Antigen-specific, MHC-restricted B cell activation by cell-free Th2 cell products. Synergy between antigen-specific helper factors and IL-4

Ag-specific and MHC-restricted Th clones of different Ag specificities and MHC haplotypes were tested for their ability to produce soluble factors capable of providing the signals required for B cell activation and IgG antibody production. Each of five Th clones tested generated significant helper activity in supernatants derived from coculture of the T cell clone with specific Ag and syngeneic APC. The same helper activity was detected in supernatants of clones stimulated with immobilized anti-CD3 antibody in the absence of Ag or APC. The secreted helper activity resembled the activity of the intact Th cells in that it was Ag-specific, carrier-hapten-linked and MHC-restricted. These T cell products functioned to activate only those B cells expressing MHC products which corresponded to the specificity of each Th clone. Thus, the specificity of the cell-free T cell product mimicked precisely that expressed by the intact Th cell and presumably mediated by the cell surface TcR. In addition to the apparent presence of specific helper factor in Th clone supernatants, a role for nonspecific lymphokines was also identified in these preparations. Although recombinant or purified IL-4 alone was not sufficient to stimulate hapten-primed B cells to secrete hapten-specific IgG antibodies, mAb specific for IL-4 blocked the induction of antibody secretion by Th cell supernatant. These results indicate that stimulation of B cells to produce hapten-specific IgG antibody requires at least two distinct signals: an Ag-specific T cell signal which is restricted by MHC products expressed on the B cells, and a nonspecific signal mediated at least in part by the lymphokine IL-4.     

Activation of B cells by autoreactive T cells: cloned autoreactive T cells activate B cells by two distinct pathways

Although the existence of autoreactive T cells has been widely reported, the functional capacities of these populations have been less well defined. Studies were therefore carried out to characterize the relationship of autoreactive T cells to antigen-specific major histocompatibility complex (MHC)-restricted T cells in their ability to act as helper cells for the induction of immunoglobulin synthesis by B cells. A number of autoreactive T cell lines and clones were isolated from antigen-primed spleen and lymph node cell populations. Autoreactive T cells were found to proliferate in response to direct recognition of syngeneic I-A or I-E subregion-encoded antigens in the absence of any apparent foreign antigen. It was shown that cloned autoreactive T cells were capable of activating B cell responses through two distinct pathways. After appropriate stimulation by syngeneic cells, autoreactive T cells polyclonally activated primed or unprimed B cells to synthesize IgM antibodies. These activated T cells functioned in these responses through an MHC-unrestricted pathway in which polyclonal responses were induced in both syngeneic and allogeneic B cells. These cloned autoreactive T cells were also able to activate IgG responses by primed B cells through a different activation pathway. In contrast to the polyclonal activation of IgM responses, the induction of IgG antibodies by the same cloned T cells required primed B cells and stimulation with the priming antigen. The activation of B cells to produce IgG was strongly MHC restricted and required the direct recognition by the autoreactive T cells of self MHC determinants expressed on the B cell surface, with no bystander activation of allogeneic B cells. These results indicate that cloned autoreactive T cells resemble antigen-specific MHC-restricted T cells in their ability to function as T helper cells through distinct MHC-restricted and MHC-unrestricted pathways.     

Peripheral T cells re-enter the thymus and interfere with central tolerance induction

The thymus mainly contains developing thymocytes that undergo thymic selection. In addition, some mature activated peripheral T cells can re-enter the thymus. We demonstrated in this study that adoptively transferred syngeneic Ag-specific T cells can enter the thymus of lymphopenic mice, where they delete thymic dendritic cells and medullary thymic epithelial cells in an Ag-specific fashion, without altering general thymic functions. This induced sustained thymic release of autoreactive self-Ag-specific T cells suggested that adoptively transferred activated T cells can specifically alter the endogenous T cell repertoire by erasing negative selection of their own specificities. Especially in clinical settings in which adoptively transferred T cells cause graft-versus-host disease or graft-versus-leukemia, as well as in adoptive tumor therapies, these findings might be of importance, because the endogenous T cell repertoire might be skewed to contribute to both manifestations.     

Characterization of the proliferative response of a CD4-8- thymic T lymphoma cell line to stimulation by thymic cellular elements

E710.2 is a cloned T cell line that was isolated from an AKR/J thymic tumor. This clone expresses Thy-1, heat-stable Ag, and the CD3/TCR complex but does not express CD4 or CD8. When the E710.2 cell line is injected into syngeneic mice, it grows as a malignant tumor in lymphoid organs and the thymus. In contrast, this cell line does not grow in vitro under standard culture conditions. This latter property allowed us to analyze the in vitro responsiveness of this CD4-CD8- cell line to stimulation by pharmacologic agents and cellular elements from the spleen and thymus. E710.2 cells proliferate when stimulated with phorbol esters or when cocultured with thymocytes or splenocytes. We could not detect soluble stimulatory factors in cultures of E710.2 and/or lymphoid cells, suggesting that cell contact might be required for this response. The stimulatory activity in thymus and spleen appears to be broadly expressed, because all cell subsets that were examined from these tissues stimulate this cell line. The stimulation of E710.2 cells is not MHC-restricted and is not inhibited by anti-MHC mAb. Furthermore, the responsiveness of these cells is not decreased when the TCR/CD3 complex is modulated from the cell surface. Similarly, TCR/CD3-deficient E710.2 variant clones retain their responsiveness to thymic and splenic cell stimulation. These findings suggest that there is a TCR-independent pathway of activation in E710.2 that is stimulated by a broadly expressed, non-MHC-encoded molecules(s).     

Thymic and postthymic regulation of diabetogenic CD8 T cell development in TCR transgenic nonobese diabetic (NOD) mice

Natural development of diabetes in nonobese diabetic (NOD) mice requires both CD4 and CD8 T cells. Transgenic NOD mice carrying alphabeta TCR genes from a class I MHC (Kd)-restricted, pancreatic beta cell Ag-specific T cell clone develop diabetes significantly faster than nontransgenic NOD mice. In these TCR transgenic mice, a large fraction of T cells express both transgene derived and endogenous TCR beta chains. Only T cells expressing two TCR showed reactivity to the islet Ag. Development of diabetogenic T cells is inhibited in mice with no endogenous TCR expression due to the SCID mutation. These results demonstrate that the expression of two TCRs is necessary for the autoreactive diabetogenic T cells to escape thymic negative selection in the NOD mouse. Further analysis with MHC congenic NOD mice revealed that diabetes development in the class I MHC-restricted islet Ag-specific TCR transgenic mice is still dependent on the presence of the homozygosity of the NOD MHC class II I-Ag7.     

Lymphokine-independent induction of macrophage membrane IL-1 by autoreactive T cells recognizing either class I or class II MHC determinants

Here we report that autoreactive T cell clones and T cell hybridomas that recognize class I or class II MHC determinants can induce IL-1 expression on cultured macrophages in an MHC-restricted manner. This genetic restriction of membrane IL-1 (mIL-1) induction is not absolute, however; it is manifest only in macrophages that have been cultured for several days before stimulation. Macrophages that are evaluated within 24 h after adherence display a basal level of mIL-1, and the T cell-induced augmentation of basal mIL-1 expression is not MHC-restricted. It appears that T cells of both Th1 and Th2 type have the capacity to induce mIL-1, suggesting that this function is not limited to the T cell subset (Th2) that is able to use IL-1. Most importantly, the ability of T cells to induce IL-1 on macrophages seems to occur by virtue of direct cellular interactions, and is independent of lymphokine secretion. The induction event is rapid enough (2 to 4 h) to allow T cells to interact with both antigen and IL-1 during the initial T cell/macrophage contact. These findings thus reveal an efficient mechanism for the induction of IL-1 during Ag presentation to T cells.     

Tolerance induction in T helper (Th1) cells by thymic macrophages.

Most macrophages in the peripheral tissues present Ag optimally to a variety of functionally distinct Th cells. Although thymic macrophages have been implicated in deleting autoreactive thymocytes, their role in influencing the functional capacities of mature T cells is not clear. We have established a normal untransformed macrophage cell line, named TMC, from the mouse thymus. The TMC line presents protein Ag to an IL-4-producing Th2 type Th clone after IFN-gamma treatment as evidence by T cell proliferation and the release of IL-3 and IL-4. However, these thymic macrophages are inefficient at stimulating a well characterized cytochrome C-specific IL-2-producing Th1 clone, A.E7. Ag presentation by TMC results in the production of IL-3 but not IL-2 production or proliferation of A.E7 cells. This selective Ag presentation defect to Th1 cells is corrected by the addition of live but not fixed allogeneic irradiated spleen cells, suggesting that the thymic macrophages lack the expression of costimulatory activity required for Th1 activation. This is further demonstrated by the failure of live thymic macrophages to provide costimulatory activity to A.E7 cells stimulated with fixed spleen cells plus the antigenic peptide 81-104. Exposure of A.E7 cells to paraformaldehyde-treated TMC in the presence of 81-104 peptide induces specific hyporesponsiveness, anergy. These data demonstrate that thymic macrophages can have a profound influence on the response of selected T cells to Ag. Furthermore, the nature of the T cell stimulus is also critical because Th1 and Th2 cells responded equally well to the T cell mitogen, Con A, and a bacterial superantigen presented by the thymic macrophages.     

Role of glycosaminoglycans in the regulation of T cell proliferation induced by thymic stroma-derived T cell growth factor

The present study investigates the regulatory effects of glycosaminoglycans such as heparin and heparan sulfate on T cell proliferation induced by thymic stromal cell monolayer or its derived T cell growth factor (TCGF). A thymic stromal cell clone (MRL104.8a) supported the growth of Ag-specific, IL-2-dependent Th cell clone (9-16) in the absence of Ag and IL-2 by producing a unique TCGF designated as thymic stroma-derived T cell growth factor (TSTGF). The addition of heparin to cultures in which the growth of 9-16 Th cells was otherwise stimulated by the MRL104.8a monolayer or a semipurified sample of the TSTGF resulted in heparin dose-dependent inhibition of 9-16 Th proliferation. The dose of heparin required for inducing 50% reduction of TSTGF-induced proliferation of Th at a given cell number was found to be proportional to the magnitude of the TSTGF added to cultures, suggesting that heparin exerted its inhibitory effect by binding to the TSTGF rather than by acting on Th cells. A similar growth-inhibiting effect of heparin was observed in IL-7-dependent proliferation of pre-B cell line or Th, but not in IL-2-dependent T cell proliferation or IL-3-dependent myeloid cell proliferation. A strong affinity of TSTGF and IL-7 for heparin was confirmed by the fact that both TSTGF and IL-7 adhered to columns of heparin-agarose and were eluted by salt. When various glycosaminoglycans were tested for the heparin-like Th growth-regulatory capacity, heparan sulfate exhibited Th growth-inhibiting ability comparable to that observed for heparin. These results indicate that the activity of thymic and/or bone marrow stroma-derived lymphocyte growth factor (TSTGF/IL-7) but not of Th-producing TCGF (IL-2) is negatively regulated by heparin or heparan sulfate, which would represent major glycosaminoglycans in the extra-cellular matrix of stromal cells.     

Distinct proliferative T cell clonotypes are generated in response to a murine retrovirus-induced syngeneic T cell leukemia: viral gp70 antigen-specific MT4+ clones and Lyt-2+ cytolytic clones which recognize a tumor-specific cell surface antigen

After immunization of B6 mice with the syngeneic retrovirus-induced T cell leukemia/lymphoma FBL-3, two major tumor-specific proliferative T cell clonotypes were derived. T cell clones derived from long-term lines propagated by in vitro culture with irradiated tumor cells and syngeneic spleen cells were exclusively of the Lyt-2+ phenotype. Such clones were cytolytic, retained their proliferative phenotype indefinitely when expanded by repeated cycles of reactivation and rest, and recognized a tumor-specific cell surface antigen in association with class I MHC molecules. This tumor cell antigen was not present on nontransformed virus-infected cells. Class II MHC-restricted MT4+ clones specific for the viral antigen gp70 were derived from lymph node T cells of FBL-3 tumor-immune mice only by in vitro culture with purified Friend virus in the presence of syngeneic splenic APC. Once derived, however, such clones could be stimulated in the presence of FBL-3 tumor cells and syngeneic spleen cells, demonstrating the reprocessing of tumor-derived gp70 antigen by APC in the spleen cell population. In contrast, no reprocessing of the tumor cell surface antigen by splenic APC for presentation to the class I MHC-restricted T cell clones could be demonstrated. Evidence is presented that FBL-3 T leukemia/lymphoma cells function as APC for Lyt-2+ class I MHC-restricted clones, and that no concomitant recognition of Ia molecules is required to activate these clones. Both Lyt-2+ and MT4+ clones were induced to proliferate in the presence of exogenous IL2 alone, but this stimulus failed to result in significant release of immune interferon. In contrast, antigen stimulation of both clones resulted in proliferation as well as significant immune interferon release. Immune interferon production is not required for the generation of MHC-restricted cell-mediated cytolytic function.     

T cells from tumor-immune mice nonspecifically expanded in vitro with anti-CD3 plus IL-2 retain specific function in vitro and can eradicate disseminated leukemia in vivo.

The therapeutic efficacy of adoptive immunotherapy of cancer has been shown to positively correlate with the dose of tumor-immune T cells transferred. Therefore, the success of this therapy is critically dependent on the ability to procure large numbers of functionally active T cells. Previous studies in animal models have shown that the limited therapeutic efficacy of a small number of immune T cells can be greatly enhanced by expansion of T cells in vitro to greater numbers before transfer in vivo. Optimal regimens for T cell expansion in vitro have generally employed the use of intermittent stimulation of the TCR with specific Ag followed by exogenous IL-2. The use of IL-2 alone does not provide for requisite episodic up-regulation of IL-2R. Stimulation of the invariant CD3 portion of the TCR/CD3 complex with antibody to CD3 (anti-CD3) represents an alternative method of up-regulating IL-2R and has been used to nonspecifically induce the growth of Ag-specific T cell lines and clones long-term in vitro with maintenance of function and specificity. The current study examined whether resting T cell populations containing small numbers of memory tumor-specific T cells could be rendered more effective in tumor therapy by nonspecific expansion in vitro with anti-CD3 plus IL-2. Spleens from C57BL/6 mice previously immunized to FBL-3, a syngeneic virus-induced leukemia, were nonspecifically stimulated with anti-CD3 plus IL-2. The resultant T cells were expanded in number, were nonlytic to FBL-3 but retained the ability to become lytic upon specific stimulation by FBL-3, and were effective in specific tumor therapy. The Ag-specific anti-tumor immune function declined on a per cell basis after each cycle of anti-CD3-induced T cell expansion. However, the approach resulted in a substantial increase in total T cell number and an overall net increase in the function of the effector T cell population. Thus, stimulation of tumor-immune T cell populations with anti-CD3 plus IL-2 represents a nonspecific method for expanding the number of specific effector T cells for cancer therapy.     

Tolerance induction by transcutaneous immunization through ultraviolet-irradiated skin is transferable through CD4+CD25+ T regulatory cells and is dependent on host-derived IL-10

UV radiation of the skin impairs immune responses to haptens and to tumor Ags. Transcutaneous immunization (TCI) is an effective method of inducing immune responses to protein and peptide Ag. We explore the effect of UV irradiation on TCI. The generation of Ag-specific CTL to OVA protein, but not class I MHC-restricted OVA peptide, is inhibited by TCI through UV-irradiated skin. Consequently, the induction of protein contact hypersensitivity and in vivo Ag-specific CTL activity following OVA protein immunization is prevented. Application of haptens to UV-exposed skin induces hapten-specific tolerance. We demonstrate that application of protein or class II MHC-restricted OVA peptide to UV-irradiated skin induces transferable Ag-specific tolerance. This tolerance is mediated by CD4(+)CD25(+) T regulatory (T(reg)) cells. These Ag-specific T(reg) cells inhibit the priming of CTL following protein immunization in the presence of CpG adjuvant. IL-10 deficiency is known to prevent hapten-specific tolerance induction. In this study, we demonstrate, using IL-10-deficient mice and adoptive T cell transfer, that IL-10 is required for the direct inhibition of CTL priming following immunization through UV-irradiated skin. However, IL-10 is not required for the induction of T(reg) cells through UV-irradiated skin as IL-10-deficient T(reg) cells are able to mediate tolerance. Rather, host-derived IL-10 is required for the function of UV-generated T(reg) cells. These experiments indicate that protein and peptide TCI through UV-irradiated skin may be used to induce robust Ag-specific tolerance to neo-Ags and that UV-induced T(reg) cells mediate their effects in part through the modulation of IL-10.     

IL-10-dependent suppression of experimental allergic encephalomyelitis by Th2-differentiated, anti-TCR redirected T lymphocytes

We previously showed that transgenically expressed chimeric Ag-MHC-zeta receptors can Ag-specifically redirect T cells against other T cells. When the receptor's extracellular Ag-MHC domain engages cognate TCR on an Ag-specific T cell, its cytoplasmic zeta-chain stimulates the chimeric receptor-modified T cell (RMTC). This induces effector functions such as cytolysis and cytokine release. RMTC expressing a myelin basic protein (MBP) 89-101-IAs-zeta receptor can be used therapeutically, Ag-specifically treating murine experimental allergic encephalomyelitis (EAE) mediated by MBP89-101-specific T cells. In initial studies, isolated CD8+ RMTC were therapeutically effective whereas CD4+ RMTC were not. We re-examine here the therapeutic potential of CD4+ RMTC. We demonstrate that Th2-differentiated, though not Th1-differentiated, CD4+ MBP89-101-IAs-zeta RMTC prevent actively induced or adoptively transferred EAE, and treat EAE even after antigenic diversification of the pathologic T cell response. The Th2 RMTC both Th2-deviate autoreactive T cells and suppress autoantigen-specific T cell proliferation. IL-10 is critical for the suppressive effects. Anti-IL-10R blocks RMTC-mediated modulation of EAE and suppression of autoantigen proliferation, as well as the induction of IL-10 production by autoreactive T cells. In contrast to IL-10, IL-4 is required for IL-4 production by, and hence Th2 deviation of autoreactive T cells, but not the therapeutic activity of the RMTC. These results therefore demonstrate a novel immunotherapeutic approach for the Ag-specific treatment of autoimmune disease with RMTC. They further identify an essential role for IL-10, rather than Th2-deviation itself, in the therapeutic effectiveness of these redirected Th2 T cells.     

Functional modulation of hepatitis B core antigen-specific T lymphocytes by an autoreactive T cell clone

Hepatitis B core (HBc)Ag-specific T cells present in the peripheral blood of a patient with chronic active hepatitis B were expanded by co-cultivation for 7 days with rHBcAg. After cloning at 1 cell/well in the presence of PHA and IL-2, five HBcAg-specific CD4+ cloned lines were obtained. All five lines proliferated and produced IL-2, IFN-gamma, and TNF in a dose-dependent fashion in response to HBcAg, but not to HBV envelope Ag. The cloned lines and derivative clones were HLA class II (DR1) restricted. All T cell clones were able to induce anti-HBc production by autologous B cells in response to HBcAg (helper effect). The proliferative response and the helper effect of the HBcAg-specific T cell lines and clones were augmented by co-cultivation with an autologous, autoreactive (HLA-DQ1 specific) T cell clone, even in the absence of HBcAg, and the autoreactive T cells directly stimulated anti-HBc secretion by autologous B cells, presumably due to the release of Ag-nonspecific factors. These findings define a model immunoregulatory circuit the physiologic significance of which remains to be determined.     

Increased mitogenic reactivity of normal spleen cells to T lectins induced by thymus humoral factor (THF)

When normal spleen cells were incubated for 24 hr in medium containing thymic humoral factor (THF) and then stimulated by phytohemagglutinin (PHA) or concanavalin A (Con A), a significant increase in the mitogenic reactivity of these cells was observed. When stimulation to T lectins was performed simultaneously with THF, a strong inhibition in cell reactivity was found. It seems that these opposite effects of THF on cell reactivity to T lectins are determined by the sequence of events which lead to maturation of lymphoid cells. Thymic humoral factor does not modify the response of cells to B mitogen lipopolysaccharide (LPS), thus suggesting that this maturative effect on lymphoid cells is exerted on T lymphocytes only.     

Anti-tumor activity of class II MHC antigen-restricted cloned autoreactive T cells. I. Destruction of B16 melanoma cells mediated by bystander cytolysis in vitro

Two Lyt-1+, L3T4a+ autoreactive T cell clones specific for self-class II major histocompatibility complex (MHC) gene products were established from lymph node cells and spleen cells of C57BL/6J mice, respectively, by different methods. They were stimulated to proliferate in culture in response to I-Ab antigen-bearing syngeneic spleen cells in a class II MHC-restricted manner. This stimulation was inhibited completely by the addition of anti-L3T4a (GK1.5) or anti-I-Ab (3JP) monoclonal antibodies. The autoreactive T cell clones lysed syngeneic I-Ab+ target cells such as lipopolysaccharide (LPS) blasts. They also lysed I-A- bystander cells such as Cloudman and B16 melanoma and lymphoid tumor cells in the presence of I-Ab+ stimulator cells but not I-Ad+ cells. This bystander killing was most likely mediated by soluble factors released from the autoreactive T cells in response to I-Ab antigens, because culture supernatants from activated autoreactive T cells inhibited the proliferation of B16 melanoma cells in vitro and also had significant cytolytic activity. Both lymphotoxin and interferon-gamma were released from activated autoreactive T cells, suggesting that these cytotoxic lymphokines were responsible for autoreactive T cell-mediated cytolysis. The finding that the two clones, established independently and by different methods, show self-class II MHC antigen-restricted cytolysis, and bystander cytolysis suggests that these properties are not restricted to a unique population of autoreactive T cells. These results favor the concept that in vivo, autoreactive T cells may express not only regulatory activity in regard to antibody responses, but also anti-tumor activity via bystander cytolysis.     

Characterization and function of autoreactive T-lymphocyte clones isolated from normal, unprimed mice

Self-Ia-reactive cloned T-cell lines, designated PK, were established by long-term culture of T cells from normal DBA/2 mice with irradiated syngeneic splenic adherent cells (SAC), rich in macrophages and dendritic cells. The cell lines were Thy 1+, Lyt 1+, Lyt 2-, produced IL-2 following stimulation with syngeneic spleen cells, and did not exhibit alloreactivity when screened against six different H-2 haplotypes. Of the five cloned PK cell lines tested, four were I-Ed restricted while one was I-Ad restricted as determined by genetic mapping and blocking studies carried out with monoclonal anti-Ia sera. Extensive specificity studies suggested that the PK cells reacted to syngeneic Ia molecules alone and not to foreign antigens such as fetal calf serum (FCS) used in the culture medium, in association with self-Ia. SAC pulsed with FCS or other protein antigens such as turkey gamma-globulin (TGG) were tested for their ability to induce proliferation of autoreactive T cells and other antigen-specific T cells using culture conditions consisting of serumless medium and interleukin 2 (IL-2). The data showed that the autoreactive T cells proliferated better in response to antigen-unpulsed SAC, while FCS-specific and TGG-specific cell lines, developed independently, proliferated only in response to FCS- or TGG-pulsed SAC, respectively, but not to antigen-unpulsed SAC. These results clearly distinguished the autoreactive T-cell clones from the antigen-specific T-cell clones. Preliminary studies carried out to investigate the functions of autoreactive T cells suggested that these cells helped in the in vitro differentiation of alloantigen-specific cytotoxic T lymphocytes (CTL) from CTL precursors obtained from the thymus and augmented syngeneic, allogeneic, and antigen-specific immune responses in vitro. The autoreactive T cells were also capable of inducing both proliferation and differentiation of antigen-specific populations of B cells in the absence of antigen. The present investigation suggests that autoreactive, non-antigen-reactive T cells can be cloned from normal, unimmunized mice and that such cell lines may provide a powerful tool for analyzing the role of the syngeneic mixed lymphocyte reaction in induction and maintenance of both T-and B-cell immune responses.     

Effect of Mlsa on antigen presentation to class II-restricted T cells

The nature of the gene products encoded or regulated by the minor lymphocyte-stimulating (Mls) loci remains enigmatic despite extensive experimental evaluation. This work tested the hypothesis that the Mlsa genotype, when compared to the Mlsb genotype, facilitates Ag presentation to class II-restricted T cells. Titrated numbers of H-2-identical, Mls-disparate APC were used to stimulate proliferation of autoreactive, alloreactive, or Ag-specific class II-restricted T cell clones or lines. Apparent preferential presentation by Mlsa vs Mlsb APC obtained from H-2-identical strains was seen infrequently, and when observed, analysis with the use of APC from recombinant inbred lines revealed that preferential presentation did not correlate with the Mls genotype of the APC. These studies show that the Mlsa genotype does not influence overall Ag presentation to class II-restricted T cells.     

Thymic stromal cell clone with nursing activity supports the growth and differentiation of murine CD4+8+ thymocytes in vitro

Thymic stromal cell clone, TNC-R3.1 cell, was established from spontaneous AKR/J mouse thymoma. TNC-R3.1 cell, which has the similar properties to thymic nurse cells, formed a unique complex with normal thymocyte subpopulations. Flow cytometry analysis demonstrated that CD4+8+ and CD4-8- immature thymocytes preferentially interacted with TNC-R3.1 stromal cell clone. CD4+8+ thymocytes, which interacted with TNC-R3.1 stromal cell clone, contained a higher proportion of large size and cycling T cells than did noninteracting CD4+8+ thymocytes. As is generally accepted, CD4+8+ thymocytes did not respond to any stimulation such as IL-2, anti-CD3 mAb (2C11), or IL-2 plus 2C11. However, culture of isolated CD4+8+ thymocytes on TNC-R3.1 stromal cell monolayer in the presence of suboptimal dose of IL-2 induced a significant cell growth. Moreover, the addition of 2C11 and IL-2 into this coculture system resulted in a dramatic increase of the proliferative response of thymocytes. Flow cytometry analysis showed the proliferating cells on TNC-R3.1, which originated from CD4+8+ thymocytes, were mostly TCR-alpha beta+ CD3+CD4-8+ T cells. These results provide in vitro evidence that CD4+8+ thymocytes are at an intermediate stage of T cell maturation and TNC-R3.1 stromal cell clone induces the growth and differentiation of CD4+8+ thymocytes into CD4-8+ T cells.     

Analysis of thymic stromal cell subpopulations grown in vitro on extracellular matrix in defined medium. II. Cytokine activities in murine thymic epithelial and mesenchymal cell culture supernatants

Two morphologically distinct primary cultures of murine thymic stroma were established and found to be of epithelial (MTEC) and mesenchymal (MTMC) origin. These cultures were generated by selective conditions of tissue disruption and were maintained on extracellular matrix in defined medium. Culture supernatants (CS) from these cultures (EC-CS and MC-CS respectively), were tested for cytokine production and for effects on thymocyte maturation. Both supernatants displayed the activities of IL-3 and of granulocyte/macrophage-CSF and not of IL-1, -2, -4, or IFN. In addition they were found to be mitogenic to murine thymocytes in a "spontaneous" [3H]TdR incorporation assay. The two supernatants differed, however, in their effect on Con A stimulation. EC-CS had a strong enhancing effect, both when used for preincubation (18 h) before Con A stimulation or when present simultaneously with it. MC-CS had a small inconsistent effect under these conditions. Also EC-CS enhanced IL-2 and IL-3 production by thymocytes. The responsive thymocyte subpopulation was the one that does not bind peanut agglutinin. CS of an established thymic epithelial cell line displayed only part of these activities at a considerably lower level. CS from primary kidney cell culture was completely devoid of activity. The results suggest that primary thymic stromal cell cultures, cultivated under the defined conditions described here, may better preserve physiologic secretory activities, and probably also other cell functions, compared with established cell lines. Furthermore, the results are compatible with the hypothesis that the soluble factors, secreted by thymic stromal cells, are active on either very early or late stages of thymic differentiation, whereas the main intrathymic stages of differentiation are conceivable dependent primarily on direct contact with stromal cells.     

TCR-independent activation of extrathymically developed, self antigen-specific T cells by IL-2/IL-15

Naive intrathymically developed T cells, which express foreign Ag-specific TCR, do not express IL-2R. After antigenic stimulation, they express high affinity IL-2R, which enables IL-2 to be used as an autocrine growth factor. On the contrary, extrathymically developed T cells, which express self Ag-specific TCR but are unresponsive to antigenic stimulation, spontaneously express low affinity IL-2R. In this study, we compared the responses of these two subsets of T cells to IL-2R stimulation and examined the influences of TCR-mediated signaling on the responses. IL-2 or IL-15 augmented the proliferative response of Ag-stimulated, intrathymically developed T cells. On the other hand, extrathymically developed T cells proliferated in response to IL-2 or IL-15, independently of Ag stimulation. Furthermore, both IL-2 and IL-15 induced IFN-gamma production of these T cells, which is strikingly augmented by the presence of IL-12. These results revealed functional differences between intrathymically developed, foreign Ag-specific T cells and extrathymically developed, self Ag-specific T cells. The latter can be activated by some inflammatory cytokines, in an Ag-independent manner, similar to NK cells.