CD8 Accessory Molecules Research Articles

Donor bone marrow and transplantation tolerance: historical perspectives, molecular mechanisms and future directions (review).

The induction of transplantation tolerance, defined as the indefinite existence of an intact, functioning allograft, has been a goal of transplantation immunologists for decades because of the morbid effects of immunosuppressive drugs required to maintain graft function. One promising method of tolerance induction involves the peritransplant administration of cytoablative drugs followed by administration of a low dose of donor bone marrow cells. A subpopulation of donor bone marrow cells bearing the CD8 accessory molecule cause the functional deletion of graft reactive T cells via a CD95 dependent mechanism that also involves the cytokine TGFbeta. This interaction is bi-directional in which the bone marrow cell deletes the graft reactive T cell that recognizes it. Therefore, only T cells that recognize the donor antigens are deleted, leaving the immune response to other antigens intact. This protocol has been successfully used in rodents and an outbred large animal model. Clinical trials of donor bone marrow administration are now underway and initially indicate that administration of donor bone marrow is clinically safe.

The molecular mechanisms of veto mediated regulation of alloresponsiveness.

Transplantation tolerance, as defined by the specific acceptance of allogeneic tissue without chronic immunosuppression, can be induced in adults by a variety of methods. One method that is close to clinical implementation involves transient peritransplant depletion of host T cells followed by intravenous administration of a low dose of donor bone marrow cells (BMC). Current evidence suggests that BMC play an active role in tolerance induction by establishing residence in the host and effecting deletion of graft reactive T cells. This activity, in which the BMC inactivate T cells that recognize them, has been called the "veto effect," and cells that mediate such activity have been called "veto cells." The specificity of veto activity is conferred by the T cell receptor of the donor reactive T cells that are to be deleted. Other molecules that have been directly implicated in the mechanism of deletion of graft reactive T cells by BMC include the CD8 accessory molecule, major histocompatibility class I gene products, CD95/CD95 ligand (Fas/Fas ligand), and transforming growth factor-beta. These molecules react with their respective ligands by distinct, non-exclusive pathways among which the relative contribution to the deletion of graft reactive T cells may depend on the relative abundance of different cellular subpopulations capable of mediating veto activity.

CENTRAL DELETION OF GRAFT REACTIVE T CELLS IN DONOR BONE MARROW INDUCED TOLERANCE.

938 Current evidence suggests that tolerance induced by donor bone marrow administration to graft recipients treated with antithymocyte globulin (ATG) is mediated through peripheral deletion of graft reactive T cells by donor bone marrow cells (BMC). Methods: To test this hypothesis, we determined the fate of graft reactive T cells following bone marrow administration using a murine system in which the graft recipients are female C57BL/10 mice transgenic for T cell receptor (TCR) αβ chains that confer specificity for the male H-Y histocompatibility antigen in the context of Db. These mice were treated with ATG on days −1 and +2, and transplanted with male skin on Day 0. On day +7, 2.5 × 107 male BMC were administered i.v. The fate of male skin reactive T cells in thymus, lymph node, spleen, and blood was followed by flow cytometry using monoclonal antibodies specific for the transgenic TCR α and β chains, and for the CD8 or CD4 accessory molecules. Tissues were analyzed prior to treatment with ATG, and at 7, 14, 21, 32, and 42 days post BMC infusion. Results: Full recovery from ATG treatment of T cell populations in thymus and peripheral lymphoid tissues occurred by 32 days post-infusion. Recipients injected with syngeneic (female) BMC exhibited two subpopulations each of CD4+ and CD8 + T cells as defined by expression of transgenic (Tg) TCR α-chains at high (TCRBright) and low density, a profile that is identical to normal, untreated mice. Previous studies have shown that TCRBright Tg cells are the cells that are activated by the H-Y antigen in vitro. However, graft recipients treated with male (donor) BMC exhibited an absence of TCRBright CD8+ and CD4+ cells in the peripheral lymphoid organs, blood, and the thymus. This was accompanied by a marked downregulation of TCR expression such that the percentage CD8+ cells expressing the transgenic TCRα chain was diminished by >2-fold (See Table). Similar results were obtained for CD4+ T cells.Table: % of CD8+ Cells Bearing TCR Transgenes at 32 days Post-BMC Infusion While these results do not preclude the existence of peripheral deletion in this system, the lack of TCRBright CD8+ and CD4+ cells in the thymus of recipients injected with male BMC strongly suggests that thymic deletion of graft reactive cells also occurs, either through physical deletion or downregulation of the TCR

How do endogenous proteins become peptides and reach the endoplasmic reticulum.

T lymphocytes, via specific T cell receptors (TCR), recognize antigenic peptides bound to major histocompatibility complex (MHC)-encoded molecules. The recent crystallization of TCR-MHC complexes has enriched our understanding of this recognition process (Garciaet al. 1996a; Garbocziet al. 1996). Broadly speaking, there are two types of MHC molecules, i.e., classical and nonclassical molecules, and the majority of TCR recognize classical MHC molecules. Classical MHC molecules can be further divided into two types, i.e., class I and class II. CD8+ T cells, or cytotoxic T lymphocytes (CTL), recognize MHC class I molecules bound to peptides derived from endogenous proteins (i.e., proteins that are synthesized within the cell or artificially introduced directly into the cytoplasm or nucleus) that are primarily degraded in the cytoplasm. The CD8 accessory molecule present on most CTL appears to augment the formation of TCR-MHC/peptide complexes (Garcia et al. 1996b; Gao et al. 1997). In addition to presenting peptides to T cells, expression of MHC class I molecules protects cells from lysis by natural killer (NK) cells (Lanier 1997). On the other hand, CD4+ T lymphocytes or helper T lymphocytes recognize MHC class II molecules that bind peptides derived from exogenous or membrane proteins which enter the cell via endocytosis or phagocytosis and are primarily degraded by lysosomal proteases.

T cell repertoire and clonal deletion of Mtv superantigen‐reactive T cells in mice lacking CD4 and CD8 molecules

CD4-CD8- double-negative T cells constitute a lymphocyte subpopulation within the thymus and peripheral lymphatic organs that express a unique T cell receptor (TCR) repertoire and do not undergo negative selection. To test whether these cells develop as a distinct lineage or due to altered selection in the absence of CD4 and CD8 expression, we analyzed the TCR repertoire in mice lacking both CD4 and CD8 accessory molecules after homologous recombination (CD40/0CD80/0). We show that mature T cells of CD40/0CD80/0 mice express an unbiased diverse TCR V beta repertoire comparable to wild type mice. In addition, clonal deletion of mouse mammary tumor virus superantigen-reactive T cells did occur in CD40/0CD80/0 mice. These data show that the intrinsic lack of CD4 and CD8 expression has no effect on the mature TCR repertoire and that clonal deletion of superantigen-reactive cells is independent of CD4 and CD8 co-receptors.

Intestinal T cells in CD8 alpha knockout mice and T cell receptor transgenic mice.

Intraepithelial lymphocytes (IEL) refer to the T cells located at the epithelium of the intestines. Unlike the T cells in other peripheral lymphoid organs, the majority of IEL express the CD8 cell surface protein. To study the role of CD8 in the ontogeny and the function of IEL, phenotypic analysis of IEL from CD8 alpha knockout mice and normal mice was performed. The CD8 alpha gene in CD8 alpha knockout mice was disrupted by homologous recombination. These mice are defective in thymic maturation of cytotoxic T cells. In normal mice, alpha beta T cells that were CD8 alpha alpha+ or CD4+ CD8 alpha alpha+, and gamma delta T cells that were CD8 alpha alpha+, were the distinct populations found only in IEL. In CD8 alpha knockout mice, the population size of IEL remained normal, but the majority of IEL were CD4- CD8- T cells expressing alpha beta or gamma delta T cell receptors. IEL from the H-Y transgenic mice2, which express the male H-Y antigen specific T cell receptor in a normal and in a CD8 alpha-null background, were also studied. In contrast to thymic derived T cells, CD8 alpha alpha+ IEL with the autoreactive transgenic T cell receptor were not deleted, but clonally expanded in the male transgenic mice. Interestingly, no pathological symptoms were observed in the intestines of these mice. In the absence of CD8 alpha expression, the H-Y specific autoreactive IEL did not accumulate in the intestines. The results suggest that CD8 alpha alpha+ IEL are derived extra-thymically and their responses towards antigens require the CD8 accessory molecule.

Exogenous superantigens acutely trigger distinct levels of peripheral T cell tolerance/immunosuppression: dose-response relationship.

Ligand-specific immunosuppression requires an understanding of the parameters that control peripheral T cell tolerance. T cell receptor (TcR) transgenic mice offer a clear advantage for studying post-thymic tolerance mechanisms in vivo that are operational in a monoclonal T cell population with preselected antigen specificity. Yet it is unclear whether the rules defined in monoclonal T cells of genetically manipulated mice reflect those operative in clonally diverse peripheral T cells of normal mice. To analyze acute tolerance mechanisms in unselected peripheral T cells, we challenged normal mice with the superantigen staphylococcal enterotoxin B (SEB) and analyzed ligand-reactive V beta 8+ T cells for TcR-triggered tolerance mechanisms such as anergy, TcR down-regulation, or apoptosis. Upon challenge with graded doses of SEB (0.001-10 micrograms) V beta 8+ T cells become anergic within 6-16 h. Importantly, a dosage effect of SEB in regard to the level of anergy induced was observed. Anergy induced by low concentrations of SEB (0.001-0.1 microgram) is transient and is overcome by clonal growth, while higher concentrations of SEB (0.1-10 micrograms) cause long-lasting anergy resistant to cell cycle progression. At high SEB concentrations (1-10 mg) about 50% of the anergic V beta 8+ T cells additionally down-regulate their TcR-CD3 complex, followed by a loss of CD2, CD4, CD8 accessory molecules. In parallel, T cell phenotype-negative but genotypically V beta 8+ T cells are generated. The T cell phenotype-negative cells reacquire their V beta 8+ T cell phenotype upon culture in vitro. In vivo, a subset of V beta 8+ cells, defined by an intermediate stage of TcR down-regulation, i.e. V beta 8lowCD3+ cells, but not T cell phenotype-negative cells are selectively programmed for apoptosis, which occurs within 1 h. These data suggest that SEB triggers distinct tolerance pathways which operate in a hierarchical fashion in clonally diverse ligand-reactive T cells. Specifically, the results illustrate the power of exogenous superantigens to exploit these distinct tolerance pathways, thereby achieving distinct levels of immunosuppression.

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.

Central Role of CD4+ T Cells in Avian Immune Response

Chicken αβ T cells express either CD4 or CD8 accessory molecules, whereas most of the γδ T cells do not. The functional significance of the αβ T cells is relatively well understood. The CD4+ αβ T cells function as coordinators of the immune response, and CD8+ αβ T cells are the effector cells in cytotoxic responses, killing infected target cells. In comparison, the role of γδ T cells is so far poorly known. In chicken, the γδ T cells comprise a large lymphocyte subset. They can be induced to proliferate by various stimuli, but the proliferative response is dependent on CD4+ αβ T cells. The CD4+ T cells are also essential for the generation of antibody responses by providing help for the B cells and can influence cytotoxic responses as well. Thus, the CD4+ αβ T cells have a central role in the avian immune system, and their activation is a prerequisite for responses by other types of cells, including γδ T cells.

Intraepithelial lymphocytes and their recognition of non-classical MHC molecules.

Recent studies of the TCR alpha and beta chains expressed by normal human IELs suggest that these intestinal lymphocytes are directed at a limited set of antigens, presumably on intestinal epithelial cells in view of their anatomic location. The direct sequence analysis of these cells has indicated that they are oligoclonal and cannot, therefore, be responding to the complex mixture of antigens which are present in the lumen. The abundant expression of the CD8 accessory molecule by the IELs, in addition, indicates that these putative intestinal epithelial cell antigens are presented by MHC class I or I-like molecules. The expression of CD8 also suggests that these cells function biologically in part as cytolytic T lymphocytes which is consistent with a variety of functional studies. Taken together with their expression of the CD45RO isoform, these phenotypic and functional observations suggest that iIELs are cytolytic, memory cells which are responsive to an extremely limited number of antigens bound to major histocompatibility complex (MHC) class I or class I-like molecules. Several non-polymorphic MHC class I-like molecules such as Qa, the thymus leukemia antigen (TL) and CD1 in the mouse and CD1 in human represent important candidate ligands for these oligoclonal iIELs. TL and CD1 are expressed specifically by murine intestinal epithelial cells. In humans, CD1d is constitutively expressed by intestinal epithelial cells. In addition, we have isolated iIEL T cell clones which specifically recognize members of the CD1 gene family when expressed on a transfected B cell line that lacks HLA-A and B and have shown that the proliferation of peripheral blood T cells to intestinal epithelial cells is CD1d dependent. Thus, the evidence to date strongly implicate the nonpolymorphic, class Ib molecules as novel restriction elements for unique populations of lymphocytes within the intestinal epithelium.

Human intestinal intraepithelial lymphocytes are derived from a limited number of T cell clones that utilize multiple V beta T cell receptor genes.

Intestinal intraepithelial lymphocytes (IEL) are a phenotypically distinct T cell population of unknown function. The majority of human intestinal IEL express the TCR-alpha beta, the CD8 accessory molecule, and the CD45RO Ag, suggesting that they are MHC class I-restricted memory T cells. Recent analyses of the TCR alpha- and beta-chains expressed by these cells have shown marked skewing toward one or several V region genes in individual donors and revealed the presence of clonally expanded cells. In addition, functional data has suggested that the MHC class I-like CD1 molecules may be the target ligands for some human intestinal IEL clones. This report examines in detail the TCR-beta repertoire of human jejunal IEL to determine what fraction of these cells are clonally expanded and to determine whether a particular subset of V beta genes are utilized by the clonally expanded cells. The results demonstrate that the majority of IEL are derived from the expansion of a relatively few T cell clones and that these clones can utilize a large number of different V beta genes. Oligoclonal expansion is also demonstrated among lamina propria lymphocytes (LPL), with overlapping but distinct clones detected in the LPL vs the IEL populations. These results indicate that most intestinal IEL-alpha beta, and a subpopulation of LPL, are specific for a limited number of Ag and place constraints on the possible roles played by IEL in the defense against diverse environmental pathogens or in the generation of oral tolerance.

Increased percentage of CD3+, CD57+ lymphocytes in patients with rheumatoid arthritis. Correlation with duration of disease.

To determine whether a small CD3+ lymphocyte population expressing 110-kd CD57 antigens (HNK1) is expanded in patients with rheumatoid arthritis (RA), as it is in patients who have undergone bone marrow transplantation and patients with the acquired immunodeficiency syndrome, and to investigate whether it is involved in the pathogenesis of RA. The phenotype of CD3+, CD57+ lymphocytes was analyzed by flow cytometry, and correlations between the percentage of these cells in the blood and various clinical and biologic parameters were investigated. The percentage of CD3+, CD57+ lymphocytes was increased in RA patients compared with controls. These lymphocytes expressed T cell receptor alpha/beta. Eighty percent expressed the CD8 accessory molecule, and 20% expressed the CD4 accessory molecule. The leukocyte common antigen CD45RA isoform was expressed by these CD3+, CD57+ lymphocytes in blood. The HLA-DR antigen was expressed in synovial fluid but not in blood. Finally, the percentage of these lymphocytes in the blood correlated with the duration of RA. The expansion of the CD3+, CD57+ lymphocyte population and their activation in the synovial fluid of RA patients suggest that these cells are involved in the inflammatory process.

Contrasting effect of transforming growth factor type beta 1 (TGF-beta 1) on proliferation and interleukin-2 receptor expression in activated and rapidly cycling immature (CD3-CD4-CD8-) thymocytes.

Transforming growth factor beta (TGF-beta) is a cytokine with immunoregulatory properties that acts negatively on T lymphocyte proliferation. However, with the EL 4-6.1 variant of the murine thymoma EL 4 activated with phorbol ester and/or interleukin-1 (IL-1), we recently found that it up-regulates interleukin-2-receptor (IL-2R) expression. Since EL 4-6.1 cells share phenotypic and functional characteristics with the immature thymic subset lacking CD4 and CD8 accessory molecules (DN), we investigated the effect of TGF-beta 1 on the IL-2R 55kD alpha chain expression and proliferation of activated DN cells and especially in DN cells that do not express CD3. We observed that TGF-beta 1 was able to increase both the percentage of CD3-DN cells expressing IL-2R alpha chains and the expression of IL-2R alpha chain in these cells. This stimulatory effect of TGF-beta 1 was distal from early transduction events. In addition, TGF-beta 1 was found to modulate CD3-DN cell proliferation. During differentiation in the thymus, CD3-DN cells transiently express the IL-2R alpha chain of the IL-2R and these IL-2R+ CD3-DN cells are preprogrammed to down-regulate the IL-2R alpha chain and up-regulate the CD4 and CD8 accessory molecule. We thus also tested the effect of TGF-beta 1 on IL-2R alpha chain expression in these in vitro differentiating CD3-DN cells. We found that TGF-beta 1 neither significantly affected IL-2R expression nor changed CD4 or CD8 expression. Hence, in CD3-DN cells, the effect of TGF-beta 1 on IL-2R expression seems to be restricted to proliferating cells.

CD8 is needed for positive selection but differentially required for negative selection of T cells during thymic ontogeny.

During thymic development, immature thymocytes expressing major histocompatibility complex (MHC) class I-restricted T cell receptors (TcR) differentiate into CD8+ T cells with cytolytic functions. To evaluate the role of CD8 in positive and negative selection during thymic ontogeny, mice rendered CD8-null by gene targeting were bred with three lines of transgenic mice expressing unique MHC class I-restricted TcR. In all three instances CD8 was required for positive selection of MHC class I-restricted transgenic T cells. The efficiency of positive selection decreased in accordance with a reduced level of CD8 expression on thymocytes. Surprisingly, there was a differential requirement for CD8 expression in negative selection of MHC class I-restricted thymocytes, depending on the antigen specificity of TcR. These observations show that CD8 is essential for positive selection but is differentially required for negative selection of MHC class I-restricted T cells. Thus thymic selection, at least for negative selection, can occur in the absence of the CD8 accessory molecule.

Mechanisms of cyclophosphamide-induced tolerance to IE-encoded alloantigens--evidence of clonal deletion in MHC antigen-reactive cells for skin allograft rejection.

Transplantation tolerance across H-2D plus IE antigen barriers has been achieved when B10.Thy1.1 (Kb, IAb, IE-, Db; Thy1.1) mice were primed i.v. with 9 x 10(7) spleen cells plus 3 x 10(7) bone marrow cells from B10.A(5R) (Kb, IAb, IEb, Dd; Thy1.2) and treated i.p. with 200 mg/kg of cyclophosphamide (CP) two days later. The tolerant state was confirmed by prolonged acceptance of donor-type skin grafts, and in vitro unresponsiveness to donor antigens. From the early stage of tolerant state, V beta 11+ or V beta 5+ T cells expressing CD4 or CD8 accessory molecules were markedly decreased in the periphery of the tolerant mice. Moreover, neither CD4+CD8- nor CD4-CD8+ thymocytes bearing a high density of V beta 11 or V beta 5 were detected in the chimeric thymus. The intrathymic clonal deletion appeared to be maintained in some of the recipient mice even after the disappearance of detectable mixed chimerism in the late stage. These results suggest that the mechanisms of the CP-induced tolerance include the destruction of the IE (and probably H-2D) reactive T cells in the periphery followed by the intrathymic clonal deletion of T cells reactive against these antigens. These results directly show the strong correlation between transplantation tolerance to H-2 alloantigens and the disappearance of alloreactive T cells in both the periphery and thymus.

Expression of an unusual T cell receptor (TCR)-V beta repertoire by Ly-6C+ subpopulations of CD4+ and/or CD8+ thymocytes. Evidence for a developmental relationship between Ly-6C+ thymocytes and CD4-CD8-TCR-alpha beta+ thymocytes.

A novel thymocyte subpopulation expressing an unusual TCR repertoire was identified by high surface expression of the Ly-6C Ag. Ly-6C+ thymocytes were distributed among all four CD4/CD8 thymocyte subsets, and represented a readily identifiable subpopulation within each one. Ly-6C+ thymocytes express TCR-alpha beta, arise late in ontogeny, and appear in the CD4/CD8 developmental pathway after birth in a sequence that resembles that followed by conventional Ly-6C- cells during fetal ontogeny. Most interestingly, adult Ly-6C+ thymocytes express an unusual TCR-V beta repertoire that is identical to that expressed by CD4-CD8-TCR-alpha beta+ thymocytes in its overexpression of TCR-V beta 8 and in its expression of some potentially autoreactive TCR-V beta specificities. This unusual TCR-V beta repertoire was even expressed by Ly-6C+ thymocytes contained within the CD4+ CD8- 'single positive' thymocyte subset. Thus, expression of this unusual TCR-V beta repertoire is not limited to CD4-CD8-thymocytes, and is unlikely to be a consequence of their double negative phenotype. Rather, we think that Ly-6C+TCR-alpha beta+ thymocytes and CD4-CD8-TCR-alpha beta+ are developmentally interrelated, a conclusion supported by several lines of evidence including the selective failure of both Ly-6C+ and CD4-CD8-TCR-alpha beta+ thymocyte subsets to appear in TCR-beta transgenic mice. In contrast, peripheral Ly-6C+ T cells are developmentally distinct from Ly-6C+ thymocytes in that peripheral Ly-6C+ T cells expressed a conventional TCR-V beta repertoire and developed normally in TCR-beta transgenic mice in which Ly-6C+ thymocytes failed to arise. We conclude that: 1) expression of a skewed TCR-V beta repertoire is a characteristic of Ly-6C+TCR-alpha beta+ thymocytes as well as CD4-CD8-TCR-alpha beta+ thymocytes, and is not unique to thymocytes expressing neither CD4 nor CD8 accessory molecules; and 2) Ly-6C+ thymocytes are developmentally linked to CD4-CD8-TCR-alpha beta+ thymocytes, but not to Ly-6C+ peripheral T cells. We suggest that Ly-6C+TCR-alpha beta+ thymocytes are not the developmental precursors of Ly-6C+ peripheral T cells, but rather may be the developmental precursors of CD4-CD8-TCR-alpha beta+ thymocytes.

CD8 γδ cells: presence in the adult rat thymus and generation in vitro from CD4 −/CD8 thymocytes in the presence of interleukin 2

Three to fifteen percent of peripheral T cells in adults express the recently described γδ T-cell antigen receptor (TcR) heterodimer. A small subpopulation of γδ cells express the CD8 accessory molecule. In this study, we analyzed the potential of highly purified CD4 −/CD8 −, double negative (DN) rat precursor thymocytes to give rise to γδ cells. We observed that in the presence of interleukin 2 (IL-2) and concanavalin A (ConA), both DN and CD8 cells expressing the γδ TcR were generated in vitro. We then examined the rat thymus for these cells and confirmed the presence of a previously undescribed CD8 TcR-αβ − subset in the rat thymus, expressing high levels of TcR-γ and δ messages with no detectable TcR-α transcripts, similar to the cells generated in vitro in the presence of IL-2 and ConA.

CD4 and CD8 accessory molecules function through interactions with major histocompatibility complex molecules which are not directly associated with the T cell receptor‐antigen complex

Both the subset-specific, CD4 and CD8 T cell accessory molecules and the antigen-specific T cell receptor (TcR) interact with major histocompatibility complex (MHC) class I and class II molecules on the surface of antigen-presenting cells. We analyzed whether the CD4/CD8 molecules exert their accessory function through binding with the same MHC molecules which participate in the TcR-antigen-MHC complex. We utilized a CD4-, CD8-, class I-allospecific T cell hybridoma which functionally manifests both cytotoxic T lymphocyte (CTL) and T helper1 (Th1) phenotypes, and rendered it bispecific by transfecting it with genes encoding either a class II-restricted, 2,4,6-trinitrophenyl (TNP)-I-Ad-specific TcR or a non-MHC-restricted chimeric TcR, composed of a variable part of an anti-TNP antibody. Expression of either CD4 or CD8 transgenes in these hybridomas enhanced and augmented their reactivity towards the appropriate target cells regardless of the type of TcR-MHC interaction. Thus, class I-specific responses could be enhanced through CD4-class II interactions, and class II-restricted responses could be augmented through CD8-class I interactions. Furthermore, these accessory molecules also potentiated TNP-specific responses by the chimeric TcR which is MHC unrestricted. The accessory molecules facilitated both interleukin 2 (IL2) production and cytolytic activity by shortening the activation time and rendering the cells responsive to lower antigenic stimuli. The degree of activity of the T cell hybridomas correlated with the level of accessory molecule expression and was not related to the effector function mediated by the cells. Anti-CD4 or -CD8 antibodies completely inhibited the activity of transfectants expressing the corresponding accessory molecule, regardless of the MHC type of the TcR interaction. Such antibodies blocked direct TcR stimulation provided by either anti-T3/Ti antibodies or lectins, but could not inhibit the activation through agents that bypass the TcR such as phorbol 12-myristate 13-acetate plus ionophore. Taken together, these studies demonstrate that the CD8/CD4 molecules can exert their accessory function through interactions with MHC molecules which are not directly associated with the TcR-Ag-MHC complex, and that this accessory effect is associated with TcR-mediated triggering at an early stage of the signaling process and is not related to the effector mechanism assigned to the CD4 and CD8 T cell subsets.

The majority of CD4+8- thymocytes are functionally immature.

The thymus is the major site of T cell development and repertoire selection. During these processes, T cells segregate into two subsets that express either CD4 or CD8 accessory molecules, the phenotype of peripheral T cells. Analysis of CD4+8- thymocytes revealed that the majority of these cells express the heat-stable Ag (HSA) but not the nonclassical class I Ag, Qa-2. This HSA+, Qa-2- phenotype is similar to that of the less mature, CD4+8+ thymocytes. The remaining CD4+8- thymocytes possess the HSA-, Qa-2+ phenotype of peripheral T cells. To determine whether the Qa-2-, CD4+8- thymic subset is fully mature, we have analyzed the functional status of these CD4+8- subpopulations. The results indicate that only those thymocytes which express Qa-2 are fully responsive to anti-TCR stimulation in a manner analogous to peripheral T cells. The Qa-2- subset is nonresponsive to stimulation by anti-TCR antibodies that have been immobilized to plastic, even in the presence of lymphokines or syngeneic APC. This subset is, however, capable of proliferating to allogeneic cells or to anti-TCR on the surface of syngeneic APC, although not to the levels achieved by Qa-2+ thymocytes. Thus, the Qa-2- subset appears to require additional interactions which are not necessary for peripheral T cells or Qa-2+ thymocytes. Relevant to this issue, the Qa-2+ thymocyte subset does not appear until relatively late in development, and does not reach adult frequencies until several weeks after birth. These results would suggest that there is a progression from HSA+, Qa-2- to HSA-, Qa-2+ which parallels the maturation of functional responsiveness. These findings are important to understanding T cell selection since thymocytes with such a decreased responsiveness may have a differential capacity for tolerance induction. The results presented suggest that the bulk of CD4+8- thymocytes are not fully mature and that Qa-2 may serve as a marker for T cells with a more complete functional competence.

A major fraction of human intraepithelial lymphocytes simultaneously expresses the γ/δ T cell receptor, the CD8 accessory molecule and preferentially uses the Vδ1 gene segment

The frequency of T cell receptor (TcR) type and the variable gene segment expression in human intraepithelial lymphocytes (IEL) from the large intestinal mucosa were studied by flow cytometry and immunohistochemistry, and compared to those in peripheral blood lymphocytes (PBL) - or lamina propria lymphocytes (LPL). Employing anti-gamma/delta TcR and anti-alpha/beta TcR monoclonal antibodies (mAb), flow cytometric analysis revealed that a large fraction of IEL (37%) are gamma/delta T cells, whereas within LPL and PBL gamma/delta T cells comprise a minor population (4.6% and 3.8% respectively). At these sites the number of gamma/delta T cells labeled with anti-CD8 mAb were 58.3% (IEL), 43.3% (LPL) and 24.4% (PBL). In situ staining of serial sections of large intestine confirmed these results. Hence, these data suggest a selective accumulation of CD8+ gamma/delta T cells in the human epithelium of the large intestine. Furthermore, analysis of gamma/delta TcR bearing IEL+ disclosed a marked preponderance of cells using the V delta 1 gene segment, whereas gamma/delta TcR+ PBL preferentially express V delta 2. Strikingly, the majority of these V delta 1+ IEL bear the CD8 molecule on their surface. These results are taken as evidence for a selective localization of V delta 1+ CD8+ gamma/delta T cells in the epithelium of the large intestine.