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.

A naturally occurring soluble isoform of murine Fas generated by alternative splicing.

We report a soluble isoform of mouse Fas, which is generated by alternative splicing of Fas mRNA to a newly identified exon located between exons 2 and 3 of the previously published Fas sequence. This splicing event creates a novel Fas transcript, Fas beta, with the potential to encode a truncated form of the extracellular domain, termed Fas B. In vitro, P815 mastocytoma cells transfected with Fas B become resistant to Fas ligand-induced apoptosis, and the resistance is mediated by a secreted product of the transfected cells. In vivo, Fas beta mRNA expression is correlated inversely with apoptosis among subsets of intrahepatic T lymphocytes, a cell population in which activation-induced T cell apoptosis occurs. We propose that Fas B is a new cytokine that acts physiologically to limit apoptosis induced by Fas ligand.

Multiple rearrangements in T cell receptor alpha chain genes maximize the production of useful thymocytes.

Peripheral T lymphocytes each express surface T cell receptor (TCR) alpha and beta chains of a single specificity. These are produced after random somatic rearrangements in TCR alpha and beta germline genes. Published model systems using mice expressing TCR alpha and/or beta chain transgenes have shown that allelic exclusion occurs conventionally for TCR-beta. TCR alpha chain expression, however, appears to be less strictly regulated, as endogenous TCR alpha chains are often found in association with transgenic TCR beta chains in TCR alpha/beta transgenic mice. This finding, coupled with the unique structure of the TCR alpha locus, has led to the suggestion that unlike TCR beta and immunoglobulin heavy chain genes, TCR alpha genes may make multiple rearrangements on each chromosome. In the current study, we demonstrate that the majority of TCR-, noncycling thymocytes spontaneously acquire surface expression of CD3/TCR. Further, we show that cultured immature thymocytes originally expressing specific TCR alpha and beta chains may lose surface expression of the original TCR alpha, but not beta chains. These data provide evidence that not only must multiple rearrangements occur, but that TCR alpha gene rearrangement continues even after surface expression of a TCR alpha/beta heterodimer, apparently until the recombination process is halted by positive selection, or the cell dies. Sequential rearrangement of TCR alpha chain genes facilitates enhanced production of useful thymocytes, by increasing the frequency of production of both in-frame rearrangements and positively selectable TCR alpha/beta heterodimers.

T cells with gamma/delta T cell receptors (TCR) of intestinal type are preferentially expanded in TCR-alpha-deficient lpr mice.

Fas-mediated apoptosis is essential for activation-induced cell death of alpha/beta T cells, but it is not clear what role, if any, it plays in regulating other components of the immune system. To study the role of Fas in gamma/delta T cell development, Fas-deficient lpr mice were bred with T cell receptor alpha gene-ablated (TCR-alpha-/-) mice to generate mice deficient in one or both genes. The TCR-alpha-/-, lpr/lpr mice had a nearly 10-fold increase in total lymph node cell (LNC) number compared with Fas-intact TCR-alpha-/- mice, because of expansion of TCR-gamma/delta+ and TCR-beta+ cells. In Fas-intact TCR-alpha-/- mice, approximately one third of the LNCs expressed TCR-gamma/delta. These were evenly divided between the CD4-, CD8-alpha+ and the CD4-, CD8- subsets, and rarely expressed the B220 epitope of CD45. In contrast, in TCR-alpha-/-, lpr/lpr mice, TCR-gamma/delta+ cells comprised half of the LNCs and were primarily CD4-, CD8-, and B220+. Moreover, Fas deficiency in TCR-alpha-/- mice caused a preferential expansion of gamma/delta T cells expressing variable region genes characteristic of intestinal intraepithelial lymphocytes. These results demonstrate a role for Fas in regulating the gamma/delta T cell contribution to peripheral lymph nodes. This mechanism may be most important in limiting the access of activated intestinal intraepithelial lymphocytes to the peripheral lymphoid system.

Transient restoration of gene rearrangement at multiple T cell receptor loci in gamma-irradiated scid mice.

The developmental arrest of thymocytes from scid mice, deficient in variable, (diversity), and joining, or V(D)J recombination, can be overcome by sublethal gamma-irradiation. Since previous studies focused on restoration of rearrangement of the T cell receptor (TCR) beta locus, productive rearrangement of which is selected for, we sought to examine to what extent locus specificity and cellular selection contributed to the observed effects. We report here that irradiation of newborn scid mice induces normal V-D-J rearrangements of the TCR delta locus, which like TCR beta, is also actively rearranged in CD(4-)CD(8-) (double negative) thymocytes. In contrast, no complete V-J alpha rearrangements were detected. Instead, we detected substantial levels of hairpin-terminated coding ends at the 5' end of the J alpha locus, demonstrating that TCR alpha rearrangements manifest the effects of the scid mutation. Irradiation, therefore, transiently compensates for the effects of the scid mutation in a locus-nonspecific manner in thymocytes, resulting in a burst of normal TCR beta and delta rearrangements. Irradiation also allows the development of cells that can initiate but fail to complete V(D)J recombination events at the TCR alpha locus, which is normally inaccessible in scid thymocytes.

Preferential expression of fibronectin receptors on immature thymocytes.

Fibronectin-adherent (FNR+) thymocytes are enriched for immature (CD4-8-) and large (CD4+8+) cells, and depleted of mature (CD4-8+ and CD4+8-) and nonmature small (CD4+8+) cells. Among purified CD4-8- thymocytes, cells with the surface marker J11d and the IL-2 receptor, which can give rise to all other thymocyte subsets, showed selective attachment to fibronectin. Analysis of FNR+ thymocytes showed that such cells are greatly enriched for cells in cycle. Additionally, FNR+ cells expressed low levels of T cell receptor. These results suggest a role for the fibronectin receptor during the early, proliferative phase of thymocyte differentiation. The data suggest that loss of the fibronectin receptor is a hallmark of cells that have become committed either to functional maturation or to programmed cell death.

CD4/CD8-negative T cells with alpha beta antigen receptors.

A minor population of alpha beta T cells expresses neither CD4 nor CD8. These cells are probably heterogeneous. They are widespread in anatomical distribution, express an abnormal T-cell repertoire largely unaffected by selective processes that act on CD4+ and CD8+ T cells, and exhibit both cytotoxic and Th-2-like functions. Their role in normal immune function is completely obscure, but they are active in murine and human autoimmune disease.

Cytotoxic T-cell response following mouse liver transplantation is independent of the initial site of T-cell priming.

BACKGROUND: Liver transplantation in the mouse results in systemic induction of tolerance. The underlying mechanisms may also account for the persistence of chronic liver infections. It has therefore been hypothesized that antigen (Ag) presentation within the liver by nonprofessional antigen-presenting cells (APC) leads to incomplete T-cell activation, ultimately resulting in tolerance induction. We tested this hypothesis in an orthotopic mouse liver transplantation model. METHODS: Mouse liver transplantation was used to manipulate antigen presentation in major histocompatibility complex (MHC)-disparate donor and recipient strains. The effect of restricted Ag presentation was studied using CD8+ T-cell receptor transgenic OT-I cells. Transgenic OT-I cells were activated by injection of their cognate peptide antigen SIINFEKL, which could be presented by the MHC class I of only one of the mouse strains. Depending on the strain combination, Ag presentation was restricted to either the transplanted liver itself, the recipient (excluding the transplanted liver), or systemically throughout the recipient. Extrahepatic Ag presentation by passenger leukocytes was eliminated by using donors of chimeric bone marrow. RESULTS: OT-I cells encountering antigen only in the transplanted liver were activated, underwent extensive proliferation, and developed effector functions, based on IFN-gamma production and in vivo cytotoxicity assays. This T-cell activation and differentiation within the liver was comparable to animals with systemic Ag presentation and to animals with absent hepatic-parenchymal Ag presentation. CONCLUSIONS: The restricted presentation of antigen in the liver showed no immunosuppressive effect on activation of CD8+ T cells. In contrast, the liver may be an excellent priming site for naive CD8+ T cells.

Death and destruction of activated T lymphocytes.

The massive clonal expansion that occurs during an antigen-specific immune response results in the flooding of immune organs with activated T lymphocytes. At the end of a specific response, the vast majority of these activated T cells are cleared from the immune system. The T cells receive signals through specific death receptors that are expressed as a result of activation. Death receptors transmit their apoptotic signals through the activation of caspases. Function of the death receptors is intimately linked to cell-cycle control, and many cell-cycle control proteins are caspase substrates. Among CD8+ T cells, apoptotic death occurs at a specific site, the sinusoids of the liver. The liver appears to contain a mechanism for the trapping and killing of activated T cells, rendering it an immunologically privileged site.

Computer-assisted analysis of in vivo cytotoxic T cell responses.

An approach has been devised for analysing data generated in the study of in vivo cytotoxic T cell responses. The method calculates the total number of lytic units generated in the peritoneal cavity, or in cultures of the lymph nodes draining the rear footpad, following antigenic stimulation. The assessment of group differences is facilitated, and the method lends itself to computerised and hence objective data analysis.

The activation state of human intrahepatic lymphocytes.

The immune tolerance induced by the liver as an allograft is difficult to reconcile with the evidence that the liver selectively accumulates activated T cells from the circulation. However, much of this information is based on murine liver lymphocytes that were isolated using enzymatic digestion. In the present study we made use of a novel resource, the lymphocytes isolated during the perfusion of living donor liver lobe prior to transplantation. These healthy human liver lymphocytes displayed surface markers indicating a high degree of activation of natural killer cells, CD56(+) T cells, CD4(+) T cells and CD8(+) T cells. These properties were independent of enzymatic treatment or the details of cell isolation. We conclude that the healthy human liver is a site of intense immunological activity.

Frequency of T-cell progenitors in nude mice.

The hypothesis that prothymocytes are distinct from and regulated independently of multilineage hemopoietic progenitors was tested by enumeration of these two cell populations in normal versus congenitally athymic (nude) mice. The absence of a thymus and of peripheral T cells in nude mice had no effect on the frequency of either multilineage progenitors (day 12 CFU-S) or prothymocytes (CFU-T), suggesting that there is no feedback regulation of CFU-T frequency. Thymus seeding from the bone marrow is therefore likely to be regulated by the availability of niches for prothymocyte maturation, rather than by feedback control of prothymocyte production.

Superantigen-driven peripheral deletion of T cells. Apoptosis occurs in cells that have lost the alpha/beta T cell receptor.

Injection of lymphoid cells expressing minor lymphocyte-stimulating antigen-1 (Mls-1a) induces tolerance to the superantigen, and partial deletion of Mls-1a-reactive T cells. We have identified a transient population of T cells that have lost the alpha/beta T cell receptor at the time when Mls-1a-reactive T cells start to disappear during the process of tolerance induction. Apoptosis was directly demonstrated in this TCR-alpha/beta negative T-cell population. This indicates a peripheral T-cell deletion pathway, characterized by TCR down-regulation, apoptosis, and clonal deletion. The consequence of Mls-1a-induced TCR down-regulation appears to be different in CD4+ cells and in CD8+ cells. Although most of the CD4+ cells that have lost TCR expressing alpha- and beta-chains appear to be undergoing apoptosis, many of their CD8+ counterparts may be able to re-express the Ag receptor. This argues for the involvement of coreceptors in the induction of apoptosis during peripheral deletion.

Differential regulation of cell cycle-related proteins by CD95 engagement in thymocytes and T cell leukemic cell line, Jurkat.

CD95 engagement results in apoptosis in thymocytes and in the Jurkat human leukemic T cell line. Biochemical analyses in CD95-engaged thymocytes and Jurkat cells revealed dysregulation of the G1/S cell cycle control point. Cyclin E was upregulated upon CD95 engagement, suggesting G1-to-S progression, but there was no upregulation of cyclin A. Instead, cyclin E was degraded by caspases. In addition, c-myc that normally acts on S-phase progression through the activation of cdc25A appeared to be involved in the inhibition of S-phase progression after CD95 ligation. This implies that G1-->S progression and apoptosis are intimately linked in cells undergoing CD95 ligation. Furthermore, our data suggest that CD95-induced apoptosis occurs at the G1/S phase transition. We therefore suggest that CD95 engagement not only triggers death signals but also affects the G1/S checkpoint.

Functional flexibility in T cells: independent regulation of CD4+ T cell proliferation and effector function in vivo.

Proliferation and differentiation of CD4+ T cells are often correlated, but it is not clear whether they are mechanistically linked. When antigen-specific T cells are present at high frequency in vivo, they all respond to antigenic peptide stimulation by expressing activation markers, but only a subset begins to proliferate. However, noncycling cells may synthesize the effector cytokine IFNgamma even though their cell cycle is blocked in G1. These data show that proliferation and effector function are not rigidly linked in T cells. Instead, CD4+ T cells have the flexibility to engage in or bypass clonal expansion based on the integration of multiple signals, including the frequency of other responding T cells.

Expression and functional significance of the J11d marker on mouse thymocytes.

Subpopulations of thymocytes have been characterized phenotypically and functionally in relation to their expression of the marker defined by the monoclonal antibody J11d. Cortical-type L3T4+, Lyt-2+ thymocytes are all J11d+. Thymocytes that share the phenotype L3T4-, Lyt-2+ with peripheral Lyt-2+ T cells contain a J11d+ and a J11d- subset. These J11d- cells behave like peripheral Lyt-2+ T cells in two functional assays: they form clonal growth bursts in response to immobilized antibody against the T cell antigen receptor, and they act as precursors of alloreactive cytotoxic T cells. The J11d+ cells are inert in both of these assays. In contrast, L3T4+, Lyt-2- thymocytes do not contain a J11d+ subset.

Neonatal, moribund and undead T cells: the role of the liver in T cell development.

T lymphocytes may be isolated from the mouse liver, and these cells have been interpreted as immature T cells in an extrathymic development pathway. However, the evidence in favor of this hypothesis is indirect, and there is some evidence that is contradictory. Most significantly, the genes RAG-1 and RAG-2, which are necessary for T cell receptor gene recombination to occur, are not expressed in liver T cells. A subset of liver T cells are undergoing apoptosis, which is the basis of an alternative model in which the liver is a site of peripheral T cell deletion. This model also provides an explanation for the accumulation of abnormal T cells in mice with the lpr mutation.

TCR ligation on CD8+ T cells creates double-negative cells in vivo.

The lack of CD95 in mice is associated with an accumulation of TCR alphabeta+ CD4- CD8- (double-negative (DN)) cells in the lymph nodes (LNs) and other organs. To test the hypothesis that these DN cells arise from TCR alphabeta+ CD8+ cells after activation via the TCR, we have crossed an MHC class I-restricted TCR transgene (tg) onto the lpr genotype to generate two TCR-transgenic experimental groups, TCRtg+ lpr/+ (CD95-intact) and TCRtg+ lpr/lpr (CD95-deficient). Specific peptide administration resulted in peripheral deletion of TCR alphabeta cells from the LNs of CD95-intact and CD95-deficient mice. On day 3 after peptide administration in the CD95-deficient but not the CD95-intact mice, there was a ninefold increase in the percentage of DN cells in the LN; this increase returned to control levels by day 10. Peripheral deletion was associated with an accumulation of TCR alphabeta+ CD8high cells in the livers of mice of both genotypes by day 3, which returned to control levels by day 10 without an increase in the percentage or total number of DN cells. Our data show that the in vivo stimulation of TCR alphabeta+ CD8+ cells in the absence of CD95 results in an initial accumulation and an eventual loss of DN cells. This identifies a role for CD95 after TCR alphabeta stimulation in the efficient removal of TCR alphabeta+ CD8+ cells after the down-regulation of CD8. CD95 is not essential for this process, because other mechanisms can compensate, but such mechanisms are less efficient in the LN.

Suppression induction in vivo by a T helper clone?

We have previously described a helper T cell clone which augments in vivo cytotoxic T cell responses when injected at 10(4) cells per mouse, but not at 10(5) per mouse (Crispe, I. N. et al., Immunology 1984. 52:55). To test whether this dose-response relationship was due to the induction of suppression, naive syngeneic mice were injected with 10(5) cloned T helper cells, and their spleen cells were subsequently assayed for suppressive activity in adoptive transfer experiments. Lymphocytes from such mice indeed suppressed an antigen-specific cytotoxic response, but only in the presence of the same T helper cell clone freshly added at the time of adoptive transfer. On this basis we argue that the distinction between T helper cell activity and T suppressor-inducer activity corresponds to differences in cell numbers, rather than to two separate cell lineages.