The Journal of Experimental Medicine

Ramseier, Hansruedy

doi: 10.1084/jem.140.3.603pmid: 4547307

In vitro cultivation of murine spleen cells resulted in a spontaneous release of receptors for alloantigens. This was revealed by the capacity of cell-free culture supernates to recognize alloantigens as measured in the PAR assay. Qualitatively, recognition responses obtained with these supernates reproduced faithfully those found with the corresponding cells. Large amounts of receptors were released by untreated spleen cells and by spleen cells treated with a rabbit antimouse immunoglobulin serum and complement, smaller amounts were released by bone marrow cells, and native thymus cells released none. Spleen cells from nude mice and spleen cells from normal mice treated with anti-θ serum and complement showed no release of receptors. It was concluded that receptors active in the PAR test were of T-cell origin. Release of T-cell receptors was found to be discontinuous and proceeded in waves. The amount of released receptors depended on the number of cells cultivated. Release occurred at 37°C but not at 4°C. Interaction with antigen, however, was temperature-independent. In contrast to T-cell receptors, a release of H-2 antigens could not be detected with the culture conditions employed. Footnotes Submitted: 6 March 1974

Pedersen, Niels C.; Morris, Bede

doi: 10.1084/jem.140.3.619pmid: 4607241

Antibody which had cytotoxic and agglutinating activity against donor lymphocytes appeared in the blood stream of primary renal allograft recipients usually within 48 h of the graft being finally rejected. Appearance of the antibody See PDF for Structure in the blood was associated with severe alterations in vascular permeability and this led to increases in the numbers of red cells and in the protein content of the lymph coming from the allograft. It was possible to elute cytotoxic and agglutinating antibody from renal allograft tissue, showing that this type of antibody was bound to graft antigens during the rejection process. The transfusion of whole serum or serum globulins obtained from sheep that had previously rejected allografts led to the destruction of recently installed renal grafts and the histological changes produced in these grafts and the alterations in the composition of the lymph coming from them were similar to those seen in the terminal stages of primary rejection. These findings have led us to the conclusion that in the sheep, at least the terminal stage of primary renal allograft rejection is mediated by humoral antibody. Footnotes Submitted: 9 April 1974

Siegel, Joan; Rent, Rosemarie; Gewurz, Henry

doi: 10.1084/jem.140.3.631pmid: 4472155

Protamine sulfate was found to consume large amounts of C selectively during preincubation with sera of individuals in the "acute phase". Marked depletion of C1, C4, and C2 with minimal, if any, depletion of C3-9, was observed. The consumption was time and temperature dependent, occurring most rapidly and extensively at 37°C, 0.10 M relative salt concentration and pH 7.5–8.0; it required calcium ions. It was mediated by a heat-stable nondialyzable factor which separated with C-reactive protein (CRP) during fractionation and purification, correlated with serum CRP levels, and, like other known reactivities of CRP, was inhibited by phosphoryl choline. Preparations of CRP purified either from serum or ascites resulted in consumption of large amounts of C1, C4, and C2 when preincubated with normal serum and protamine. We conclude that CRP is a potent activator of the C system at the level of C1, and that polycations such as protamine sulfate are substrates of CRP which can bring about this activation. It seems not unlikely that one role of CRP in health and disease involves its ability to interact with the C system. Footnotes Submitted: 8 April 1974

Kapp, Judith A.; Pierce, Carl W.; Schlossman, Stuart; Benacerraf, Baruj

doi: 10.1084/jem.140.3.648pmid: 4137682

In recent studies we have found that GAT not only fails to elicit a GAT-specific response in nonresponder mice but also specifically decreases the ability of nonresponder mice to develop a GAT-specific PFC response to a subsequent challenge with GAT bound to the immunogenic carrier, MBSA. Studies presented in this paper demonstrate that B cells from nonresponder, DBA/1 mice rendered unresponsive by GAT in vivo can respond in vitro to GAT-MBSA if exogenous, carrier-primed T cells are added to the cultures. The unresponsiveness was shown to be the result of impaired carrier-specific helper T-cell function in the spleen cells of GAT-primed mice. Spleen cells from GAT-primed mice specifically suppressed the GAT-specific PFC response of spleen cells from normal DBA/1 mice incubated with GAT-MBSA. This suppression was prevented by pretreatment of GAT-primed spleen cells with anti-θ serum plus C or X irradiation. Identification of the suppressor cells as T cells was confirmed by the demonstration that suppressor cells were confined to the fraction of the column-purified lymphocytes which contained θ-positive cells and a few non-Ig-bearing cells. The significance of these data to our understanding of Ir -gene regulation of the immune response is discussed. Footnotes Submitted: 23 May 1974

Howard, Jonathan C.; Wilson, Darcy B.

doi: 10.1084/jem.140.3.660pmid: 4137934

Selected populations of thymus-derived (T) rat lymphocytes having specific immunological reactivity to chosen histocompatibility (H) alloantigens are found among the cellular products of the mixed lymphocyte interaction (MLI). Such specific selection seems to depend on ( a ) the antigen-induced proliferation of specific H antigen reactive cells (HARC), and ( b ) the disappearance of nonreactive cells from the cultures. When the surviving cells from this lymphocyte-antigen interaction are transferred into thymectomized, X-irradiated, marrow-reconstituted syngeneic recipients (B rats) which lack detectable T-lymphocyte functions, the lymphocyte populations subsequently recovered from the hosts possess the capacity to react in the MLI and in the graft-vs.-host (GVH) reaction, and the reactions have specificity for the original priming alloantigens. In addition, these findings identify the cell that reacts in the MLI with the GVH reactive cell. Footnotes Submitted: 28 October 1973

Claflin, J. Latham; Davie, Joseph M.

doi: 10.1084/jem.140.3.673pmid: 4137581

A new idiotypic determinant(s) on mouse anti-PC antibodies is described. Antibodies to the determinant(s) were raised in rabbits by immunization with HOPC 8, a PC-binding myeloma protein, and were isolated from HOPC 8 immunoadsorbent by elution with PC. These antibodies react with binding site determinants on anti-PC antibodies raised in all 15 inbred mouse strains tested regardless of histocompatibility or allotype, but fail to react with antibodies of other specificities or with anti-PC antibodies raised in other rodent species. These results correlate closely with other studies which show similar binding specificity of anti-PC antibodies raised in 17 different strains of mice. The site-associated idiotypic determinant(s) is clearly distinct from that detected by mouse anti-HOPC 8 antisera. This latter determinant(s) is present on anti-PC antibodies of only a few strains of mice and may not be in the binding site. Footnotes Submitted: 6 May 1974

Michaelides, Maria C.; Eisen, Herman N.

doi: 10.1084/jem.140.3.687pmid: 4138007

To explore the possibility that the affinity of some myeloma proteins for 2,4-dinitrophenyl (DNP) ligands is the consequence of a "strange" (i.e., unexpected) cross-reaction for more natural ligands, a variety of substances (primarily derivatives of purines, pyrimidines, naphthaquinone) were tested for ability to block the binding of 3 H-ϵ-DNP- L -lysine by protein 315, an IgA mouse myeloma protein with high affinity for DNP ligands. The most impressive inhibiting activity was observed with 2-methyl-1,4-napthaquinone (menadione, vitamin K 3 ). The affinity (intrinsic association constant) of protein 315 for menadione was 5 x 10 5 L/M (at 4°C). Because the same affinity was measured in direct-binding assays (e.g., equilibrium dialysis) and in an indirect one based on the assumption of competitive binding with DNP-lysine, it is likely that menadione and DNP bind at overlapping sites in the protein's combining region. This conclusion is supported by molecular models which reveal some common structural features in these ligands. Hence it is not surprising that antinitrophenyl antibody preparations, raised by conventional immunization procedures (anti-2,4-DNP; anti-2,6-DNP; anti-2,4,6-TNP) also bind menadione with considerable affinity. As with DNP ligands, when menadione binds to protein 315 or to conventional antinitrophenyl antibodies, some of the protein's tryptophan fluorescence is quenched, there is a change in the ligand's absorption spectrum (hypochromia and/or red shift), and the binding is temperature-dependent (exothermal). Footnotes Submitted: 30 May 1974

Cerottini, Jean-Charles; Engers, Howard D.; MacDonald, H. Robson; Brunner, K. Theodor

doi: 10.1084/jem.140.3.703pmid: 4278108

Mouse cytotoxic T lymphocytes (CTL) were generated in mixed leukocyte cultures (MLC) using spleen cells as responding cells and irradiated allogeneic spleen cells as stimulating cells. Cytotoxicity was assessed by a quantitative 51 Cr assay system and the relative frequency of CTL in individual cell populations was estimated from dose-response curves. Inclusion of 2-mercaptoethanol in the MLC medium resulted in a 20–40-fold increase in the relative number of CTL generated at the peak of the response. Under these culture conditions, cell-mediated cytotoxic activity was detectable in MLC populations as early as 48 h after the onset of the cultures. When spleen cells from mice immunized with allogeneic tumor cells 2–4 mo previously were cultured with irradiated spleen cells of the same alloantigenic specificity (MLC-Imm), it was found that the cell-mediated cytotoxic response was detectable earlier and reached higher levels than that observed in a primary MLC. At the peak of the response, MLC-Imm populations were observed to lyse up to 50% of the target cells within 3 h at a lymphocyte: target cell ratio of 0.3:1. Immunological and physical characterization of the effector cells generated in MLC-Imm indicated that they were medium to large-sized T lymphocytes. Altogether, these studies suggested the existence of an anamnestic cell-mediated cytotoxic response in MLC-Imm. Footnotes Submitted: 1 May 1974

MacDonald, H. Robson; Engers, Howard D.; Cerottini, Jean-Charles; Brunner, K. Theodor

doi: 10.1084/jem.140.3.718pmid: 4278109

Mouse cytotoxic T lymphocytes (CTL) were generated in unidirectional mixed leukocyte cultures (MLC) using normal C57BL/6 spleen cells as responding cells and irradiated DBA/2 spleen cells as stimulating cells. Cytotoxicity was assayed on 51 Cr-labeled P-815 (DBA/2) target cells, and the relative frequency of CTL in individual cell populations was estimated from dose-response curves. Upon inclusion of 2-mercaptoethanol in the culture medium, it was found that significant CTL activity could be detected for as long as 3 wk in primary MLC. Reexposure of MLC cells to the original stimulating alloantigens after 14–41 days in culture resulted in significant cell proliferation and rapid regeneration of high levels of immunologically specific cytotoxicity. CTL activity in these secondary cultures increased dramatically within the first 24 h and reached higher peak levels than those found at the peak of the primary response. Furthermore, proliferation and reappearance of CTL activity could be demonstrated following each of as many as four sequential alloantigenic stimulations of the same initial cell population at 20-day intervals. Interestingly, cells recovered from MLC at the peak of the primary response on day 4 were insensitive to further allogeneic stimulation. Taken together, these results are consistent with the hypothesis that CTL differentiate in MLC to become long-lived memory cells which gradually lose their cytotoxic activity. Upon reexposure to specific alloantigen, such memory CTL rapidly regain their functional activity and proliferate to generate an expanded CTL population. Footnotes Submitted: 1 May 1974

Binz, Hans; Lindenmann, Jean; Wigzell, Hans

doi: 10.1084/jem.140.3.731pmid: 4153293

The rat popliteal node graft-vs.-host assay was shown to depend on the presence of parental T cells in the inoculum. Antialloantisera raised in F 1 hybrid rats against alloantibodies of one parent directed at transplantation antigens of the other parent displayed some or all of the following specific effects on parental T cells: They inhibited local GvH by purified T-cell suspensions; they blocked the capacity of GvH-reactive cells to adsorb onto fibroblast monolayers of the relevant genotype; together with complement, they killed GvH-reactive cells. Footnotes Submitted: 15 May 1974