The balance between deletion and activation of CD4+8+ thymocytes is controlled by T cell receptor-antigen interactions and is affected by cyclosporin A

The sensitivity of immature thymocytes to antigen-induced deletion has been shown to correlate with their differentiation status. By using an in vitro approach we have investigated whether parameters of antigenic stimulation may also affect the response of thymocytes. Two T cell receptor (TcR)-transgenic (Tg) mouse models have been compared, both of which recognize the allo-antigen H-2K^b but are functionally CD8“-dependent” (KB5.C20-Tg) and “-independent” (BM3.3-Tg). Presentation of the antigen H-2K^b on the surface of fibroblasts, to thymocytes in vitro, resulted in the apoptosis of CD4⁺8⁺ thymocytes. In contrast to in vivo deletion, in vitro deletion was much greater for KB5.C20-Tg than for BM3.3-Tg. In the absence of engagement of CD8 (using an altered H-2K^b-α3 domain or CD8-specific antibodies), the H-2K^b-induced deletion of CD4⁺8⁺ thymocytes was decreased for KB5.C20-Tg, but no change in the pattern of deletion for BM3.3-Tg occurred. CD4⁺8⁺ thymocytes which remained viable after in vitro exposure to antigen, were shown to have been activated. Cyclosporin A (CsA), which has been reported to inhibit activation-induced cell death, did not affect antigen-induced deletion of CD4⁺8⁺ thymocytes from KB5.C20-Tg. More strikingly, deletion of CD4⁺8⁺ thymocytes from BM3.3-Tg increased, whilst activation was partially inhibited by CsA. These results provide direct evidence that presentation of antigen to thymocytes can result in deletion or activation, depending on not only the differentiation status of the cell, but also parameters of TcR-antigen interaction. Additionally, the effects of CsA suggest that activation can prevent the induction of deletion.     

CD4+8− help prevents rapid deletion of CD8+ cells after a transient response to antigen

We have followed the fate of mature CD8⁺ T cells with a male-specific transgenic T cell receptor after antigenic stimulation with hemopoietic cells in the absence or presence of help. Our data show that mature CD8⁺ T cells can be deleted after a 3-week period of transient activation and that help, e.g. in the form of interleukin-2, can considerably delay the deletion. These experiments have implications for the design of protocols aiming at the establishment of specific immunological tolerance in T cells.     

Differential induction of helper and killer T cells from isolated CD4+CD8+ thymocytes in suspension culture

Thymocytes of T cell receptor transgenic mice with nonselecting and RAG-2^−/− backgrounds were developmentally arrested at the CD4⁺CD8⁺ stage before positive selection. These thymocytes underwent lineage commitment upon transient stimulation with a combination of ionomycin, a calcium ionophore, and phorbol 12-myristate 13-acetate (PMA), a protein kinase C activator, in suspension culture. The effective drug doses were limited within narrow ranges and much lower than those which induce proliferation of mature T cells. The doses corresponded to those which inhibit glucocorticoid-induced apoptosis in these thymocytes. CD4 lineage commitment required longer duration, higher intensity of the stimulation, or both, than CD8 lineage commitment. Functional helper T cells (Th1 and Th2) were induced from the CD4 lineage-committed cells upon secondary stimulation with a combination of ionomycin and PMA followed by lymphokine treatment. Cytotoxic T cells were induced from the CD8 lineage-committed cells upon incubation with concanavalin A and irradiated splenic dendritic cells, but not with the combination of ionomycin and PMA. These results indicate that positive selection is mimicked by the pharmacological stimulation in the absence of other cell types, but that final maturation of CD8 T cells may require a different signal.     

Clonal deletion of major histocompatibility complex class I-restricted CD4+CD8+ thymocytes in vitro is independent of the CD95 (APO-1/Fas) ligand

The CD95 (APO-1/Fas) ligand (CD95L) mediates apoptosis in sensitive target cells, Ca²⁺-independent cytotoxicity of cells from perforin knock-out mice, and peripheral deletion of activated T cells through engagement of its cognate receptor CD95. Double-positive thymocytes show a high constitutive expression of CD95. Therefore, we used a model system and investigated whether negative selection through apoptosis might involve CD95/CD95L. We analyzed whether CD95L may induce antigen-specific deletion of double-positive thymocytes from mice transgenic for a lymphocytic choriomeningitis virus (LCMV)/H2^b-specific T cell receptor (TCR). These cells are deleted in vitro upon addition of the LCMV-peptide 33–41 in a major histocompatibility complex-class I-restricted fashion. Deletion was not blocked by soluble mouse and human CD95-Fc receptor decoys. CD95-Fc receptor decoys, however, were effective in blocking apoptosis induced by mouse CD95L-transfected L929 cells in sensitive CD95⁺ target cells and in thymocytes. These results suggest that TCR-induced deletion of immature thymocytes in vitro is independent of CD95L. Thus, our data argue against a role of CD95L in negative selection of MHC-class I-restricted autoreactive thymocytes.     

Thymocytes can tolerize thymocytes by clonal deletion in vitro.

Clonal deletion of thymocytes bearing TCR for self antigens is one major mechanism of T cell tolerance induction. Peptide antigen-induced deletion of thymocytes from alpha beta TCR transgenic mice has been studied using single cell suspension cultures. The results show that antigen-presenting immature CD4+CD8+ thymocytes can tolerize antigen-reactive immature thymocytes in vitro by programmed cell death (apoptosis) 6-8 h after antigen exposure. Antigen-induced apoptosis of immature thymocytes was inhibited by antibodies specific for the alpha beta TCR, CD3, CD8, and LFA-1 molecules. This implies that clonal elimination of self-reactive CD4+CD8+ thymocytes does not depend on specialized deleting cell types in the thymus and occurs whenever the TCR of immature thymocytes bind antigen fragments presented by MHC molecules.     

Deletion of CD4+ T cells and thymocytes by apoptosis in mouse mammary tumor virus (C4)-infected Vβ2 transgenic mice

Mouse mammary tumor virus MMTV(C4) encodes a Vβ2-specific superantigen. In Vβ2 transgenic (TG2) mice more than 98 % of peripheral T cells express Vβ2. Infection of Tg2 mice with MMTV(C4) at birth through their mothers' milk or at 6–8 weeks of age by intravenous injection resulted in massive deletion of peripheral CD4⁺ T cells and suppressed thymopoiesis. The number of peripheral CD8⁺ T cells was not affected in neonatally infected mice. In older mice injected with MMTV(C4), splenic CD8⁺ T cells were significantly elevated. Suppressed thymopoiesis was observed in both neonatally infected and older mice injected with MMTV(C4). Thymocytes which expressed high level CD3 or Vβ2 were deleted. To determine if T cells or thymocytes were deleted through apoptosis, DNA fragmentation was examined by flow cytometry and diphenylamine (DPA) binding assay. Approximately 31 % of CD4⁺ T cells from MMTV(C4)-infected Tg2 mice as compared to 6% from normal Tg2 mice contained fragmented nuclear DNA by flow-cytometric analysis. The DPA binding assay showed significantly increased total soluble DNA in lymph node cells and thymocytes from MMTV(C4)-infected mice. The kinetics of T cell and thymocyte apoptosis correspond to their deletion, supporting apoptosis as the mechanism of T cell and thymocyte deletion. CD4⁺ T cell and thymocyte deletion by MMTV(C4) in Tg2 mice provides a sensitive system for the analysis of retrovirus superantigen-induced apoptosis.     

Involvement of apoptosis antigen Fas in clonal deletion of human thymocytes

Apoptosis appears to play a major role in the differentiationand selection of T and B lymphocytes, but the mechanisms ofclonal deletion of T cells in thymus are not well understood.We have prepared an anti-human Fas IgM mAb with associated apoptosis-inducingactivity in Fas antigen-positive target cells including humanT cells. We analyzed the expression of apoptosis antigen Fason human thymocytes by cytofluorometry showing low, but significantamounts of Fas antigen on double-negative and double-positiveundifferentiated thymocytes. On the contrary, most of the differentiatedthymocytes (single-positive or CD3-brightest) expressed undetectablelevels of Fas antigen. About 1-2% of thymocytes expressed highamounts of Fas antigen, and these cells, which were CD3-bright,were CD4-bright and CD8-low at the stage of late double-positivelineage. Immunohlstologlcal analysis shows these Fas-brightcells on the edge of the medulla. Stimulation through the TCRcomplex was shown to induce the expression of Fas antigen onthymocytes at the late double-positive stage and prolonged stimulationthrough the TCR complex rendered the Fas-bright thymocytes sensitiveto apoptosis-induclng activity of anti-Fas. To show the involvementof the Fas system in the negative selectlon/clonal deletionof thymocytes, we organ-cultured human thymus in the presenceof the superantigen, staphylococcus enterotoxin B (SEB), andnew antagonistic anti-Fas mAb, which can inhibit the apoptosis-induclngactivity of the original anti-Fas mAb. The SEB-reactJve TCRcomplex on thymocytes was at first down-regulated by SEB, thenthe SEB-reactlve clone was deleted by apoptosis, which was inhibitedby an antagonistic anti-Fas mAb. Thus, Fas antigen is shownto be involved in the negative selection/clonal deletion ofsuperantlgen-reactive thymocytes.     

Evidence for a selective and multi-step model of T cell differentiation: CD4+CD8low thymocytes selected by a transgenic T cell receptor on major histocompatibility complex class I molecules

We have characterized a prominent (15-20 %) thymocyte population expressing CD4 at a high and CD8 at a low level “CD4⁺8^lo” in mice transgenic for a T cell receptor “TCR” restricted by major histocompatibility complex “MHC” class I molecules. The results demonstrate that the CD4⁺8^lo population is an intermediate stage between immature CD4⁺8⁺ and end-stage CD4⁺8^- thymocytes and that the survival of these cells crucially depends on the successful interaction of the transgenic TCR with self MHC class I molecules. In addition we demonstrate that the avidity of the interaction between TCR and self MHC class I molecules determines whether CD4⁺8^lo thymocytes are found in significant numbers in this transgenic model. Our findings support a selective and multi-step model of T cell differentiation in the thymus.     

Small CD4+8+TCRlow thymocytes contain precursors of mature T cells

To evaluate directly the developmental potential of cortical CD4⁺8⁺ thymocytes, highly purified populations of small, nondividing CD4⁺8⁺TCR^low and large, dividing CD4⁺8⁺TCR^high thymocytes from H-2^d mice expressing a transgenic T cell receptor restricted by H-2D^b (major histocompatibility complex class I) molecules were transferred into the thymus of normal, nonirradiated H-2^b recipient mice. The results show that both populations generate CD4^?8⁺ thymocytes under these conditions, thus providing conclusive evidence that small cortical thymocytes do not represent a “dead end” but an important intermediate stage in T cell development.     

In vivo and in vitro repertoire of CD3+CD4 CD8 thymocytes

CD4-CD8- thymocytes contain a cell subset which expresses thecomplete CD3-TCR complex (/ or /) of which the ontogeny andfiliation are unknown. One of the questions is whether thispopulation can be intrathymically selected, an obligatory stepfor the mature CD4⁺ and CD8⁺ cell differentiation pathway, orif the absence of CD4 and CD8 allows them to escape thymic selection.The repertoire of CD3 ⁺ CD4 - CD8 - (CD3 ⁺ DN) thymocytes wasanalyzed in different strains of mice with different combinationsof H-2 and Mls expression. The expression of V_ß8.1in freshly isolated CD3⁺ DN cells is the highest in Mls-l^b miceand the lowest in MIS-l^a and F₁ mice, suggesting that selectionagainst this specificity can be achieved in vivo. By contrast,a low percentage of V_ß6⁺ cells is found in all thestrains, with no correlation according to MIS expression. Invitro culture of DN thymocytes with IL-1 and IL-2 leads to theproliferation of CD3⁺ DN cells. In vitro culture amplifies thein vivo pattern of V_ß8.1 expression, while V_ß6⁺cells only expand in DN cells of 66 and 61002 Mls-l^b mice withthe same genetic background. These results show: (i) CD3⁺ DNthymic cells can be intrathymically selected but the repertoireis different from that of mature T cells; (ii) V_ß6and V_ß8.1 selection do not follow the same pattern;(iii) this repertoire can be modified by In vitro culture, towarda more mature type; and (iv) comparison of DN cell repertoireof BALBlc, BALB.D2 (congenic for MLs), and other strains ofmice suggests a multigenic control for this selection, and aninvolvement of background genes.     

Induction of CD4+ T cell depletion in mice doubly transgenic for HIV gp120 and human CD4

It has been suggested that loss of uninfected T cells in HIV infection occurs because of lymphocyte activation resulting in cell death by apoptosis. To address the question of whether cross-linking of CD4/HIV gp120 complexes by antibodies were sufficient to induce T cell depletion in vivo, we developed an animal model of continuous interaction between human CD4 (hCD4), gp120 and anti-gp120 antibodies in the absence of other viral factors. Double-transgenic mice have been generated in which T cells express on their membrane hCD4 and secrete HIV gp120. Although these mice have hCD4/gp120 complexes present on the surface of T cells, they do not show gross immunological abnormalities, and they are able to produce anti-gp120 antibodies following immunization with denaturated gp120. However, double-transgenic mice with antibodies to gp120, when immunized with tetanus toxoid, mount an IgG response that is significantly lower than that of double-transgenic mice without antibodies to gp120. Furthermore, the presence of anti-gp120 antibodies leads to CD4⁺ T cell depletion and immunodeficiency in the absence of HIV infection. Thus, the antibody response to gp120 can lead to CD4⁺ T cell attrition in vivo.     

Peripheral deletion of autoreactive CD8+ T cells in transgenic mice expressing H-2Kb in the liver

The response of T cells specific for liver antigens was examined in transgenic mice expressing the allogeneic major histocompatibility complex class I molecule H-2K^b (K^b) under the control of the sheep metallothionein promoter (Met-K^b mice). To follow the fate of K^b-specific T cells, and to prevent any aberrant thymic expression of the K^b transgene, the mice were thymectomized, lethally irradiated, protected with bone marrow cells from transgenic mice expressing in their T cells a K^b-specific T cell receptor identifiable by a clonotypic antibody, and given syngeneic non-transgenic thymus grafts. Although K^b-specific CD8⁺ T cells were produced in the thymus grafts of these manipulated Met-K^b mice, only small numbers of such cells could be detected in the spleen and lymph nodes. The livers, however, showed signs of damage and were heavily infiltrated by actively dividing CD8⁺ T cells. We provide strong evidence that the hepatocytes, not generally regarded as antigen-presenting cells, activated the K^b-specific CD8⁺ T cells and that these disappeared after a vigorous autoimmune response that resulted in deletion.     

Evidence for down-regulation of highly expressed TCR by CD4 and CD45 on non-selected CD4+CD8+thymocytes

Immature CD4+CD8+ double-positive (DP) thymocytes are positivelyselected for further development if they express TCR reactingwith thymic ligands of low affinity. However, the majority ofDP thymocytes express low TCR levels. This low level of TCRmay be insufficient to recognize thymic ligands. To understandthe basis for the low expression of TCR on DP thymocytes, wedetermined the density of TCR expression at various stages oftheir development using TCR transgenic (TCR-Tg) mice. We foundthat TCR expression was high in the thymocytes that had recentlytransited into the DP stage but then gradually decreased onDP cells if they were not selected by TCR interaction with MHCmolecules. However, such TCR suppression was not observed inpositively selected DP cells and in the non-selected DP cellsobtained from CD45 deficient mice or from mice receiving anti-CD4mAb. These findings suggest that the once highly expressed TCRat the DP stage is suppressed by CD45 and/or CD4 on non-selectedthymocytes. Furthermore, TCR suppression is prevented by TCR-mediatedsignals. The maintenance of high TCR levels on positively selectedDP thymocytes may facilitate their selection.     

Tolerance is overcome in beef insulin-transgenic mice by activation of low-affinity autoreactive T cells

To gain insight into the factors controlling the maintenance or loss of T cell self tolerance we produced beef insulin (BI)-transgenic BALB/c mice. Transgenic mice express BI under control of the human insulin promoter and secrete physiological amounts of beef insulin. Although these mice are tolerant to BI, as evidenced by the lack of insulin-specific IgG antibody production following intraperitoneal immunization, tolerance is not complete. Footpad immunization results in a weak antigen-specific T cell proliferative response, indicating the presence of self- reactive BI-specific T cells in the periphery. These T cells are functional in vivo, providing support for IgG1, IgG2a, and IgG2b BI-specific antibody production, but require higher concentrations of antigen than nontransgenic T cells (both in vivo and following recall responses in vitro) to become activated. In vitro, BI-specific T cell proliferation in BI-transgenic mice can be largely restored by addition of interleukin-2, indicating that a significant component of T cell tolerance is mediated by anergy. To characterize the autoreactive T cells that become activated when tolerance is broken, BI-specific T cell hybridomas were generated from transgenic mice and compared to a panel of hybridomas previously derived from nontransgenic BALB/c mice. The majority of BI-transgenic hybridomas recognized the immunodominant A1–14 beef insulin peptide but with lower avidity than BALB/c hybridomas. Consistent with this, none of the dominant T cell receptor rearrangements found in the BALB/c BI-specific T cell receptor repertoire were found in the transgenic hybridomas. These results indicate that, despite evidence for clonal inactivation of many BI-specific T cells in BI-transgenic mice, loss of tolerance results from activation of low-affinity antigen-specific T cells that appear to have escaped this process.     

Interleukin‐4 downregulates CD127 expression and activity on human thymocytes and mature CD8+ T cells

Signaling via the IL‐7 receptor complex (IL‐7Rα/CD127 and IL‐2Rγ/CD132) is required for T‐cell development and survival. Decreased CD127 expression has been associated with persistent viral infections (e.g. HIV, HCV) and cancer. Many IL‐2Rγ‐sharing (γ_C) cytokines decrease CD127 expression on CD4⁺ and CD8⁺ T cells in mice (IL‐2, IL‐4, IL‐7, IL‐15) and in humans (IL‐2, IL‐7), suggesting a common function. IL‐4 is of particular interest as it is upregulated in HIV infection and in thyroid and colon cancers. The role of IL‐4 in regulating CD127 expression and IL‐7 activity in human thymocytes and mature CD8⁺ T cells is unknown and was therefore investigated. IL‐4 decreased CD127 expression on all thymocyte subsets tested and only on naïve (CD45RA⁺) CD8⁺ T cells, without altering membrane‐bound CD127 mRNA expression. Pre‐treatment of thymocytes or CD8⁺ T cells with IL‐4 inhibited IL‐7‐mediated phosphorylation of STAT5 and decreased proliferation of CD8⁺ T cells. By downregulating CD127 expression and signaling on developing thymocytes and CD8⁺ T cells, IL‐4 is a potential contributor to impaired CD8⁺ T‐cell function in some anti‐viral and anti‐tumor responses. These findings are of particular consequence to diseases such as HIV, HCV, RSV, measles and cancer, in which CD127 expression is decreased, IL‐7 activity is impaired and IL‐4 concentrations are elevated.     

Developmental status of CD4-CD8+ and CD4+CD8- thymocytes with medium expression of CD3

In normal mice, more than 10% of thymocytes in the CD4+CD8- and CD4-CD8+ single-positive (SP) subsets express a medium level of CD3 on the cell surface. However, the fate of CD3medium cells is unclear. The CD3medium SP subpopulations might contain (i) cells in an immature stage of the pathways leading to CD3high cells, (ii) cells in developmental pathways that do not lead to CD3high cells, or (iii) cells that have been negatively selected. We found that sorted CD3medium CD4+CD8- thymocytes from adult mice up-regulated CD3 to high levels in reaggregation thymus organ culture. Unlike their CD3high counterparts, CD3medium CD4+CD8- thymocytes were unable to undergo chemotaxis towards the chemokines CCL19 and CCL21. CD3medium thymocytes of both CD4+CD8- and CD4-CD8+ subsets were also considerably more responsive than CD3high SP cells to apoptotic signals induced in vitro by ligation of CD95 (Fas/APO-1) or by dexamethasone. In both SP subsets, a higher frequency of thymocytes expressing forbidden Vbeta+ T cell receptors reactive with endogenous mammary tumor virus superantigens was found in CD3medium subpopulations than in CD3high subpopulations. These findings argue that the CD3medium SP thymocyte subpopulations contain apoptosis-susceptible precursor cells of CD3high SP cells and are subject to negatively selecting pressures.     

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.     

Peripheral clonal deletion of antiviral memory CD8+ T cells

Antiviral cytotoxic memory CD8⁺ T cells adoptively transferred to mice which are persistently infected with lymphocytic choriomeningitis virus WE or DOCILE initially proliferated extensively; they either caused the death of the recipient or, alternatively, disappeared within a few days. Apparently, the complete and coordinated induction and stimulation by widely distributed viral antigen caused these memory T cells to die before virus had been eliminated from the host. Thus memory T cells are as susceptible to peripheral exhaustion/deletion as unprimed T cells. These results indicate possible limitations of exclusively CD8⁺ T cell-mediated adoptive immunotherapy against viral infections or tumors.     

Epithelial expression of human papillomavirus type 16 E7 protein results in peripheral CD8 T‐cell suppression mediated by CD4+CD25+ T cells

The role of thymic versus peripheral epithelium in the regulation of the antigen‐specific CD8 T‐cell repertoire is still largely unresolved. We generated TCR‐β chain transgenic mice in which an increased frequency of peripheral CD8 T cells recognizes an epitope from a viral oncoprotein (HPV16E7) in the context of H‐2D^b MHC class I. When T cells from these mice developed through the thymus of mice expressing functional E7 protein from a keratin 14 promoter, no major perturbation to transgenic T‐cell development in the thymus was observed in these double‐transgenic mice. In contrast, peripheral CD8 T‐cell responses in the single‐transgenic, K14E7 mice, including those unrelated to E7 antigen, are reduced whereas CD4 T‐cell responses and antibody production are unchanged in these mice. Peripheral non‐responsiveness among CD8 T cells was mediated largely by CD4⁺CD25⁺ T cells. This suggested that epithelium expressing HPV16E7 protein induces Treg that specifically down‐regulate CD8 T‐cell responses in the periphery. This may have important consequences for the treatment of cervical pre‐cancers and provides a model for understanding differential suppression of T and B lymphocyte subsets by Treg.     

The role of CD4 in regulating homeostasis of T helper cells

Intrathymic T cell selection and peripheral activation of mature T cells are crucial for self-recognition and the general immune response to viral, bacterial, and tumor antigens. The T cell coreceptors, CD4 and CD8, contribute to the regulation of these processes. The importance of interactions between CD4 and moleculesencoded by the class II major histocompatibility complex (MHC) for thymic T cell selection has been clearly established, however, the role of CD4-MHC class II interactions in T helper (Th) cell differentiation, in the maintenance of homeostasis in the peripheral immune system, and in the generation of memoryTh cells is largely unclear. Here, we present evidence for a role of CD4 in controlling homeostasis in the peripheral immune system. We also demonstrate the importance of CD4-MHC class II interactions in inducing these previously not recognized functions of CD4.