Plaque-forming Cell Precursors Research Articles

Requirement for three signals in B cell responses. II. Analysis of antigen- and Ia-restricted T helper cell-B cell interaction.

We have recently reported that resting B cells must receive at least three different signals in a T helper cell (TH)-dependent as well as in a lipopolysaccharide (LPS)-induced B cell response (3), i.e., a specific TH signal (that can be bypassed by LPS), a nonspecific TH signal (mediated by Ia or antigen-nonspecific B cell helper factor), and an antigen (hapten) signal. In a system using male (H-Y) antigen-specific cloned TH of C57BL/6 origin and male (or female) B cells, we now confirm and extend these findings by demonstrating that H-Y-specific TH must see both H-Y and Ia determinants on the B cells (and not only on macrophages) to provide the first specific TH signal required for a plaque-forming cell (PFC) response. This signal was interfered with by a monoclonal anti-I-Ab antibody at the B cell level, was not mediated by detectable soluble factors (in contrast to the nonspecific signal also provided by the TH), and could be bypassed by LPS, in which case anti-I-Ab antibody had no effect. However, although the H-Y-specific TH induced a polyclonal PFC response (B cell differentiation) in the apparent absence of an antigen seen by the B cells, significant clonal expansion of PFC precursors occurred only when the B cells also recognized an antigen (hapten).

Specificity, avidity, and size of B-lymphocyte populations during ontogeny

The size and specificity of plaque-forming cell precursors (PFC) in murine fetal liver, neonatal, and adult spleen were studied in an adoptive transfer system. In this system, anti-4-hydroxy-3-iodo-5-nitrophenylacetic acid (anti-NIP) and anti-2,4,6-trinitrobenzene sulfonic acid (anti-TNP) direct PFC are generated from bone marrow-derived (B) cell precursors in fetal liver between 17 and 20 days of gestation and in 6- or 14-day neonatal spleen. PFC generated from fetal liver and neonatal and adult spleen cells are specific in that they lyse either NIP-coupled SRBC or TNP-coupled SRBC but not both. The generation of specific anti-NIP and anti-TNP PFC from precursors in fetal liver is primarily independent of antigenic stimulation. In contrast, the anti-NIP and anti-TNP responses generated from neonatal and adult spleen are antigen dependent. Both high-avidity PFC (detected with SRBC indicators coupled at low hapten density) and low-avidity PFC (detected with SRBC coupled at high hapten density) are generated from fetal liver and neonatal spleen cells; however, the proportion of high-avidity PFC precursors in adult spleen is at least threefold greater than in fetal liver or neonatal spleen. Analysis by velocity sedimentation indicates that most high-avidity PFC precursors are small lymphocytes in fetal liver, medium lymphocytes in 6-day neonatal spleen, and small lymphocytes in 14-day-old and adult spleen. Low-avidity PFC precursors are primarily medium-sized lymphocytes in fetal liver and 6-day neonatal spleen. In 14-day-old and adult spleen almost all high- and low-avidity PFC precursors are small lymphocytes. The results are discussed in terms of relative changes in the pool sizes of these lymphocyte populations.

In vitro studies of the rabbit immune system : I. Hemolytic plaque-forming response of dissociated spleen cells from normal and primed rabbits

The conditions for the in vitro generation of primary and secondary immune responses by rabbit spleen cells to sheep red blood cell (SRBC) antigen have been examined. Spleen cells from many normal and all previously immunized rabbits are capable of producing in vitro plaque-forming cell (PFC) responses when cultured as dissociated cell suspensions in the presence of antigen. Primed spleen cells generate approximately 100 times the number of PFCs obtained in normal cultures with a shorter lag period. Both types of cultures demonstrate a period of exponential increase in PFCs during which the doubling time is 12–14 hr. This increase occurs after 1 day of culture of spleen cells from primed rabbits and after 4 days of culture of spleen cells from unprimed rabbits. The PFCs which arise in cultures of primed cells appear not to be the progeny of those generated in vivo but to be derived from an increased number of PFC precursors. Repeated immunization of the spleen cell donor is required to produce significant numbers of indirect (IgG) PFC or indirect precursors; most of the PFC found after a single immunization in vivo or in vitro are direct (IgM). There is no evidence for conversion of IgM to IgG PFC in vitro. This system should provide a means for further identification of the cellular interactions involved in the immune response of the rabbit.

In vitro immune response of partially purified spleen cells: Nutritionally distinct stages in differentiation

Partially purified populations of mouse spleen cells have been previously separated by equilibrium centrifugation on bovine serum albumin gradients and it has been suggested that separate fractions which are unique by several criteria contain precursors of plaque-forming cells (PFC) at different stages of differentiation. One of the unique features of these populations was the medium requirements. By making use of permissive and nonpermissive culture conditions, differentiation stages were demonstrable and a mechanism by which cultures regulate the amount of immunogen was detectable. These result are interpreted as being consistent with normal spleen cells having precursors at different stages of differentiation. It is also suggested that there is competition between the triggering of cells by antigen and the sequestration of antigen, presumably by macrophages.

Cell-cooperation in anti-sheep red blood cell antibody response in mouse spleen cell cultures. Use of antilymphocyte globulin for selective suppression of the antigen reactive cells.

Restoration of impaired antibody response to sheep red blood cells (SRBC) in spleen cell cultures from mice treated with heterologous antilymphocyte globulin (ALG) was studied by adding normal cells from various sources, to explore the problems of cell-cooperation in anti-SRBC antibody response and the target of ALG. When spleen cells from ALG-treated mice were separated into macrophage-rich and lymphoid cell-rich subpopulations, only the latter was found to be impaired in the ability for anti-SRBC antibody response. Addition of even a small number of normal allogeneic spleen cells sufficiently restored the impaired anti-SRBC antibody response of the spleen cells from ALG-treated mice. By use of allo-antisera, most hemolysin plaque-forming cells (PFC) generated in such cultures were proved to be derived from the cells of ALG-treated mice. Restoration was also achieved by adding thymus-derived cells, which were obtained from spleens of mice heavily irradiated and repopulated with syngeneic thymus cells, or lymphoid cells directly collected from thymuses. All results indicate that ALG selectively depletes the thymus-derived antigen reactive cells (ARC) in the spleen cell population, and that ARC supplied from normal spleen or thymus can interact with plaque-forming cell precursors (PFCP) that remain intact in the spleen cell population of ALG-treated mice. The results also suggest that a single ARC interacts with more than one PFCP and makes them develop into PFC.

Immunological memory in mice. 3. Memory to heterologous erythrocytes in both T cell and B cell populations and requirement for T cells in expression of B cell memory. Evidence using immunoglobulin allotype and mouse alloantigen theta markers with congenic mice.

Using anti-allotype sera and AKR anti thetaC3H sera, a requirement for two cell types has been demonstrated in the adoptive secondary response of mice to heterologous erythrocytes. The cell types have been designated B cells [precursors of plaque-forming cells (PFC)] and T cells (thymus-influenced cells, not providing precursors of detectable PFC). The in vivo indirect PFC response of spleen cells from primed mice is markedly reduced by in vitro treatment of the cells with a mixture of anti-theta serum and guinea pig serum (Anti theta + GPS). This B cell response is fully restored to control levels by thymus cells from normal mice which do not themselves provide precursors of indirect PFC. Thus memory is carried by the B cell lineage but the expression of this memory is dependent on the presence of a cell population which is sensitive to Anti theta + GPS and which is replaced functionally by unprimed T cells. When assayed for T cell activity, thoracic duct cells from specifically primed mice are better than cells from nonspecifically primed mice in restoring the B cell response of spleen cells from immunized mice. Moreover, the T cell activity of a reconstitutive cell population from primed mice is reduced by incubation with Anti theta + GPS. We conclude that memory to heterologous erythrocyte antigens is carried by the T cell lineage as well as the B cell lineage even though unprimed T cells are sufficient for expression of B cell memory.

Fractionation of specific antigen-reactive cells in an in vitro system of cell-mediated immunity.

ACCORDING to present concepts the diversity of antibodies is determined by a similar diversity of the precursors of antibody-producing cells. The existence of a diversified cell population in the lymphoid organs was most directly demonstrated by specific adherence of antigen-reactive cells on antigen columns. Antigen-binding cells were specifically eliminated from lymphoid cell populations of both preimmunized1,2 and non-immunized donors3–5. The non-bound cells were incapable of producing antibody to the antigen applied on the column, yet they could produce antibody to non-related antigens. Plaque forming cell precursors, plaque forming cells and memory cells towards various antigens were separated1–5. In all these cases the cells which specifically adhered to the antigenic column were most probably bone marrow-derived lymphocytes (B cells). On the other hand, no such specific adherence was achieved with thymus-derived lymphocytes (T cells), such as those involved in carrier recognition during immunization with hapten carrier conjugates6 and in cell-mediated immunity.

Antigen-specific cells in mouse bone marrow. II. Fluctuation of the number and potential of immunocyte precursors after immunization.

Quantitative and qualitative changes of mouse bone marrow cells were studied by limiting dilution assays 2-3.5 months after immunization of donors with sheep erythrocytes or unrelated antigens (Salmonella typhimurium, horse and chicken erythrocytes). Irradiated (C3H x C57BL/10)F(1) mice were reconstituted with an excess of nonprimed thymocytes and small graded numbers of primed bone marrow cells. Direct and indirect plaque-forming cells (PFC) were induced by secondary stimulation with SRBC and enumerated on the 9th day after cell transplantation. Marrow precursors of PFC (P-PFC) cooperated with thymocytes in the production of direct and indirect PFC after SRBC priming. The limiting dilution plots, which were not compatible with predictions of the Poisson model before immunization, changed and conformed to this model afterwards, as if the population of P-PFC had become functionally more homogeneous. The concentration of marrow P-PFC increased up to the 3rd month after priming, and decreased during the 4th, varying over two logarithms of nucleated marrow cells. The fluctuation was simultaneous and of the same order of magnitude for precursors of direct and indirect PFC, which were class restricted. A third effect of immunization was detected at 3.5 months: individual precursor units generated 3-4 times more direct and indirect PFC than at earlier intervals. Qualitative and quantitative changes of marrow P-PFC participating in anti-sheep responses were specific, since antigens unrelated to SRBC failed to induce them. The data suggested that marrow-derived cells were the major carriers of immunologic memory, but that they functioned in cooperation with thymus-derived inducer cells during secondary anti-sheep responses.

Cellular differentiation of the immune system of mice. VI. Strain differences in class differentiation and other properties of marrow cells.

Marrow cells and 5 x 10(7) thymocytes of unprimed (C57BL/6 x DBA/2)F(1), (C57BL/10 x WB)F(1) and (C3H x C57BL)F(1) donor mice were mixed in vitro and transplanted into X-irradiated syngeneic hosts. Upon injection of sheep erythrocytes, splenic plaque-forming cells (PFC) secreting IgM (direct PFC or IgG (indirect PFC) hemolytic antibody were enumerated at the time of peak responses. By grading the numbers of marrow cells, inocula were found that contained few immunocompetent cells reaching the recipient spleens, interacting with thymocytes or other accessory cells (or both), and generating PFC. The frequency of responses in BDF(1) mice conformed to Poisson statistics, indicating that immunocompetent marrow cells participated in a single-hit interaction limiting PFC responses. The marrow cells assayed were not restricted for the antibody class (IgM versus IgG) to be secreted by mature PFC. Unrestricted marrow cells could have been either the precursors of PFC or accessory cells. Different results were obtained in BWF(1) and C3BF(1) mice. The frequency of responses in relation to the number of marrow cells grafted did not follow Poisson statistics, and the limiting cells were restricted for antibody class. Presumably, immunocompetent cells of these strains were more heterogeneous than those of BDF(1) mice and participated in a multiplicity of cell-to-cell interactions. The strain differences reflected inherent properties of marrow cells and not influences of the environment in which PFC were produced. The results confirmed for bone marrow the heterogeneity of immunocompetent cells reported by others for spleen, and suggested that genetic factors such as "immune response" genes regulate cellular differentiation also for functions other than those related to antibody specificity.

DISTINCT EVENTS IN THE IMMUNE RESPONSE ELICITED BY TRANSFERRED MARROW AND THYMUS CELLS

Marrow cells and thymocytes of unprimed donor mice were transplanted separately into X-irradiated syngeneic hosts, with or without sheep erythrocytes (SRBC). Antigen-dependent changes in number or function of potentially immunocompetent cells were assessed by retransplantation of thymus-derived cells with fresh bone marrow cells and SRBC; of marrow-derived cells with fresh thymocytes and SRBC; and of thymus-derived with marrow-derived cells and SRBC. Plaque-forming cells (PFC) of the direct (IgM) and indirect (IgG) classes were enumerated in spleens of secondary host mice at the time of peak responses. By using this two-step design, it was shown (a) that thymus, but not bone marrow, contained antigen-reactive cells (ARC) capable of initiating the immune response to SRBC (first step), and (b) that the same antigen complex that activated thymic ARC was required for the subsequent interaction between thymus-derived and marrow cells and/or for PFC production (second step). Thymic ARC separated from marrow cells but exposed to SRBC proliferated and generated specific inducer cells. These were the cells that interacted with marrow precursors of PFC to form the elementary units for plaque responses to SRBC, i.e. the class- and specificity-restricted antigen-sensitive units. It was estimated that each ARC generated 80-800 inducer cells in 4 days by way of a minimum of 6-10 cell divisions. On the basis of the available evidence, a simple model was outlined for cellular events in the immune response to SRBC.

Cellular differentiation of the immune system of mice. V. Class differentiation in marrow precursors of plaque-forming cells.

Marrow cells and thymocytes of unprimed donor mice were mixed in vitro and transplanted into X-irradiated syngeneic hosts. 18 hr later sheep erythrocytes were injected to induce immune responses. Splenic plaque-forming cells (PFC) secreting IgM (direct PFC) or IgG (indirect PFC) hemolytic antibody were enumerated at the time of peak responses. By transplanting graded and limiting numbers of marrow cells with 5 x 10(7) thymocytes, inocula were found that contained few precursors of PFC (P-PFC) reaching the recipient spleens, interacting with thymocytes, and generating PFC. However, the frequency of responses in relation to the number of grafted marrow cells did not follow Poisson statistics, presumably because the interaction of marrow cells with thymocytes was more complex than a single or a one-to-one cell event. The frequency of direct PFC responses was greater than that of indirect PFC responses in 13 of 15 groups of mice tested. This was interpreted as evidence for the existence of two classes of P-PFC, each of which was restricted to generate either direct or indirect PFC. The precursors of direct PFC were approximately 15 times more frequent than those of indirect PFC. Since thymic antigen-reactive cells were not differentiated for antibody class, it follows that antigen-sensitive units reactive to sheep erythrocytes owe their class restriction to specialized marrow cells. Specialization of P-PFC may have arisen within marrow cell lines by differentiation, or may have been conferred upon P-PFC by interaction with other cells, including those of the irradiated host.

Cellular differentiation of the immune system of mice. IV. Lack of class differentiation in thymic antigen-reactive cells.

Thymocytes and marrow cells of unprimed donor mice were mixed in vitro and transplanted into X-irradiated syngeneic mice. 18 hr later, sheep erythrocytes were injected to induce immune responses. Splenic plaque-forming cells (PFC) secreting IgM (direct PFC) or IgG (indirect PFC) hemolytic antibody were enumerated at the time of peak responses. By transplanting graded and limiting numbers of thymocytes with 4 x 10(7) marrow cells, inocula were found which contained one or a few thymic antigen-reactive cells (ARC) reaching the recipient spleens, interacting with marrow cells, and inducing PFC formation. The frequency values of ARC inferred from direct and indirect plaque assays were very similar, 1 in approximately 10(7) thymocytes. Furthermore, statistical analysis indicated that the formation of direct PFC was not independent of the formation of indirect PFC. This was interpreted to mean that ARC were not specialized themselves and did not determine the molecular class of antibody to be secreted after interaction with marrow cells. Spleens of thymus-marrow grafted mice containing one or two ARC and non-limiting numbers of marrow precursors of PFC (P-PFC), had direct and indirect PFC clustered in several focal areas. Assuming that each focal area represented the progeny of one P-PFC that had interacted with ARC, these results confirmed the statistical evidence for lack of class differentiation in thymic ARC, and also indicated that each ARC or its progeny cells interacted with more than one P-PFC of either class.

Cellular differentiation of the immune system of mice. 3. Separate antigen-sensitive units for different types of anti-sheep immunocytes formed by marrow-thymus cell mixtures.

Marrow cell suspensions of unprimed donor mice have been transplanted into X-irradiated syngeneic hosts. 5-46 days later, bone cavities and spleens contained regenerated cells of the immune system which required interaction with thymocytes (from intact donors) and antigen (SRBC) to form antigen-sensitive units (ASU) and to generate mature immunocytes. These cells were capable of differentiating either into direct or indirect hemolytic plaque-forming cells (PFC). The precursors of PFC regenerated earlier than the other cell type necessary for immunocompetence, the antigen-reactive cell (ARC). The latter was not found until 10 or more days after transplantation. Availability of ARC was inferred from PFC responses elicited by grafted mice challenged with SRBC at varying intervals. In a second series of experiments, graded numbers of marrow cells (ranging from 10(7) to 5 x 10(7)) were transplanted with 5 x 10(7) or 10(8) thymocytes into irradiated mice, and SRBC were given 18 hr later. After 9-12 days the recipient spleens contained all or some of the following immunocytes: direct and indirect PFC, and hemagglutinating cluster-forming cells. The frequency of each immune response varied independently of the others, but in relation to the number of marrow cells grafted. This was interpreted to indicate that ASU formed in irradiated mice by interaction of marrow and thymus cells were similar to those of intact mice. In particular, they were specialized for the molecular class (IgM or IgG) and function (lysis or agglutination) of the antibody to be secreted by their descendent immunocytes. Hence, class-differentiation appeared to be conferred upon ASU by their marrow-derived components.