Enterocyte expression of interleukin 7 induces development of gammadelta T cells and Peyer's patches.

The intestinal mucosa is suggested to support extrathymic T cell development, particularly for T cell receptor (TCR)-gammadelta intraepithelial lymphocytes (IELs). TCR-gammadelta cell development requires interleukin (IL)-7; IL-7(-/)- or IL-7 receptor(-/)- mice lack TCR-gammadelta cells. Using the intestinal fatty acid binding protein (iFABP) promoter, we reinstated expression of IL-7 to mature enterocytes of IL-7(-/)- mice (iFABP-IL7). In iFABP-IL7 mice, TCR-gammadelta IELs were restored, as were cryptopatches and Peyer's patches. TCR-gammadelta cells remained absent from all other tissues. Likewise, T cell development in thymus and B cell maturation in the bone marrow and spleen retained the IL-7(-/)- phenotype. Thus, IL-7 expression by enterocytes was sufficient for extrathymic development of TCR-gammadelta cells in situ within the intestinal epithelium and was crucial for organization of mucosal lymphoid tissue.

Proinflammatory roles of T-cell receptor (TCR)gammadelta and TCRalphabeta lymphocytes in a murine model of asthma.

The role of lymphocytes bearing alphabeta or gammadelta T-cell receptors (TCRs) was assessed during the acute allergic response in a mouse model of asthma. The inflammatory immune response to ovalbumin (OVA) was characterized in wild-type C57BL/6J mice and congenic TCRbeta(-/-) and TCRdelta(-/-) mice by evaluation of airway eosinophilia, histopathology, serum immunoglobulin (Ig)E levels, and in vivo airway responsiveness to methacholine. OVA-challenged wild-type mice demonstrated marked pulmonary inflammation, evidenced by airway eosinophilia (68 +/- 7 x 10(4) cells), peribronchial lympho-plasmocytic infiltration, and elevated serum IgE (4.9 +/- 0.6 microg/ml). These responses were markedly attenuated in TCRdelta(-/-) animals (5.0 +/- 1.0 x 10(4) eosinophils and 1.6 +/- 0. 3 microg/ml IgE) and were completely absent in TCRbeta(-/-) mice (< 1 x 10(3) eosinophils and 0.38 +/- 0.21 microg/ml IgE). Similar results were observed in mice treated with anti-TCRgammadelta or anti-TCRalphabeta monoclonal antibodies. Airway responsiveness to aerosolized methacholine was also reduced in challenged TCRdelta(-/-) animals relative to challenged wild-type mice. These results demonstrate that acute allergic airway responses are dependent upon intact TCRalphabeta and TCRgammadelta lymphocyte function and that TCRgammadelta cells promote acute airway sensitization.

Adoptively transferred EAE in gamma delta T cell-knockout mice.

Recently there has been evidence suggesting that gamma delta receptor-bearing T cells may play a role in both multiple sclerosis (MS) and experimental allergic encephalomyelitis (EAE). We have recently described approaches for the generation of encephalitogenic T-cell populations from EAE-resistant strains of mice. Using encephalitogenic T-cell lines and clones generated from wild-type C57BL/6 mice we have studied adoptively transferred EAE in C57BL/6-TCR delta-knockout mice. We now report that the adoptive transfer of encephalitogenic T cells into TCR delta T-knockout mice leads to clinical EAE that is not significantly different in severity or time course than that seen after transfer into wild-type C57BL/6 mice. We conclude that gamma delta T cells do not play an integral role in the mediation or regulation of the effector-phase mechanisms in EAE.

Adoptively transferred EAE in mice bearing the lpr mutation.

We have recently developed approaches for the generation of encephalitogenic T cell clones from mouse strains considered resistant to experimental allergic encephalomyelitis (EAE). By allowing for the direct use of knockout and mutant strains of mice, such clones allow for the efficient characterization of the relevance of specific gene products in the effector phase of EAE. Recent studies have suggested that Fas/FasL-mediated cell death may play a role in the pathogenesis of MS. To assess the role of Fas/FasL in EAE, we have tested the ability of wild-type C57BL/6-derived, encephalitogenic T cell clones to mediate adoptively transferred EAE in Fas-deficient C57BL/6-lpr mice. We now report that mice with the lpr mutation are fully susceptible to the adoptive transfer of EAE. Our results suggest that Fas/FasL-mediated cell death in the central nervous system does not play an integral role in the effector phase of acute EAE.

The generation of encephalitogenic T cell lines from experimental allergic encephalomyelitis-resistant strains of mice.

While only a few strains of mice are susceptible to the primary induction or passive transfer of experimental allergic encephalomyelitis (EAE), the basis of EAE resistance remains unclear. In the present studies, we have defined two approaches that allow for the generation of encephalitogenic, myelin basic protein-reactive, T cell lines from EAE-resistant strains of mice. The first approach, based on the putative relevance of apoptosis to autoimmune disease, involves repeat antigenic stimulation of recently initiated T cell lines. The second approach involves the initiation of lymph node cultures in the absence of exogenous splenocytes as antigenic-presenting cells and the use of a higher antigen concentration. Both approaches lead to the generation of encephalitogenic T cell lines from EAE-resistant mouse strains and will be useful for identifying factors relevant to the pathogenesis of EAE.

Elevation of intracellular cyclic AMP induces an anergic-like state in Th1 clones.

Because elevated intracellular cAMP suppresses T cell receptor (TCR)-mediated effector activity and/or proliferation in response to antigen but does not always affect IL-2-stimulated proliferation, the effects of cAMP on a T lymphocyte response to antigen resemble antigen-induced anergy. To test the hypothesis that elevated cAMP induces anergy in T lymphocytes, we have precultured murine Th1 clones responsive to porcine myelin basic protein (PMBP) with dibutyryl cyclic AMP (dbcAMP) or forskolin and subsequently removed the dbcAMP or forskolin and measured the proliferative response of the clones to antigen and antigen-presenting cells (APC) in the presence or absence of exogenously added interleukin-2 (IL-2). Cells precultured with dbcAMP or forskolin for 3 days did not proliferate or produce IL-2 in response to antigen and APC, but did proliferate to antigen and APC in the presence of IL-2. Cells that had not been stimulated recently with antigen/APC or IL-2 were not affected by dbcAMP, while cells stimulated recently with antigen/APC and IL-2 were susceptible to the anergizing effect of dbcAMP. These observations support the hypothesis that elevation in intracellular cAMP in antigen-activated Th1 clones, prior to subsequent culture with antigen, induces a state of anergy.

Identification of encephalitogenic V beta-4-bearing T cells in SJL mice. Further evidence for the V region disease hypothesis?

Experimental allergic encephalomyelitis (EAE) is an autoimmune disease of the central nervous system mediated by T cells bearing TCR of restricted heterogeneity. Thus, in the murine PL strain, V beta-8.2 is used by 80% of the encephalitogenic T cells. This observation has led to the successful prevention and reversal of EAE by the in vivo use of mAb directed to these restricted gene products. In SJL mice, the V beta-17a gene product has been shown to be used by approximately 50% of encephalitogenic T cells subsequent to immunization with a myelin basic protein (MBP)-derived peptide. However, the other V beta genes used by encephalitogenic T cells in SJL EAE have remained uncharacterized. We now report, for the first time, the beta-chain-encoding DNA sequence of two encephalitogenic, MBP-reactive, SJL-derived T cell clones. These clones which are specific for H-2s and the carboxyl-terminus (amino acid 92-103) of MBP, use TCR encoded by V beta-4. In addition, we demonstrate that the transfer of EAE by a heterogenous SJL-derived encephalitogenic T cell line can be prevented using an anti-V beta-4 antibody in vivo. V beta-4 usage has been previously described in a H-2u/MBP amino-terminus-reactive encephalitogenic T cell. The present findings may thus further support the "V region-disease" hypothesis.

An antibody to lymphotoxin and tumor necrosis factor prevents transfer of experimental allergic encephalomyelitis.

Uncertainty regarding pathogenic mechanisms has been a major impediment to effective prevention and treatment for human neurologic diseases such as multiple sclerosis, tropical spastic paraparesis, and AIDS demyelinating disease. Here, we implicate lymphotoxin (LT) (tumor necrosis factor beta [TNF-beta]) and TNF-alpha in experimental allergic encephalomyelitis (EAE), a murine model of an autoimmune demyelinating disease. In this communication, we report that treatment of recipient mice with an antibody that neutralizes LT and TNF-alpha prevents transfer of clone-mediated EAE. LNC-8, a myelin basic protein-specific T cell line, produces high levels of LT and TNF-alpha after activation by concanavalin A, antibody to the CD-3 epsilon component of the T cell receptor, or myelin basic protein presented in the context of syngeneic spleen cells. LNC-8 cells transfer clinical signs of EAE. When LNC-8 recipient mice were also treated with TN3.19.12, a monoclonal antibody that neutralizes LT and TNF-alpha, the severity of the transferred EAE was reduced, while control antibodies did not alter the disease. The effect of anti-LT/TNF-alpha treatment was long lived and has been sustained for 5 mo. These findings suggest that LT and TNF-alpha and the T cells that produce them play an important role in EAE.

Murine T helper cell clones secrete granulocyte-macrophage colony-stimulating factor (GmCSF) by both interleukin-2-dependent and interleukin-2-independent pathways.

Granulocyte-macrophage colony-stimulating factor (GmCSF) is a lymphokine secreted by class II major histocompatibility complex (MHC)-restricted T cells after lectin or antigen stimulation. To investigate the relationship between interleukin-2 (IL-2) and GmCSF production, we utilized long-term cultures of porcine myelin basic protein (PMBP)-specific T helper cell clones that were maintained with IL-2 in the absence of antigen or irradiated antigen-presenting cells (APC). We have found that supernatants of these T cell clones contained GmCSF activity after IL-2 stimulation. Inhibition of cell proliferation by irradiation failed to stop GmCSF production. When these clones were stimulated with PMBP and irradiated APC in the presence of anti-IL-2 receptor antibody, the T cell supernatants still contained GmCSF activity. These results indicate that (1) GmCSF production by T helper clones after IL-2 stimulation is independent of cell proliferation and (2) antigen/MHC-stimulated GmCSF production by T cell clones can occur by an IL-2-independent pathway.

Effects of H-7 are not exclusively mediated through protein kinase C or the cyclic nucleotide-dependent kinases.

Culturing murine T cell tumor lines in the presence of the protein kinase inhibitor H-7 for 4 days led to their dependence on H-7 for maximal constitutive proliferation. Withdrawal of H-7 from H-7-conditioned cells led to inhibition of proliferation and cell death. The mechanism underlying this H-7 dependence does not appear to be related to clonal selection or to effects on protein kinase C or the cyclic nucleotide-dependent kinases. This suggests that all the effects of the widely used H-7 may not be completely understood, and that H-7 may be useful in the dissection of the complex patterns of growth regulation in T cell malignancies.

Subtypes of helper cells. Non-inflammatory type 1 helper T cells.

Class II MHC-restricted T cells recently have been characterized as being either type 1 (Th1) or type 2 (Th2) based on their ability to both secrete different lymphokines and perform different functions. Characterization of these subtypes to date have indicated that Th1 cells secrete IL-2, IFN-gamma, lymphotoxin, and IL-3, whereas Th2 cells secrete IL-4, IL-5, and IL-3. Functionally, Th1 cells mediated cytotoxicity and delayed-type hypersensitivity, and have been termed "inflammatory cells," whereas Th2 cells mediate helper function for Ig secretion and have been termed, "regulatory cells." We now present evidence that not all Th1 clones are inflammatory and capable of mediating cutaneous delayed-type hypersensitivity. We have generated a number of myelin basic protein-specific Th1 clones that do not mediate swelling when injected together with myelin basic protein directly into the footpads of syngeneic mice. These results suggest that Th1 cells can be further subdivided based on their ability to mediate delayed-type hypersensitivity, and that the Th1/Th2 characterization of Th cells may be insufficient to adequately characterize all functional subtypes of class II MHC-restricted T cells.

T cell receptor beta chain gene rearrangement shared by murine T cell lines derived from a site of autoimmune inflammation.

Advances in our understanding of the structure and molecular biology of the T lymphocyte antigen-receptor have now made it feasible to study human autoimmune diseases using new approaches. One such approach involves cloning of T cells from sites of autoimmune pathology followed by identification of putative disease-related T cell oligoclonality at the level of the T cell receptor gene rearrangements. We have now tested the feasibility of this approach in an animal model of autoimmunity, murine experimental allergic encephalomyelitis (EAE). Spinal cord-derived, self (murine) myelin basic protein (MBP)-reactive T cell lines and sublines were analyzed at the level of their receptor beta chain rearrangements using Southern blots. We now report that the MBP-reactive T cell lines and sublines derived from the spinal cords of four of five SJL/J mice with EAE share a 14.5-kb rearranged T cell receptor beta 1 band on Southern blots. A spinal cord-derived T cell line that was reactive to purified protein derivative of tuberculin (PPD), several lymph node-derived ovalbumin- and PPD-reactive T cell lines, as well as one MBP-reactive spinal cord-derived T cell line did not share this 14.5-kb rearranged beta 1 band. These results suggest that analysis of the antigen receptors used by T cells cloned from sites of inflammation may be a useful initial approach for identifying pathogenetically relevant T cells in the study of certain human autoimmune diseases.

The protein kinase C inhibitor H-7, inhibits antigen and IL-2-induced proliferation of murine T cell lines.

Activation of protein kinase C has been shown to be involved in the activation pathway of many cell types. Recently, a number of investigations have suggested that protein kinase C plays an essential role in T lymphocyte activation. The recent synthesis of the protein kinase inhibitors, H-7 and HA1004, have now made possible a new approach for testing the relevance of protein kinase C in T cell activation and proliferation. We now report that the antigen-induced and interleukin-2-induced proliferation of murine T cell lines can be consistently inhibited by the protein kinase C inhibitor, H-7. HA1004, a somewhat more potent inhibitor of cyclic nucleotide-dependent protein kinases, but a significantly weaker inhibitor of protein kinase C than H-7, demonstrated no consistent inhibition of these T cell responses. These results represent a further demonstration that protein kinase C plays an essential role in the activation of T cells.

T cell lines derived from the spinal cords of mice with experimental allergic encephalomyelitis are self reactive.

Experimental allergic encephalomyelitis (EAE) is an animal model of T cell-mediated, central nervous system neuropathology that may be a relevant animal model for multiple sclerosis. EAE is usually induced by sensitization of animals with a xenogeneic myelin basic protein (MBP). Recently, MBP-reactive T cell lines and clones derived from lymphoid tissue of animals with EAE have proved very useful in elucidating certain aspects of the pathogenesis in EAE. However, questions relating to how T cells actually mediate the pathologic changes seen in EAE remain unresolved. We now report for the first time the derivation of long-term, interleukin 2-dependent T cell lines and sublines from a site of pathology in murine EAE--the spinal cord. All of the spinal cord-derived T cell lines and sublines were found to be "autoreactive" in that they responded to self (murine) MBP as well as to the xenogeneic immunogen, porcine MBP. The ability to derive T cell lines and sublines from the spinal cords of mice with EAE should now aid in the elucidation of pathogenetic mechanisms in EAE by allowing for a characterization of those T cells found at the site of pathology.

Compartmentalized immune responses: antigen-specificity of cerebrospinal fluid T-cell lines maintained in the absence of antigen.

The generation of long-term interleukin 2-dependent T-cell lines from anatomically compartmentalized sites of pathology offers a unique approach to the investigation of certain autoimmune diseases. However, it is generally believed that antigen-specific T-cell lines and clones lose antigen reactivity and specificity when propagated in the absence of antigen. Therefore, the optimal application of this approach to such diseases in which the pathogenetic antigens are unknown may be difficult. In approaching this problem, we have recently demonstrated that a proportion of antigen-specific T-cell lines derived from the peripheral circulation can maintain antigen specificity if propagated with antigen-presenting cells alone or with antigen-presenting cells together with OKT3 antibody, but in either case in the absence of antigen. In this report we describe the use of this approach to maintain the antigen specificity of T cells obtained from an anatomically compartmentalized site of pathology--the cerebrospinal fluid from a patient with tuberculous meningitis. We report here that a proportion of the T-cell lines generated from such cerebrospinal fluid lymphocytes can be maintained as antigen specific in the absence of antigen if propagated with either antigen-presenting cells alone or with antigen-presenting cells and OKT3 antibody. The approach illustrated in this report should now find broad applicability in the investigation of a number of autoimmune disease.

Maintenance of antigen specificity by human interleukin-2-dependent T cell lines. Use of antigen-presenting cells and OKT3 antibody in the absence of antigen.

The in vitro growth of T cells obtained from localized anatomic sites of pathology may offer a new approach to the investigation of certain human autoimmune diseases. However, if interleukin-2-dependent T cell cloning is to be useful in helping to elucidate putative pathogenetic antigens in these diseases, the expansion of the small number of T cells obtainable from localized anatomic sites of pathology will often have to be accomplished in the absence of these, as yet undetermined, antigens. At present, it is a generally held belief that antigen-responsive, interleukin-2-dependent T cell lines and clones will lose antigen responsiveness if propagated in the absence of specific antigen. Thus, the use of T cell cloning might be viewed as being of limited usefulness in the investigation of certain human autoimmune diseases. In this report we demonstrate that, when propagated in the absence of antigen, human tetanus toxoid-specific, interleukin-2-dependent T cell lines will indeed lose antigen reactivity. However, if propagated in the absence of antigen but in the presence of antigen-presenting cells, the tetanus toxoid reactivity of a subset of such lines can be maintained. Moreover, the propagation with OKT3 antibody, in addition to antigen-presenting cells, may be even more effective in maintaining antigen reactivity. These results may suggest a new approach to the use of T cell cloning technology in the investigation of certain autoimmune diseases.