Gait analysis in a murine model of collagen-induced arthritis

Murine collagen-induced arthritis (CIA) has become a valuable animal model for elucidating pathogenic mechanisms and evaluating therapeutic effects for rheumatoid arthritis. Recent advances in digital imaging and computer technology have enabled gait analysis to develop into a powerful tool for objectively detecting functional deficits in human and animal models. The present study explored the use of non-invasive video-capture gait analysis in the evaluation of a murine CIA model. CIA was induced in 45 female DBA/1LacJ mice (8 to 10 weeks old) by immunization with lyophilized bovine articular type II collagen. Gait parameters were determined by ventral plane videography and were correlated to traditional arthritis clinical scores. Our results showed that increases in clinical scores that measure the severity of CIA corresponded to changes in multiple gait parameters that reflect both morphologic (increases in paw area) and functional (increase in stride frequency, decrease in stride length, hind-limb paw placement angle, as well as stride, stance, and braking times) deficits. Our work indicated that the non-invasive video-capture device may be used as a simple and objective data acquisition system for quantifying gait disturbances in CIA mice for the investigation of mechanisms and the evaluation of therapeutic agents.     

Inhibition of fractalkine ameliorates murine collagen-induced arthritis

Rheumatoid arthritis (RA) is a chronic inflammatory disease associated with massive infiltration of inflammatory cells in the synovium of multiple joints. We and others have shown that fractalkine (FKN/CX3CL1), a chemokine expressed on fibroblast-like synoviocytes and endothelial cells in RA synovium, may contribute to the accumulation of T cells, macrophages, and dendritic cells, which express CX3CR1, the receptor for FKN. This interaction might be involved in adhesion of the inflammatory cells to endothelial cells, migration into the synovium, and cytokine production. In this study, we examined the effect of FKN inhibition on murine collagen-induced arthritis. Anti-FKN mAb significantly lowered clinical arthritis score compared with control Ab, and reduced infiltration of inflammatory cells and bone erosion in the synovium. However, anti-FKN mAb did not affect the production of either serum anti-collagen type II (CII) IgG or IFN-gamma by CII-stimulated splenic T cells. Furthermore, treatment with anti-FKN mAb inhibited migration of adoptively transferred splenic macrophages into the inflamed synovium. Our results suggest that anti-FKN mAb ameliorates arthritis by inhibiting infiltration of inflammatory cells into the synovium. Thus, FKN can be a new target molecule for the treatment of RA.     

Nucleosides from Phlebotomus papatasi salivary gland ameliorate murine collagen-induced arthritis by impairing dendritic cell functions

Among several pharmacological compounds, Phlebotomine saliva contains substances with anti-inflammatory properties. In this article, we demonstrated the therapeutic activity of salivary gland extract (SGE) of Phlebotomus papatasi in an experimental model of arthritis (collagen-induced arthritis [CIA]) and identified the constituents responsible for such activity. Daily administration of SGE, initiated at disease onset, attenuated the severity of CIA, reducing the joint lesion and proinflammatory cytokine release. In vitro incubation of dendritic cells (DCs) with SGE limited specific CD4(+) Th17 cell response. We identified adenosine (ADO) and 5'AMP as the major salivary molecules responsible for anti-inflammatory activities. Pharmacologic inhibition of ADO A2(A) receptor or enzymatic catabolism of salivary nucleosides reversed the SGE-induced immunosuppressive effect. Importantly, CD73 (ecto-5'-nucleotidase enzyme) is expressed on DC surface during stage of activation, suggesting that ADO is also generated by 5'AMP metabolism. Moreover, both nucleosides mimicked SGE-induced anti-inflammatory activity upon DC function in vitro and attenuated establishment of CIA in vivo. We reveal that ADO and 5'AMP are present in pharmacological amounts in P. papatasi saliva and act preferentially on DC function, consequently reducing Th17 subset activation and suppressing the autoimmune response. Thus, it is plausible that these constituents might be promising therapeutic molecules to target immune inflammatory diseases.     

Suppression of murine collagen-induced arthritis by targeted apoptosis of synovial neovasculature

Because angiogenesis plays a major role in the perpetuation of inflammatory arthritis, we explored a method for selectively targeting and destroying new synovial blood vessels. Mice with collagen-induced arthritis were injected intravenously with phage expressing an RGD motif. In addition, the RGD peptide (RGD-4C) was covalently linked to a proapoptotic heptapeptide dimer, _D(KLAKLAK)₂, and was systemically administered to mice with collagen-induced arthritis. A phage displaying an RGD-containing cyclic peptide (RGD-4C) that binds selectively to the αvβ3 and αvβ5 integrins accumulated in inflamed synovium but not in normal synovium. Homing of RGD-4C phage to inflamed synovium was inhibited by co-administration of soluble RGD-4C. Intravenous injections of the RGD-4C–_D(KLAKLAK)₂ chimeric peptide significantly decreased clinical arthritis and increased apoptosis of synovial blood vessels, whereas treatment with vehicle or uncoupled mixture of the RGD-4C and the untargeted proapoptotic peptide had no effect. Targeted apoptosis of synovial neovasculature can induce apoptosis and suppress clinical arthritis. This form of therapy has potential utility in the treatment of inflammatory arthritis.     

Type II collagen-reactive T cell clones from mice with collagen-induced arthritis

Certain strains of mice develop a symmetrical polyarthritis after immunization with type II collagen. The incidence of arthritis after such immunization is variable. To study the arthritogenic potential of T cells reactive with type II collagen, we isolated draining lymph node cells from mice that had developed arthritis after immunization with bovine type II collagen. From these immune lymph node cells we were able to clone T cells reactive with type II collagen. Two separate sets of T cell clones were isolated. The first set reacted with either native bovine or native chick type II collagen, but did not react with type I collagen. The second set of T cell clones reacted with bovine type II collagen, but did not respond to either native chick type II collagen or type I collagen. These clones will be tested for their influence on the development of arthritis in vivo.     

A novel therapy of murine collagen-induced arthritis with soluble T1/ST2

Rheumatoid arthritis is characterized by chronic inflammatory infiltration of the synovium, leading to eventual cartilage and bone destruction. Previously, we have reported that soluble T1/ST2 (sST2), a member of the IL-1R gene family, inhibits LPS-induced macrophage proinflammatory cytokine production. In this study, we report the therapeutic effect of sST2-Fc in the murine model of collagen-induced arthritis. A short term administration of sST2-Fc fusion protein significantly attenuated disease severity compared with controls treated with normal IgG. Histological examination revealed that while control IgG-treated mice developed severe cellular infiltration in the joints, synovial hyperplasia, and joint erosion, this pathology was profoundly reduced in sST2-Fc-treated animals. Treatment of sST2-Fc also down-regulated serum levels of IL-6, IL-12, and TNF-alpha. Spleen cells from the sST2-Fc-treated mice produced significantly less IFN-gamma, TNF-alpha, IL-6, and IL-12 compared with cells from the control mice when cultured with collagen in vitro. Finally, pretreatment with ST2-Fc markedly inhibited the ability of human monocytic THP1 cells to release TNF-alpha when cocultured with peripheral blood T cells from rheumatoid patients. Together these results demonstrate that sST2-Fc may provide a novel approach in treating chronic autoimmune conditions by inhibiting the release of proinflammatory cytokines.     

Isolation of T cell line capable of protecting mice against collagen-induced arthritis

A T cell line specific to human type II collagen (CII) was selected and propagated from DBA/1J mice immunized with human CII. The line cells were not reactive to type I or type III collagen of human origin, but they were cross-reactive to bovine, rat, and rabbit CII and they recognized both native and heat-denatured human CII. The cells were reactive to an N-terminal three-quarters fragment of human CII, produced by tadpole collagenase digestion of human CII, but not to a C-terminal one-quarter fragment of human CII. The cells showed Thy-1+, Lyt-1+, Lyt-2-, and L3T4+ phenotypes characteristic of T helper cells or delayed-type hypersensitive cells, determined by the immunofluorescence method. To clarify the role of T cells in the pathogenesis of collagen-induced arthritis, we inoculated this cell line into DBA/1J mice and found that they developed clinical arthritis, albeit at a low incidence. The cells attenuated by x-ray were capable of inducing resistance to the subsequent induction of collagen-induced arthritis of DBA/1J mice. The sera from mice protected by inoculation of the cell line exhibited anti-idiotypic antibody response against conventional and monoclonal anti-CII antibodies. Anti-T cell receptor response may be involved in the mechanism for the protective effect of the cell line against autoimmune murine arthritis.     

Mechanisms of inhibition of collagen-induced arthritis by murine IL-18 binding protein

IL-18 is an important cytokine in autoimmune and inflammatory diseases through the induction of IFN-gamma, TNF-alpha, and IL-1. We report herein that collagen-induced arthritis (CIA) in mice is inhibited by treatment with murine IL-18 binding protein (mIL-18BP). CIA was induced in DBA/1J mice by the injection of bovine type II collagen (CII) in IFA with added Mycobacterium tuberculosis on days 0 and 21. The mice were then treated for 3 wk with PBS or with two doses of mIL-18BP (0.5 and 3 mg/kg) as a fusion protein with the Fc portion of murine IgG1. Both the clinical disease activity scores and the histological scores of joint damage were reduced 50% in mice treated with either dose of mIL-18BP. Proliferation of CII-stimulated spleen and lymph node cells as well as the change in serum levels of IgG1 and IgG2a Ab to collagen between days 21 and 42 were decreased in mice treated with mIL-18BP. The production of IFN-gamma, TNF-alpha, and IL-1beta in cultured spleen cells was reduced by in vivo treatment with low dose, but not high dose, mIL-18BP. FACS analysis showed a slight decrease in NK cells and an increase in CD4(+) T cells in spleens of mice treated with mIL-18BP. The steady state mRNA levels of IFN-gamma, TNF-alpha, and IL-1beta in isolated joints were all decreased in mice treated with both doses of mIL-18BP. The mechanisms of mIL-18BP inhibition of CIA include reductions in cell-mediated and humoral immunity to collagen as well as decreases in production of proinflammatory cytokines in the spleen and joints.     

Glucocorticoids alter the lipid and protein composition of membrane rafts of a murine T cell hybridoma

Glucocorticoids (GC) are widely used anti-inflammatory agents known to suppress T cell activation by interfering with the TCR activation cascade. The attenuation of early TCR signaling events by these compounds has been recently attributed to a selective displacement of key signaling proteins from membrane lipid rafts. In this study, we demonstrate that GC displace the acyl-bound adaptor proteins linker for activation of T cells and phosphoprotein associated with glycosphingolipid-enriched microdomains from lipid rafts of murine T cell hybridomas, possibly by inhibiting their palmitoylation status. Analysis of the lipid content of the membrane rafts revealed that GC treatment led to a significant decrease in palmitic acid content. Moreover, we found an overall decrease in the proportion of raft-associated saturated fatty acids. These changes were consistent with a decrease in fluorescence anisotropy of isolated lipid rafts, indicating an increase in their fluidity. These findings identify the mechanisms underlying the complex inhibitory effects of glucocorticoids on early TCR signaling and suggest that some of the inhibitory properties of GC on T cell responses may be related to their ability to affect the membrane lipid composition and the palmitoylation status of important signaling molecules.     

T hybridoma alpha/beta gene transfected in a murine T cell hybridoma: role of CD4 molecule in vitro and in vivo--engraftment in SCID mice induces T cell maturation.

In the present work, we tested in SCID and Balb/c mice the activity of T hybridoma transfected with T cell receptor (TCR) alpha/beta chain genes. A T cell hybridoma denoted D011107 was used as recipient for transfection of cytotoxic KB5C20 TCR alpha/beta heterodimer genes by protoplast fusion or electroporation. After transfection, the parental D011107 T cell line reexpressed CD5 and CD4 surface molecules. In vitro, we noted strong proliferation and unusual cytotoxic reactivities against H-2k target cells although the transfected cell line does not express the CD8 molecule. The fate of parental and transfected cells was examined in severe combined immunodeficient (SCID) and Balb/c mice at Day 16 after intravenous injection. Cells from bone marrow, thymus, and spleen tissues were analyzed by immunofluorescence. The transfected T cell hybridoma was CD3+ Desire 1+ CD4+ Thy1.2. The SCID mice grafted with the transfected T cell hybridoma presented a high percentage of CD3+ (15%), CD4+ (27%), Thy1.2+ (27.52%), and Desire 1+ (8.74%) cells in the spleen. The percentages of CD3+ (6.2%) and Thy1.2+ (5.06%) cells in the spleen from SCID mice grafted with parental T cell D011107 and from untreated SCID were similar and lower (CD3+, 3.52%; Thy1.2+, 4.34%). It seems that transfected T cells hybridoma grafted in the SCID mice induce significant expression of CD4+ Thy1.2+ Desire 1- cells (17%) in the spleen. These results indicate that transfected T cells graft may allow T cell differentiation. In Balb/c mice, the percentage of different T cell subsets in bone marrow, thymus, or spleen cells in mice injected with transfected T cells was similar to that in untreated mice. We did not observe any cytotoxic or significant allogeneic proliferation in vitro.     

Differential activation of phosphotyrosine protein phosphatase activity in a murine T cell hybridoma by monoclonal antibodies to CD45.

The effect of two anti-CD45 (T200, LCA, Ly5) antibodies on the activation of the murine T-cell hybridoma 13.13 has been evaluated. These studies have been carried out in a system that did not require cross-linking or coclustering of antibodies. Activation of 13.13 cells with the anti-CD3 monoclonal antibody, 145.2C11, gave rise to rapid increases in intracellular calcium and interleukin-2 production. Additionally, within 1 min, phosphorylation on tyrosine of four major proteins of about 130,000, 110,000, 80,000, and 37,000 daltons could be seen. Pretreatment of the cells with the anti-CD45 mAb M1/89.18.7.HK markedly inhibited all three biological responses, while an alternate anti-CD45 antibody, M1/9.3.4.HL.2, had little effect. The two antibodies bound to CD45 with similar affinities, and no differences in the lateral mobility of antibody-CD45 complexes in the cell membrane were observed. The inhibition of activation of the cells by M1/89.18.7.HK was abrogated significantly both by the phosphotyrosine protein phosphatase inhibitor orthovanadate and by excess M1/9.3.4.HL.2. If M1/89.18.7.HK was added to the 13.13 cells after they had already been activated with anti-CD3, it very effectively stimulated dephosphorylation of substrates that had been phosphorylated on tyrosines prior to adding the anti-CD45 antibody. These results indicate that the phosphotyrosine protein phosphatase activity of CD45 is critical to its biological function and that bivalent (i.e. uncross-linked) anti-CD45 antibodies can give rise to markedly different responses. One of the antibodies, M1/89.18.7.HK, appears to behave much like a receptor ligand and is able to activate the enzymatic activity associated with the CD45 transmembrane protein.     

Constitutive production of a factor supporting B lymphocyte differentiation by a T cell hybridoma

To investigate the role of soluble T cell products during B cell differentiation more fully, we have produced T cell hybridomas by the fusion of normal helper T cells with the T cell lymphoma BW5147. In this report we describe the production of one such hybrid, 14G3, the subclone 14G3.1F2, and the functional activity of the constitutive product. The hybrid supernatant acts exclusively in antigen-nonspecific, but antigen-dependent, promotion of B cell differentiation. It is optimally effective in the presence of small amounts of EL4 supernatant. It does not itself contain any detectable IL 2 or BCGF or interferon activity, however. 14G3.1F2 activity is probably an important component of the conventional TRF preparations produced by mixed lymphocyte populations, and will be useful in further dissection of the contributions of different soluble T cell products to B cell differentiation.     

Mechanism of phorbol myristate acetate-induced lymphotoxin production by a human T cell hybridoma

Various hydroxyl radical scavengers markedly inhibited phorbol myristate acetate (PMA)-induced lymphotoxin (LT) production by a human T cell hybridoma, AC5-8. Among those we tested, tetramethylurea (TMU) was the most potent scavenger, and it was revealed that TMU must be added before 2 h have elapsed after PMA addition in order for LT production to be inhibited. In concordance with this fact, soluble NADPH dependent O2- forming enzyme(s) were activated several fold by PMA. PMA also induced DNA strand breaks, a process markedly inhibited by TMU. As expected, ADP-ribosyl transferase (ADPRT), which is well known to require DNA strand breaks for its enzymatic activity, was activated by PMA treatment. In addition, specific inhibitors for ADPRT, namely 3-amino-benzamide and nicotinamide, inhibited PMA-induced LT production. Taken together, these three successive events, activation of soluble NADPH dependent O2- forming enzyme(s), DNA strand breaks and activation of ADPRT, may be required for PMA-induced LT production by AC5-8.     

Vascular endothelial growth factor expression and regulation of murine collagen-induced arthritis

We have examined the expression and function of the angiogenic factor, vascular endothelial growth factor (VEGF) during the evolution of type II collagen-induced arthritis (CIA). Biologically active VEGF was expressed along a time course that paralleled the expression of two specific VEGF receptors, Flk-1 and Flt-1, and the progression of joint disease. Moreover, levels of VEGF expression correlated with the degree of neovascularization, as defined by vWF levels, and arthritis severity. Macrophage- and fibroblast-like cells, which infiltrated inflamed sites and were then activated by other inflammatory mediators, are probably important sources of VEGF and may thus regulate angiogenesis during the development of CIA. Administration of anti-VEGF antiserum to CIA mice before the onset of arthritis delayed the onset, reduced the severity, and diminished the vWF content of arthritic joints. By contrast, administration of anti-VEGF antiserum after the onset of the disease had no effect on the progression or ultimate severity of the arthritis. These data suggest that VEGF plays a crucial role during an early stage of arthritis development, affecting both neovascularization and the progression of experimentally induced synovitis.     

Constitutive and mitogen-induced production of T cell growth factor by stable T cell hybridoma lines

Stable T cell growth factor- (TCGF; IL 2) producing cloned T cell hybridoma lines were constructed by fusing murine alloantigen-activated T cells with the 8-azaguanine-resistant lymphoma line, BW5147. Many, but not all, clones of one of these hybridomas, i.e., hybridoma 24, secreted TCGF constitutively, but production was markedly enhanced by stimulation with T cell mitogens. Large numbers of TCGF-secreting hybridoma cells in a stable functional state could be obtained from histocompatible mice inoculated with cloned T cell hybridomas. Moreover, such in vivo-derived hybridoma cells could be stimulated sequentially with mitogen at least twice to secrete their biologically-active product, resulting in larger TCGF yields from the same cells. The secreted product of these T cell hybridoma lines resembled TCGF isolated from other cellular sources in that it: a) supported the growth of a TCGF-dependent T cell line; b) provided help for the induction of alloantigen-reactive cytotoxic T lymphocytes from thymocyte precursors; c) facilitated concanavalin A-induced mitogenic responses of low thymocyte numbers; d) had an apparent m.w. of 30,000 to 40,000 by gel filtration chromatography; and e) was eluted from DEAE-Sephacel ion-exchange chromatography columns by salt concentrations of 30 to 150 mM NaCl. The ability of these T cell hybridomas to grow in vivo and retain their functional characteristics in a stable form should prove useful in terms of providing large numbers of TCGF-secreting cells and studying in vivo aspects of the production of TCGF as well as other immunoregulatory mediators.     

Kirenol exerts a potent anti-arthritic effect in collagen-induced arthritis by modifying the T cells balance

Rheumatoid arthritis is characterized by the imbalance of T cells, which leads to increased pro-inflammatory and reduced anti-inflammatory cytokines. Modulating the balance among T cells is crucial for the treatment of RA. Kirenol is a major diterpenoid components of Herba Siegesbeckiae, which has been applied for arthritic therapy for centuries. Since prior research showed Kirenol exhibited anti-inflammatory effect in rats, in this study we have evaluated the effect and mechanism of bioactive Kirenol in a rat model of collagen-induced arthritis (CIA) on modulation of T cells. After immunization with bovine type II collagen (CII), Wistar rats were orally administered saline (CIA group), 2 mg/kg Kirenol or 2 mg/kg prednisolone daily for 30 days. The severity of arthritis was clinically and histologically assessed. The numbers of CD4?CD25?Foxp3? T regulatory cells (Tregs) and IFNγ?CD4? and IL4?CD4? T cells were determined by flow cytometry, the mRNA expression level of Foxp3 was quantified by RT-PCR, cytokine levels were measured by ELISA and CII-induced cell proliferation was quantified in vitro. Kirenol significantly delayed the occurrence and reduced the disease severity of CIA. Histological analysis confirmed Kirenol suppressed joint inflammation and inhibited cartilage and bone destruction, compared to the CIA group. Kirenol also upregulated the mRNA expression of Foxp3, increased the numbers of CD4?CD25?Foxp3? and IL4?CD4? T cells, and reduced the number of IFNγ?CD4? T cells. Kirenol reduced the levels of TNF-α, IL-17A and IL-6 in synovial fluid and TNF-α, IL-17A and IFN-γ in serum, and increased the serum levels of IL-4, IL-10 and TGF-β1. In addition, Kirenol inhibited the ability of CII to induce splenocyte, PBMC and lymph node cell proliferation in vitro, compared to cells from CIA rats. In conclusion, these results suggest that Kirenol may be a potential immunosuppressant for the treatment for rheumatoid arthritis.     

Characterization of a tolerogenic T cell epitope of type II collagen and its relevance to collagen-induced arthritis.

A synthetic peptide representing sequences of type II collagen, (CII 245-270), has previously been used to induce tolerance and suppress arthritis in DBA/1 mice. To determine important residues, a series of peptides, each containing one or two site-directed substitutions, was generated. Mononuclear cells from DBA/1 mice immunized with CII were cultured in the presence of each peptide and the T cell response determined by measuring IFN-gamma in culture supernatant fluids. Substitutions within the region CII 260-270 led to significant decreases in IFN-gamma responses, identifying this sequence as a T cell epitope. To determine the effects of substitutions within this epitope on arthritis, substituted peptides were administered to neonatal mice as tolerogens. Five site-directed substitutions, four of which included the insertion of a residue found in type I collagen to replace its type II counterpart, abrogated the ability of the peptides to induce tolerance and suppress arthritis. These substitutions were located at residues 260, 261, 263, 264, and 266. Two patterns of T cell reactivity were observed. Peptides containing individual substitutions at positions 261, 264, or 266 were capable of generating a significant T lymphokine response, although those containing substitutions at residues 260 or 263 were ineffective Ag. Systematic analysis of the fine structures of T cell determinants important for autoimmune arthritis can lead to strategies for therapeutic intervention.     

Lymphokine-mediated induction of cytolytic activity in a T cell hybridoma

Functionally inducible CTL hybridomas were constructed by fusing alloantigen-specific T cells (C57BL/6 alpha-DBA/2) with cells from the rat thymoma line W/FU (C58NT)D. A cloned hybridoma line (KSH.4.13.6) that was specifically cytolytic in the presence of activated rat spleen cell supernatant fluid (rat Con A SN) lost activity when transferred to normal medium. However, a cytolytic activity could be reinduced by culturing KSH.4.13.6 cells in medium containing rat Con A SN or secondary mixed leukocyte culture SN. By using various sources of SN, it was found that cytolytic induction required two different factors. PMA-induced EL-4 SN and SN from antigen-activated cloned T cells, neither of which were capable of inducing cytolytic activity alone, were able to synergize in the cytolytic induction of KSH.4.13.6 IFN-gamma and IL 1 failed to induce cytolytic activity even in the presence of EL-4 SN. Furthermore, this hybridoma produced macrophage activating factor (MAF) upon culture in rat Con A SN, although MAF production could not be induced by either specific antigen or lectins. The kinetics of induction and loss of cytolytic activity mediated by rat Con A SN were similar to those of the induction of MAF production. However, EL-4 SN, which by itself was incapable of inducing cytolytic activity, was able to induce MAF production in the KSH.4.13.6 hybrid to an extent similar to that induced by rat Con A SN. These results suggest that the induction of cytolytic activity and of MAF production in this cloned hybridoma cell line are regulated by different mechanisms. Such a functionally inducible T cell hybrid may provide a tool for biochemical and molecular analysis of T cell function and regulation, and of the characterization of cytokines required for CTL differentiation.     

Isolation and characterization of a tumor-specific T suppressor factor from a T cell hybridoma

In our laboratory we have described a monoclonal antibody, B16G, which has been shown to bind to suppressive T cell factors (TsF) in DBA/2 mice. Therefore, B16G was used as a probe to identify T cell hybridomas secreting putative TsF. Hybridomas were obtained by the fusion of DBA/2 thymocytes stimulated in vivo by P815 tumor membrane extracts with the thymoma BW5147. One such hybridoma, A10, was selected and used for additional studies. From both the supernatants and ascites fluid of this hybrid a factor could be obtained that could specifically bind to both B16G and P815 antigen immunoadsorbent columns, and that scored positively with B16G in an ELISA after elution. Such reactivity could not be obtained from A10 supernatants or ascites absorbed over irrelevant columns, nor was it obtained from supernatants or ascites from other T cell hybrids that had scored B16G nonreactive in the original screening. In vivo studies indicated that affinity-purified A10 material injected into DBA/2J mice enhanced significantly the growth of P815 tumor cells, but not the growth of other DBA/2 syngeneic tumor lines such as L1210 or M-I. Additionally, this material did not inhibit the in vitro mixed leukocyte reaction (MLR) between DBA/2 splenocytes and allogeneic B10.BR target cells (unlike B16G purified material from whole DBA/2 spleens, which has been demonstrated to be suppressive in this type of MLR). Biochemical analysis of this tumor-specific TsF from A10 was undertaken; the native m.w. was found to be in the region of 80,000 and 90,000. Under reducing conditions, affinity-purified A10 TsF was found to resolve in SDS-PAGE as what appeared to be a heterodimer of 45,000 and 43,000. In most preparations, an associated molecule resolving at about 25,000 was observed. The implications of these observations are discussed.