Expression of inflammatory cytokines following acute spinal cord injury in a rodent model.

Many therapies that have been developed for acute spinal cord injury (SCI) either influence or are influenced by posttraumatic inflammation. Many such therapies have reportedly produced promising neurologic benefits in animal models of SCI, but demonstrating convincing efficacy in human clinical trials has remained elusive. This discrepancy may be related in part to differences in the inflammatory response to SCI between human patients and the widely studied rodent models. Our objectives were, therefore, to establish the time course of inflammatory cytokine release in the spinal cord of rats after a thoracic contusion, to determine whether the cytokine release was injury dependent, and to correlate these findings with those that we have recently reported for the cerebrospinal fluid (CSF) of human SCI patients. After rodent SCI, GRO (the rat equivalent of IL-8), IL-6, IL-1α, IL-1ß, IL-13, MCP-1, MIP1α, RANTES, and TNFα were elevated within the spinal cord, whereas IL-12p70 was decreased. In human SCI, IL-6, IL-8, and MCP-1 were also elevated within the cerebrospinal fluid but at later times than those observed in the rodent spinal cord. IL-6, IL-8, and MCP-1 were released in an injury-dependent manner in both the rodent model of SCI and the human condition. In this regard, similar patterns of expression were observed for a number of inflammatory cytokines after SCI in rodent spinal cords and in human CSF. Such proteins may therefore have potential utility as biomarkers and surrogate outcome measures for evaluating biological response to therapeutic interventions.

Preadministration of a T-suppressor factor enhances tumor immunity in DBA/2 mice.

Previously we have described the isolation and characterization of a T-suppressor factor (TsF) from a T cell hybridoma (A10F), which has a degree of specificity for the DBA/2 mastocytoma P815. Administration of A10F intravenously at the time of tumor cell injection resulted in an accelerated rate of tumor growth, decreased cytotoxic T lymphocyte antitumor activity, and reduced survival time. In the work reported here, we have shown that administration of affinity-enriched A10F 7-14 days prior to tumor cell injection causes what appear to be reverse effects, in that an enhanced resistance to the P815 tumor is observed in vivo, an effect which we can correlate with the demonstration of antitumor cytotoxic T lymphocyte activity in vitro. These effects are dose-dependent since only doses of TsF at 20 micrograms or greater are effective. A similar effect was found when A10F was administered to DBA/2 mice 10 days prior to challenge with two unrelated tumors (L1210 and M-1). However, when another TsF (Fd11F) with apparent specificity for a nominal antigen was tested in this system, it had no effect on tumor growth.

Identification of a T cell subset using a rabbit antibody raised against a T suppressor molecule.

Rabbit antiserum to a unique component of an Ag-binding Ts-factor was generated by repeated immunization with purified 30-kDa protein isolated from Fd11 Ts factor (11). This antiserum (anti-p30) was shown to recognize cell surface determinants expressed on the Ts hybridomas Fd11 and A10 but not the fusion partner BW5147. Furthermore, this antiserum was shown to bind to approximately 4% of thymocytes and 10% of nylon wool-purified splenic T cells from all strains of mice tested. Sorting nylon wool-purified T cells from DBA/2 mice for the CD4+ and CD8+ subsets revealed both populations contained cells that bound anti-p30. In addition, when CD4-8- thymocytes were examined for anti-p30 labeling, it was found that about 30% of this enriched population also expressed p30 molecules. In a functional study, anti-p30 was able to neutralize the suppressive effects of Fd11 on a specific assay for in vitro antibody synthesis against ferredoxin.

Suppressor deletion therapy: selective elimination of T suppressor cells in vivo using a hematoporphyrin conjugated monoclonal antibody permits animals to reject syngeneic tumor cells.

A MAb (B16G) which recognizes a constant epitope on TsC and their soluble factors in DBA/2 mice has been described previously. In this study, we show that when this MAb is covalently linked to the photoactivable molecule Hp, and injected i.v. into P815 tumor-bearing mice which were subsequently exposed to light, tumors undergo permanent regression in 10%-40% of these mice (depending on the individual experiment). All control animals died within an average of 22-24 days after tumor cell injection. It is suggested that tumor regression is attributable to immune mechanisms facilitated by the elimination of a population of TsC. When splenocytes of B16G-Hp-treated mice were assayed in vitro for the generation of CTL active against P815 tumor cells, it was found that 24 h after treatment, a significant increase in killer cell activity was noted but that this effect was gone by 48h. We also show that B16G-Hp conjugates are capable in vitro of specifically killing cells of a TsC hybridoma, A10 (which has been shown previously to secrete a T suppressor factor reactive with P815 cell surface antigens). This conjugate had no cytotoxic effect on P815 cells under conditions in which A10 cells were killed.

Immunization of DBA/2 mice with a T cell hybridoma-derived TsF increases immune resistance to the syngeneic tumors P815 and L1210.

The ability of a tumor-specific T suppressor factor (TsF) isolated from a T cell hybridoma, A10, to act as an immunogen in DBA/2 mice was investigated. The TsF was affinity purified from ascites over an immunoadsorbent column containing a monoclonal antibody (B16G) that has specificity for the TsF molecule, or over columns containing membrane extracts of the P815 mastocytoma (the tumor for which A10 is specific). The specificity control was BW5147 (the fusion partner for A10) membrane extracts treated in the same way as A10. DBA/2 mice were immunized with the affinity-purified material or PBS and were subsequently challenged with either the P815 tumor or the L1210 DBA/2 thymoma. When mice were immunized with material affinity purified over B16G, eluted material from both A10 ascites and BW5147 membrane extracts enhanced resistance to both P815 and L1210 challenge, indicating that B16G was binding immunogenic material derived from both preparations, which exerted a tumor-protective effect. However, when a P815 affinity column was used, protective material was eluted only from A10 ascites, and this bestowed resistance to both P815 and L1210. When irradiated whole cells were used as immunogens, only A10 cells stimulated anti-tumor immunity, and this appeared to be directed specifically to the P815 tumor. The implications of these findings in terms of the potential for immune modulation with anti-suppressor therapy, and the specificity of the B16G monoclonal, are discussed. The demonstration of B16G binding material (TsF) in the membranes (but not the ascites) of the BW5147 line is also of significance to investigators using BW5147 fused suppressor hybridomas.

Isolation of functional polyclonal TsF from MRL-lpr spleens using monoclonal antibody absorbance.

We have previously described a monoclonal antibody, B16G, which has been found to be specific for T-cell derived suppressor factors (TsF). B16G has been shown to react with T-suppressor cells, TsF in the spleens of normal or tumor-bearing mice, the TsF produced by a tumor-specific T-cell hybridoma, and with polyclonal whole human TsF isolated from tonsilar tissue. This panreactivity inherent to the B16G MAb has made it clear that it recognizes some common, shared epitope of the TsF molecule. In this study we have used B16G as a probe to isolate TsF from the spleens of MRL-lpr mice and compare the activity with these factors isolated from the spleens of an MHC compatible nonautoimmune strain, CBA. We find that equivalent quantities of functional TsF are isolable from both strains and thus, it can be concluded that the associated oligoclonal B-cell activation characteristic of MRL-lpr mice is not due to a polyclonal T-suppressor cell deficit, nor to the ability of TsC in these mice to produce soluble, functional TsFs. The molecular and biochemical characteristics of these TsFs are discussed.

Preliminary characterization of human T cell suppressor factor (HTsF) isolated from tonsil cells by monoclonal antibody immunoadsorbence.

We have shown that a murine monoclonal antibody, B16G, recognizes a constant region determinant of a T cell suppressor factor (TsF) in DBA/2 mice. The molecule recognized by B16G was shown to be a heterodimer with a native m.w. in the region of 80,000. We now show that B16G also reacts with a similar molecule derived from human lymphoid tissue. Yields of about 100 micrograms could be obtained from the solubilized membranes and cytosol from about 10(10) tonsillar cells by elution of adsorbed materials from B16G immunoadsorbent columns. As with the murine system, the human TsF (HTsF) thus derived was capable of suppressing the mixed leukocyte reaction (MLR) of homologous effector T lymphocytes. However, this same material was not suppressive across the HL-A barrier, that is, when allogeneic effector cells were used in the MLR. Preliminary characterization of the HTsF showed it to have a native m.w. of 80,000 to 90,000 to be composed of a heterodimer with subunit m.w. in the region of 45,000 to 50,000, and to have an associated peptide of approximately 25,000. These observations provide evidence for the conserved nature of genes encoding TsF and correlate with observations of other investigators on the considerable homology between genes encoding the murine and human T cell receptor.

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.

Preliminary characterization of a soluble immunosuppressive molecule from DBA/2 spleen cells using monoclonal antibody immunoadsorbence.

In a previous publication a monoclonal antibody (B16G) which appeared to recognize T suppressor cells and a T-suppressor factor (TsF) in the spleens of DBA/2 mice was described. B16G appears to be directed to a public specificity of DBA/2 TsF and therefore has been shown to inhibit a variety of immunological reactions. The present study involves preliminary characterization of the material with which B16G reacts. It was found that the B16G-reactive protein (putative TsF) could be absorbed and eluted specifically from a B16G immunoadsorbent column. Material eluting from the B16G column reacted with B16G in an ELISA and appeared to run as two or more bands of 40-45 kDa in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The eluted material was biologically active (i.e., suppressive) in the standard assay (mixed leukocyte reaction of DBA/2 splenocytes with B10.BR targets), and its suppressive activity was abrogated by the addition of B16G to the mixed leukocyte reaction cultures. Sephadex G-150 chromatography of the B16G-reactive material showed that under these conditions, its native molecular mass was between 80-90 kDa, indicating that it might occur as a dimer under natural conditions.

Characterization of a monoclonal antibody directed to a T cell suppressor factor.

A monoclonal antibody raised by fusion of immune BALB/c splenocytes with NS1 cells is described. BALB/c mice were immunized with a T suppressor factor (TsF) raised in DBA/2 mice with specificity for the P815 tumor. The TsF had been purified by affinity chromatography before its use as an immunogen. The monoclonal antibody (B16G) was shown to affect the course of P815 growth in DBA/2 mice if administered i.v. on days -2 to 0 before tumor cell inoculation. Mice treated with B16G demonstrated slower tumor development and growth than controls, and a small number of animals (about 10%) did not develop tumors; all controls did. It was also shown that B16G did not exhibit total specificity for the P815 tumor in that it had similar effects on the growth of an unrelated myosarcoma (M-1) of DBA/2 mice. The general immunopotentiating effect of the B16G monoclonal was demonstrated by the fact that spleen cells of DBA/2 mice that had received B16G showed significantly higher MLC than did equivalent controls. When spleen cells of DBA/2 mice were depleted of B16G-reactive cells by panning in vitro, it was also found that cells so treated gave rise to elevated MLC reactions in comparison to appropriate controls. When an immunoadsorbent was prepared with B16G and cell lysates from splenocytes of immunosuppressed mice were passed over it, the immunosuppressive properties of the lysate in MLC were eliminated. The conclusion that the B16G recognizes a marker common to a group of regulatory T cells in DBA/2 mice is discussed.