Characterization of CD4−CD8− T cell receptor αβ+ T cells appearing in the subarachnoid space of rats with autoimmune encephalomyelitis

Inflammation of the central nervous system (CNS) in experimental autoimmune encephalomyelitis (EAE) starts in the subarachnoid space (SAS) and spreads later to the adjacent CNS parenchyma. To characterize the nature of lesion-forming T cells in situ in more detail, T cells were isolated from the SAS and their surface phenotype and the nucleotide sequence of the junctional region of the T cell receptor (TCR) was determined and compared with those of the lymph node (LN) and spinal cord (SC) T cells. Characteristically, more than 70% of SAS TCR αβ⁺ T cells isolated at the early stage of EAE lacked both CD4 and CD8 molecules, whereas those from LN and SC were either CD4⁺ or CD8⁺. Analysis of nucleotide sequences of the junctional region of TCR revealed that T cells bearing a sequence identical to that for encephalitogenic T cell clones were found in both SAS and SC. Furthermore, purified CD4^?CD8^? T cells expressed CD4 molecules after culture. At the same time, these T cells acquired reactivity to myelin basic protein and induced passive EAE in naive animals after adoptive transfer. Our results suggest that CD4^?CD8^? T cells in the SAS are precursors of lesion-forming T cells in the SC and that phenotype switching takes place during the process of T cell infiltration into the CNS parenchyma. The double-negative nature of these T cells may explain an escape of encephalitogenic T cells from negative selection in T cell differentiation.     

Frequent occurrence of in vivo clonal expansion of CD4− CD8− T cells bearing T cell receptor αβ chains in adult humans

We have previously reported 2 cases of healthy men showing in vivo monoclonal expansion of mature CD4^? CD8^? αβ T cells. In the present study, an additional 3 adults were found to exhibit such an expansion, among a total 464 adult donors studied. These 5 individuals were otherwise physiologically normal, with no history of severe illness and autoimmune disease at the time of examination. To investigate the mechanisms of the clonal expansion, further characterization of the clonal cells was attempted. No apparent preference for usage of the Tcell receptor β chain variable region was observed in the clonal T cells. These clonal T cells showed lectin-dependent or redirected antibody-dependent cell-mediated cytotoxicities, whereas they could not lyse autologous lymphoblastoid cell lines. Failure of Fas antigen expression was not observed for any of these clones. These results suggest that clonal expansion of CD4^? CD8^? αβ T cells frequently occurs in the periphery without any T cell abnormalities.     

Early increase of CD4+ CD45RA+ and CD4+ CD95− cells with conserved repertoire induced by anti-retroviral therapy in HIV-infected patients

Administration of anti-retroviral drugs induces a decrease of viral load associated with increase of CD4⁺ cell count in most HIV-infected patients. To investigate the early changes in CD4⁺ cell phenotype induced by anti-retroviral therapy, six patients with CD4⁺ cell count > 100/mm³ and never treated with anti-HIV therapy were enrolled and blood samples collected several times within 14 days from the initiation of therapy with Zidovudine plus Didanosine. CD4⁺ cell count and HIV viraemia were investigated at each time point, as well as the expression of CD45RA, CD45RO and CD95/Fas molecules on CD4⁺ cells, and the T cell receptor (TCR) Vβ repertoire of CD4⁺ cells. All patients showed a rapid and dramatic decrease in viral load with a corresponding increase of CD4⁺ cell count. The main remodelling of CD4⁺ cell subpopulations took place in the first 14 days of therapy, and consisted of: (i) increased CD4⁺ CD45RA⁺/CD4⁺ CD45RO⁺ ratio; (ii) decrease of CD95/Fas expression. The rise in absolute number of CD4⁺ CD45RA⁺ cells was paralleled by an increase of CD4⁺ CD95/Fas^? cells and accounted for most of the early increment of CD4⁺ cell count. The TCR Vβ repertoire of CD4⁺ cells was conserved after anti-HIV therapy, with the exception of two patients with expanded CD4⁺ Vβ12⁺ cells, which also tested CD45RA⁺ and CD95/Fas^?. These experiments show that newcomer CD4⁺ lymphocytes are CD45RA⁺ CD95/Fas^? cells, suggesting that blocking HIV replication causes an early and antigen-independent proliferation of possibly ‘naive’ cells unprimed for CD95/Fas-mediated apoptosis. These cells expressed a conserved and widespread TCR repertoire, suggesting that their capability for antigenic recognition is intact.     

Thymus-derived cytokine(s) including interleukin-7 induce increase of T cell receptor α/β+ CD4−CD8− T cells which are extrathymically differentiated in athymic nude mice

Extrathymic T cell differentiation pathways have been reported, although the thymus is the main site of T cell differentiation. The thymus is also known to produce several cytokines that induce proliferation of thymocytes. In the present study, we investigated the influence of thymus-derived cytokines on extrathymic T cell differentiation by intraperitoneal implantation with a diffusion chamber which encloses fetal thymus (we named it fetal thymus-enclosed diffusion chamber, FTEDC) in athymic BALB/c nu/nu mice. Increase in number of T cells bearing T cell receptor (TcR) α/β was detected in lymph nodes and spleens of FTEDC-implanted nude mice 1 week after implantation, whereas no such increase was detected in control nude mice implanted with a diffusion chamber without thymus. The FTEDC-induced increase of T cells was suppressed by intraperitoneal injection of anti-interleukin-7 monoclonal antibody (mAb). The TcR α/β T cells in FTEDC-inplanted BALB/c nu/nu mice preferentially expressed Vβ11, although Vβ11-positive T cells are deleted in the thymus of euthymic BALB/c mice by clonal elimination of self-superantigen Dvb 11-specific T cells. TcR α/β T cells in FTEDC-implanted nude mice were of CD4^?CD8^? phenotype and showed no proliferative response against anti-TcR monoclonal antibody stimulation. These results suggest that the thymus can induce extrathymic T cell differentiation through the influence of thymus-derived cytokine(s) including interleukin-7, and that such extrathymically differentiated T cells have acquired only a little or no ability for proliferation when they recognize antigen by their TcR.     

Antigen dose‐dependent suppression of murine IgE responses is mediated by CD4−CD8− double‐negative T cells

Background The IgE response against protein antigens is profoundly influenced by the dose used for sensitization. Objective The aim of the study was to identify immune cells that are involved in antigen dose‐dependent regulation of IgE formation. Methods Wild‐type mice as well as T helper (Th)1‐deficient IL‐12p40^?/? and IFN‐γ^?/? mice were immunized by repeated intraperitoneal injection of either low doses (K01 mice) or high doses (K100 mice) of keyhole limpet haemocyanin adsorbed to aluminium hydroxide. Splenocytes of immunized mice were restimulated in vitro and antigen‐dependent T cell proliferation and cytokine production were measured. The frequency of regulatory T cell subsets among splenocytes from K01 and K100 mice was compared using fluorocytometry and RT‐PCR analysis. Splenocytes or T cell subpopulations were transferred into naïve mice and the effect of lymphocyte transfer on IgE production after priming of recipients with low antigen doses was determined. Results Specific IgE production was considerably impaired in K100 mice. Antigenic restimulation revealed hypoproliferation of K100 splenocytes and reduced production of Th2 cytokines IL‐4, IL‐5 and IL‐13, but no induction of IFN‐γ production. Moreover, lymphocytes from K01 and K100 mice did not show significant differences in the expression of molecules associated with the phenotype or activity of conventional regulatory T cells. Transfer of splenocytes or purified T cells from K100 mice substantially suppressed the induction of IgE production in the recipients in an antigen‐ and isotype‐specific manner. Neither CD4⁺ nor CD8⁺ T cells from K100 mice were able to inhibit IgE formation; instead, we identified CD4^?CD8^? double‐negative T cells (dnT cells) as the principal T cell population, which potently suppressed IgE production. Conclusion Our data demonstrate that CD4^?CD8^? dnT cells play a major role in the regulation of IgE responses induced by high antigen doses.     

CD57+ T lymphocytes are derived from CD57− precursors by differentiation occurring in late immune responses

CD3⁺ T cells expressing the 110-kDa CD57 antigen are found in survivors of renal, cardiac and bone marrow transplants, in patients with acquired immune deficiency syndrome and in patients with rheumatoid arthritis. They are also present in normal individuals and expand upon ageing. They do not grow in culture and their role in the immune response is poorly understood. The expression of the various isoforms of the leukocyte common antigen (CD45) identifies a spectrum of differentiation in CD4⁺ and CD8⁺ T cells ranging from naive (CD45RA⁺CD45RB^brightCD45RO^?) through early primed cells (CD45RA^?RB^brightRO^dull) to highly differentiated memory cells which are CD45RA^?RB^dullRO^bright. CD45 isoforms expressed by CD57⁺ T cells showed distinct differences between CD4⁺ and CD8⁺ populations, but in each case indicated an advanced state of differentiation. The expression of T cell receptor Vβ families was highly variable between individuals, but both CD57⁺ and CD57^? cells show a full range of the specificities tested. Vβ expression was more closely related within either the CD4⁺ or the CD8⁺ subsets, irrespective of CD57 expression, than between these subsets, suggesting a relationship between CD57⁺ and CD57^? cells within the same T cell pool. This possibility was supported by experiments showing that CD3⁺CD57⁺ lymphocytes were similar to CD3⁺CD57^? T cells in terms of the production of basic T cell cytokines [interleukin (IL)-2, IL-4, and interferon-γ]. Furthermore, in vitro stimulation of CD3⁺CD57^? T cells in secondary mixed leukocyte reaction or by co-culture with IL-2 and IL-4 induced the appearance of CD3⁺CD57⁺ cells with phenotypic and functional similarities to in vivo CD3⁺CD57⁺ cells. These data strongly suggest that the expression of CD57 is a differentiation event which occurs on CD57^? T cells late in the immune response.     

The role of the interleukin-2 (IL-2)/IL-2 receptor pathway in MRL/lpr lymphadenopathy: The expanded CD4−8− T cell subset completely lacks functional IL-2 receptors

Autoimmune MRL/MP-lpr/lpr (MRL/lpr) mice spontaneously develop a systemic lupus erythematosus-like disease accompanied by a profound lymphadenopathy that consists of CD4^?8^?B220⁺ a P T cells. By the use of cross-linking experiments with radiolabeled interleukin-2 (IL-2), these abnormal T cells have been reported to constitutively express the IL-2 receptor β chain (IL-2Rα), a signal transducing component of IL-2R, in the absence of the a chain (IL-2Rα).To critically reevaluate the role of the IL-2/IL-2R pathway in the pathogenesis of lymphadenophathy we examined expression of the IL-2Rα and IL-2Rβ in MRL/lpr mice by ¹²⁵I-IL-2 binding analysis and also by flow cytometric analysis using monoclonal antibodies against each component of the receptor. We found that, contrary to the previous report, the CD4^?8^?B220⁺ α β T cells in lymph node (LN) of MRL/lpr mice were negative for both IL-2Rα and IL-2Rβ expression. The lpr liver CD4^?8^?B220⁺ a P T cells that had been implicated in the genesis of these abnormal LN T cells were also negative for IL-2Rβ expression. Therefore, our results indicate that the IL-2/IL-2R system plays little role, if any, in the expansion of abnormal CD4^?8^? B220⁺ α β T cells in MRL/lpr mice.     

In vitro responses of human CD45R0brightRA− and CD45R0−RAbright T cell subsets and their relationship to memory and naive T cells

The cellular basis of immunological memory, particularly with respect to T cells is not understood. In humans, monoclonal antibodies to CD45 have been used to identify memory (CD45R0) and naive (CD45RA) T cells. However, this identification has been called into question by various studies which suggest that high molecular weight CD45 isoforms may be re-expressed by previously activated cells. In the present study, using cultures which supported responses of naive T cells, we examined the responses of purified CD45R0^brightRA^? or CD45R0^?-RA^bright T cell subsets. The former subset was found to respond preferentially to recall antigens with minimal responses apparent to neo-(or non-recall)-antigens. The inverse pattern was found for CD45R0^?RA^bright T cells, which converted to CD45R0^brightRA^? after stimulation with a neo-antigen. Moreover, the two populations of T cells exhibited distinct response kinetics with a faster response evident from the CD45R0^brightRA^?T cells compared to the CD45R0^?RA^bright subset. The poor responses of CD45R0^?RA^bright T cells to recall antigens compared to neo-antigens suggests that this putative naive population is specifically depleted of reactive T cells following an encounter with antigen. We propose that T cell priming results in the stimulation of many CD45R0^?RA^bright T cells with various T cell receptor specificities from which memory T cells are selected for survival. If re-expression of higher molecular weight isoforms does occur in humans in vivo, our results suggest that R0 expression would be retained (CD45R0⁺RA⁺). Alternatively, if primed CD45R0^?RA^bright T cells exist, they are not prevalent in peripheral blood and thus may be sequestered within lymphoid tissues. Our data support the view that in human peripheral blood, CD45R0^bright and CD45RA^bright expression identify memory and naive CD4⁺ T cells, respectively.     

CD4-negative cytotoxic T cells with a T cell receptor α/βintermediate expression in CD8-deficient mice

Targeted disruption of the CD8 gene results in a profound block in cytotoxic T cell (CTL) development. Since CTL are major histocompatibility complex (MHC) class I restricted, we addressed the question of whether CD8–/– mice can reject MHC class I-disparate allografts. Studies have previously shown that skin allografts are rejected exclusively by T cells. We therefore used the skin allograft model to answer our question and grafted CD8–/– mice with skins from allogeneic mice deficient in MHC class II or in MHC class I (MHC-I or MHC-II-disparate, respectively). CD8–/– mice rejected MHC-I-disparate skin rapidly even if they were depleted of CD4⁺ cells in vivo (and were thus deficient in CD4⁺ and CD8⁺ T cells). By contrast, CD8+/+ controls depleted of CD4⁺ and CD8⁺ T cells in vivo accepted the MHC-I-disparate skin. Following MHC-I, but not MHC-II stimulation, allograft-specific cytotoxic activity was detected in CD8–/– mice even after CD4 depletion. A population expanded in both the lymph nodes and the thymus of grafted CD8–/– animals which displayed a CD4^?8^?3^intermediateTCRα/β^intermediate phenotype. Indeed its T cell receptor (TCR) density was lower than that of CD4⁺ cells in CD8–/– mice or of CD8⁺ cells in CD8+/+ mice. Our data suggest that this CD4^?8^?T cell population is responsible for the CTL function we have observed. Therefore, MHC class I-restricted CTL can be generated in CD8–/– mice following priming with MHC class I antigens in vivo. The data also suggest that CD8 is needed to up-regulate TCR density during thymic maturation. Thus, although CD8 plays a major role in the generation of CTL, it is not absolutely required.     

A human CD4 transgene rescues CD4−CD8+ cells in β2-microglobulin-deficient mice

The specificity of the αβ T cell receptor for class I or class II major histocompatibility complex (MHC) molecules determines whether a mature T cell will be of the CD4^?CD8⁺ or CD4⁺CD8^? phenotype, respectively. We show here that a human CD4 transgene can rescue a significant fraction of CD4^?CD8⁺ T cells in β2-microglobulin-deficient mice. Cells with this phenotype could be induced to become potent killers of targets expressing allogeneic MHC antigens, indicating that lineage commitment can precede the rescue of developing cells by the T cell receptor for antigen and the CD4 coreceptor.     

Neoplastic thymic epithelial cells of human thymoma support T cell development from CD4−CD8− cells to CD4+CD8+ cells in vitro

Human thymoma is a thymic epithelial cell tumour which often contains a large number of immature T cells and is frequently associated with autoimmune diseases. Since thymic epithelial cells play key roles in the development and selection of T cells in the normal thymus, we hypothesized that the neoplastic thymic epithelial cells of thymoma may support T cell differentiation in the tumour. We characterized CD4^?CD8^? cells in thymoma and applied an in vitro reconstitution culture system using the CD4^?CD8^? cells and the neoplastic epithelial cells isolated from thymoma. CD34, a stem cell marker, was expressed on 29.9 ± 12.2% of CD4^?CD8^? cells in thymoma. TCRγδ was expressed on 27.4 ± 15.1% of CD4^?CD8^? cells and CD19, a B cell marker, was expressed on 14.1 ± 23.1% of CD4^?CD8^? cells. CD4^?CD8^? cells expressed both IL-7R α-chain and common γ-chain. Purified CD4^?CD8^? cells from thymomas were cultured with the neoplastic epithelial cells, and their differentiation into CD4⁺CD8⁺ cells via CD4 single-positive intermediates was observed within 9 days' co-culture in the presence of recombinant IL-7. Furthermore, we examined the reconstitution culture using CD34⁺CD4^?CD8^? cells purified from normal infant thymus. The CD34⁺CD4^?CD8^? cells in normal thymus also differentiated to CD4⁺CD8⁺ cells in the allogeneic co-culture with the neoplastic epithelial cells of thymoma. These results indicate that the tumour cells of thymoma retain the function of thymic epithelial cells and can induce differentiation of T cells in thymoma.     

The expansion and selection of T cell receptor αβ intestinal intraepithelial T cell clones

In conventional mice, the T cell receptor (TCR)αβ⁺ CD8αα⁺ and CD8αβ⁺ subsets of the intestinal intraepithelial lymphocytes (IEL) constitute two subpopulations. Each comprise a few hundred clones expressing apparently random receptor repertoires which are different in individual genetically identical mice (Regnault, A., Cumano, A., Vassalli, P., Guy-Grand, D. and Kourilsky, P., J. Exp. Med. 1994. 180: 1345). We analyzed the repertoire diversity of sorted CD8αα and CD8αβ⁺ IEL populations from the small intestine of individual germ-free mice that contain ten times less TCRαβ⁺ T cells than conventional mice. The TCRβ repertoire of the CD8αα and the CD8αβ IEL populations of germ-free adult mice shows the same degree of oligoclonality as that of conventional mice. These results show that the intestinal microflora is not responsible for the repertoire oligoclonality of TCRαβ⁺ IEL. The presence of the microflora leads to an expansion of clones which arise independently of bacteria. To evaluate the degree of expansion of IEL clones in conventional mice, we went on to measure their clone sizes in vivo by quantitative PCR in the total and in adjacent sections of the small intestine of adult animals. We found that both the CD8αα and the CD8αβ TCRαβ IEL clones have a heterogeneous size pattern, with clones containing from 3 × 10³ cells up to 1.2 × 10⁶ cells, the clones being qualitatively and quantitatively different in individual mice. Cells from a given IEL clone are not evenly distributed throughout the length of the small intestine. The observation that the TCRαβ IEL populations comprise a few hundred clones of very heterogeneous size and distribution suggests that they arise from a limited number of precursors, which may be slowly but continuously renewed, and undergo extensive clonal expansion in the epithelium.     

A novel peripheral CD4+CD8+ T cell population: Inheritance of CD8α expression on CD4+ T cells

In this study we show the inheritance of a CD4⁺CD8⁺ peripheral T cell population in the H.B15 chicken strain. A large proportion of αβ T cells in peripheral blood (20–40%), spleen (10–20%) and intestinal epithelium (5–10%) co-express CD4 and CD8α, but not CD8β. CD4⁺ CD8αα cells are functionally normal T cells, since they proliferate in response to mitogens and signals delivered via the αβT cell receptor as well as via the CD28 co-receptor. These cells induce in vivo a graft versus host-reaction, providing further evidence for their function as CD4⁺ T cells. The CD4⁺CD8αα T cell population was found in 75% of the first progeny and in 100% of further progenies, demonstrating that co-expression of CD4 and CD8 on peripheral T cells is an inherited phenomenon. In addition, cross-breeding data suggest a dominant Mendelian form of inheritance. The hereditary expression of CD8α on peripheral CD4⁺ T cells in chicken provides a unique model in which to study the regulation of CD4 and CD8 expression.     

CD3-induced apoptosis of CD4+CD8+ thymocytes in the absence of clonotypic T cell antigen receptor

Clonal selection of T cells mediated through the T cell antigen receptor (TCR) mostly occurs at the CD4⁺CD8⁺ double positive thymocyte stage. Immature CD4⁺CD8⁺ thymocytes expressing self-reactive TCR are induced to die upon clonotypic engagement of TCR by self antigens. CD3 engagement by antibody of the surface TCR-CD3 complex is known to induce apoptosis of CD4⁺CD8⁺ thymocytes, a process that is generally thought to represent antigen-induced negative selection in the thymus. The present study shows that the CD3-induced apoptosis of CD4⁺CD8⁺ thymocytes can occur even in TCRα^? mutant mice which do not express the TCRαβ/CD3 antigen receptor. Anti-CD3 antibody induces death of CD4⁺CD8⁺ thymocytes in TCRα^? mice either in cell cultures or upon administration in vivo. Interestingly, most surface CD3 chains expressed on CD4⁺CD8⁺ thymocytes from TCRα^? mice are not associated with clonotypic TCR chains, including TCRβ. Thus, apoptosis of CD4⁺CD8⁺ thymocytes appear to be induced through the CD3 complex even in the absence of clonotypic antigen receptor chains. These results shed light on previously unknown functions of the clonotype-independent CD3 complex expressed on CD4⁺CD8⁺ thymocytes, and suggest its function as an apoptotic receptor inducing elimination of developing thymocytes.     

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 (CD4^0/0CD8^0/0). We show that mature T cells of CD4^0/0CD8^0/0 mice express an unbiased diverse TCR Vβ repertoire comparable to wild type mice. In addition, clonal deletion of mouse mammary tumor virus superantigen-reactive T cells did occur in CD4^0/0CD8^0/0 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.     

T cell development and repertoire of mice expressing a single T cell receptor α chain

We examined T cell development and T cell repertoire in transgenic mice expressing a single T cell receptor (TCR) α chain derived from the H-2D^b -lymphocytic choriomeningitis virus (LCMV)-specific cytolytic T lymphocyte (CTL) clone P14. To generate these α P14 mice, mice transgenic for the P14 TCR α chain were backcrossed to TCR α-deficient mice. Thymi from α P14 mice exhibited a marked decrease of mature CD4⁺8^? and CD8⁺4^? single-positive thymocytes comparable to thymi from TCR α-deficient mice. Correspondingly, the number of peripheral T cells was reduced in the CD4 (tenfold) and in the CD8 (twofold) subsets when compared to normal mice. T cells from α P14 mice generated a primary anti-LCMV CTL response when stimulated in vitro with LCMV in contrast to normal mice which require priming in vivo; elimination of LCMV in vivo was, however, not improved. Flow cytometric analysis of T cells with Vβ-specific antibodies showed a diverse endogenous TCR Vβ repertoire. Functional analysis of the T cell repertoire, however, revealed a strongly reduced (30-fold) allogeneic and the absence of a vesicular stomatitis virus-specific CTL response and an impaired ability to provide T cell help for antibody isotype switching. Thus, T cell selection in the thymus was impaired and the T cell repertoire was limited in mice expressing only one type of TCR α chain.     

Are primed CD4+ T lymphocytes different from unprimed cells?

Primed and unprimed lymphocytes are usually classified as separate subsets of cells, based on phenotypic and functional distinctions. In the case of CD4⁺ T lymphocytes, primed cells are thought to proliferate more vigorously, quickly and easily, and to release a different profile of cytokines, than their naive equivalent. However, most of these data were obtained from studies in which populations of lymphocytes were compared before and after antigenic stimulation, and therefore did not distinguish between the effects resulting from the clonal expansion of specific precursor cells within such populations and those due to cell differentiation per se. We have investigated the contribution of precursor cell frequency to some of the functional changes observed in populations of CD4⁺ T cells following antigenic stimulation, using approaches in which antigen-specific precursor frequencies are high in both primary and secondary stimulations: mixed leukocyte reaction responses and cells from αβ T cell receptor transgenic mice. Our data suggest that when equivalent numbers of antigen-specific naive and previously primed CD4⁺ responder T cells are compared, there is no difference in their potency to proliferate but only the previously activated subset can generate cytokines such as interferon-γ.