Bertie F. Argyris

Mastocytoma-mediated suppression of mixed lymphocyte culture and mitogen responsiveness☆

Abstract Murine spleen cells, stimulated in vitro by allogeneic spleen, display a strong proliferative response with the subsequent development of cytotoxic cells. This proliferation and sensitization can be abrogated by the addition of mitomycin-treated or X-irradiated murine DBA/2 mastocytoma cells (P-815). The substance required for this depression of lymphocyte responsiveness is present in the cell-free supernatant fluids of P-815 cultures. The suppression appears to be due to interference with cell proliferation in the mixed lymphocyte culture, because the P-815 also prevents spleen cells from proliferating in response to the mitogens concanavalin A (Con A), lipopolysaccharide (LPS), and phytohemagglutinin (PHA). The significance of these findings is discussed.

Prolongation of allograft survival with antimacrophage serum.

Heterologous antimaerophage serum (AMS) and antilymphoeyte serum (ALS) were prepared by immunizing rabbits with mouse peritoneal cells and lymphocytes, respectively. In vitro tests demonstrate the specific cytotoxic effect of AMS on peritoneal cells and of ALS on lymphocytes. In vivo peripheral leukocyte counts are reduced after a single injection of AMS or ALS. Treatment of allograft recipients with AMS enhances allograft survival to the same degree as does treatment with ALS.

Stimulation of hair growth during skin regeneration

Abstract Wound healing in mice results in the stimulation of surrounding resting hair follicles to undergo normal growth and differentiation. This stimulation is first apparent microscopically about 7–8 days after injury and grossly at 12–15 days. Neither liver regeneration nor the growth of subcutaneously inoculated Ehrlich ascites tumor result in the stimulation of hair growth. It is concluded, therefore, that the stimulation of hair growth by regenerating skin may be organ specific. It has been possible to demonstrate that the stimulation of resting follicles is best associated with the hyperplastic epithelium situated between the resting hair follicles and the wound, and not with the initial tissue necrosis or fibroblastic proliferation. The results of this investigation are analogous to other studies which demonstrate the stimulation of one tissue by another, such as the stimulation of embryonic or adult tissues by tumors, and the stimulation of one tumor by another. It is suggested that in all such investigations three factors seem to be present, namely, necrosis, epithelial hyperplasia, and a competent target organ or tissue. What varies is the topographical location of each of these factors.

The role of macrophages, thymus- and bone marrow-derived cells in the graft-versus-host reaction.

SUMMARY Specific antisera are used to delineate the role of macrophages, T and B cells in the expression of graft-versus-host activity. Treatment of unfractionated C57BL/6 spleen with antimacrophage serum (AMS) or anti-0 serum (A0S) decreases the graft-versus-host activity in newborn (C3H X C57)F1 mice. Treatment of spleen with anti- ß serum AßS) has no effort on the graft-versus-host activity of spleen. Density gradient centrifugation of spleen on a discontinuous bovine serum albumin gradient results in a subpopulation of cells, of medium density, which is enriched in graft-versus-host activity. The graft-versus-host reactive cells in this subpopulation are sensitive to the cytotoxic action of A0S, but no of AMS or AßS. The results suggest that the T cell is required and the macrophage can contribute to the graft-versus-host reaction. The B cells in the donor spleen are not essential for the expression of graft-versus-host reactivity. The results also suggest the existence of two subpopulations of 0-positive cells, one of light density, enriched in rosette-forming cells, and another of medium density, enriched in graft-versus-host-reactive cells.

Role of suppressor cells in immunological maturation.

Neonatal mice exhibit high suppressor cell activity that can be measured by the ability of their spleen cells to suppress the in vitro response of adult mouse spleen cells to alloantigens in mixed lymphocyte culture. In this paper we have compared the changes in splenic suppressor cell activity after birth with the changes in immunological maturity, the latter being measured by mixed lymphocyte reactivity of the neonatal spleen cells. The data indicate that there are significant strain differences in the postnatal changes of suppressor cell activity. The results further indicate that four of the five mouse strains tested (C57BL/6, B10.A(5R), B10.A, and DBA/2) demonstrate a good correlation between the decrease in suppressor cell activity and increase in mixed lymphocyte reactivity of mouse spleen cells after birth. In one strain (B10.A(3R)), however, the suppressor cell activity drops to adult levels before full mixed lymphocyte reactivity is achieved. The results suggest that, although suppressor cells may have a role in immunological maturation, other factors may contribute to the development of immunocompetence.

Immunologic unresponsiveness of mouse spleen sensitized to allogeneic tumors.

Abstract CS7BL/6 mice were sensitized with an ip injection of allogeneic P-815 mastocytoma cells. Fifteen days later the spleen cells of the tumor allosensitized mice were cultured and tested for their responsiveness to mitogens and alloantigens, and for their ability to generate cytotoxic cells in vitro. The results indicate that 15 day tumor-sensitized spleen cells are hypo-responsive in mixed lymphocyte culture (MLC) with DBA/2 or AKR as stimulating spleen cells. The cells which are hypo-responsive in MLC can proliferate in response to mitogens and they also can generate cytotoxic cells in vitro. MLC reactivity recovers in about 2–3 months which is 1 1 2 –2 1 2 months after the mice have rejected their tumors. The mechanism of MLC hypo-responsiveness was investigated. The results suggest the presence of a suppressor cell which does not appear to be a macrophage or a B-cell. The suppressor cell can be separated from the cytotoxic cell and therefore appears to be a noncytotoxic T-cell.

Interleukin-2 production in the neonatal mouse

Neonatal mouse spleen and thymus cells fail to produce interleukin-2 (IL-2) in vitro. In vitro IL-2 production by spleen cells begins to develop between 20 and 27 days of age and reaches adult levels at 40 days of age. Thymus cells do not develop the ability to produce IL-2 in vitro. Addition of neonatal spleen, neonatal thymus, or adult thymus cells to adult spleen cell cultures inhibits IL-2 production. Treatment of the added cells with mitomycin does not abrogate their suppressor activity. Soluble factors, obtained from three-day cell cultures of neonatal spleen, neonatal thymus, or adult thymus also suppress IL-2 production in adult spleen cell cultures. Addition of interleukin-raises IL-2 production in adult thymus, neonatal spleen, or neonatal thymus cell cultures slightly, but the IL-2 productivity is still considerably lower than that of adult spleen in the absence of IL-1. The results indicate that suppressor cells for IL-2 production are present in neonatal spleen and thymus. The splenic suppressor cell activity disappears after birth, but thymic suppressor cell activity is retained into adulthood.

Density gradient fractionation of mouse lymphoid tissues: II. Effects of anti-thymus and anti-bone marrow sera

Abstract The origin of antigen-binding (rosette-forming) and antibody-forming (plaque-forming) cells in mouse spleen is determined with the use of anti-thymus (anti-theta) and anti-bone marrow (anti-beta) sera. Evidence is presented that anti-theta serum is specific for thymus and thymus-derived cells. Anti-beta serum acts only on bone marrow and bone marrow-derived cells and does not affect the viability or functional activity of thymus cells. The cytotoxic activity of anti-theta and anti-beta serum does not overlap. Mouse spleen is fractionated, by density gradient centrifugation on bovine serum albumin, into subpopulations of cells. The top (light density) fractions are enriched in plaque-forming (PFC) and rosette-forming (RFC) cells. Cells sensitive to the cytotoxic action of anti-beta serum (bone marrow-derived cells) are found in all fractions of the gradient. The bone marrow-derived cells in the top bands appear to be antibody-forming cells and can be assayed with the plaque test. The bone marrow-derived cells in the bottom bands seem to be antibody-forming precursor cells and can be assayed by transfer to irradiated mice. Cells sensitive to the cytotoxic action of anti-theta serum (thymus-derived cells) are found in the top “A” and bottom “D” bands of the gradient. Our results suggest that a considerable portion of RFC in the top band are thymus derived. The evidence for this is that (1) anti-theta serum reduces the number of RFC but not PFC, (2) displacement of RFC from the top gradient fraction is accompanied by a corresponding displacement of cells sensitive to the cytotoxic action of anti-theta serum and, (3) RFC are present in the spleen of irradiated, thymus-repopulated mice.

Suppressor factor from tumor-allosensitized spleen cells—Its effect on in vitro proliferation of tumor cells and in vivo skin allograft survival

Abstract C57BL/6 mice are sensitized ip with allogeneic P-815 mastocytoma cells. Fifteen days later the spleen cells of the sensitized mice are used in the production of suppressor factor or treated with mitomycin and used as suppressor cells. Sensitized spleen cells incubated with the specific alloantigen (DBA/2 m-treated spleen cells) release suppressor factor (SF) 2 which inhibits cell proliferation in mixed lymphocyte culture (MLC) as well as the in vitro generation of cytotoxic cells (CML). SF is most effective when added eary during MLC. SF also inhibits mitogen responsiveness of normal spleen cells. In addition to inhibiting lymphocyte function in vitro , suppressor cells as well as SF inhibit the in vitro proliferation of tumor cells. This inhibition is specific for the tumor to which the suppressor cells are induced. The inhibition of tumor cell proliferation is not due to the presence of cytotoxic cells in the spleen of the tumor-allosensitized mice. Suppressor cells from neonatal mice do not inhibit the in vitro proliferation of tumor cells. SF injected iv into C57BL/6 mice decreases the mixed lymphocyte reactivity of the host spleen cells and decreases the ability of the host to reject skin allografts. We interpret these data to suggest that tumor-allosensitized spleen cells, and the SF they produce, not only affect lymphocyte function but also inhibit tumor cell proliferation. This dual effect of suppressor cells could be an important part of the immune surveillance against tumors.

Adsorption of suppressor cell activity on mouse macrophage monolayers.

Suppressor cell activity is high in the spleen of C57BL/6 mice sensitized to the allogeneic P-815 mastocytoma. The suppressor cells inhibit proliferation of normal mouse spleen cells in mixed lymphocyte cultures. Preincubation of tumor-allosensitized spleen cells on monolayers of syngeneic or allogeneic macrophages reduces suppressor activity. Preincubation on monolayers of spleen cells does not affect suppressor activity. Suppressor cells can be retrieved from the macrophage monolayers used for adsorption. Heat-killed macrophages fail to adsorb out suppressor cell activity. Incubation of tumor-allosensitized spleen cells on syngeneic macrophage monolayers does not reduce cytotoxic cell activity. Removal of suppressor cell activity by macrophage adsorption restores helper activity of tumor-allosensitized spleen cells. These results demonstrate that suppressor cell activity can be reduced by contact with macrophages and point to an additional role for the macrophage in regulating the immune response to tumors.