Anti-theta Serum Research Articles

In vitro sensitization to transplantation antigens : V. Suppression of T-cell and of B-cell function in vitro

A previous report from this laboratory indicated that thymus-bone marrow cooperation occurs during the in vitro sensitization to transplantation antigens. The present report continues our investigation of this T and B cell interaction by attempting to control each of these cell populations in vitro. Two lines of evidence indicate that the T cell is required during the in vitro sensitization to alloantigens: (1) Anti-theta serum pretreatment of lymphoid cells interferes with their ability to become sensitized in vitro to transplantation antigens; (2) Lymphoid cells from neonatally thymectomized donors do not become allosensitized in vitro. In vitro suppression of B cell function was accomplished with an antibone marrow serum (anti-beta serum). Anti-beta serum treatment of lymph node or bone marrow cells impairs the ability of such cells to partcipate in the in vitro sensitization to transplantation antigens. The active cell subpopulations of lymph node cells, obtained by density gradient fractionation, can likewise be suppressed in vitro by pretreatment with anti-theta or anti-beta serum. These results confirm the necessity for thymus-marrow cooperation in vitro, and in addition suggest the existence, in the lymph node, of subclasses of T and B cells participating in the in vitro allograft reaction.

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

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.

The allogeneic effect in inbred mice. II. Establishment of the cellular interactions required for enhancement of antibody production by the graft-versus-host reaction.

Experimental conditions have been established for the elicitation of an allogeneic effect on the adoptive transfer secondary anti-2,4-dinitrophenyl (DNP) antibody response in mice. Thus, spleen cells from DNP-keyhole limpet hemocyanin (KLH)-primed mice manifest good secondary anti-DNP responses to a challenge with DNP-KLH, but not with DNP-bovine gamma globulin (BGG), after adoptive transfer to irradiated syngeneic recipients. However, a good adoptive transfer secondary anti-DNP response of such cells can be elicited with DNP-BGG when a second transfer of allogeneic lymphoid cells, in appropriate numbers, is carried out 24 hr before secondary challenge. The advantage to this system is that the DNP-primed cell population as well as the population of allogeneic lymphoid cells are accessible to experimental manipulation such that the T lymphocytes of one or the other can be removed. Utilizing this model, we have established that the allogeneic effect on antibody production can operate on a population of primed B lymphocytes which have been depleted of their isologous T lymphocytes by in vitro incubation with anti-theta serum plus complement. The potential cellular interactions involved in the mechanism of this phenomenon are considered and discussed in detail.

Selective induction of DNA synthesis in T and B lymphocytes

The ability of concanavalin A (Con A) and a lipopolysaccharide from E. coli bacteria (LPS) to induce DNA synthesis in various types of mouse lymphocytes cultivated in vitro was investigated. The results demonstrate that Con A is selectively active on T cells, being capable of activating DNA synthesis in thymocytes, cortisone-treated thymocytes, peripheral “educated” T cells, spleen cells and to a smaller degree bone marrow cells. When T cells are removed from spleen cell suspensions by treating them with antitheta serum, the effect of Con A is markedly reduced. Spleen cells from thymectomized and lethally irradiated mice repopulated with antitheta serum treated bone marrow (T × B mice) or spleen cells from congenitally athymic (nude) mice do not respond to Con A. Cortisone-treated thymocytes and spleen cells responded equally well to Con A; whereas untreated thymocytes respond 10 to 20 times less, suggesting that the cortisone-resistant thymocytes are the responsive cells both in the thymus and in the spleen. Pure T cells exhibited a very narrow dose response profile to Con A, 5 μg being optimal and 1 or 10 μg giving a small, or no, response. Contrarywise, a mixed T and B cell population, such as the spleen, showed a broad dose response profile to Con A. It is suggested that Con A-activated T cells can influence B cells to respond to Con A; whereas B cells by themselves cannot be activated by Con A. LPS cannot activate thymocytes, cortisone-treated thymocytes, or educated peripheral T cells, but stimulates DNA synthesis to an equal degree in normal spleen cells, antitheta-treated spleen cells, spleen cells from T × B or nude mice and normal bone marrow cells. LPS can induce DNA synthesis in spleen cells from animals tolerant to LPS to the same degree as in normal spleen cells. Another thymus-independent antigen (PVP) cannot activate DNA synthesis in normal spleen cells, suggesting that LPS exerts a non-specific stimulatory effect on B cells.

Density gradient fractionation of mouse lymphoid tissues: I. Plaque-forming and rosette-forming cells in normal, sensitized and tolerant spleen

Spleen from non-sensitized, primary sensitized, secondary sensitized and tolerant mice was separated by density gradient centrifugation on bovine serum albumin (BSA) into 5 subpopulations of cells. These subpopulations were analyzed for the presence of antigen-reactive cells (ARC), with the rosette-test, and for the presence of antibody-forming cells (AFC), with the plaque test. The thymic origin of the cells was tested with anti-theta serum. Sheep red blood cells were used as antigen. The results indicate that: 1. (1) Spleen cells are distributed in a characteristic pattern in the gradient, with most of the cells localizing in the middle “C” fraction. 2. (2) The top “A” fraction (light density) contains relatively more macrophages and large lymphocytes. The bottom “D” fraction (high density) contains relatively more small lymphocytes. 3. (3) Non-sensitized spleen has very few plaque-forming cells (PFC). Most of the rosette-forming cells (RFC) are localized in the “C” fraction but enrichment of RFC is highest in the “A” fraction. 4. (4) In primary sensitized spleen, the number of RFC and PFC has increased. The PFC secrete IgM antibody. Most of the RFC are localized in the top 3 fractions (“A”, “B”, “C”) of the gradient. Most of the PFC are localized in the top 2 fractions (“A”, “B”) of the gradient. The enrichment of both RFC and PFC is greatest in the top “A” fraction. 5. (5) In secondary sensitized spleen, some of the RFC and PFC (IgG-secreting) have increased in density. Most of the RFC and PFC are localized in the “B” and “C” fraction. The enrichment of RFC is still greatest in the “A” fraction. The enrichment of PFC is equally divided among the “A” and “B” fraction of the gradient. 6. (6) Tolerant spleen lacks both RFC and PFC. 7. (7) Anti-theta serum is cytotoxic only to cells in the “A” fraction of non-sensitized, primary sensitized and secondary sensitized spleen. Antitheta serum is not cytotoxic for any fraction of tolerant cells. 8. (8) Anti-theta serum only reduces the RFC in the top “A” fraction of the gradient. Anti-theta serum does not affect the PFC. The results indicate that RFC and PFC from primary and secondary sensitized spleen localize primarily in the light density fraction(s) of a BSA gradient and that the RFC in the top (light density) fraction are thymusderived. The results also indicate that tolerant spleen lacks thymus-derived cells.

Effects of anti-T cell (theta) and anti-B cell (beta) serum on the immune response in mice.

Heterologous anti-B cell (anti-beta) serum was prepared in rabbits against the spleen from neonatally thymectomized mice. The anti-beta serum, after absorption with thymus, is cytotoxic for bone marrow, bone marrow-derived cells, fetal liver and peritoneal lymphocytes. The cytotoxicity to the B cell can be absorbed out with bone marrow.The cytotoxic effects of anti-beta serum on spleen and lymph node cells is compared to that of anti-theta serum. The data suggest that spleen has relatively more B than T cells, while lymph node has relatively more theta-positive cells.To test the effect of anti-beta and anti-theta serum on the functional activity of lymphoid cells, C57 spleen or thymus was pre-incubated with the antiserum, in the presence of complement, and tested in vivo for graft-vs-host activity or transfer of an adoptive immune response to SRBC.Treatment with anti-beta serum does not decrease the graft-vs-host activity of thymus or spleen cells. Anti-theta serum does decrease the graft-vs-host activity...

Antigen recognition by T lymphocytes: II. Similar effects of azathioprine, antilymphocyte serum, and anti-theta serum on rosette-forming lymphocytes in normal and neonatally thymectomized mice

Anti-thêta serum inhibits a portion of spleen spontaneous rosette-forming cells (sRFC) and all of thymus sRFC (as seen after in vivo hydrocortisone treatment). Bone marrow sRFC are also partially sensitive to anti-theta serum but at much higher concentrations. The rosette inhibiting activity of anti-theta serum is absorbed by thymus cells and brain; it is also absorbed by bone marrow cells but to a much lesser degree. Theta-positive sRFC are inhibited by low and noncytotoxic concentrations of azathioprine (10 −6 M) and ALS ( 1 2000 – 1 16,000 ) in the presence of complement. Bone marrow sRFC are much less sensitive to azathioprine and ALS than spleen and thymus sRFC. Spleen sRFC from neonatally thymectomized and Nude mice lose their susceptibility to anti-theta serum, azathioprine and ALS. Conversely, thymosin (a cell free thymus extract) confers on normal bone marrow sRFC a sensitivity identical to that of spleen sRFC.

Immune Response in Vitro to Salmonella H-Antigens, Not Affected by Anti-Theta Serum

Summary Treatment of mouse spleen cells with antiserum cytotoxic to theta antigens does not affect the subsequent immune response in vitro to Salmonella H-antigens. Similarly treated cells fail to initiate an immune response to sheep erythrocytes. This finding represents a strong argument for the existence of immune responses to certain antigens which do not require thymus-derived cells.

Studies on immunological paralysis: VI. Thymic-independence of tolerance and immunity to type III pneumococcal polysaccharide

The rosette-forming cell (RFC) response to type III pneumococcal polysaccharide (SIII) was not impaired in thymectomised syngeneic radiation chimaeras as compared with normal CBA mice, whereas the direct plaque-forming cell (PFC) response was greatly diminished in both these and intact (restored) chimaeras. Paucity of antibody-secreting cell precursors also characterised bone marrow transferred from these animals. No evidence for T B lymphocyte synergism with respect to SIII could be obtained from repopulated mice. Addition of thymocytes to bone marrow did not augment the PFC response to SIII in recipients and neither did pretreatment of the marrow with anti-theta serum abolish its efficacy in transferring reactivity to SIII. Irradiated mice repopulated with marrow from SIII-tolerant donors were unresponsive. Prior thymectomy did not retard the spontaneous loss of tolerance induced by 50 μg SIII, and the ensuing immune PFC response was not impaired. It is concluded that induction of immunity and tolerance to SIII is independent of T lymphocyte involvement, although there was some indication that immunity to SIII is less well maintained in the absence of the thymus.

Appearance of T-cell markers in bone marrow rosette-forming cells after incubation with thymosin, a thymic hormone.

After incubation with thymosin, a thymic hormone, normal bone marrow rosette-forming cells acquire T-cell characteristics, including increased sensitivity to azathioprine, anti-lymphocyte serum, and anti-theta serum. This activity of thymosin provides a new sensitive and reproducible bioassay for thymosin, and is well correlated with an in vivo graft-versus-host assay. In addition, incubation of spleen cells from adult thymectomized mice with thymosin in vitro restores to normal their diminished sensitivity to azathioprine and anti-lymphocte serum.