Specifically depleted spleen cell populations recover responsiveness by a concomitant unrelated delayed hypersensitivity reaction.

Spleen cells from nonimmunized CBA mice were specifically depleted of cells able to react spontaneously to pigeon erythrocytes (PRBC) by the formation of either rosettes (RFC) or hemolytic plaques (PFC). Spontaneous RFC were eliminated by centrifugation on a Ficoll-Isopaque gradient whereas spontaneous PFC were removed by filtration through a PRBC-coated column. RFC-depleted populations transferred into lethally irradiated syngeneic recipients and stimulated with PRBC failed to develop any significant response during the first 7 days after transfer but developed a definite anti-PRBC reaction on the eighth day. PFC- depleted populations remained unresponsive to PRBC throughout the 12-day observation period. When the spleen cells were taken from mice whose skin had been painted with picryl (trinitrophenyl, TNP) chloride 12–15 days before and the recipients of cell populations depleted of anti-PRBC were challenged with picryl chloride and stimulated with PRBC, they recovered the responsiveness to PRBC in an accelerated fashion. Under these conditions both anti-PRBC and anti-TNP RFC and PFC were found, and some cells simultaneously reacting to both PRBC and TNP were also detected.     

Different effects of the capsular polysaccharide of Klebsiella pneumoniae on the functions of various subpopulations of B cells in the immune response of mice to T-dependent antigen.

Using the capsular polysaccharide of Klebsiella pneumoniae (CPS-K) as a polyclonal B-cell activator (PBA) and sheep red blood cells (SRBC) as a T-dependent antigen, we studied the effects of PBA on the functions of various subpopulations of B cells in the immune response of mice to T-dependent antigen. Antibody-forming cells (AFC) of IgM and IgG types were estimated as anti-SRBC direct and indirect plaque-forming cells (PFC), and the B cells with precursor activities involving generation of AFC and supplementing new B cells as rosette-forming cells (RFC) of the B-cell type. Stimulation of normal mice by CPS-K caused a definite increase in the number of direct PFC but not in that of indirect PFC and RFC in the spleens. The responsiveness of spleen cells of CPS-K-treated mice to generate PFC and RFC responses to a subsequent injection of SRBC was lower than that of CPS-K-untreated normal mice. In this case, the responsiveness to generate RFC and indirect PFC was inhibited more strongly by CPS-K than that to generate direct PFC. When CPS-K was injected into normal mice simultaneously with SRBC, CPS-K never decreased but increased the levels of PFC and RFC responses to SRBC. In the spleens of SRBC-primed mice, the number of RFC was markedly decreased following injection of CPS-K, the number of direct PFC was increased only slightly and the number of indirect PFC was increased very slightly. The responsiveness of spleen cells of these CPS-K-treated SRBC-primed mice to generate secondary PFC and RFC responses to a subsequent injection of SRBC was much lower than that of CPS-K-untreated SRBC-primed mice. In this case, the responsiveness to generate the secondary RFC and indirect PFC responses was more strongly inhibited by CPS-K than that to generate the secondary direct PFC response. When CPS-K was injected into SRBC-primed mice simultaneously with the secondary injection of SRBC, there were marked decreases in the level of the secondary RFC response and slight decreases in that of the secondary indirect PFC response, but little change in that of the secondary direct PFC response. From these results it has been concluded that CPS-K provides the positive signal (the minor action) and the negative signal (the major action) to various subpopulations of B cells functioning at various stages of the immune response to T-dependent antigen in different ways, and acts to regulate the levels of B-cell responses to the antigen-mediated positive signal.     

Antigen-binding cells in central and peripheral lymphoid tissues of the rabbit

The rosette assay was used to study antigen-binding activity by cells in lymphoid tissues of rabbits immunized with sheep red blood cells and in unimmunized controls. Percentages of rosette-forming cells (RFC) observed were compared with those of cells which secreted antibody (plaque-forming cells, PFC) and cells which both bound antigen and secreted antibody. Rosette-forming cells and PFC were shown to be two distinct reactive cell populations. Thus, in the spleen less than 1% of RFC also formed plaques. Immediately following antigen stimulation, the number of RFC in the bone marrow decreased to below detectable limits. After an initial rise, the number of RFC in the appendix declined similarly. In contrast, RFC levels in the spleen rose steadily from the time of immunization. These patterns suggest that bone marrow and appendix may function as a reservoir of antigen-binding cells which are released to other sites following antigenic stimulation. Rosette-forming cells were rarely observed in the thymus. Rosette-inhibition studies using antisera specific for bone marrow-derived cells (anti-B) and thymus-derived cells (anti-T) revealed a markedly greater proportion of T-RFC in the appendix than in the spleen.     

Induction of an immune response in athymic nude mice to thymus-dependent antigens by Pseudomonas aeruginosa exotoxin A

Exposure of spleen cells from athymic nude mice to Pseudomonas aeruginosa exotoxin A induces these cells to respond to the thymus-dependent (TD) antigen sheep erythrocytes (SRBC). The response induced by toxin is dose dependent, antigen specific, and not due to polyclonal B-cell activation. Enhancement of the anti-SRBC response can be observed when toxin addition precedes antigen stimulation by 24-48 hr, which decreases when toxin administration follows antigen stimulation. A significant response is also observed when toxin and antigen are added simultaneously. A significant anti-SRBC response can be observed out to Day 10 postantigen and toxin stimulation after attaining a peak response at Day 5. Cultures exposed to toxin in the presence or absence of antigen exhibited a higher cell number and relative number of B cells as compared to control cultures. Exposure of T-cell depleted B cells from euthymic +/nu mice to toxin plus antigen does not result in an anti-SRBC response indicating that exotoxin A alone is not sufficient to induce B-cell responsiveness to T-dependent antigens and that other cells and/or factors are involved in the toxin-induced responsiveness of nude mice to T-dependent antigens.     

Comparative study of rosette and plaque formation in rats infected with Mycoplasma arthritidis and Acholeplasma laidlawii]

M. arthritidis suppresses rosette- and plaque-formation in rats infected with this strain of mycoplasma. On the 15th day the immune response was restored in the control and experimental groups of animals. In later stages a strong stimulation of RFC and PFC was recorded, which levelled on day 150. On the contrary, A. laidlawii stimulated RFC and PFC in all stages of infection. A relationship between the phenomena described and pathogenic properties of the mycoplasma is discussed.     

Migration and differentiation of bone marrow lymphocytes: development of surface immunoglobulin, Fc receptor, complement receptor, and functional responsiveness as studied with anti-allotype serum

Immunofluorescent studies using fluorescein isothiocyanate-conjugated mouse anti-allotype antibody were carried out to study the migration pattern and the development of surface Ig (SIg), Fc receptor for IgG (FcR gamma), and complement receptor (CR) or mouse bone marrow lymphocytes following intravenous injection into congenic mice. After transfer of bone marrow cells from CSW mice into untreated congenic CWB mice, the absolute number of donor-type SIg-bearing (SIg+) cells and the proportion of either FcR gamma- or CR-bearing (FcR gamma+ or CR+) cells in donor-type SIg+ cells were evaluated in the recipient spleen and the results were compared with those obtained after the transfer of CSW spleen cells. After injection of donor bone marrow cells, detectable donor-type SIg+ cells, although few initially, increased from day 1 to Day 2 and reached a plateau thereafter. The proportion of FcR gamma+ cells in donor-type SIg+ cells, although very low in the donor marrow inoculum, increased progressively after 1 day to reach a maximum at Day 5 (90%). On the other hand, following the transfer of spleen cells, the proportion of FcR gamma+ cells remained at high levels (90%) for 5 days after transfer. Likewise, the proportion of CR+ cells in donor-type SIg+ cells was very low (less than 1%) in the original donor bone marrow cells but high (60%) in the donor spleen cells. However, in transferring bone marrow cells this proportion also increased in the recipient spleen to reach a maximum (49%) at Day 5 although it was lower compared to the percentage of FcR gamma+ cells in donor SIg+ cells. Furthermore, the ability of functional responsiveness to antigen was also examined in the same system by detecting plaque-forming cells (PFC) from donor origin. In transferring donor bone marrow cells into recipient, the participation of donor cells in the PFC response was very low when the recipients were primed with sheep red blood cells at Day 3 after transfer. However, when the recipients were primed at Days 7 to 21 after transfer, increasing numbers of the donor marrow-derived cells were involved in the PFC response. Thus, the present study demonstrates that the bone marrow-derived lymphocytes, albeit lacking both distinctive surface receptors (IgM, FcR gamma, CR) and the functional responsiveness to antigen, continue their development along the B-cell lineage after migrating into the spleen, as evidenced by the surface receptor expression and participation in the antibody response.     

Relationship of lymphotoxin secretion and DNA synthesis in the human mixed lymphocytes reaction in vitro.

Influenza virus particles, inactivated with formalin, have been covalently bound to cyanogen bromide-activated Sepharose beads (Se-vi beads). Preservation of the hemagglutination properties of the viral particles enabled a strong binding of pigeon or human group O erythrocytes (PRBC or HoRBC) to these Se-vi beads. The conditions for preparation of PRBC- or HoRBC-Se-vi columns are described.Spleen cell suspensions from mice immunized with the above erythrocytes were considerably depleted of cells forming hemolytic plaques (PFC) against the corresponding erythrocytes after passage through these columns. In the case of cells from nonimmunized mice, the depletion is still greater and reaches up to 95–100%. However, the number of PFC reactive to unrelated erythrocytes is not affected in the filtered population. Specifically attached cells recovered from the Se-vi-RBC columns passed with normal spleen cells are considerably enriched in the number of PFC against homologous erythrocytes. Syngeneic irradiated hosts transferred with filtered cells are able to give a normal primary PFC response against heterologous, but not against homologous RBC up to the 12th day after immunization. These results are discussed in relation to the problem of precommitment of specific PFC precursor cells.     

Immune response of (C57BL/6xA/Sn)F1 mice in mycoplasm-virus infection

Mixed Rauscher leukemia virus (RLV) and M. arthritidis infection of (C57BL/6xA/Sn)f1 mice-hybrids, highly resistant to RLV, was accompanied by a progressive inhibition of rosette-forming cells (RFC) and plaque-forming cells (PFC), resulting in the induction of malignant erythroblastosis identical by cytology to Rauscher leukemia. The mice-hybrids infected with A. laidlawii and RLV developed significant splenomegaly on the 21st day of the infection, and their immune response was almost entirely suppressed, but both RFC and PFC populations as well as the spleen weight returned to the initial level by the 62d day the infection. A possible role of mycoplasm in the induction and development of Rauscher leukemia is discussed.     

Formation of heterophile antibodies by human tonsilar lymphocytes: II. In vitro stimulation with allogeneic cells

Short-term cultures of human tonsilar lymphocytes (HTL), 5 × 10⁶ cells/culture, in medium RPMI 1640 supplemented with human group AB serum were studied for the production of plaque-forming cells (PFC) against sheep (SRBC) and bovine (BRBC) red blood cells following in vitro stimulation by various allogeneic lymphoid cells. Of 55 HTL specimens examined, 48 produced a significant number (50–300/culture) of PFC against SRBC and/or BRBC following the in vitro stimulation. The optimal doses of the stimulator HTL and peripheral blood lymphocytes (PBL) were 10⁷ and 5 × 10⁶/culture, respectively. After the stimulation, PFC appeared in significant numbers on the third day, reached the peak number on the sixth day, and decreased sharply in number thereafter. Removal of E-rosetting cells from both stimulator and responder populations abolished the PFC formation. PFC formation against SRBC was inhibited by solubilized Forssman antigen, while PFC formation against BRBC was inhibited strongly by Hanganutziu-Deicher antigen, hardly by Paul-Bunnell antigen and not at all by Forssman antigen. Supernatants of mixed lymphocyte culture of PBL were shown to enhance PFC formation of HTL cultures stimulated by allogeneic lymphocytes. The results of this study indicated that in vivo primed B cells of the HTL were triggered in vitro by allogeneic stimulation for the heterophile antibody formation. Since these antibodies are apparently directed against Forssman and Hanganutziu-Deicher antigens, the “allo” nature of these antigens as well as their relationship to the previously described heterophile transplantation antigens have to be clarified.     

Antigen-induced and polyclonal B-cell responses in human peripheral blood lymphocyte cultures

This work was designed to delineate the anti-hapten antibody (Ab) response induced by trinitrophenol-polyacrylamide (TNP-PAA) beads from the nonspecific B-cell response which concomitantly occurs in human peripheral blood mononuclear cell (PBMC) cultures. Indeed human PBMC produce consistent amounts of immunoglobulins when cultured at high cell density in the presence of fetal bovine serum, regardless of the presence of antigen. In contrast, the stimulation of such cultures by TNP-PAA leads to an Ab response characterized by the following: cells secreting anti-hapten Ab at a high rate (detected by a plaque-forming cel (PFC) assay); a 10-30 times enhancement in the number of hapten-specific binding cells (detected by a rosette-forming cell (RFC) assay); the production of anti-TNP IgM Ab (detected by an ELISA assay). The anti-TNP response is specifically triggered by the particulate antigen, as shown by the following: The TNP-PAA antigen induces a clear-cut increase in the amount of anti-TNP Ab whereas it only marginally increases that of total IgM. The anti-TNP Ab response is specifically abolished when anti-TNP RFC are depleted from the PBMC preparation before the initiation of the cultures. The anti-TNP Ab response is specifically abolished when PBMC are triggered by TNP-PAA in the concomitant presence of a soluble TNP-protein conjugate. These results demonstrate the ability of polymeric antigens to specifically activate human peripheral blood B cells.     

In vitro immune response to sheep erythrocytes in macrophage depleted cultures. Restoration with interleukine 1 or a monokine from resident macrophages and stimulation by N-acetyl-muramyl-L-alanyl-D-isoglutamine (MDP)

The involvement of macrophages in the adjuvanticity of N-acetyl-muramyl-L-alanyl-D-isoglutamine (MDP) has been examined. The stimulation of the in vitro primary immune response to sheep red blood cells (SRBC) has been studied, because it is known that macrophages cooperate through the mediation of soluble compounds for the induction of the anti-SRBC response. The cultures depleted of macrophages by passing spleen cells on Sephadex G-10 were unable to give any response to SRBC. Their immune responsiveness was fully restored by the addition of either Interleukine 1 (IL 1) obtained from P388D1 cells or a factor able to replace macrophages (FRM) obtained from resident peritoneal macrophages. MDP alone, at any dose, was unable to induce any response in such macrophage depleted cultures, but it was able to enhance the antibody response of these cultures reconstituted with monokines, with the same characteristics in dose effect and timing dependence than in whole spleen cells.     

Influence of a sublethal irradiation on the immune response of the mouse to sheep erythrocytes]

In the CBA mice, the immunological response of the spleen cells (RFC and PFC direct and indirect) against the sheep erythrocytes is highly depressed by a 400 R dose of X rays. The recovery is not complete at the 30th day after irradiation. The response of the bone marrow cells either irradiated or unirradiated to the antigenic stimulation is very low.     

Effects of the methanol extract residue (MER) tubercle bacillus fraction on the production of antibodies in vitro. II. Effects on macrophage and lymphocyte populations

The effect of the methanol extract residue (MER) fraction of BCG tubercle bacilli on the generation of primary antibody responsiveness in vitro to sheep red blood cells (SRBC) was ascertained in cell reconstitution experiments, employing enriched populations of mouse macrophages and of T and B lymphocytes. In each of the antibody generation cultures one or another of the cell fractions had been exposed to MER, either by treatment of the donor animals or by preincubation with the agent for 48 hr in vitro. In some experiments, supernatants of MER-preincubated cells were employed in place of the cells. Macrophages and T cells that had been exposed to MER in vivo or in vitro and their supernatants demonstrated a markedly greater effect than nonexposed cells in the generation of direct specific plaque-forming cells (PFC) upon antigenic stimulation of the cultures with SRBC. In contrast, PFC production was not stimulated in B-lymphocyte populations that had been in contact with the agent.     

Stimulation of the in vivo dinitrophenyl antibody response to the DNP conjugate of L-glutamic acid60-L-alanine30-L-Tyrosine10 (GAT) polymer by a synthetic adjuvant, muramyl dipeptide (MDP): target cells for adjuvant activity and isotypic pattern of MDP-stimulated response

Specific anti-dinitrophenyl (DNP) response to DNP-conjugated L-glutamine60-L-alanine30-L-tyrosine10 (DNP-GAT) was obtained in GAT-responder mice by using synthetic N-acetylmuramyl-L-alanyl-D-isoglutamine (MDP) as adjuvant. Significant levels of anti-DNP antibodies were observed during a secondary response to DNP-GAT, when both antigen and MDP were used for priming. In this system, MDP was able to prime the carrier-specific T cells but not the hapten specific B cells. The study of the isotypic pattern of the anti-DNP response shows that MDP stimulates only the appearance of specific anti-DNP IgG1 plaque-forming cells. Anti-DNP plaque-forming cells were stimulated in animals primed with DNP-GAT in Freund's complete adjuvant or in Maalox-pertussis and used as control IgG1, IgG2a, and IgG2b.     

Regulation of the secondary in vitro antibody response by endogenous natural killer cells: kinetics, isotype preference, and non-identity with T suppressor cells

Our previous studies had demonstrated that depletion of endogenous natural killer (NK) cells resulted in an augmented primary antibody response in vivo and in vitro. We have now examined the effect of NK cell depletion on the in vitro secondary response to antigen. Treatment of primed murine spleen cells with anti-NK-1.1 allo-antibody and complement before culture resulted in a significant increase in the magnitude of the antigen-specific plaque-forming cell (PFC) response. This treatment did not affect the proportions of Lyt-2+, L3T4+, or sIg+ cells in the population, however, indicating that the augmentation in PFC was not due to changes in the ratio of T to B cells. Removal of endogenous NK cells had a greater effect on the IgG (indirect) PFC response (100 to 200% increase) than on the IgM (direct) PFC response (25 to 50% increase). In contrast, removal of Lyt-2+ cells before culture affected the IgM and IgG responses similarly. Moreover, the kinetics of augmentation differed between cultures depleted of Lyt-2+ cells and those depleted of NK-1.1+ cells. NK cells appeared to act earlier in the response than did T suppressor cells. The NK-1.1+ cells involved in antibody regulation were not involved in the generation of the in vitro derived T suppressor cells. The conclusion that the regulation of the antibody response by NK-1.1+ cells is distinct from that involving T suppressor cells was confirmed in experiments in which removal of both regulatory cell populations resulted in an increase in PFC that was greater than in cultures depleted of either NK or T suppressor cells.     

Enhancement of carrier-specific helper T cell function by the synthetic adjuvant, N-acetyl muramyl-L-alanyl-D-isoglutamine (MDP).

The adjuvant effect of a synthetic peptidoglycan, muramyl dipeptide (N-acetyl muramyl-L-alanyl-D-isoglutamine, MDP), was studied by using the anti-Tnp PFC and hemagglutinin responses of BALB/c mice to hapten-carrier conjugates. Administration of Tnp-OVA and MDP in saline to mice, followed 2 weeks later by a boost of Tnp-OVA in saline, led to significantly higher IgM and IgG anti-Tnp PFC and total anti-Tnp-hemagglutinin responses than those obtained in mice not treated with MDP in the initial immunization. A similar adjuvant effect by MDP on anti-hapten PFC responses was seen if mice were primed with KLH together with MDP and challenged with Tnp-KLH 2 weeks later. This apparent effect on carrier priming for helper function was confirmed and quantitated by double adoptive transfer experiments with graded numbers of spleen cells from KLH +/- MDP-primed mice and a fixed number of hapten-primed spleen cells from syngeneic Tnp-OVA immunized animals. These data suggest that at least one mode of action of the synthetic adjuvant MDP is via the enhanced stimulation of the helper T cell function.     

In vitro studies of the rabbit immune system: III. T-cell requirement for initiation but not maintenance of anti-sheep erythrocyte plaque-forming cells

The in vitro anti-sheep erythrocyte plaque-forming-cell (anti-SRBC PFC) response of primed rabbit spleen cells can be abrogated by prior treatment with anti-thymocyte serum plus complement (ATS + C). The direct addition of ATS without C to stimulated cultures also reduces the response 60–90%, if the antiserum is added before the initiation of antigen-stimulated PFC production. However, the PFC response becomes refractory to ATS inhibition by 48 hr. This loss of ATS sensitivity is not caused by T cells themselves becoming refractory to ATS for the following reasons: (i) ATS + C treatment or ATS addition at 48 hr causes cytotoxic effects comparable to similar treatments at 0 hr; (ii) The responsiveness of cells incubated 48 hr before the addition of SRBC remains sensitive to ATS inhibition at that time. The loss of ATS sensitivity is also not the result of soluble T-cell factors which have accumulated in the medium, because the number of PFC in stimulated cultures continues to increase after treating with ATS + C and reculturing in fresh medium at 48 hr. We conclude that T cells are required to initiate the production of antibody by B cells but are not required to maintain the later proliferative events.     

Enhancement of the in vivo antibody response by an 8-derivatized guanine nucleoside

The capacity of the C8-substituted guanine ribonucleosides to enhance the in vivo humoral immune response to the protein antigen, human gamma globulin (HGG), in A/J mice was evaluated. It has been shown recently that the C8-substituted guanine ribonucleosides are a new class of potent adjuvant for humoral immune responses to the sheep erythrocyte antigen. The current studies extend these findings to HGG with 8-bromoguanosine (8BrGuo), a representative of this group of nucleosides. The adjuvant activity of 8BrGuo in this system is highly dose and time dependent. Although 8BrGuo enhanced responses when injected either early (Day 0) or late (Day 4 or 5) after immunization, its administration on Day 1 or 2 most often led to no enhancement, suggesting that 8BrGuo may act on two events separated by a resistant stage in an ongoing immune response. The plaque-forming cell (PFC) response to HGG was enhanced optimally at doses as low as 1 mg 8BrGuo/mouse administered either on the day of immunization or 4 days thereafter. In contrast, however, serum anti-HGG antibody concentration assayed by enzyme-linked immunosorbent assay (ELISA) was enhanced only at doses of 10 mg or more, injected on the day of immunization, but doses as low as 1 mg were effective on Day 4. 8BrGuo was also an effective adjuvant when injected after antigen administration in incomplete Freund's adjuvant or when administered by several different routes (intraperitoneal, subcutaneous, oral).     

Role of T Cells in the Mitogen-Induced Proliferation and Polyclonal Antibody Response of Murine B Cells

The effect of thymus-derived lymphocytes (T cells) on the responsiveness of bone marrow-derived lymphocytes (B cells) to bacterial lipopolysaccharide (LPS) was determined in in vitro experiments. Radiation resistant splenic T cells obtained from euthymic nu/+ mice increased the number of proliferating cells in the cultures of splenic B cells from athymic nu/nu mice even in a nonstimulated state. The radiation resistant T cells augmented significantly the responsiveness of B cells to LPS, as determined by an increase in proliferating cells and polyclonally induced anti-sheep erythrocyte (SRBC) IgM hemolysin plaque-forming cells (PFC). Addition of the T cells to B cell cultures not only augmented the responsiveness of B cells to suboptimal doses of LPS but also enabled B cells to respond to supraoptimal doses of LPS. As is well documented, the radiation resistant T cells were unable to induce the generation of anti-SRBC PFC in B cell cultures, unless the cultures were simultaneously stimulated with SRBC. Colcemid, a specific inhibitor of cell mitosis, blocked almost completely the exponential generation of anti-SRBC PFC in B cell cultures responding to SRBC with the aid of radiation resistant T cells. In contrast, colcemid did not affect the exponential generation of anti-SRBC PFC of a polyclonal nature in B cell cultures responding to LPS, either in the presence or absence of radiation resistant T cells.     

Antigen-antibody complex binding and cell interaction in stimulating normal rabbit lymphocytes

Complexes of antigen with specific antibody have been shown to enhance or suppress the specific antibody response in vivo. In vitro, antigen-antibody (Ag-Ab) complexes prepared in a slight antigen excess with rabbit antibody induced proliferation of unprimed rabbit lymphocytes. The Ag-Ab stimulated cells from a number of different normal lymphoid organs, including peripheral blood, bone marrow, spleen, and lymph node, but not thymus. Cells exposed to Ag-Ab for 1 hr and washed, bound Ag-Ab through Fc and complement receptors (CR), but were not induced to proliferate unless additional Ag-Ab was added. Specific antigen, which was otherwise unstimulatory, interacted with Ag-Ab-coated cells to activate them, probably through the cross-linking of membrane-bound ligands. Proliferation stimulated by Ag-Ab involved the interaction of three bone marrow cell subpopulations; a macrophage-enriched, a B-cell-enriched, and an mIgM- cell-enriched population. The separated subpopulations were poorly responsive to Ag-Ab stimulation, even though Ag-Ab bound to cells in each of the populations. Low levels of responsiveness to Ag-Ab also resulted when any two of the three subpopulations were combined. Only when all three subpopulations were mixed, was stimulation equivalent to the levels of stimulation reached by unseparated cells.