR signals that fix TCR specificity prior to lineage commitment.

During positive selection, immature thymocytes commit to either the CD4+ or CD8+ T cell lineage ("commitment") and convert from short-lived thymocytes into long-lived T cells ("rescue"). By formal precursor-progeny analysis, we now identify what is likely to be the initial positive selection step signaled by alpha beta TCR, which we have termed "induction". During induction, RAG mRNA expression is downregulated, but lineage commitment does not occur. Rather, lineage commitment (which depends upon the MHC class specificity of the alpha beta TCR) only occurs after downregulation of RAG expression and the consequent fixation of alpha beta TCR specificity. We propose that positive selection can be viewed as a sequence of increasingly selective developmental steps (induction-->commitment-->rescue) that are signaled by alpha beta TCR engagements of intrathymic ligands.

Developmentally regulated expression of peanut agglutinin (PNA)-specific glycans on murine thymocytes.

Intrathymic maturation of T lymphocytes is characterized by variable expression of O-linked Gal beta 1,3GalNAc glycans reactive with peanut agglutinin (PNA) lectin. Recent studies on human thymocytes show that conversion from PNA+ to PNA- phenotype is correlated with increased expression of alpha 2,3 O-linked sialyltransferase (ST), which sialylates Gal beta 1,3GalNAc glycans, masking their binding sites for PNA. Interestingly, alpha 2,3 O-linked ST expression is highest within the regions of the thymus containing the most immature and most mature thymocyte subsets, suggesting that PNA-specific glycans are intermittently masked by sialylation during thymic selection processes. Here, we studied expression of PNA receptors on developing thymocytes in the murine system using thymocytes from both normal mice and transgenic mice that are genetically arrested at the early phases of T cell development. Our results confirm and extend recent findings in the human system by showing that murine T cells sequentially progress from PNAlo-->PNAhi-->PNAlo stages during their differentiation within the thymus. In addition, our data demonstrate that a similar set of polypeptides is variably masked by sialylation throughout T cell development.

Surface molecules that drive T cell development in vitro in the absence of thymic epithelium and in the absence of lineage-specific signals.

Differentiation of immature double positive (DP) CD4+ CD8+ thymocytes into single positive (SP) CD4+ and CD8+ T cells is referred to as positive selection and requires physical contact with thymic cortical epithelium. We now have identified "coinducer" molecules on DP thymocytes that, together with TCR, signal DP thymocytes to differentiate into SP T cells in vitro in the absence of thymic epithelium. A remarkable number of different molecules on DP thymocytes possessed "coinducing" activity, including CD2, CD5, CD24, CD28, CD49d, CD81, and TSA-1. Interestingly, in vitro differentiation occurred in the absence of lineage-specific signals, yet resulted in the selective generation of CD4+CD8- T cells. Thus, the present study has identified surface molecules that can signal DP thymocytes to differentiate into SP T cells in the absence of thymic epithelium and has characterized a default pathway for CD4+ T cell differentiation.

Lineage commitment in the thymus: only the most differentiated (TCRhibcl-2hi) subset of CD4+CD8+ thymocytes has selectively terminated CD4 or CD8 synthesis.

Lineage commitment is a developmental process by which individual CD4+CD8+ (double positive, DP) thymocytes make a decision to differentiate into either CD4+ or CD8+ T cells. However, the molecular event(s) that defines lineage commitment is controversial. We have previously proposed that lineage commitment in DP thymocytes can be molecularly defined as the selective termination of CD4 or CD8 coreceptor synthesis. The present study supports such a molecular definition by showing that termination of either CD4 or CD8 synthesis is a highly regulated event that is only evident within the most differentiated DP subset (CD5hiCD69hiTCRhibcl-2hi). In fact, essentially all cells within this DP subset actively synthesize only one coreceptor molecule. In addition, the present results identify three distinct sub-populations of DP thymocytes that define the developmental progression of the lineage commitment process and demonstrate that lineage commitment is coincident with upregulation of TCR and bcl-2. Thus, this study supports a molecular definition of lineage commitment and uniquely identifies TCRhibcl-2hi DP thymocytes as cells that are already committed to either the CD4 or CD8 T cell lineage.

Identification of CD8 as a peanut agglutinin (PNA) receptor molecule on immature thymocytes.

Differentiation of most T lymphocytes occurs within the thymus and is characterized by variable expression of CD4/CD8 coreceptor molecules, increased surface density of T cell antigen receptor (TCR) alpha beta proteins, and decreased expression of glycan chains recognized by the galactose-specific lectin peanut agglutinin (PNA). Although appreciated for several decades that PNA agglutination is useful for the physical separation of immature and mature thymocyte sub-populations, the identity of specific PNA-binding glycoproteins expressed on immature thymocytes remains to be determined. In the current report, we studied the expression of PNA-specific glycans on immature and mature T cells and used lectin affinity chromatography and immunoprecipitation techniques to characterize PNA-binding glycoproteins on thymocytes. Our data demonstrate that PNA-specific glycans are localized on a relatively small subset of thymocyte surface proteins, several of which were specifically identified, including CD43, CD45, and suprisingly, CD8 molecules. CD8 alpha and CD8 alpha' proteins bound to PNA in the absence of CD8 beta expression showing that O-glycans on CD8 beta glycoproteins are not necessary for PNA binding and that glycosylation of CD8 alpha and CD8 alpha' proteins proceeds effectively in the absence of CD8 beta. Finally, we demonstrate that PNA binding of CD8 is developmentally regulated by sialic acid addition as CD8 proteins from mature T cells bound to PNA only after sialidase treatment. These studies identify CD8 as a PNA receptor molecule on immature thymocytes and show that PNA binding of CD8 on immature and mature T cells is developmentally regulated by sialic acid modification.

Thymic-independent T cell regeneration occurs via antigen-driven expansion of peripheral T cells resulting in a repertoire that is limited in diversity and prone to skewing.

Thymic regenerative capacity in humans decreases with age, suggesting that thymic-independent pathways of T cell regeneration may predominate during adulthood. Using a murine bone marrow transplantation model, we present evidence that thymic-independent T cell regeneration occurs primarily via expansion of peripheral T cells and is Ag driven since significant expansion of CD4+ or CD8+ transgenic (Tg+)/TCR-bearing cells occurs only in the presence of Ag specific for the TCR. Such expansion resulted in skewing of the regenerated repertoire with 40 to 65% of the regenerated CD4+ or CD8+ T cells expressing the Tg+/TCR in thymectomized hosts after bone marrow transplantation. In experiments in which nontransgenic population are used as T cell inocula, we noted decreased CD4 expansion when Class II MHC was blocked by mAb treatment in vivo, an CD8 expansion failed to occur in Class I MHC-deficient hosts providing evidence that T cell regeneration in thymic-deficient hosts largely occurs via TCR-MHC-mediated selection of peripheral T cell populations. This process results in a T cell repertoire comprised exclusively of T cells recently activated by the antigenic milieu of the host, with negligible numbers of residual "naive" cells bearing TCRs for Ags absent at the time of expansion. These findings have important implications for approached to enhance T cell regeneration in humans and provide evidence that vaccine strategies could skew the T cell repertoire toward a specific antigenic target if administered to thymic-deficient hosts during immune reconstitution.

Disruption of intrathymic CD4-Ia interactions on immature CD4+CD8+ thymocytes results in diminished TCR expression on mature CD8+ T cell progeny.

Cell surface TCR expression by developing thymocytes is actively regulated during ontogeny. Whereas most immature CD4+CD8+ thymocytes express low levels of TCR alpha beta, mature CD4+ and CD8+ thymocytes express significantly higher levels of surface TCR. Low TCR expression on CD4+CD8+ thymocytes is due, at least in part, to inhibitory signals generated by CD4 interactions with MHC class II ligands in the thymus. In the present study we wished to determine whether levels of TCR expressed on mature thymocytes were also influenced by CD4-Ia interactions that had occurred on their CD4+CD8+ precursors. To do so, we examined TCR expression on mature CD8+ T cells from animals in which CD4-ligand interactions had been disrupted experimentally by in vivo administration of anti-CD4 mAb or by targeted disruption of an MHC class II gene. We found that TCR expression was significantly diminished on all mature CD8+ T cells from both anti-CD4 mAb-treated mice and MHC class II-deficient animals. These results demonstrate that TCR expression by mature CD8+ T cells, as well as that of immature CD4+CD8+ thymocytes, is regulated by CD4-mediated signals acting on CD4+CD8+ thymocytes. Because the effects of disrupting CD4-Ia interactions on TCR expression by CD8+ T cells were independent of TCR specificity, these findings directly support the concept that CD4-CD8+ T cells arise from precursor CD4+CD8+ cells.

Programmed differentiation of murine thymocytes during fetal thymus organ culture.

Fetal thymus organ culture (FTOC) has become widely used to investigate the impact of immunomodulators on T cell development. However, these studies have given variable results among different laboratories. In this study, we have found that fetal tissue age and mouse strain differences can affect the development of T cell phenotypes in this system. T cell development in FTOC occurred in two 'waves', defined as peaks of cell recovery. The first wave consisted initially of CD4-CD8- double negative (DN) cells and CD4-CD8+ single positive (SP) T cells expressing gamma delta T cell receptor (TCR). CD4+CD8+ double positive (DP) cells expressing low levels of alpha beta TCR were produced soon thereafter; and these cells dominated the cultures for the balance of the first wave. Prolonged FTOC resulted in the production of another wave of T cells which were relatively enriched for CD4 or CD8 SP cells expressing high levels of alpha beta TCR, as well as DN cells and CD4-CD8+ SP T cells expressing high levels of gamma delta TCR. As defined by cell number and differentiation of alpha beta TCR SP cells, development was delayed in FTOC using fetal thymus tissue from younger fetuses relative to that observed when older fetal thymus tissue was used. The degree of development of T cells in FTOC was also strain dependent. Organ cultures derived from 14 gestation days (gd) C.B-17 scid/scid fetal thymus did not generate TCR-bearing mature SP cells, but they did produce TCR-negative CD4 and CD8 SP cells likely to be precursors of DP thymocytes. Such cultures made from 18 gd tissue did not produce SP cells. Negative selection in FTOC was also evaluated. Mtv-specific V beta 3 cells were deleted in FTOC of C3H/HeN tissue. Deletion occurred only in late FTOC, suggesting a late encounter between the Mtv deleting elements and susceptible T cells during ontogeny. These results show that while FTOC recapitulates normal thymic development by a variety of criteria, results can be influenced by the length of culture, as well as by the age and strain of fetal thymus tissue utilized.

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.

Engagement of the external domains of CD45 tyrosine phosphatase can regulate the differentiation of immature CD4+CD8+ thymocytes into mature T cells.

Immature precursor cells are induced in the thymus to express clonotypic T-cell antigen receptors (TCRs) and to differentiate into mature T cells. Perhaps the least understood event which occurs during intrathymic development is the positive selection of immature CD4+CD8+ thymocytes for differentiation into mature CD4+ and CD8+ T cells based on the TCR specificity individual thymocytes express. TCR expression by CD4+CD8+ thymocytes is quantitatively regulated by CD4-mediated activation of p56lck protein-tyrosine kinase whose activity can in turn be regulated by the membrane-bound protein-tyrosine-phosphatase CD45. Here we show that antibody engagement of CD45 external domains enhances Lck tyrosine kinase activity in CD4+CD8+ thymocytes, inhibits TCR expression, and inhibits differentiation of immature CD4+CD8+ thymocytes into mature T cells. Thus, engagement of the external domains of CD45 tyrosine phosphatase can regulate the ability of immature CD4+CD8+ thymocytes to undergo positive selection, suggesting an important regulatory role for intrathymic ligands that are capable of engaging CD45 within the thymus.

Quantitative differences in cell surface expression of class I MHC antigens on murine epidermal Langerhans cells.

Epidermal Langerhans cells are derived from cells of bone marrow origin and, as the primary APC population in the skin, are responsible for initiation of many immune responses. Consequently, cell surface expression of MHC Ags by Langerhans cells is central to their function. Although murine Langerhans cells express class II MHC Ags at high levels, their level of expression of class I MHC has been controversial. In this study, cell surface expression of multiple individual class I MHC Ags on murine epidermal Langerhans cells was analyzed using quantitative immunofluorescence. It was found that Langerhans cells differentially express products of distinct class I genes. Langerhans cells expressed low cell surface amounts of H-2K and Qa-2, whereas expression of surface H-2D and H-2L by the same cells was high. Murine epidermal Langerhans cells therefore express low cell surface amounts of some but not all class I MHC Ags. Differential surface expression of products of distinct class I MHC genes by Langerhans cells may have a profound effect on cutaneous immune responses.

Expression of the homotypic adhesion molecule E-cadherin by immature murine thymocytes and thymic epithelial cells.

Cadherins mediate homotypic adhesion between lineage-related cells in epithelia and other tissues. One cadherin, E-cadherin, is also responsible for adhesion of murine epidermal Langerhans cells to keratinocytes in vitro, and may play a role in the localization of Langerhans cells in epidermis. The thymus is another tissue in which important adhesive interactions between bone marrow-derived cells and keratinizing epithelia occur. To determine whether cadherins might be involved in interactions between thymocytes and thymic epithelial cells, we examined thymocytes from C57BL/6 mice of various gestational ages for cadherin expression. Most day 14 (D14) and essentially all D16 isolated fetal thymocytes expressed cell surface E-cadherin. After D16, the proportion of fetal thymocytes expressing E-cadherin and the level of E-cadherin expressed by individual thymocytes decreased with increasing gestational age. A minority of neonatal thymocytes and very few adult thymocytes expressed E-cadherin. E-cadherin was maximally expressed by the least mature (CD4-CD8-, HSA (J11d)high, CD5 (Ly-1)low, CD25 (IL-2R alpha)+) thymocytes. P-cadherin, another epithelial cadherin, was not detected on thymocytes at any stage of development. Immunohistologic studies revealed that thymic epithelial cells also expressed E-cadherin. Similar levels of E-cadherin were expressed by neonatal and adult thymic epithelial cells in situ, and E-cadherin was easily demonstrable on the thymic epithelial cell line, TE-71. In contrast, P-cadherin was transiently expressed by thymic epithelial cells in situ, and only small amounts of P-cadherin were detected on TE-71 cells. These studies demonstrate that thymocytes and thymic epithelial cells each have the capacity to express the homotypic adhesion molecule E-cadherin. E-cadherin may play a role in developmentally regulated interactions between early thymocytes and thymic stromal cells.

Negative selection of CD4+CD8+ thymocytes by T cell receptor-induced apoptosis requires a costimulatory signal that can be provided by CD28.

CD4+CD8+ thymocytes expressing self-reactive T cell antigen receptors (TCR) are deleted in the thymus as a consequence of TCR/self-antigen/major histocompatibility complex interactions. However, the signals that are necessary to initiate clonal deletion have not yet been clarified. Here we demonstrate that TCR engagement does not efficiently induce apoptosis of CD4+CD8+ thymocytes, although it generates signals that increase expression of CD5, a thymocyte differentiation marker. In fact, TCR signals fail to induce thymocyte apoptosis even when augmented by simultaneous engagement with CD4 or lymphocyte function 1-associated molecules. In marked contrast, signals generated by engagement of both TCR and the costimulatory molecule CD28 potently induce apoptosis of CD4+CD8+ thymocytes. Thus, the present results define a requirement for both TCR and costimulatory signals for thymocyte apoptosis and identify CD28 as one molecule that is capable of providing the necessary costimulus. These results provide a molecular basis for differences among cell types in their ability to mediate negative selection of developing thymocytes.

Murine dendritic epidermal T cells (DETC) express the homophilic adhesion molecule E-cadherin.

Cadherins mediate homotypic intercellular adhesion in epidermis and other epithelia. E-cadherin is also involved in interactions between murine epidermal Langerhans cells (LC) and keratinocytes (KC). Dendritic epidermal T cells (DETC) comprise another subpopulation of epidermal leukocytes. Using flow cytometry, we determined that DETC expressed levels of E-cadherin similar to those expressed by LC and KC. DETC also adhered congruent to three-fold better to KC and E-cadherin-transfected fibroblasts than to normal fibroblasts. Treatment of DETC with trypsin in the absence of calcium caused a loss of E-cadherin and resulted in an congruent to 80% decrease in DETC-KC adhesion whereas treatment of DETC with trypsin in the presence of calcium did not significantly affect E-cadherin expression or DETC-KC binding. Thus, E-cadherin may be involved in adhesion of DETC to KC. DETC are derived from TCR V gamma 3+ thymocytes that transiently populate embryonic murine thymus. We determined that TCR V gamma 3+ thymocytes as well as other early (fetal day 16) TCR gamma/delta+ thymocytes expressed E-cadherin; TCR gamma/delta+ (TCR V gamma 3-) thymocytes that developed later did not. These results indicate that cells of the T cell lineage can express E-cadherin, and suggest that E-cadherin may play a role in adhesion of DETC (and/or DETC precursors) to KC.

T cell receptor V alpha-V beta combinatorial selection in the expressed T cell repertoire.

This study has evaluated whether preferential pairing occurs between TCR alpha- and beta-chains expressing specific V alpha and V beta gene products in the mature peripheral T cell population, as a result of either thymic selection or of structural constraints on chain pairing. The association of specific V alpha products with specific V beta products on individual T cells was found, in multiple instances, to be highly selective. Moreover, patterns of preferential V alpha-V beta association were highly strain-specific and were independently expressed in CD4+ and CD8+ T cell subsets. Although these findings do not exclude the possibility that structural constraints may limit V alpha-V beta pairing in other instances, they indicate that the observed instances of skewed expression are not caused by structural constraints in chain pairing. Rather, they suggest that strain-specific selective events alter the expressed V alpha V beta repertoire as a result of recognition of self or environmental Ag during T cell repertoire selection.