Anti-alpha 1-6 epitope, specificity of a T-cell hybrid-secreted factor: affinity- and ion-exchange chromatographic separation.

We have studied the specificity of the products of a "T-cell" hybridoma, Th 1, a fusion product of AKR thymoma BW 5147 with spleen cells from Dx-hyperimmunized mice, which has been shown to affect the anti-Dx but also the anti-SRC response from culture supernatants and ascitic fluids. The anti-Dx-affecting material was separated from unspecific effector molecules by Sephadex affinity chromatography combined with HPLC DEAE chromatography and gel filtration. The activities of fractions were tested for their effects on anti-Dx, anti-SRC, and anti-SSS-III IgM responses. We show that the anti-Dx response-affecting material binds to Sephadex. Its Ig contamination can be reduced by two DEAE chromatographies at pH 6 and 8.1. At pH 8.1 it starts eluting with 0.13 M NaCl, but is still contaminated with materials that affect the anti-SRC and to a smaller extent the anti-SSS-III response. On gel filtration it localizes in the area of 100-40 kDa. The effects of the active material on anti-Dx IgM varied from suppression to enhancement. The details of that effect are largely unknown but three other findings further confirm the Dx specificity of Th 1 products. The growth of Th 1 in mice induces the production of anti-Dx IgA, detectable in their sera with ELISA. The priming of mice with Th 1 products affects the magnitudes of anti-Dx IgM PFC responses to the subsequent immunization with Dx with or without the product. The binding to Dx of material from in vivo active fractions can be verified in the ELISA with an antiserum produced against Th 1.

Purification of an IgM antibody response-enhancing factor to the alpha 1-6 epitope of native dextran from serum and supernatants from T-cell hybridomas.

We have found that some hybrids produced by fusing AKR thymoma BW 5147 with spleen cells from Dx-hyperimmunized CBA mice produce factors that affect the primary CBA IgM PFC response. By the use of HPLC-DEAE chromatography, sometimes combined with affinity chromatography, we have purified from hyperimmune anti-Dx serum, from ascites-growing T hybrids, and from normal CBA serum a family of factors that specifically enhance the anti-Dx IgM response. They show specificity for either Dx or anti-Dx, they may contain an Iak determinant, and they are genetically restricted in their function. The factors from all three sources are found in the same fractions after separation and move in fractions without Ig contamination. The affinity-purified factor was eluted at an NaCl concentration of 0.15-0.20 M. On the basis of these characteristics we believe that we have separated a class of T cell-derived helper factors from serum. The concentration of the factor in serum increases after hyperimmunization with Dx. As a specificity control we show that the same sera contain a natural anti-SRBC IgM response-enhancing factor. It co-separates with the anti-Dx-enhancing factor, but they can be distinguished by their preferential binding to their own antigens.

ELISA assay for the detection of a dextran-binding product secreted by a T-cell hybrid Th 1.

When CBA X BALB/c mice are immunized with Th 1 hybrid prepared by fusing spleen cells from Dx-immunized mice with the AKR thymoma BW 5147, which secretes material that affects the anti-Dx response in CBA mice, antibodies are produced that in the ELISA are shown to bind to the material secreted by Th 1 but not to the material secreted by parent BW 5147. A fraction of the Th 1 secreted material that starts eluting on DEAE chromatography in 20 nM Tris with 0.13 M NaCl is shown to bind to Dx but not SRC or uncoated ELISA plates. This binding, like that of anti-Dx Ig, can be inhibited with 1-10 micrograms/ml concentrations of free Dx. However, the affinity of the product of Th 1 for Dx is apparently lower than that of anti-Dx Ig, since high concentrations of anti-Dx interfere with the binding of Th 1 products to Dx. On gel permeation chromatography, a similar Dx binding material elutes in two molecular weight areas, a high area of the molecular weight of IgG and less and a low area of the molecular weight of 43,000 and above, and both are found in samples of Th 1 ascites and hyperimmune CBA anti-Dx serum, but not (or less so) in normal CBA serum and not in the hyperimmune nude C57B1 anti-Dx serum. The Th 1-derived material that affected the anti-Dx response in previously shown experiments had similar elution characteristics. Since we also show that these fractions need not to contain Ig and that anti-Th 1 minimally cross-reacts with anti-Dx IgM or IgG, it is likely that that fraction of Th 1-secreted material represents a Dx-binding, non-Ig, anti-Dx response-regulating factor.

In vitro enhancement of natural cytotoxicity by tumour necrosis serum.

Cells cytotoxic for syngeneic, allogeneic and xenogeneic cultured target cells are shown to be induced spontaneously in cultures of murine lymphoid cells. Serum containing bacillus Calmette-Guérin and endotoxin-induced tumour necrosis factor accelerates the appearance of observed natural cytotoxic cells.