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.     

αβ Lineage-committed thymocytes can be rescued by the γδ T cell receptor (TCR) in the absence of TCR β chain

Commitment of the αβ and γδ T cell lineages within the thymus has been studied in T cell receptor (TCR)-transgenic and TCR mutant murine strains. TCRγδ-transgenic or TCRβ knockout mice, both of which are unable to generate TCRαβ-positive T cells, develop phenotypically αβ-like thymocytes in significant proportions. We provide evidence that in the absence of functional TCRβ protein, the γδTCR can promote the development of αβ-like thymocytes, which, however, do not expand significantly and do not mature into γδ T cells. These results show that commitment to the αβ lineage can be determined independently of the isotype of the TCR, and suggest that αβ versus γδ T cell lineage commitment is principally regulated by mechanisms distinct from TCR-mediated selection. To accommodate our data and those reported previously on the effect of TCRγ and δ gene rearrangements on αβ T cell development, we propose a model in which lineage commitment occurs independently of TCR gene rearrangement.     

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.     

T cell receptor α gene rearrangement and transcription in adult thymic γδ cells

T cells belong to two separate lineages based on surface expression of αβ or γδ T cell receptors (TCR). Since during thymus development TCR β, γ, and δ genes rearrange before α genes, and γδ cells appear earlier than αβ cells, it has been assumed that αδ cells are devoid of TCR α rearrangements. We show here that this is not the case, since mature adult, but not fetal, thymic γδ cells undergo VJα rearrangements more frequently than immature αβ lineage thymic precursors. Sequence analysis shows VJα rearrangements in γδ cells to be mostly (70 %) nonproductive. Furthermore, VJα rearrangements in γδ cells are transcribed normally and, as shown by analysis of TCR β-/- mice, occur independently of productive VDJβ rearrangements. These data are interpreted in the context of a model in which precursors of αβ and γδ cells differ in their ability to express a functional pre-TCR complex.     

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.     

T lymphocyte development in p56lck deficient mice: allelic exclusion of the TcR β locus is incomplete but thymocyte development is not restored by TcR β or TcR αβ transgenes

The protein tyrosine kinase, p56^lck, is involved in signal transduction in mature T cells and in the molecular events controlling early thymocyte differentiation. Thymuses of mice deficient for p56^lck expression (p56^lck-/-) consist of immature CD4-CD8- double-negative (DN) and CD4⁺CD8⁺ double-positive (DP) thymocytes and are severely reduced in total cell number. In this report we have studied DN thymocytes from p56^lck-/- mice and found an increase in the proportion of the CD44^?CD25⁺ subset, suggesting that transit through this stage, which is known to require T cell receptor (TcR) β expression, may be delayed in the absence of p56^lck expression. In addition, the expression of a transgenic TcR β chain or TcR αβ pair did not restore thymic development in p56^lck-/- mice. However, in contrast to mice expressing a dominant negative isoform of p56^lck in which DP thymocytes do not develop, DP thymocytes still develop in nontransgenic and TcR transgenic p56^lck-/- mice. These results demonstrate that expansion of the DP subset is impaired in p56^lck-/- mice. In contrast, allelic exclusion is not severely compromised. Although there was an increase in the number of peripheral T cells expressing more than one Vβ chain in TcR transgenic p56^lck-/- mice, we found that inhibition of endogenous TcR β gene rearrangement was almost complete in thymocytes of Vβ transgenic p56^lck-/- mice and we could not detect any peripheral T cells that expressed more than one Vβ chain in non-transgenic p56^lck-/- mice.     

Evidence that productive rearrangements of TCR γ genes influence the commitment of progenitor cells to differentiate into αβ or γδ T cells

Two models have been considered to account for the differentiation of γδ and αβ T cells from a common hematopoietic progenitor cell. In one model, progenitor cells commit to a lineage before T cell receptor (TCR) rearrangement occurs. In the other model, progenitor cells first undergo rearrangement of TCRγ, δ, or both genes, and cells that succeed in generating a functional receptor commit to the γδ lineage, while those that do not proceed to attempt complete β and subsequently α gene rearrangements. A prediction of the latter model is that TCRγ rearrangements present in αβ T cells will be nonproductive. We tested this hypothesis by examining Vγ2-Jγ1Cγ1 rearrangements, which are commonly found in αβ T cells. The results indicate that Vγ2-Jγ1Cγ1 rearrangements in purified αβ T cell populations are almost all nonproductive. The low frequency of productive rearrangements of Vγ2 in αβ T cells is apparently not due to a property of the rearrangement machinery, because a transgenic rearrangement substrate, in which the Vγ2 gene harbored a frame-shift mutation that prevents expression at the protein level, was often rearranged in a productive configuration in αβ T cells. The results suggest that progenitor cells which undergo productive rearrangement of their endogenous Vγ2 gene are selectively excluded from the αβ T cell lineage.     

T cell receptor-γδ-expressing fetal mouse thymocytes are generated without T cell receptor Vβ selection

We investigated whether fetal mouse T cell receptor (TCR) γδ cells have been subjected to so-called TCRβ selection at the CD25 stage of thymus development. To this end, we carried out a comparative three-color flow microfluorimetric analysis of TCRβδ cells developing in the fetal, neonatal and adult thymus using monoclonal antibodies to CD2, CD8, CD24, CD25 and CD44. Day-15 fetal TCRγδ cells were CD2⁺, suggesting an origin at a post-CD25 stage. Molecular analysis of TCRβ rearrangements were also carried out. Thus, by semi-quantitative polymerase chain reaction (PCR) amplification of Vβ6 and Vβ8 to Jβ2 rearrangements day-15 fetal TCRγδ showed extensive TCRβ rearrangements, a finding confirmed by PCR amplification from single micromanipulated cells. Finally, sequencing analysis of 104 PCR-amplified TCR VDJβ2 fragments showed that the majority (58%) were rearranged out of frame. Taken together, these phenotypic and molecular analyses suggest that fetal TCRγδ cells have not been subject to TCRβ selection.     

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.     

T cell receptor β chain dimers on immature thymocytes from normal mice

During T cell development the T cell receptor (TCR) β chain is expressed before the TCRα chain. Experiments in TCRβ transgenic severe combined immune deficiency (SCID) mice have shown that the TCRβ protein can be expressed on the cell surface of immature thymocytes in the absence of the TCRα chain and that the TCRβ protein controls T cell development with regard to cell number, CD4/CD8 expression and allelic exclusion of the TCRβ chain. Subsequent experiments have shown that on the surface of thymocytes fromTCRβ transgenic SCID mice the TCRβ protein can be expressed in a monomelic and dimeric form whereas only the dimeric form was found on the surface of a TCRβ-transfected, immature T cell line. The results presented here show that normal thymocytes from 16-day-old fetuses likewise express only the dimeric form and that the monomelic form on the surface of thymocytes from transgenic mice results from glycosyl phosphatidylinositol linkage. Our results show for the first time that under physiological conditions a TCRβ dimer can be expressed on the cell surface without the TCRα chain.     

Encephalitogenic,myelin basic protein-specific T cells from naive rat thymus: preferential use of the T cell receptor gene Vβ8.2 and expression of the CD4− CD8− phenotype

Using a primary limiting dilution approach to generate T cell lines, we compared myelin basic protein (MBP)-specific T cell clones from naive unprimed Lewis rat thymuses with the corresponding T cell repertoire of primed rats. We found that in the naive thymus repertoire MBP-specific, encephalitogenic T cell clones preferentially use T cell receptor Vβ8.2 genes, along with CDR3 sequences typical for the primed Lewis anti-MBP response. In contrast to T cells from primed immune organs, which all display the CD4⁺ CD8^? phenotype, the majority of naive thymus-derived T cell clones expressed reduced levels of the CD4 co-receptor. Some clones were completely CD4^?CD8^?, while others included CD4^? CD8^? subpopulations along with CD4⁺CD8^? T cells. In the one mixed population examined in detail, the CD4^?CD8^? and CD4⁺CD8^? T cell subpopulations used a T cell receptor with identical β chain sequence. The data suggest that in the Lewis rat the biased T cell receptor gene usage by encephalitogenic T cells is a property of the natural thymic T cell repertoire, possibly as a consequence of positive selection. The unusually low expression of CD4 in the major histocompatibility complex class II-restricted autoreactive T cells could be related to their escape from negative selection within the thymus.     

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.     

Helper activity of CD4+ αβ T cells is required for the avian γδ T cell response

We have studied the in vitro activation of chicken γδ T cells. Both splenic αβ and γδ T cells obtained from complete Freund's adjuvant-primed chickens proliferated in vitro when stimulated with mycobacterial sonicate or purified protein derivative of Mycobacterium tuberculosis. When CD4⁺ cells or αβ T cell receptor (TcR)-positive cells were removed, both the proliferation and the blast formation of γδ T cells in response to mycobacterial antigens were abrogated. The response was restored if supernatant from concanavalin A (Con A)-activated lymphocyte cultures (CAS) as a source of helper factors was added together with the specific antigen purified protein derivative. The CD4- or αβ TcR-depleted cells still proliferated in response to Con A, although a decrease of the response was observed. To analyze the γδ T cell response more specifically we stimulated peripheral blood cells with immobilized monoclonal antibodies against T cell receptor. Anti-γδ TcR antibody alone did not induce significant proliferation. When CAS was added together with the anti-γδ TcR monoclonal antibody, a strong proliferation of γδ T cells was observed. In contrast, both Vβ1- and Vβ2-expressing αβ T cells proliferated in vitro in response to stimulation with the relevant anti-TcR monoclonal antibody alone. Depletion of either Vβ1⁺ or Vβ2⁺ T cell subset alone had no negative effect on the proliferation or blast formation of γδ T cells stimulated with mycobacterial antigens. Taken together our results suggest that CD4⁺ αβ T cells (both Vβl- and Vβ2-expressing) play a role in the activation and response of chicken γδ T cells.     

Developmental expression of the αIELβ7 integrin on T cell receptor γδ and T cell receptor αβ T cells

A novel monoclonal antibody, 2E7, was shown by immunoprecipitation to be reactive with the α_IELβ7 integrin and was employed to analyze the expression of this integrin in lymphocyte subsets and during T cell ontogeny. In adult lymph nodes, α_IEL was expressed at low levels by 40–70% of CD8⁺ T cells and < 5% of CD4⁺ T cells. However, virtually all intestinal intraepithelial lymphocytes and ?20% of lamina propria CD4⁺ T cells were 2E7⁺, indicating a preferential expression of this integrin on mucosal T cells. Examination of α_IEL integrin expression during thymus ontogeny revealed that ?3–5% of fetal or adult thymocytes were 2E7⁺. Interestingly, early in fetal thymus ontogeny, ?40% of 2E7⁺ cells expressed T cell receptor (TcR)-γδ and this subset persisted through birth. A developmental switch occurred such that 2E7⁺ TcR^? CD4^?8⁺ cells detected on fetal day 19 were followed by 2E7⁺ TcR-αβ CD4^?8⁺ cells in the neonatal thymus. The latter population persisted throughout thymus ontogeny into adulthood. Interestingly, a subset of TcR-γδ Vγ3⁺ day 16 fetal thymocyte dendritic epidermal cell (DEC) precursors were 2E7⁺, but all mature DEC expressed high levels of α_IEL integrin, suggesting that the α_IEL integrin was acquired late in DEC maturation. This possibility was strenghthened by immunohistochemical localization of the majority of 2E7⁺ γδ and αβ T cells to the medullary regions of the thymus. Overall, the results demonstrate a developmentally ordered expression pattern of the α_IELβ₇ integrin that suggests a common function for this integrin during TcR-γδ and -αβ CD4^?8⁺ T cell thymocyte development or perhaps in effector functions for these subsets.     

T cells expressing the γδ T cell receptor are not required for egg granuloma formation in schistosomiasis

Immunopathology in schistosomiasis consists of a granulomatous response around parasite eggs. It has been established that granuloma formation is mediated by CD4⁺ T helper cells. However, the role of T cells bearing the γδ T cell receptor (TCR) has not been determined. In this study we utilized mutant mice that lack either αβ or γδ T cells as a result of gene targeting to investigate the relative roles of αβ and γδ T cells in the induction of immunopathology related to schistosomiasis. Mutant and control mice were infected with Schistosoma mansoni and granuloma formation as well as lymph node cell proliferative responses to egg antigens were analyzed after 8 weeks. TCR δ mutant mice (lacking γδ T cells) displayed vigorous formation of egg granulomas that were not significantly different from those observed in normal controls, both in terms of granuloma size and cellular composition. In contrast, TCR α and TCR β mutant mice (lacking αβ T cells) were unable to form granulomas. Moreover, mesenteric lymph node cells from TCR δ mutant and control mice responded strongly to egg antigens in vitro, while TCR α and β mutant mice did not. Our studies show that in schistosomiasis granuloma formation and proliferative responses to egg antigens are strictly dependent on αβ T cells. They also suggest that γδ T cells by themselves can neither mediate a granulomatous inflammation, nor significantly modify one mediated by αβ T cells.