Age dependency of spontaneous anti-autologous erythrocyte plaque-forming cells among cultured rabbit appendix cells.

The development in rabbit appendix cell cultures of plaque-forming cells (PFC) against autologous erythrocytes (RRBC), which we have reported recently (1), is now shown to depend strongly upon the age of the rabbit. Appendix cells from rabbits younger than 6 weeks produce no anti-autologous RRBC PFC. Cells from rabbits 8–16 weeks of age are most active. But, only a few anti-autologous RRBC plaque-forming cells developed in appendix cell cultures from the 6-month- to 5-year-old rabbits we have tested.     

Appendix and γM-antibody formation: VII. Rosette-forming cells in rabbits immunized with sheep erythrocytes

After intravenous immunization with sheep red blood cells the rabbit spleen shows a sharp rise in the number of plaque-forming cells but there is no detectable rise in PFC in the appendix or mesenteric lymph node of the same animals. Repeated immunization via an appendicostomy blind loop results in virtually no local PFC and only a small rise in splenic PFC.In lethally irradiated animals neither thymocytes nor appendix cells alone restore the splenic PFC response. Simultaneous injection of the two cell types restores both direct and indirect plaque formation. The injected cells were labeled with tritiated adenosine and a standard rosette assay for specific antigen-binding cells applied to recipients' spleen cells following immunization. Rosettes appeared by 3 days after immunization whether thymocytes or appendix cells were labeled. Labeled rosettes were observed only in animals receiving labeled appendix cells.This result demonstrates the presence of rosette forming cell precursors in rabbit appendix cell populations and suggests that the cells of gut-associated lymphoid tissues include antibody-forming cell precursors which are normally seeded to the spleen and draining lymph nodes.     

Primary in vitro antibody response of rabbit lymphoid cells and T-B cell collaboration in the absence of detectable mitogens

To investigate whether the antibody response and T-B-cell collaboration in vitro can be obtained in the absence of mitogens, a method of obtaining an in vitro primary anti-sheep red blood cell antibody response by rabbit spleen and lymph node cells was developed. We used Marbrook culture vessels and a specially prepared medium containing 10% autologous serum and maintained at pH 7.4–7.6. The system was shown to be devoid of any polyclonal mitogens as assessed by [³H]thymidine incorporation and by direct examination for blast cells in stained smears. The primary response increased continuously over the 5-day cultivation period and only IgM but not IgG plaque-forming cells (PFC) were detected. In over 20 experiments, the response ranged from 357 ± 17 to 4425 ± 110 PFC/10⁷ cultured cells with a median stimulation index of 52. The spleen cells required less antigen than the lymph node cells and 2-mercaptoethanol inhibited the response of the spleen cells but not that of the lymph node cells. Lymphocytes were separated into highly pure T- and B-cell populations by negative selection using antibody-coated human erythrocytes to rosette either T or B cells and Ficoll-Hypaque centrifugation to remove rosetted cells. Upon cultivation, B cells alone gave a low IgM response, whereas B cells reconstituted with T cells gave a response similar to that obtained with unseparated lymphoid cells. We concluded that: (a) optimal conditions for obtaining primary in vitro antibody responses using rabbit spleen and lymph node cells were established, (b) T-B-cell collaboration was demonstrated in the rabbit primary antibody response to sheep erythrocytes, and (c) the primary antibody response in vitro and T-B-cell collaboration may occur in the absence of detectable polyclonal mitogens.     

Fluorescence-activated cell sorter (FACS) analysis of rabbit cells

Monoclonal antibodies RB1, RB2, RT1, RT2 and RB3 were prepared against rabbit lymphoid cells by immunization with various fractions of rabbit lymphoid cells. The antigens detected by the antibodies are found on B and T cells in different densities. High proportions of polymorphonuclear and bone marrow cells which do not carry the RABELA and RTLA antigens carry the antigens of the RB and RT series. A subpopulation of appendix sIg-negative, RTLA-negative cells has a relatively high concentration of RT2. In general, B and T cells of the appendix show relatively small differences in the membrane densities of RB and RT antigens.     

Phylogeny of lymphocyte heterogeneity. II. Differential effects of temperature on fish T-like and B-like cells.

Lymphocytes from the bluegill, a freshwater fish, were observed to undergo in vitro mitogenic responses to a variety of “classical” mitogens. Using cell fractionation approaches based upon surface markers and in vitro mitogenesis, bluegill lymphocytes could be divided into two populations. One population responded to PHA and Con A but not to LPS, contained surface antigens in common with bluegill brain, and did not form spontaneous rosettes with rabbit erythrocytes. The other population responded to LPS but not to PHA or Con A, did not appear to contain surface antigens in common with brain, and did form rosettes with rabbit erythrocytes. The former population responded to mitogenic stimulation very well at 32 °C, whereas the latter population responded better at 22 than at 32 °C. The pattern of mitogenic responses and brain antigen distribution coupled with the observation that mixed lymphocyte responses were obtained at 32 but not at 22 °C makes it likely that the 32 °C responsive population represents the fish equivalent of T cells. The other population may be B cells. These data suggest that the immunosuppressive effects of low temperatures on cold-blooded animals may be effects on the generation of functional T cells and not on B cells.     

In vitro development of a primary antibody response with lymph node cells.

Utilizing a variety of lymphoid tissues from three common laboratory species, comparative studies were performed to investigate the competence of the dissociated cells to respond to a heterologous erythrocyte with the development of specific plaque-forming cells. Dissociated spleen cells harvested from BDF1 mice consistently developed specific plaque-forming cells (PFC) to sheep red blood cells (SRBC), while hamster spleen cells inconsistently developed specific antibody-forming cells to SRBC. Under identical conditions, guinea pig spleen cells did not develop significant numbers of PFC to SRBC. However, lymph node cell cultures of all three species tested yielded specific PFC. In the mouse and hamster lymph node cell cultures, the yield of PFC per culture or per 10⁶ recovered viable cells was always greater than the yield from companion spleen cell cultures. Guinea pig mesenteric lymph node cell cultures developed the major PFC response to SRBC, while both mesenteric and peripheral lymph node cell cultures from hamsters were equivalent in their response to SRBC. The data demonstrate that it is possible to develop a primary antibody response to SRBC in vitro utilizing normal endogenous hamster or guinea pig lymphoid cells, if lymph nodes are the source of cells.     

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.     

Studies on mouse autoreactive cells: I. Role of H-2 antigens in mouse autologous rosette formation

A minority (1–2%) of normal mouse lymphoid cells bind autologous erythrocytes and form rosettes. In this study we examined the antigenic specificity involved in the formation of such rosettes. A significant difference in the incidence of rosettes formed, respectively, with autologous and allogeneic mouse erythrocytes is found. Moreover, preincubation of lymphoid cells with low concentrations of syngeneic erythrocytic ghosts causes significant competitive inhibition of subsequent rosette formation. Allogeneic ghosts obtained from nonrelated or from congenic resistant strains of mice do not display this inhibitory effect under the same conditions. It is thus suggested that mouse autologous rosette-forming cells bear receptors for syngeneic H-2 antigens that are involved in the binding of autologous erythrocytes. More precisely, compatibility between lymphocyte and erythrocyte restricted to K or D only is sufficient to ensure a level of rosettes similar to that obtained when complete identity occurs for K, I, and D regions.     

The in vitro production of anti-DNA antibody by cultured peripheral blood or tonsillar lymphoid cells from normal donors and SLE patients

Anti-DNA antibody responses by cultured circulating lymphocytes from SLE patients and by the tonsillar lymphoid cells of normal donors were detected and enumerated by a sensitive specific ELISA of culture supernatants, or by a hemolytic anti-DNA PFC assay. Although spontaneous IgM and IgG anti-DNA and anti-ssDNA responses were characteristic of SLE lymphocytes and spontaneous IgM anti-ssDNA responses were characteristic of tonsillar lymphocytes, the circulating lymphocytes of normal controls never produced anti-DNA antibodies spontaneously, and rarely after PWM stimulation. The anti-DNA antibody PFC response of tonsil lymphocytes correlated directly with the total number of immunoglobulin-producing cells measured by a reverse hemolytic PFC assay. Mixing experiments in which we employed cultures of comparable numbers of separately enriched autologous circulating and tonsillar B and T cells revealed that tonsillar tissue contained an enriched population of anti-DNA antibody precursor B cells and/or helper T cells.     

Germinal centers and the B-cell system

Summary The migration pattern of germinal center cells of the rabbit appendix was studied and compared with that of appendix dome cells, spleen cells, thymus cells and thoracic duct lymphocytes. To discriminate T-and B-cell migration pathways, normal or T-cell-depleted rabbits were used as donors. Cell suspensions were labeled in vitro with ³H-leucine followed by intravenous transfer. The migration of labeled cells in lymphoid organs was studied using autoradiography, particular attention being paid to the spleen of the recipient. B-cells from the appendix dome, spleen and thoracic-duct lymph migrate to primary follicles or the corona of secondary follicles via thymus-dependent areas of peripheral lymphoid organs. In contrast, a B-cell subpopulation from the germinal centers of the appendix migrates to the center of splenic primary follicles and into germinal centers. The migration of germinal center cells to splenic follicle centers is not enhanced by specific antigens. The migration properties of B-cells, possibly changing during differentiation, may be instrumental in the two types of immune reactions, i.e., plasma-cell reaction and germinal-center reaction.     

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.     

Spontaneous helper factor production by nonadherent rabbit lymphoid cells and its feedback regulation by adherent cells

Normal, unstimulated rabbit lymphoid cells, when depleted of adherent cells, produced soluble helper factor activity that augmented antibody formation by rabbit spleen cells primed against sheep red blood cells (SRBC). Adherent cells inhibited the production of the helper factor by nonadherent cells via a soluble product. Thus unseparated (adherent cell-containing) appendix, lymph node, and spleen cell cultures did not produce the helper factor. On the other hand, the activity of the helper factor required the presence of adherent cells in the assay cultures. Peritoneal exudate cells, predominantly esterase positive, also inhibited the production of the helper factor if they were first exposed to the helper factor-containing culture supernatant. These results imply that a helper factor may participate in the feedback regulation of its own production via an adherent cell population.     

Rosette formation of guinea pig lymphoid cells with rabbit erythrocytes—Differences between thymocytes and peripheral blood lymphocytes

Rosette formation of guinea pig thymocytes (Th) and thymus-derived peripheral blood lymphocytes (TBL) was tested under different experimental conditions. Up to 93% of Th and 38% of TBL showed an affinity to rabbit red blood cells (RRBC). Treatment with metabolic inhibitors like sodium azide and sodium cyanide or freezing and thawing nearly abolished rosette formation by TBL but was ineffective with respect to Th. Heating the cells destroyed rosette-forming capacity of both cell types. These results indicate that spontaneous rosette formation with RRBC by Th does not require the live cell.     

Effects of antibodies to MHC gene products on different Fc-receptor-mediated cell functions

The effects of rabbit anti-HLA-DR and anti-β₂-microglobulin (anti-β₂m) antibodies on three different Fc-receptor-mediated cell functions of peripheral blood mononuclear leukocytes were studied. Both IgG and F(ab′)₂ fragments of anti-HLA-DR antibodies inhibited the cytotoxicity against a monolayer of antibody-sensitized sheep erythrocytes (plaque formation) whereas no effect was observed on the cytotoxicity against antibody-sensitized chicken erythrocytes in suspension (⁵¹Cr release), or on EA-rosette formation. On the other hand, all the three functions were inhibited by the IgG fraction of anti-β₂m antibodies but not by the corresponding F(ab′)₂ fragments. The results demonstrate that the plaque-forming cells (PFC) carry HLA-DR-like antigens. Furthermore, a closer association exists between the HLA-DR-like antigens and the Fc receptors than between the β₂m molecules and the Fc receptors on the PFC. The results further support our earlier investigations suggesting that the PFC are of monocytic origin.     

Apparent T cell function of bone marrow cells from mice experiencing a graft-versus-host reaction.

The graft-versus-host (GVH) reaction, induced in adult F₁ mice by the injection of parental strain lymphoid cells (GVH mice), suppressed the in vitro plaque-forming cell (PFC) response to sheep erythrocytes (SRBC) of spleen cells obtained from the GVH mice (GVH-SC). In vitro restoration of the PFC response of GVH-SC was carried out employing a modified Marbrook culture chamber consisting of an inner culture compartment (IC) separated from an outer culture compartment (OC) by a cell-impermeable membrane. Thymus cells (TC) and lymph node cells (LNC) but not bone marrow cells (BMC) from normal mice placed in the IC restored the PFC response of GVH-SC cultured with SRBC in the OC. The restoring ability of TC and LNC was markedly reduced following treatment with anti-theta serum plus complement. BMC taken from GVH mice 3 or more days post-GVH induction (GVHBMC) and placed in the IC restored the PFC response of GVH-SC as well as TC and LNC. Treatment of GVH-BMC with anti-theta serum plus complement did not affect their restoring ability; furthermore, the number of theta-bearing cells in the bone marrow did not increase as a consequence of the GVH reaction. Two possible explanations are proposed for the T-like function of GVH-BMC.     

C-Reactive protein (CRP)-mediated inhibition of the induction of in vitro antibody formation. I. T-cell dependence of the inhibition.

Purified human C-reactive protein (CRP) inhibited the in vitro anti-hapten antibody plaque-forming cells (PFC) response of both carrier keyhole limpet hemocyanin (KLH)-primed and unimmunized Balb/c spleen cells to TNP-KLH. The inhibitory effect was neutralized by the CRP-substrate, C-polysaccharide. The response to the T-independent antigens, TNP-T4 and DNP-lys-Ficoll, was not inhibited by CRP. A cell population that was suppressive for the in vitro PFC response was generated by incubating normal spleen cells with CRP. These cells suppressed the PFC response of syngeneic KLH-primed cells to TNP-KLH in proportion to the number of added lymphoid cells with bound CRP. Selective depletion of B cells, T cells or macrophages before incubation with CRP revealed that T cells were required for the induction of suppressive cells. Treatment of spleen cells after incubation with CRP, with T cell-specific antisera and C abolished suppressor-cell activity. Mitomycin-C treatment of the CRP-binding cells did not alter their suppressive activity. These results indicated that CRP mediates suppression of antibody induction to T-dependent antigens by interacting with T cells and generating a suppressive T-cell population.     

Development of responsiveness and incidence of bispecific cells as revealed by in vitro assessment of the maturation of mouse bone marrow cells.

The kinetics of the maturation of B cells were studied in adult thymectomized, lethally irradiated, and bone marrow-reconstituted mice. The spleen cells of the recipients were taken at various intervals after transfer and cultured in vitro with trinitrophenylated sheep erythrocytes (TNP-SRBC). The cultures were supplemented with either allogeneic culture supernatant or educated T-cell helper activity. Appearance of functional B cells in the bone marrow inoculum was assessed by the number of hemolytic plaque-forming cells (PFC) on the fourth day of culture. In a parallel series the frequency of surface Ig-bearing cells was determined by using fluorescent rabbit anti-mouse Ig serum. When helped by allogeneic culture supernatants, differentiating bone marrow cells showed a slower rate of maturation into functional B cells than when helped by specifically educated T cells. But in both cases the recovery of responsiveness reached the same level (number of PFC/10⁶ cells) as that of normal spleen cells 55 to 60 days after transfer. During the initial periods of recovery, bispecific PFC (reacting both to TNP and to native SRBC determinants) were detected regularly in numbers far exceeding their frequency in normal spleen cell cultures; in some experiments, the number of bispecific PFC amounted to as much as 30% of the total PFC, whereas, when the bone marrow cells completely recovered (sixtieth day), the frequency of bispecific PFC was similar to that found in normal spleen cell cultures. The number of surface Ig-bearing cells also reached a normal level after the fiftieth day following transfer. In general, the degree of functional maturation was better correlated with the cells bearing surface Ig in the shape of rings or caps, whereas the predominance of spot-bearing cells indicated immaturity of the population.     

Polyclonal B lymphocyte activation during Trypanosoma cruzi infection

Infection of A/J mice with Trypanosoma cruzi results in the polyclonal activation of B lymphocytes in vivo as assessed by the spontaneous plaque-forming cell (PFC) response to trinitrophenyl and to goat, equine, and sheep erythrocytes. The peak response to these antigens is found at 5 to 6 days of infection. Additionally, a polyclonal response to syngeneic erythrocytes can be detected in infected mice by using aged but not fresh indicator cells. Polyclonally stimulated PFC to human gamma-PFC found late in infection during a period of marked splenomegaly and parasitemia. This trypanosoma-induced polyclonal B cell activation may well be responsible for the abnormalities in immunoglobulin synthesis and secretion that have been reported to occur during human infection with T. cruzi.     

Ontogenical studies on kinetics of lipopolysaccharide-induced response to bromelain-treated mouse erythrocytes in mouse spleen cells. II. Response of spleen cells with or without Fc receptors, C3 receptors, or Ia antigens

Lipopolysaccharide (LPS)-induced plaque-forming cells secreting IgM (IgM-PFC) and antibodies against bromelain-treated mouse erythrocytes (anti-BrMRBC PFC) on Days 1 and 2 of cultures were quantitatively estimated in spleen cells from mice of various ages. The concentrations of the four groups of PFC changed independently with age. The LPS dose dependency of the PFC response was markedly different between PFC on Days 1 and 2, but not different between anti-BrMRBC PFC and IgM-PFC or between 2- and 10-week-old mice. In a second experiment, spleen cells from 2- and 10-week-old mice were separated into subpopulations with or without Fc receptors, C3 receptors, or Ia antigens, and the LPS-induced PFC responses were quantitatively assessed in each subpopulation. Both the receptor-bearing and -lacking populations included LPS-reactive B cells, and the percentages of the LPS-reactive B cells recovered in the receptor-bearing population increased with age. However, the percentages of anti-BrMRBC PFC recovered in each receptor-bearing or -lacking population were different from those of IgM-PFC. In Ia- populations, the percentages of IgM-PFC on Day 2 were obviously higher than those on Day 1, and both of the percentages increased with age. These results suggest that the four groups of LPS-reactive B cells can be discriminated from each other by their LPS dose dependency and their cell surface markers, and that they develop differently during ontogeny.     

Studies of the immunological capacity of germ-free mouse radiation chimeras: I. Chimerism and humoral immune response

The genetic origin of both the functional lymphoid cell and progenitor cell populations of germ-free mouse radiation chimeras was assessed by indirect immunofluorescence (IIF), anti-H-2 cytotoxicity, and survival of lethally x-irradiated secondary recipients of chimera cell populations. These studies demonstrated that germ free C3H/He mice given 1000 R and 10⁷ DBA/2 bone marrow cells express H-2 antigens on their lymphoid and bone marrow cell populations characteristic of the DBA/2 donor. These cells persist for at least 14 months postirradiation and bone marrow transplantation. However, these allogeneic mouse radiation chimeras have a reduced humoral immune response to sheep erythrocytes (SRBC). This decreased humoral immune capacity as assessed by kinetic studies of the spleen plaque-forming cell (PFC) response is present throughout the life span of the chimera. The γ₁ PFC response shows extreme depression. The reduced humoral immune responsiveness to the thymusdependent SRBC antigen is considered to be due to the absence or malfunctioning of a thymocyte population.