The Role of Thymocytes and Bone Marrow Cells in Defining the Response to the Dinitrophenyl Hapten Attached to Positively and Negatively Charged Synthetic Polypeptide Carriers

Abstract

An inverse relationship exists between the net electrical charge of immunogens and the antibodies they elicit (1). Results of an earlier study have demonstrated that the net charge phenomenon has a cellular basis, since the immune response potential of murine spleen cells to 2,4-dinitrophenyl (DNP) on a negatively charged synthetic polypeptide carrier was reduced by cell fractionation over negatively charged glass beads, whereas the response to the same hapten on a positively charged carrier was unaffected (14). To verify that the net charge correlation is expressed at the cellular level, spleen cells were fractionated over positively charged poly-L-lysine-coated glass bead columns, and their immunocompetence to DNP on positively and negatively charged carriers was tested by cell transfers in irradiated recipient mice. In this case, the fractionated cells showed reduced response potential to DNP on the positively charged carrier only. Thus, the cellular basis of the net charge phenomenon has been demonstrated for both positively and negatively charged immunogens (for the same specificity) by cell separation techniques over columns of opposite charge. In order to establish whether the cell population relevant for the charge properties of immunogens was of thymus or marrow origin, thymocytes and bone marrow cells were selectively passed over positively or negatively charged columns and mixed with unfractionated cells of the complementary type. Transfers of the filtered and unfiltered cell mixtures in irradiated recipient mice immunized with DNP on either a positive or a negative synthetic polypeptide carrier indicated that fractionation of thymocytes, but not of marrow cells, correlated with the spleen population. Thus, thymocytes fractionated over negatively charged columns and mixed with unfractionated marrow cells exhibited reduced response to DNP on the negative carrier, but normal responses to DNP on the positive carrier. The opposite result was obtained when thymocytes were passed over positively charged columns. No effect on the anti-DNP response was detected by filtration of bone marrow cells over columns of either charge. These findings indicate that it is possible to distinguish between thymocytes on the basis of their capacity to react with more acidic or more basic surfaces and that a population of thymus-derived cells may recognize immunogens on the basis of their overall electrical charge. No evidence was found by these techniques that marrow-derived cells contribute to the net charge phenomenon.

Inverse relationship between net electric charge on the antigen and that on the sensitized cell in cellular immune response: demonstration with basic encephalitogen of the brain.

“…Whereas the transfer of EAE by LNC does not in itself rule out the contribution of B cells in the pathogenic mechanisms, indirect evidence tends to implicate primarily T cells (14)(15)(16) . This would also gain credence from the previous finding that thymocytes rather than bone marrow cells account for the charge effect of whole spleen populations (8), though in that study helper cell function was assayed rather than a cellular immune response by itself. Recently, it has been demonstrated that there is a dissociation between the T-helper-cell function and delayed hypersensitivity (17,18).…”

“…This inverse net charge phenomenon has a cellular basis for both positively and negatively charged immunogens as demonstrated by cell separation techniques over columns of opposite charge (7,8) . Mouse spleen cells eluted from glass bead columns or poly-L-lysine-coated glass bead columns comprised 35 or 20% of the whole cell suspension, respectively .…”

“…The cellular basis ofthe net charge phenomenon has been established for both positively and negatively charged immunogens, by cell separation techniques over columns of opposite charge (7,8). To establish whether this phenomenon can be extended to include cell-mediated immunity, the response to basic encephalitogenic protein (BE) which induces experimental allergic encephalomyelitis (EAE) was now investigated .…”

“…It was established that the cell population relevant for the charge properties of immunogens was of thymus and not of marrow origin, by experiments in which thymocytes and bone marrow cells were selectively passed over positively or negatively charged columns and mixed with unfractionated cells of the complementary type (8). Thus, thymocytes fractionated over negatively charged columns and mixed with unfractionated marrow cells exhibited reduced antibody response to the hapten on the negative carrier but normal responses to hapten on the positive carrier.…”

“…These hapten-specific antibodies differed in net electrical charge, but were indistinguishable with respect to their specificity and affinity (6). It has been previously established that the inverse net charge phenomenon has a cellular basis (7,8) . Thus, the immune response potential ofmouse spleen to the 2,4-dinitrophenyl hapten attached to a negatively charged synthetic polypeptide carrier was reduced by cell fractionation over negatively charged glass bead columns, whereas the response to the same hapten on a positively charged carrier was unaffected (7).…”