The role of T-lymphocytes in the development of a humoral immune response to the corpuscular antigen of Staphylococcus aureus

The dependence of humoral immune response and the formation of immunological memory to corpuscular staphylococcal antigen (CSA) on the T-system of immunity was studied in experiments on B-mice and on mice with the congenital absence of the thymus (nude). Primary and secondary immune response to CSA in athymic mice was found to be considerably less than in normal animals. After the repeated immunization of genetically athymic mice the pronounced secondary reaction of the formation of antibodies to CSA was observed. As shown in this investigation carried out with the use of adoptive transfer techniques, the induction of memory B-cells to CSA may occur in animals with congenital or experimentally induced T-immunodeficiency. The conclusion was made on the T-dependence of humoral immune response to CSA, the formation of immunological memory to this antigen being relatively T-independent.     

Specificity of suppressor cells of the delayed hypersensitivity type for Staphylococcus studied by immunoadsorption

The immunological specificity of T-suppressors obtained from mice after intravenous immunization with corpuscular antigen was shown. The splenocytes of such a mice suppressed DH to staphylococcal antigens, but not to sheep red blood cells. The suppressor cells under study were specifically adhesive to staphylococci.     

Patterns of the forming of an immunologic memory to staphylococcal corpuscular antigen

The experiments carried out on inbred mice have revealed that the level of the immunological memory to staphylococci depends on the intensity of the antigenic stimulation; high priming dose of antigen proving to be the most effective one. The opposite character of immune responsiveness observed during primary antibody response to particulate staphylococcal antigen in C3H and A/Sn mice increased after the second immunization. It is established that immunological memory to staphylococci may be induced in genetically athymic mice. Many antibody-forming cells are found in the bone marrow of the secondary immunized mice. This phenomenon may be due to the repopulation of the bone marrow tissue by recirculating memory cells.     

Immunosuppressor role of staphylococcal protein A

The removal of protein A from the surface of staphylococci by means of proteolytic enzymes increases the immunogenic properties of staphylococci. Staphylococci containing protein A are less effective in mediating the immunological memory than those treated with proteolytic enzymes. The conjugation of protein A with staphylococci treated with proteolytic enzymes leads to the decrease of the immunogenic properties of staphylococci. Protein A not bound to staphylococci also suppresses antistaphylococcal immune response. The protective properties of corpuscular staphylococcal antigen are increased after the removal of protein A from the surface of staphylococci by proteolysis.     

The role of the bone marrow in forming an immunological memory to Staphylococcus

The dynamic study of antibody formation in the bone marrow tissue of mice immunized with corpuscular staphylococcal antigen was made. A single intravenous injection of this antigen to the animals produced no essential changes in the number of antibody-forming cells in the bone marrow, their repeated immunization led to the involvement of the bone marrow in the anamnetic reaction, which was manifested by the accumulation of a great number of plasmocytes synthesizing antibodies to staphylococci in bone marrow tissue. The relative contribution of the bone marrow to systemic antibody formation essentially increased at later stages of secondary immune response. The possible biological significance of the regularities and the practical importance of the data revealed in this investigation are discussed.     

Antigen-initiated B lymphocyte differentiation. XIV. Nonspecific effects of antigen stimulation cause proliferation in the "pre-progenitor" subset of primary B cells.

The effect of specific and nonspecific stimuli on the cycle status of subsets of primary B lymphocytes was assessed by preinjecting donor CBA mice 1 to 2 days previously with various substances, and then incubating the isolated spleen cells with high specific activity 3H-TdR before assay. AFC-progenitor activity was assessed as a response to NIP-POL antigen, either by adoptive transfer to irradiated recipients or by cell culture. Previous studies showed these assays reflected the activity of different subsets of B cells, termed "pre-progenitors" (adoptive assay) and "direct progenitors" (culture assay). Most functional primary B cells, whether assayed in culture or by adoptive transfer, were not initially in rapid cell cycle in normal adult mice. However, nonspecific stimulation for 1 day caused NIP-specific adoptive transfer IgM AFC-progenitors to enter rapid cell cycle. This effect was independent of T cells and not related to the antigenicity of the stimulus: particulate peritoneal irritants were the most effective stimulants. In contrast to adoptive transfer results. AFC-progenitors assayed in cell culture were unaffected by nonspecific stimuli, but were activated into cell cycle by specific antigen.     

Antigen modulation of the secondary immune response. VI. Contribution of cells recruited from precursor pool to expression of memory.

The adoptive transfer system has been used extensively to study the ability of antigen triggered memory cells to become antibody forming cells and/or to proliferate and expand the memory cell population. Selective antigen triggering of the memory cells for low and high affinity antibody formation has also been studied in this way. One of the main counter-arguments to the interpretation of these data is that the presence of antigen in the adoptive host may lead to recruitment of new memory cells from either a host or donor precursor population. In this paper we examined the contribution of both host and donor precursor cells to the total antibody response in adoptive secondary recipients. The following donor-host combinations were used in which the recipients were given 1 mg fluid antigen intravenously: (A) normal (non-immune) donors to normal irradiated recipients; (B) normal donors to carrier primed irradiated recipients; (C) carrier primed donors to normal irradiated recipients; (D) normal donors to carrier primed recipients with challenge and subsequent transfer to additional carrier primed recipients; (E) carrier primed donor to normal recipients to carrier primed recipients; (F) repeat of B and C above with multiple antigen administration; (G) purified immune (DNP-BGG) donor T cells mixed with normal B cells transferred to normal irradiated recipients. In most cases recruitment was seen but this represented less than 4% of the responses seen with immune cells. Thus we conclude that this level of recruitment does not compromise the use of the adoptive transfer system for studying selective antigen triggering of memory cells.     

The extracorporeal immunostimulating effect of terrilytin in staphylococcal infection]

The intravenous injection of terrilytin-treated lymphocytes into rats infected with staphylococci enhances the formation of staphylococcal alpha antitoxin in the animals and the development of immune response to T-dependent antigen, such as sheep red blood cells (SRBC), but produces no effect on the development of immune response induced by T-independent antigen (lipopolysaccharide). Terrilytin-treated lymphocytes induce the release of the factor promoting the development of immune response to staphylococcal antigens and SRBC by spleen cells, incapable of adherence to plastic, but have no influence on the development of immune response to lipopolysaccharide in rats infected with staphylococci. At the same time in such rats spleen cells adhering to plastic take part in the transfer of signals from terrilytin-treated lymphocytes to nonadhering spleen cells of recipients.     

Transition in the Character of Immunological Memory in Mice after Immunization

The immunological memory in antibody response of mice to bovine serum albumin (BSA) was investigated at the level of antibody-producing cells or their precursor B cells and thymus-dependent helper T cells. Spleen cells obtained from mice previously primed with alum-precipitated BSA at various times were transferred to irradiated syngeneic mice. Spleen cells from mice immunized 8 days or 64 days before presented a high degree of adoptive secondary response, whereas the adoptive response of cells from mice immunized 2 days previously was found to be inferior even to that of unprimed spleen cells. Primed spleen cells treated with anti-mouse thymocyte rabbit serum plus complement were supplemented with normal thymus cells and the restoration of the responsiveness was examined. It was suggested that the memory was carried mainly by T cells in the earlier phases of the immunological memory (2 days or 8 days after the primary immunization). On the other hand, the immunological memory in the B-cell population was shown to grow gradually toward the later phase (later than 40 days).     

Kinetics of B cell memory development during a thymus "independent" immune response

Some parameters of the development of immunological memory to B. abortus (BA) and sheep erythrocytes (SE) in the mouse have been compared. The thymus-independence of the BA response allowed evaluation of B-cell memory in vivo and in adoptive immune responses. A reduced responsiveness to BA was seen during the first few days after the primary injection, whereas enhanced ability to give responses to SE (thymus dependent) occurred at that time.The ability of primed spleen cells to transfer 19S and 7S memory responses to SE developed in parallel. In contrast, the earliest appearance of 19S memory to BA on Days 5–7 after priming was not yet accompanied by memory for the 7S response, but by Day 10 both 19S and 7S memory were present. At 1–2 months after priming, 100-fold fewer cells than needed for transfer of the primary response still transferred excellent 19S and 7S memory responses to BA. Anti-θ treatment of long-term memory 19S and 7S spleen cells did not affect their ability to respond to challenge even with limiting BA doses. It is suggested, however, that the T-independency of the response to BA applies only to the specific induction by antigen of preexisting B cells into antibody secreting cells, whereas optimal B cell memory formation to any antigen may be a separate T-dependent function.Serial spleen cell transfers into lethally irradiated recipients at 1–2 week intervals with antigen challenge at each transfer, appeared to interfere with the development of memory to BA, particularly for the 7S response. No such effect was seen on the responses to SE.     

Mechanism of formation of antibody production stimulating ability of bone marrow cells of mice immunized with staphylococci

The mechanism of the increase in immune response to particular staphylococcal antigen was studied in CBA and BALB/c mice injected by primed bone marrow cells (BMC). It was found that immunostimulatory effect of immune BMC is not mediated by macrophages or T cells, but is associated with staphylococcus-specific B memory cells present in the pool of primed BMC. Splenectomy performed in donor animals prior to immunization did not abolish the induction of stimulating BMC activity. It was concluded that primed B lymphocyte migration from spleen into bone marrow is not obligatory for the induction of staphylococcus-specific immunological memory in the bone marrow.     

Mitogenic activity of the antigens isolated from different Staphylococcus aureus strains in relation to mouse and guinea pig splenocytes

The mitogenic effect of corpuscular antigens with respect to the splenocytes of animals was found to depend on the strain of Staphylococcus aureus. The maximum synthesis of DNA in the cells was induced by corpuscular antigen Smith and the minimum synthesis, by Wood-46. The synthesis of DNA was activated in both B- and T-splenocytes in response to corpuscular antigens Wood-46, Cowan-1 and Smith, as well as to the cell wall and protein A. Peptidoglycan produced a mitogenic effect only in B-lymphocytes, and teichoic acid showed no mitogenic activity in mouse splenocytes. The mitogenic effect of staphylococcal antigens on splenocytes depended on the dose of the antigen and the time of cultivation. After 48-hour cultivation the incorporation of 3H-thymidine into the DNA of mouse cells was 5 times higher than into the DNA of guinea-pig cells. The optimum mitogenic dose in thymectomized BALB/c mice with respect to splenocytes was higher than in normal BALB/c mice practically by one order.     

Transition in the character of immunological memory in mice after immunization. II. Memory in T and B cell populations.

Teh immunological memory in antibody response of mice to bovine serum albumin (BSA) was investigated at the level of antibody-producing cells or their precursor B cells and thymus-dependent helper T cells. Spleen cells obtained from mice previously primed with alum-precipitated BSA at various times were transferred to irradiated syngeneic mice. Spleen cells from mice immunized 8 days or 64 days before presented a high degree of adoptive secondary response, whereas the adoptive response of cells from mice immunized 2 days previously was found to be inferior even to that of unprimed spleen cells. Primed spleen cells treated with anti-mouse thymocyte rabbit serum plus complement were supplemented with normal thymus cells and the restoration of the responsiveness was examined. It was suggested that the memory was carried mainly by T cells in the earlier phases of the immunological memory (2 days or 8 days after the primary immunization). On the other hand, the immunological memory in the B-cell population was shown to grow gradually toward the later phase (later than 40 days).     

Mechanism of the intensification of the immune response to Staphylococcus in mice after the transplantation of primed donor splenocytes

Experiments on CBA, C57Bl/6 mice and (CBA X X C57Bl/6)F1 hybrids were made to study the mechanism of stimulation of the immune response to staphylococci after injection of primed splenocytes. The stimulating action of immune splenocytes was reversed after their in-vitro treatment with anti-immunoglobulin serum and complement. The stimulant effect was also seen in a semi-allogeneic system (adoptive transfer of CBA mice immune cells to (CBA X C57Bl/6)F1 recipients). Preincubation of splenocytes with CBA-anti-C57Bl/6-serum and complement prior to demonstration of antibody-forming cells did not influence their number in the spleen of hybrid recipients injected with immune cells carrying parent genotype but decreased this indicator of the immune response in control mice. It is concluded that stimulation of the immune response to staphylococci after transplantation of primed splenocytes is due to the anamnestic response of donor's cells repeatedly stimulated by antigen in the recipient's host.     

Regulation of antigen induced changes in cyclic nucleotide levels in NZB/wf1 mice.

Normal C57BL/6J mice respond to the iv injection of antigen with an increase in splenic cAMP at 2 min. NZB/WF₁ mice are predisposed to autoimmune and immunological disorders upon aging. The ability of NZB/WF₁ mice to respond to antigen with an increase in their splenic cAMP level was found to diminish with age. This loss of responsiveness is antigen specific and not due to a loss of adenylate cyclase activity in spleen cells of old NZB/WF₁ mice. The adoptive transfer of spleen cells from unresponsive old mice into responder young mice inhibited the cAMP response to antigen by the recipients. Spleen cells from young responsive mice, on transfer into old nonresponsive NZB/WF₁ recipients, resulted in restoration of the cAMP response to antigen. In both cases, the activity of donor cells was dependent on the transfer of T cells. These results indicate that populations of T cells participate in the regulation of the cAMP response to antigen by NZB/WF₁ mice. The response of old mice could also be restored by treatment with indomethacin, and also the spleen cells of such treated donors failed to suppress the cAMP response of young recipients. Together, the results suggest a role for prostaglandins in regulating the cAMP response to antigen.     

Maintenance and amplification of cell-associated immunological memory in in vivo culture system

By employing bovine serum albumin as antigen and the capsular polysaccharide of Klebsiella pneumoniae as adjuvant, maintenance and amplification of immunological memory were analyzed in an in vivo culture system in mice. For this purpose, the double cell transfer technique was employed to minimize the influence of regulatory factors on memory expression. Memory associated with primed cells is maintained at the original level during in vivo culture for at least a month in the absence of antigen. In contrast, memory is amplified more than 30 times during this period by stimulation with antigen. This secondary increase in memory does not require the action of adjuvant. Neither the residual primary antigen nor preformed primary antibody seems to play a significant role in the maintenance and amplification of memory of the primed cells. From these results it is probable that the enduring immunological memory in actively immunized mice is conveyed by long-lived memory cells, and additional antigenic stimulating on once-established memory cells serve to amplify (not simply to maintain) memory in a secondary fashion.     

Antigenic markers on mouse lymphoid cells: origin of cells mediating immunologic memory

Specific antisera were used for the purification of thymus dependent and thymus independent or bursa equivalent lymphoid cells in the mouse. Spleen cells from mice immune to sheep erythrocytes, a thymus dependent antigen, or to E. coli 055:B5 lipopolysaccharide, a thymus independent antigen, were treated with anti-θ (C3H) serum or anti-MBLA serum and complement prior to their adoptive transfer into lethally irradiated syngeneic recipients. Syngeneic thymocytes, bone marrow cells, or spleen cells from nonimmune donors were appropriately added to antiserum treated cells prior to transfer. The secondary response to these antigens was assayed in recipient spleens six days after cell transfer. The kinetics of the primary response to SRBC was investigated as to its effect on origin of specific hyper-reactive T or B lymphoid cells.The adoptive response to CPS originated in the B lymphoid cell population. Immunologic memory to CPS was demonstrated in recipients of immune cells, compared to recipients of normal cells, by a five fold increase in antibody forming cells.The IgM and IgG adoptive immune response to high doses of SRBC depended upon an increased number of specifically hyper-reactive T-lymphoid cells to facilitate cooperation between T and B lymphocytes. High doses of SRBC initially stimulated T cell memory but at 42 days after priming an increased number of specifically hyper-reactive B lymphoid cells were present.     

Secondary delayed type hypersensitivity to sheep red blood cells in mice: Dependence on long-lived memory cells

Secondary delayed type hypersensitivity (DTH) to sheep red blood cells (SRBC) in mice is a long-lived memory phenomenon which is characterized by the accelerated reappearance of the state of DTH after a booster injection of the antigen. In this paper the nature of the DTH-related T memory cells accounting for secondary DTH was investigated. Parabiosis of primed and nonprimed mice for a period of 4 weeks resulted in an equally large secondary DTH responsiveness in both partners. This ability was maintained in both members for at least 6 months after termination of the parabiosis. These results indicate that (a) DTH-related T memory cells are potentially circulating cells, and (b) the persistence of these memory cells is not dependent on the presence of the antigen which induced their generation. Subcutaneous (sc) injection of intravenously (iv) primed mice with a small dose of antithymocyte serum before boosting did prevent the development of secondary DTH responsiveness in sc boosted mice, but not in iv boosted mice. Treatment of primed mice with vinblastine or azathioprine did not decrease the capacity of adoptive transfer of secondary DTH by means of spleen cells, but passive transfer of secondary DTH was completely abolished by this treatment. These results suggest that (a) SRBC-induced DTH-related T memory cells are nonproliferating, partially sessile, partially recirculating cells, and (b) these memory cells proliferate before they become DTH-related effector cells.     

The role of complement receptor positive and complement receptor negative B cells in the primary and secondary immune response to thymus independent type 2 and thymus dependent antigens

Both complement receptor positive (CR+) and complement receptor negative (CR-) B cells have been shown to be involved in the primary immune response to PC-Hy (phosphocholine conjugated hemocyanin), a thymus dependent (TD) antigen which preferentially induces antibody secretion in Lyb-5+ B cells during a primary adoptive transfer assay. CR+ and CR- B cells also responded in a primary adoptive transfer assay to TNP-Ficoll, a thymus independent type 2 (TI-2) antigen which activates only Lyb-5+ B cells. When the secondary immune response to PC-Hy and TNP-Ficoll were analyzed, it was found that most of the immune memory to both antigens was present in the CR- B cell subset. The CR- B cell subset also dominated the secondary immune response to PC-Hy in immune defective (CBA/N X DBA/2N)F1 male mice. These data indicate that CR- B cells dominate the memory response in both the Lyb-5+ and Lyb-5- B cell subsets of normal and xid immune defective mice and suggest that Lyb-5+ and Lyb-5- B cells can be subdivided into CR+ and CR- subsets.     

Antigen modulation of the immune response. III. Evaluation of the hypothetical short-lived memory cell

The putative short-lived memory cells, whose existence has been suggested by the results of secondary adoptive transfer experiments, was investigated. On the basis of the following evidences we have concluded that the short-lived memory cell is probably an artifact of the adoptive transfer technique: (a) when immune thoracic duct lymphocytes, known to consist predominantly of long-lived memory cells, were transferred to irradiated recipients and challenged at various times after transfer, approximately 80–90% of the initial response was absent by Day 14 challenge; (b) Preirradiating adoptive recipients with increasing dose of X-irradiation tended to lengthen the observed half life of memory cells; (c) single or multiple treatments of immune donors with 0.3 mg Vinblastin before transfer resulted in neither a depression of the initial secondary response nor an alteration in the rate of decline of the memory potential; (d) reconstitution of irradiated hosts with normal spleen cells one day before transfer of memory cells and challenge resulted in inhibition of the adoptive secondary response; and (e) the transfer of memory cells to antigen free intermediate hosts, in which they were allowed to reside for one day or fourteen days before transfer to irradiated recipients, resulted in only a slight decline in their capacity to respond.We propose that the rapid decline of memory potential in adoptive recipients challenged at various times after transfer is due to modulating effects by the hosts as it recovers from irradiation. These effects may be the result of cell crowding or the loss of irradiation-produced stimulatory factors. The relevance of these findings to adoptive transfer systems in general and the secondary response of intact animals is discussed.