Cytotoxicity mediated by soluble antigen and lymphocytes in delayed hypersensitivity. I. Characterization of the phenomenon.

In the presence of specific antigen, lymph node cells from inbred rats with delayed hypersensitivity to tuberculoprotein, bovine gammaglobulin, and egg albumin produced progressive destruction of monolayers of rat embryo fibroblasts in tissue culture, first apparent at 48 hr and maximal at 72 hr. The effect was specific and did not depend on a genetic difference between the lymph node cells and target cells. It required antigen concentrations equal to or greater than 1.25 microg/ml and lymphocyte: target cell ratios of approximately 10 or 20:1. It could be evaluated both by a plaquing technique and by cell enumeration with an electronic particle counter.

Cytotoxicity mediated by soluble antigen and lymphocytes in delayed hypersensitivity. II. Correlation of the in vitro response with skin reactivity.

Damage of rat embryo fibroblasts in the presence of sensitized lymph node cells reacting with specific antigen was shown to be closely correlated with delayed hypersensitivity in the animals from which the lymph node cells were taken. The phenomenon was not correlated with Arthus reactivity. In. animals sensitized with picryl conjugates of ovalbumin or human serum albumin, skin reactivity and the in vitro cytotoxic effect could be elicited only with the homologous conjugate or the protein carrier alone and not with picryl conjugates of heterologous proteins. Lewis rats developed more intense delayed sensitivity than BN rats, and Lewis lymph node cells were correspondingly more effective in producing specific damage of both syngeneic and allogeneic fibroblasts.

Cytotoxicity mediated by soluble antigen and lymphocytes in delayed hypersensitivity. 3. Analysis of mechanism.

The cytopathic effect of lymph node cells from tuberculin-sensitized rats on rat embryo fibroblasts in the presence of PPD was not enhanced by admixture of normal (nonsensitized) lymph node cells. Preincubation studies showed that this in vitro response is initiated by the reaction of lymphocytes with specific antigen, beginning within 30 min, rather than uptake of antigen by the fibroblasts. The supernatant fluids from suspensions of sensitized cells incubated with PPD for 17 hr or more possessed cytotoxic activity. The target fibroblasts showed a marked increase in acid phosphatase content within 48 hr after the addition of sensitized lymph node cells and antigen.

A critical role for lymphotoxin in experimental allergic encephalomyelitis.

The lymphotoxin (LT)/tumor necrosis factor (TNF) family has been implicated in the neurologic inflammatory diseases multiple sclerosis (MS) and experimental allergic encephalomyelitis (EAE). To determine the role of individual family members in EAE, C57BL/6 mice, LT-alpha-deficient (LT-alpha-/- mice), or LT-beta-deficient (LT-beta-/- mice), and their wild-type (WT) littermates were immunized with rat myelin oligodendrocyte glycoprotein (MOG) peptide 35-55. C57BL/6 and WT mice developed chronic, sustained paralytic disease with average maximum clinical scores of 3.5 and disease indices (a measure of day of onset and sustained disease scores) ranging from 367 to 663 with central nervous system (CNS) inflammation and demyelination. LT-alpha-/- mice were primed so that their splenic lymphocytes proliferated in response to MOG 35-55 and the mice produced anti-MOG antibody. However, LT-alpha-/- mice were quite resistant to EAE with low average clinical scores (<1), an average disease index of 61, and the negligible CNS inflammation and demyelination. WT T cells transferred EAE to LT-alpha-/- recipients. LT-beta-/- mice were susceptible to EAE, though less than WT, with an average maximum clinical score of 1.9 and disease index of 312. These data implicate T cell production of LT-alpha in MOG EAE and support a major role for LT-alpha3, a minor role for the LT-alpha/beta complex, and by inference, no role for TNF-alpha.

Chronic inflammation caused by lymphotoxin is lymphoid neogenesis.

In presenting a unifying concept for chronic inflammation and lymphoid organogenesis, we suggest that lymphotoxin's (LT, LT-alpha, TNF-beta) crucial role in these processes is pivotal and similar. Chronic inflammatory lesions that developed in the kidney and pancreas at the sites of transgene expression in rat insulin promoter-LT (RIP-LT) mice resembled lymph nodes with regard to cellular composition (T cells, B cells, plasma cells, and antigen-presenting cells), delineated T and B cell areas, primary and secondary follicles, characteristic morphologic and antigenic (ICAM-1, VCAM-1, MAdCAM-1, and PNAd) features of high endothelial venules, and ability to respond to antigen and undergo Ig class switching when obtained from mice immunized with SRBC. The vascular changes, with the exception of PNAd, appear to be the direct consequence of transgene derived LT expression, as they were also observed in RIP-LT mice lacking mature T and B cells. These data show that LT-induced chronic inflammation has the characteristics of organized lymphoid tissue.

Expression of endogenous murine leukemia viruses during the course of a protracted immunological disorder.

Mice of the low leukemia (BALB/cJ x A/J)F1 hybrid (CAF1) strain express B-and N-tropic infectious murine leukemia virus (MuLV) after the age of 6 mo. Initation of a protracted immunological disorder, the graft-versus-host reaction (GVHR), at 7 wk of age, accelerates the induction of both these mouse-tropic endogenous viruses, and preferentially enhances the replication of B-tropic MuLV. The earlier appearance of B-tropic MuLV in a greater proportion of mice and in higher titer is thought to be casually related to the eventual development of lymphoreticular tumors in the GVHR mice, since previous studies have shown that these same tumors can be reproduced by inoculating syngeneic recipients with serially passaged GVHR extracts containing B-tropic MuLV.

Surface expression of alpha 4 integrin by CD4 T cells is required for their entry into brain parenchyma.

Cloned CD4 T cell lines that recognize the Ac1-16 peptide of myelin basic protein bound to I-Au were isolated and used to analyze the immunopathogenesis of experimental autoimmune encephalomyelitis (EAE). T helper type 1 (Th1) clones induced disease, while Th2 clones did not. Using variants of a single cloned Th1 line, the surface expression of alpha 4 integrins (very late antigen 4 [VLA-4]) was identified as a major pathogenic factor. Encephalitogenic clones and nonencephalitogenic variants differ by 10-fold in their level of surface expression of alpha 4 integrin and in their ability to bind to endothelial cells and recombinant vascular cell adhesion molecule 1 (VCAM-1). The alpha 4 integrin-high, disease-inducing cloned Th1 T cells enter brain parenchyma in abundance, while alpha 4 integrin-low, nonencephalitogenic Th1 cells do not. Moreover, antibodies to alpha 4 integrin, its ligand VCAM-1, and intercellular adhesion molecule 1 all influence the pathogenicity of this encephalitogenic clone in vivo. The importance of the expression of VLA-4 for encephalitogenicity is not unique to cloned T cell lines, as similar results were obtained using myelin basic protein-primed lymph node T cells. alpha 4 integrin levels did not affect antigen responsiveness or production of the Th1 cytokines interleukin 2, interferon gamma, and lymphotoxin/tumor necrosis factor beta; and antibodies against alpha 4 integrin did not block antigen recognition in vitro. Thus, we conclude that surface expression of alpha 4 integrin is important in CD4 T cell entry into brain parenchyma. A general conclusion of these studies is that alpha 4 integrins may be crucial in allowing activated effector T cells to leave blood and enter the brain and other tissues to clear infections.

Tumor induction by immunologically activated murine leukemia virus.

A graft-vs.-host reaction (GVHR) was induced in young male CAF(I) and CB6F(1) mice by the administration of BALB/cJ spleen cells. A proportion of such mice subsequently developed lymphoreticular rumors. Cell-free extracts (CFEs) prepared from the reticular tissues of CAF(1) mice killed at intervals after the induction of the GVHR were tested for their capacity to produce the same tumors in a litter of syngeneic mice inoculated at birth. 12 of 29 (41.4%) such extracts were positive, causing lymphoreticular tumors in one or more littermate recipients. The positive CFEs came from donors killed at all stages of the GVHR, from tumor-bearing mice as well as from non-tumor-bearing mice. However, whereas less than 30% of CFEs from mice killed within 12 mo of GVHR induction were oncogenic, the incidence of oncogenic extracts from mice killed 12-15 mo after GVHR induction rose to 75%. None of the CFEs prepared from nine normal uninjected male CAF(1) mice killed between the ages of 8 and 18 mo transmitted tumors to recipients. CFEs prepared from CAF(1) mice with the GVHR were tested for infectious murine leukemia virus (MuLV) using the XC assay and also for complement-fixing (CF) group-specific MuLV antigen. Substantial titers of B-tropic MuLV and CF antigen were detected in at least half the extracts from mice killed 11-14 mo after GVHR induction. During the first few months of GVHR induction, MuLV titers were low and CF antigen was absent. Neither infectious MuLV nor CF antigen were detected in CFEs prepared from normal control mice. Serially passed CFEs originating from a CB6F(1) GVHR-induced RCN caused similar tumors in successive generations of syngeneic recipient mice. These lymphoreticular tumors were shown to contain infectious MuLV, CF MuLV antigen, and C-type particles. These data together provide evidence that MuLV is activated during the GVHR and that it is responsible for the eventual development of lymphoreticular tumors.

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.

Assignment of the receptor for ecotropic murine leukemia virus to mouse chromosome 5.

The gene for the receptor for ecotropic murine leukemia virus (Rev) has been assigned to mouse chromosome 5. This determination was made possible by an analysis of somatic cell hybrids between mouse and Chinese hamster cells. The parents of these hybrids were A/HeJ or Mus poschiavinus peritoneal exudate cells or BALB/c primary embryo fibroblasts and E36, a Chinese hamster lung fibroblast deficient in hypoxanthine guanine phosphoribosyltransferase. Segregation of mouse chromosomes in these hybrids was analyzed by chromosome banding and isozyme expression. Cells were tested for their ability to absorb and replicate vesicular stomatitis virus (murine leukemia virus [MuLV]) pseudotype particles and ecotropic MuLV as measured by the XC test. The presence of chromosome 5 was essential for receptor expression as determined by three statistical procedures. Segregation of the receptor for ecotropic murine leukemia virus was also followed in two series of subclones. In both, receptor expression was syntenic with phosphoglucomutase-1, an isozyme which has been mapped to mouse chromosome 5.

Cloning and expression of a Leishmania donovani gene instructed by a peptide isolated from major histocompatibility complex class II molecules of infected macrophages.

The studies reported here describe the isolation of peptides from MHC class II molecules of murine macrophages infected with Leishmania donovani, and the use of the derived peptide sequences to rescue the pathogen peptide donor protein. The isolation of the peptides was carried out by comparing the RP HPLC profile of peptides extracted from infected macrophages with the peptides extracted from noninfected cells. Several distinct HPLC peaks unique to infected macrophages were sequenced. One of the peptides that was not homologous to any known protein was used to instruct the designing of an oligonucleotide sense primer that was used in combination with an oligo dT nucleotide (anti-sense primer) to amplify by PCR a DNA fragment from L. donovani cDNA. The amplified DNA fragment was cloned and used as a probe to screen a L. donovani cDNA library. The cloned gene (Ld peptide gene) has an open reading frame of 525 bp and has no homology with any known protein/gene sequence. Northern blot analyses indicated that the Ld peptide/gene is broadly distributed and expressed among species of the Leishmania genus, in both the amastigote and promastigote life cycle forms. Using the pGEX 2T vector, the gene was expressed and the relationship of the purified recombinant protein with L. donovani was confirmed using both antibody and T cell responses from immunized or infected animals. The gene encodes a 23-kD molecule (Ldp 23) associated with the cell surface of L. donovani promastigotes. In addition, T cells purified from the lymph nodes of BALB/c mice immunized with L. donovani or infected with L. major, and from CBA/J mice infected with L. amazonensis were stimulated to proliferate by the recombinant Ldp 23 and produced high levels of IFN-gamma and no IL 4. This observation suggests that the Ldp 23 is an interesting parasite molecule for the studies concerning the host/parasite interaction because the Th1 pattern of cytokine response that it induces is correlated with resistance to Leishmania infections. These results clearly point to an alternative strategy for the purification of proteins useful for the development of both vaccines and immunological diagnostic tools not only against leishmaniasis but also for other diseases caused by intracellular pathogens.

Cytotoxic effect of lymphocyte-antigen interaction in delayed hypersensitivity.

Lymph node cells from inbred rats having delayed sensitivity to soluble proteins inhibit growth of syngeneic or allogeneic fibroblasts in the presence of specific antigen. A relation is suggested between this in vitro phenomenon and other systems believed to be specific manifestations of delayed hypersensitivity.

Abnormal development of peripheral lymphoid organs in mice deficient in lymphotoxin.

Mice rendered deficient in lymphotoxin (LT) by gene targeting in embryonic stem cells have no morphologically detectable lymph nodes or Peyer's patches, although development of the thymus appears normal. Within the white pulp of the spleen, there is failure of normal segregation of B and T cells. Spleen and peripheral blood contain CD4+CD8- and CD4-CD8+ T cells in a normal ratio, and both T cells subsets have an apparently normal lytic function. Lymphocytes positive for immunoglobulin M are present in increased numbers in both the spleen and peripheral blood. These data suggest an essential role for LT in the normal development of peripheral lymphoid organs.

Tumor necrosis factor (TNF-alpha) and lymphotoxin (TNF-beta).

Distinctions between tumor necrosis factor, TNF-alpha, and lymphotoxin, TNF-beta, have previously been based on the differences between their protein sequences, biological activity, and molecular regulation. In the past year, elucidation of the molecular nature of the two molecules and the interactions with common receptors has emphasized their similarities, although profound differences continue to emerge with regard to their mode of production, transcription rates, mRNA half-lives, and the importance of various DNA regulatory sequences. A role for both TNF-alpha and TNF-beta has recently been suggested with regard to disease, particularly multiple sclerosis. The past year has also seen the description of an extensive microsatellite polymorphic system which should provide a more definitive understanding of the association of the TNF locus with disease.

Mediators of inflammation.

Therapeutic studies and genetically engineered animals have elucidated the inflammatory roles of cytokines and chemokines in autoimmune disease. Most unexpected has been a continuum of recent evidence demonstrating that inflammatory mediators are crucial in lymphoid organ development, thus suggesting that these hitherto unrelated processes have common elements with implications for determinant spreading.

A poly(dA-dT) upstream activating sequence binds high-mobility group I protein and contributes to lymphotoxin (tumor necrosis factor-beta) gene regulation.

Lymphotoxin (LT; also known as tumor necrosis factor-beta) is a pleiotropic cytokine whose expression is tightly regulated in most cells and is repressed prior to activation signals. In some early B cells and Abelson murine leukemia virus-transformed pre-B-cell lines, LT mRNA is constitutively expressed. To examine the molecular regulation of the LT gene in a constitutively expressing cell line, we studied the Abelson murine leukemia virus-transformed lines PD and PD31. As demonstrated by primer extension analysis, constitutively expressed pre-B-cell-derived and inducibly expressed T-cell-derived LT mRNA were initiated at the same cap sites and predominant cap site utilization was conserved. Furthermore, we delineated an upstream activating sequence that was an important functional component of lymphotoxin transcriptional activation in PD and PD31 cells. The upstream activating sequence was localized to an essentially homopolymeric A + T-rich region (LT-612/-580), which was bound specifically by recombinant human high-mobility group I protein (HMG-I) and a PD/PD31 nuclear extract HMG-I (HMG-I-like) protein. The nuclear extract-derived HMG-I-like protein was recognized by anti-HMG-I antibody and bound to LT DNA to effect an electrophoretic mobility shift identical to that of bound recombinant human HMG-I. These findings implicate HMG-I in the regulation of constitutive lymphotoxin gene expression in PD and PD31 cells. HMG-I and HMG-I-like proteins could facilitate the formation of active initiation complexes by altering chromatin structure and/or by creating recognition sites for other activator DNA-binding proteins, some of which may be unique to or uniquely modified in these constitutive LT mRNA producers.

Role of endogenous murine leukemia virus in immunologically triggered lymphoreticular tumors. I. Development and use of oncogenic cellfree preparations serially passaged in vivo.

Cellfree extracts (CFEs) prepared from (BALB/cJ X A/J)F1 (CAF1) and (BALB/cJ X C57BL/6J)F1 (CB6F1) mice in which a graft-versus-host reaction (GVHR) has been induced are known to be oncogenic, but only after a protracted latent period (mean, 16 mo). Serial passage of such CFEs in successive generations of syngeneic mice inoculated at birth led to the development of two separate oncogenic preparations, the CA serioes in CAF, mice and the CB series in CB6F, mice, in which the mean latent period was reduced to 6 and 12 months, respectively. Both oncogenic preparations contained infectious B-tropic murine leukemia virus (MuLV) and particles with the ultrastructural characteristics of MuLV. No other kind of virus particle was seen. When these preparations were injected into infant syngeneic mice, B-tropic MuLV could be detected in the reticular tissues as early as 2 weeks thereafter. The virus persisted in the reticular tissues and was present in the lymphoreticular tumors that subsequently developed. However, if the same preparation was injected into young adult recipients, there may have been transient MuLV replication, but the virus subsequently disappeared from the reticular tissues and no lymphoreticular tumors developed. Previous experiments showed that MuLV was present in CFEs prepared from CAF, animals with the GVHR but absent in those of normal control mice. Since the lymphoreticular tumors arising in mice with the GVHR were the same as those induced by the CA and CB MuLV preparations, it was concluded that tumorigenesis in mice with the GVHR was caused by endogenous B-tropic MuLV activated by the immunologic disturbance.

The contribution of insulitis to diabetes development in tumor necrosis factor transgenic mice.

The inflammatory response mediated by cytokines such as TNF can promote recruitment of lymphocytes to a tissue. Moreover, if other conditions are met, this can provide a predisposing role to autoimmune disease. TNFs induce the appearance of adhesion molecules (and presumably, therefore, extravasation of lymphocytes into tissue from the vasculature) and increase the levels of MHC class I on tissue. However, it is not clear which of these effects plays the key role in induction of disease. This should be the subject of further study. The data substantiate the hypothesis that chronic inflammation might play a precipitating role in autoimmunity and could be one of the environmental factors of importance in the development of so many autoimmune diseases.

Lymphoid neo-organogenesis: lymphotoxin's role in inflammation and development.

Lymphoid organ development and inflammation have previously been considered as distinct mechanistically and functionally. In recent years, it has been realized that these phenomena have much in common. This insight has been gained from the recognition that cytokines of the lymphotoxin (LT)/tumor necrosis factor (TNF) family are involved in both processes. The members of the family, LT-alpha, LT-beta, and TNF-alpha, and their multiple receptors participate combinatorially in lymphoid organ development and chronic inflammation. When inflammation that arises in microbial infection or autoimmune disease becomes chronic, it can take on the appearance of organized lymphoid tissue and has been called a tertiary lymphoid organ. Data with transgenic and knockout mice suggest that the process is cytokine-mediated and could be called "lymphoid neo-organogenesis." LT as LT-alpha3 and LT-alpha1beta2 plays a key role in these processes. Data obtained in vitro in an endothelial cell line and in vivo in transgenic and knockout mice indicate that LT influences these events through induction of adhesion molecules such as E-selectin adhesion molecule (ELAM), vascular cell adhesion molecule (VCAM), intercellular adhesion molecule (ICAM), mucosal addressin cellular adhesion molecule (MAdCAM), and peripheral node addressin (PNAd), and chemokines.

A critical analysis of the T cell hybrid technique.

The T cell hybridization technique can be used to prepare continuous cell lines which express the antigen specificity and function of T cells within the milieu of a proliferating lymphoma. Technical details for the preparation and maintenance, selection and analysis of T cell hybrids are defined. Techniques for cloning of hybrid cells and production of hybrid-derived tumors are also presented. Parameters influencing the hybridization frequency and the production of functional hybrids are discussed. A variety of T cell subsets, including suppressor cells and delayed-type hypersensitivity effector cells as well as T cells maintained on TCGF, are excellent sources of primary parents for hybridization. When BW 5147 is used as the T lymphomas parent in these experiments, the resulting hybridization frequencies range between 10 and 434 X 10(-7). We have had moderate success using YAC-1; however, additional lines such as L5178Y, BALENTL 5, EL4 BU and S491TB.2 have proved ineffective as sources of T lymphoma parents. The technique of T cell hybridization is evaluation in terms of retention of differentiated functions and the stability and growth characteristics of the resultant hybrids.