CBA Spleen Cells Research Articles

The immunological properties of haptens coupled to thymus-independent carrier molecules. II. The influence of the graft-versus-host reaction on primary antibody responses to hapten-coupled polysaccharides and proteins.

AbstractPrevious work from our laboratory has shown that hapten‐coupled pneumococcal polysaccharide (SIII) and levan (LE) (DNP‐lys‐SIII and DNP‐lys‐LE) elicit thymus‐independent IgM anti‐DNP responses in mice. In the present study the effects of the graft‐versus‐host (GVH) reaction on primary antibody responses to these two antigens and to various hapten‐protein conjugates was investigated.Injection of (CBA × C57BL)F1 mice with CBA spleen cells at the time of immunization significantly enhanced primary anti‐hapten responses, irrespective of whether the carrier stimulates T cells (proteins) or not (polysaccharides). Injection of allogeneic cells increased the IgM and elicited a significant IgG anti‐DNP response to DNP‐lys‐LE and DNP‐lys‐SIII. Contrary to the observations of others, we found that allogeneic spleen cells enhanced primary antibody responses to hapten‐protein conjugates. In contrast, we have been unable to demonstrate an IgG response to the polysaccharide moieties of DNP‐lys‐SIII or DNP‐lys‐LE, thus suggesting that the effects of activated T cells on antibody responses are related to the nature of the epitope involved.Isoelectric focusing studies of IgG anti‐DNP antibodies elicited in response to DNP‐lys‐LE via the GVH reaction indicate that allogeneic T cells can cooperate with and stimulate a heterogeneous population of B cells.

Differences in genetic specificity are observed when spleen and lymph node leucocytes are used as the responsive cell population in mixed leucocyte cultures.

SummaryIn mixed leucocyte culture, CBA lymph node cells respond to stimulation by irradiated (CBA x C57Bl) F1 spleen cells, but (CBA x C57Bl) F1 lymph node cells are not stimulated by irradiated CBA spleen cells. However, when spleen cells are the responsive cell population. (CBA x C57Bl) F1 cells respond to stimulation by irradiated CBA spleen cells and CBA spleen cells respond to stimulation by irradiated (CBA x C57Bl) F1 spleen cells.

Immunization against abolition of transplantation tolerance*

AbstractAttempts were made to break the tolerance of murine transplantation antigens of strain A in CBA mice by adoptive transfer of: 1) normal, syngeneic (CBA) lymphoid cells, or 2) moderate doses of sensitized, syngeneic (CBA anti‐A) lymphoid cells followed later by high doses of CBA anti‐A cells. Both kinds of attempt failed. Tolerance could be broken when a large enough dose of sensitized cells was given. Prior “immunization” of CBA mice tolerant to A with small doses of CBA spleen cells protected against doses of sensitized cells otherwise successful in breaking tolerance.Determinations of anti‐recognition structure (anti‐RS) antibody and of alloantibody activities in sera of tolerant mice, of mice which were successful in defending tolerance, and of mice in which tolerance could be abolished revealed an important role for anti‐RS antibodies. No clear role could be assigned to alloantibodies present in the same sera. The function of anti‐RS antibodies was shown by the failure of spleen cells from immunized tolerant mice to recognize tolerated antigen. This suggests that anti‐receptor antibodies are active in neutralizing receptors of responding lymphoid cells emerging from a stem cell reservoir.

Functional Development of the Interacting Cells in the Immune Response

Abstract Young CBA spleen cells respond initially to sheep red blood cells (SRBC) in vitro at 15 days of age but the response is less than 10% of the adult response. The magnitude of the response reaches levels comparable to those in the adult at 7 weeks of age. Spleen cells of 1-week-old mice can reconstitute the anti-SRBC response of lethally x-irradiated adult mice but cannot respond to SRBC when placed in diffusion chambers implanted in the peritoneal cavity of sub-lethally irradiated adult syngeneic recipients. In an attempt to determine the cellular basis of the “immunologic immaturity,” reciprocal recombinations of adult and young macrophage-rich adherent (A) cells and lymphocyte-rich non-adherent (NA) cells were made. It was found that young A cells are functional, but young NA cells are not. The implications of these findings in terms of the mechanism of maturation and the acquisition of immunocompetence by the young mouse are discussed. It is suggested that the cause of incompetence in the young spleen cells is the presence of pluripotent stem cells but not of committed lymphoid precursors. This may be due to the lack of functional lymphocyte-directed-inducing-microenvironments which would induce stem cells to differentiate into committed lymphoid precursors.

Cell Interactions in Antibody Formation in Vitro

Abstract Spleen cells from DBA/2J and CBA/J mice were separated into adherent and nonadherent cell populations; adherent cells obtained from either strain may be recombined with DBA nonadherent cells and the kinetics and magnitude of the plaque-forming cell response to SRBC are equivalent. An alloantiserum directed against CBA spleen cells was used to study the length of time that the adherent population was required for a maximum in vitro response. The alloantiserum abolished the response when added during the first 2 days of culture, indicating that the adherent cell was required at least during the inductive phase of the response. This was true whether adherent cells were prepared from either spleen cells or peritoneal exudate cells of CBA mice. Nonadherent cells from mice immunized 1 week earlier required the cooperative participation of adherent cells for a shorter period than nonadherent cells from normal or remotely immunized mice. This result is consistent with the idea that most of the adherent cell dependent events have occurred in the nonadherent population following exposure to antigen in vivo. The present study demonstrates that a cell type in the adherent population is an active participant in the cell interactions leading to antibody formation in both primary and secondary in vitro responses. It is proposed that the adherent cell is an “antigen-focusing” cell which functions by providing foci of increased cell density thereby promoting interactions between thymus and bone marrow-derived cells.

Responsiveness of thymus cells to syngeneic and allogeneic lymphoid cells.

THE thymus is necessary for the normal development of cell-mediated immunity in mice as shown by the immunological defects after neonatal thymectomy1. Thymus cells themselves can be stimulated by allogeneic lymphoid cells in mixed leucocyte reaction (MLR)2 and become killer cells or cytotoxic lymphocytes after stimulation with allogeneic spleen cells in vitro (H. Wagner and M. Feldmann, unpublished work) and in vivo3,4. This suggests that the thymus as well as peripheral lymphoid tissues contain T cells which can be stimulated by foreign histocompatibility antigen to divide and differentiate into the cytotoxic lymphocytes which mediate cellular immunity. There have been suggestions that thymus cells might be stimulated to divide by “self” antigen, as well as foreign cells: incorporation of 3H-thymidine above background levels has been found in cultures with syngeneic spleen and thymus cells of adult rats5, although the experiments do not determine whether thymus or spleen cells have been stimulated. In contrast to these experiments, Howe et al. reported that only thymus cells of neonatal CBA mice reacted to allogeneic and syngeneic spleen cells of adult animals in “one way” MLR cultures6,7. Whether the reaction of neonatal thymus cells to syngeneic adult spleen cells is recognition of “self” antigens is uncertain, since spleens of adult mice could carry antigens which do not occur in neonatal animals and are therefore “unknown” for neonatal thymus cells. We demonstrate here that neonatal thymus cells do not react to 4-day-old CBA spleen cells, but adult thymus cells do react against both allogeneic and syngeneic adult spleen cells.

Participation of three cell types in the anti-sheep red blood cell response in vitro. Separation of antigen-reactive cells from the precursors of antibody-forming cells.

Spleen cells of unprimed CBA mice were shown to produce anti-sheep red blood cell antibodies comparable in amount in vivo and in vitro. Under identical culture conditions spleen cells of C57BL mice did not respond. CBA spleen cells, passed through columns of cotton wool (CBAf), were equally inactive in vitro. However combined cultures containing both CBAf and C57BL cells yielded as many or more plaque-forming cells than the same number of unfractionated CBA spleen cells. Analysis of the contribution of each cell population to the synthesis of antibody in the combined cultures has disclosed the participation of three cell types. A thymus-dependent, radiosensitive cell was derived from the CBAf population, while the C57BL was the source of the precursor of the antibody-forming cell and of a radioresistant cell. The latter two were partially separated in a Staput apparatus.

A COMPARISON OF THE CHANGES PRODUCED IN RETICULOENDOTHELIAL ORGANS OF MICE DURING HOST‐VERSUS‐GRAFT AND GRAFT‐VERSUS‐HOST REACTIONS

SummaryA host‐versus‐graft reaction initiated by the intravenous injection of F1 (Balb/c × C57 Black) spleen cells into Balb/c mice is characterized by hepatosplenomegaly and a marked increase in the phagocytic activity of the reticuloendothelial system. Although the host‐versus‐graft reaction is accompanied by changes in reticuloendothelial organs which are also found in the graft‐versus‐host reaction, viz., phagocytic stimulation, splenomegaly and hepatomegaly, significant differences exist in the onset, duration and nature of the functional and morphological changes produced. They occur much earlier in the host‐versus‐graft reaction and persist for shorter periods. No increase in the relative number of liver macrophages occurs in the host‐versus‐graft reaction, and splenic enlargement is due to white pulp hyperplasia with prominent germinal centre development. In a situation where the potential exists for host‐versus‐graft and graft‐versus‐host reactions to occur simultaneously, such as that created by injecting CBA spleen cells into Balb/c mice, the host‐versus‐graft reaction develops and is of sufficient intensity to prevent the transplanted cells from mounting a graft‐versus‐host reaction against the recipient animal.

Requirement of a nonproliferating class of cells for generation of immune responses in cell culture.

SummaryNumbers of unimmunized CBA spleen cells, too small to give a PFC response in culture by themselves, can produce PFC if they are cultured in the presence of a large number of heavily irradiated cells. The survival after irradiation of the PFC response by normal spleen cells has a D0 of 95–105 rads. In contrast, inhibition of the capacity of heavily irradiated spleen cells to facilitate PFC production by normal cells requires doses greater than 2.5 krads. Attempts to demonstrate the provision of a conditioning factor by the heavily irradiated cells were not successful. However, mixtures of normal and heavily irradiated cells in limiting dilution did result in PFC production. These results are in keeping with the hypothesis that cell—-cell interaction between the two cell populations is required for generation of PFC responses in culture. Since it is known that in thymus-marrow synergism in intact animals proliferation of both the thymus- and marrow-derived cell populations is required for PFC product...

Role of Thymus in Adoptive Tolerance

Summary Specific homograft tolerance was induced in neonatal C3H mice with CBA spleen cells. These mice were test-grafted with CBA skin. After bearing successful CBA skin grafts for 2 months or longer they served as donors for “tolerant” thymus and bone marrow transplants. Adult intact and thymectomized C3H mice were irradiated and injected with 10 million “tolerant” bone marrow cells. The thymectomized mice were, in addition, supplied with a subcutaneous “tolerant” thymus graft. The irradiated recipients of “tolerant” thymus and “tolerant” bone marrow accepted CBA skin, demonstrating adoptive tolerance. They also rejected C57B1/6 skin, demonstrating that “tolerant” thymus can reconstitute immune recovery of thymectomized, irradiated bone marrow treated mice. Nontolerant thymus grafts were unable to abolish the transfer of adoptive tolerance by “tolerant” bone marrow, both in intact and thymectomized irradiated recipients. “Tolerant” thymus was capable of imposing specific homograft tolerance on irradiated recipients of nontolerant bone marrow, especially if these recipients were thymectomized at the time of irradiation. These results support the evidence of others that thymus plays an important role in immunologic tolerance.

Suppression of Transferred Allogeneic Spleen Cells in the Mouse: Active Induction and Passive Transfer by Antiserum

Summary Splenic cells of inbred mice were transferred to irradiated recipient mice of the same or a different inbred strain following stimulation with sheep erythrocyte or Shigella O antigen. In syngeneic recipients, the corresponding antibodies rose to a maximum on the 10th to 14th day and then declined slowly at approximately log2=1 per month over a maximum observation period of 3 months. In allogeneic recipients, maximal titers attained were lower, the decrease began earlier, and titers fell rapidly to below measurable levels before the 20th day following transfer. In various strain combinations, involving differences at the H2 locus, a wide range could be found between maximal titers attained in syngeneic and allogeneic recipients, this difference varying between log2 0 and 6. In an allogeneic transfer of spleen cells, CBA to Balb, the prior injection of CBA cells into the recipients caused rejection of subsequently transferred CBA cells. The rejection was demonstrated both by failure of the appearance of antibody in the recipient and by the absence of fatal graft-vs.-host reactions. The extent of the rejection reflected the immunizing dose of spleen cells and the interval between immunization and cell transfer. Rejection of transferred antigen-stimulated CBA spleen cells was also conferred adoptively by spleen cells of Balb mice, previously injected with CBA cells. Antiserum of Balb mice to CBA spleen cells suppressed antibody production by transferred CBA cells whether injected into the recipient animal or incubated with the spleen cells prior to transfer.

INHIBITORY EFFECT OF PRE-IMMUNIZED CBA SPLEEN CELLS ON TRANSPLANTS OF A-STRAIN MOUSE MAMMARY CARCINOMA IN (CBA X A) F1 HYBRID RECIPIENTS.

Inhibitory Effect of Pre-Immunized CBA Spleen Cells on Transplants of A-strain Mouse Mammary Carcinoma in (CBA × A) F 1 Hybrid Recipients

Acquired Tolerance to Skin Homografts in Mice

Summary Newborn C3H mice were rendered tolerant to CBA skin homografts by neonatal injection of CBA spleen cells. The offspring of these tolerant C3H mice were slightly more susceptible to a tolerance-inducing stimulus of CBA spleen cells than were the offspring of nontolerant C3H mice. The immunologic competence of normal C3H spleen and lymph node cells, as measured by the graft-versus-host assay in newborn C57BL/6J mice, continued to increase with age, exhibiting peaks of immunologic reactivity at 3 and 6 months for spleen, and at 7 months for lymph node cells. The increased immune potential of C3H mice with age could, at least in part, be responsible for the delayed loss of tolerance in C3H mice. The immunologic reactivity of C3H spleen and lymph node cells, during the various phases of tolerance, as measured by the graft-versus-host assay in newborn CBA mice, was subnormal but increased during graft contraction and graft rejection. The prolonged survival of second and third CBA skin grafts and the relative ease by which tolerance could be reinduced, were additional evidence for the subnormal immunologic status of post-tolerant C3H mice.

Acquired Tolerance to Skin Homografts in Mice

Summary A quantitative analysis was carried out on the induction and maintenance of homograft tolerance in the CBA → C3H strain combination of mice. These strains share the H2-histocompatability locus. The results indicate that the incidence of tolerance, as measured by the ability of neonatally injected C3H mice to accept CBA skin grafts for 2 months or longer, decreases proportionally with spleen cell doses of less than 2 million. One-eighth million CBA spleen cells will induce 22% tolerance. CBA lymph node cells are slightly less capable in inducing tolerance than spleen cells. F1 hybrid lymph node cells are equally as effective in inducing tolerance as parental homologous lymph node cells. When the duration of tolerance is determined the results suggest that the length of the tolerant period cannot be correlated to the initial size of the donor cell population. The duration of tolerance is variable, even within groups of mice injected with equal cell doses of homologous spleen cells. Tracing the fate of donor cells by radioautography during the early phases of tolerance and by the “chimera” test during the later stages, indicates that the injected donor cells settle primarily in the spleen and lymph nodes of the tolerant host. Donor cells can be detected by chimera analysis, during all stages of graft acceptance, graft contraction, and even shortly after graft rejection. The results are discussed in the light of the hypothesis that in the CBA → C3H strain combination of mice, induction and maintenance of homograft tolerance is dependent on a critical balance between the immune potential of the host and the size of the donor lymphoid cell population.

Decreased Immunogenicity of a Transplantation Antigen in Hosts Sensitized to Other Isoantigens of its Cellular Vehicle

Summary Accelerated rejection of male skin isografts by C57BL/6 female mice occurs as a consequence of previous injection of 20 million male CBA spleen cells by either the i.p. or i.v. route. However, if the test mice are immunized against CBA isoantigens from female donors 20 days before inoculation of the male CBA cells, rejection of the test grafts is significantly delayed. The inhibitory effect of prior CBA isoimmunization on sensitization to the male factor does not occur when isologous male cells are mixed with CBA female spleen cells and inoculated. These results are interpreted as evidence that the secondary response to the CBA isoantigens is capable of blocking access of the male antigen to those sites in which the primary response can be initiated.

FURTHER ANALYSIS OF ELIMINATION OF RUNT DISEASE AND INDUCTION OF TOLERANCE BY X-IRRADIATED SPLEEN CELLS.

A quantitative estimate was made of the decrease in proliferative capacity and immunologic activity of spleen cells exposed to 400 r of total-body x irradiation. The decreased proliferative capacity of irradiated CBA spleen cells was inferred from a decreased ability to induce homograft tolerance in newborn C3H mice as compared to the ability of various doses of nonirradiated CBA spleen cells to induce homograft tolerance. The decreased immunologic activity of CBA spleen cells was estimated from a decreased capacity to induce a graft- versus-host reaction in newborn C57BL/6 mice as compared to the capacity of standard doses of nonirradiated CBA cells to elicit a graft-versus-host reaction. The results indicate that 400 r of total-body x irradiation reduces the proliferative capacity of CBA spleen cells by about 95 to 96% and the immunologic activity by about 98 to 99%.

Acquired tolerance to skin homografts in mice. I. Histological analysis of lymphoid tissues before, during, and after the loss of tolerance.

Acquired tolerance to CBA skin homografts is lost in a large number of C3H mice neonatally injected with CBA spleen cells. The skin homografts persist for at least 2 months but then exhibit a chronic rejection pattern which may last up to 7 months. Histological analysis of the lymphoid tissues reveals the onset of an immune response in the axillary lymph nodes of many 4-month-old tolerant mice. This immune response which appears before external indications of graft rejection are evident, is manifested as an increase in number of germinal centers and plasma cells in the cortex and medulla, respectively. In older tolerant mice, an even larger proportion show these histological indications of immunological activity. During graft contraction and shortly after graft rejection, the immune response is still limited to the lymph nodes. After rejection of a second graft by post-tolerant mice, histological indications of an immune response are not only found in lymph nodes but also in spleen. The data suggest the development of a host versus graft reaction in seemingly tolerant C3H mice, which increases in severity with the age of the animal. The results are discussed from the point of view that tolerance is dependent upon a critical balance between the immune potential of the host and the population of donor cells. As mice mature, their immune potential may increase. The resulting host versus graft reaction increases, culminating in the rejection of skin graft and donor lymphoid cells.