Antigen-binding B cells and polyreactive antibodies

The present experiments were initiated to see if cells capable of binding antigens could make polyreactive antibodies. Fluorescein isothiocyanate-labeled self and non-self antigens were incubated with B cells from normal individuals. Antigenbinding cells were separated from non-antigen-binding cells by flow cytometry, immortalized with Epstein-Barr virus and analyzed at the clonal level for their capacity to make polyreactive antibodies. Four to six times more cells making polyreactive antibodies were found in the B cell subset that bound antigens than in the B cell subset that did not bind antigens. The majority of the polyreactive antibodies were of the immunoglobulin (Ig)M isotype. Immunoflow cytometry revealed that cell lines making polyreactive antibodies bound a variety of antigens (e.g., insulin, IgGFc and β-galactosidase), whereas cell lines making monoreactive antibodies bound only a single antigen. The binding of antigens to B cell lines that made polyreactive antibodies could be inhibited (range, 28%–57%) by both homogeneous and heterogeneous antigens. Both CD5⁺ and CD5^? antigen-binding B cells made polyreactive antibodies, but the frequency was slightly higher in the CD5⁺ antigen-binding (85%) as compared to the CD5^? antigen-binding (50%) population. Comparison of CD5⁺ B cells that bound antigens with CD5⁺ B cells that did not bind antigens showed that approximately 86% of the former, but only 15% of the latter, made polyreactive antibodies. It is concluded that cells capable of binding a variety of different antigens can make polyreactive antibodies and that antigen binding is a good marker for identifying polyreactive antibody-producing cells.     

The development of antigen-binding lymphocytes in foetal tissues

The time of appearance and counts of antigen-binding cells, using radioiodine-labelled flagellin and haemocyanin, was studied in the human and mouse foetus at different gestational ages: the capacity for binding radioiodine-labelled antigens was equated with acquisition of immunological funciton. Cells resembling lymphocytes from human and mouse liver and bone marrow showed antigen binding at early gestational ages, but this binding could not be prevented with species-specific antisera to immunoglobulins. Specific antigen binding to lymphocytes was detected first with thymic lymphocytes, at gestational ages of 12 weeks in humans and 14 days in mice, then with splenic lymphocytes, at 16 weeks in humans and 17 days in mice, and still later with gut lymphocytes. Relative counts of antigen-binding cells in human foetal thymus were maximal at 16–22 weeks and decreased thereafter. Lymphocyte immunocompetence, as judged by the capacity specifically to bind antigen, develops rapidly after the appearance in thymus of cells with the morphology of lymphocytes; this seemed to occur at the equivalent foetal stage in the species studied.     

Detection of T and B cell-specific heteroantigens on electrophoretically separated lymphocytes of the mouse

Mouse spleen lymphocytes were electrophoretically separated into pools of T and B lymphocytes. Heteroantisera were raised in rabbits against these cell pools, the IgG fractions were isolated and their lymphocytotoxicity tested. After appropriate absorption, the antisera were specifically directed against lymphocyte antigens. Further cross-absorption with T and B lymphocytes made the antisera specific for antigens which were mutually exclusively present on T and B cells and were related to neither alloantigens nor immuno-globulins. These anti-B and anti-T cell antisera killed antibody-forming cells and graft-versus-host reactive cells, respectively, in vitro. Furthermore, anti-B cell antiserum was cytotoxic to lymphocytes of low electrophoretic mobility in bone marrow and peripheral organs, except in the thymus, whereas anti-T cell antiserum was cytotoxic to lymphocytes of high electrophoretic mobility in all organs and, in addition, killed all thymocytes of low electrophoretic mobility, which had not been affected by anti-B cell antiserum. This distribution of lymphocytes in the electrophoretic distribution profiles, together with the described properties, gives evidence that heteroantigens were found to be exclusively present on T and B cells. They were designated “mouse B cell-specific” and “mouse T cell-specific” antigens and are part of mouse lymphocyte-specific antigen.     

Isolation of antigen-binding cells from unprimed mice. II. Evidence for monospecificity of antigen-binding cells.

Spleen cells from unimmunized mice were exposed to two contrastingly fluorescent antigens simultaneously. Antigen-binding cells of either specificity were isolated using a fluorescence-activated cell sorter (FACS). Purified cells binding one or the other of the antigens were then examined by fluorescence microscopy for the presence of bound antigen of the alternate specificity. No double binding cells were seen. If cells bear receptors of two or more specificities and these receptors are randomly distributed among antigen-binding cells, then of the 13 000 binding cells examined 82 were expected to bind both antigens. These results strongly suggest that antigen-binding cells bear receptors of only one specificity. In addition, by inference from the functional correlation between antigen-binding cells and precursor cells, the data support the contention that precursors of antibody-forming cells are monospecific.     

B cell clonal elimination induced by membrane-bound self-antigen may require repeated antigen encounter or cell competition

Transgenic mouse experiments indicate that autoreactive B cells are eliminated upon encounter with membrane self-antigen. In this study we tested how B cell tolerance to MHC class I antigens is affected by altering the frequency of antigen-carrying cells in mixed bone marrow (BM) chimeras. When antigen-bearing cells are present at low frequency, the reactive B cells and their antigens may coexist in the peripheral lymphoid organs, but under these conditions the B cells are functionally anergic and have a shortened lifespan. Such putative anergic cells are strongly deleted in the presence of additional, non-antigen-bearing, non-transgenic B cells. Since the antigen concentration on the surface of each antigen-bearing cell should be high, these results suggest that for efficient deletion of autoreactive B cells multiple antigen encounters may be required, particularly when cellular competition is weak. These results have implications for the therapeutic use of BM chimerism to induce B cell tolerance to grafts.     

Information carried by the DNA released by antigen-stimulated lymphocytes.

Both antigen-stimulated and non-stimulated human blood lymphocytes release in vitro a DNA-containing complex which is not the product of dying or disintegrating cells. Lymphocytes obtained from different PPD or HBs positive or negative donors were incubated with one of these antigens and the DNA released in the culture medium was tested for its information content using, successively, two cell-free systems. The ability of the resulting protein product to bind specifically to the stimulating antigen was examined by immunoadsorption chromatography. Results show that DNA excreted by stimulated lymphocytes was transcribed into an RNA which coded for an antigen-binding protein, whereas DNA released by unstimulated lymphocytes did not. The protein produced in this system, using as template the DNA released after cell stimulation, bound specifically to PPD or HBs Sepharose 4B coated columns, depending on the stimulating antigen and on the cell response to this antigen. After elution from the column the protein sedimented at 19S in a linear sucrose gradient.     

Selective binding of chemically defined antigenic peptides to mouse lymphocytes

Immunologically active peptides containing either two different (N? 10? C) or two identical (N? 8? N) antigenic amino acid sequences, bridged by 10 and 8 glycine residues, respectively, were used to study antigen-binding lymphocyte populations in BALB/c mice. Spleen cells from mice immunized with either N? 10? C or N? 8? N were treated with either normal rabbit serum or brain associated anti-thymus (BA Θ) serum and subsequently examined by autoradiography for the numbers of cells binding ¹²⁵I-labeled N? 10? C, and ¹²⁵I-labeled conjugates of either the C antigenic determinant or the N antigenic determinant with poly? D? glutamic acid. In unimmunized mice there were more cells binding the N determinant than the C determinant. After anti-BA Θ treatment, the relative numbers of each population increased, implying that many of these cells were bone marrow-derived. In N? 10? C or N? 8? N immunized mice, the numbers of cells binding the N determinant remained virtually unchanged after anti-BA Θ treatment, indicating an equal distribution of these cells in both bone marrow or thymus-derived populations. However, the C determinant binding population in N? 10? C immunized animals appeared to be mainly of bone marrow origin. Since the C determinant exhibited mainly B cell reactivity, the role of thymus cells in immune responsiveness to a peptide (C? mal? 10? C) containing two C determinants was examined. Only those mice which were reconstituted with both bone marrow and thymus cells after thymectomy and irradiation were capable of responding normally to this antigen, demonstrating thymus involvement even with antigens that appear to be mainly reactive with B cells.     

Surface markers of small lymphocytes appearing in the mouse Ehrlich ascites tumour, host spleen and blood

Small lymphocytes sampled from intraperitoneally growing Ehrlich ascites tumour in CBA/H-T₆ mice as well as host spleen and blood at different days of tumour development were characterized radioautographically on the basis of two surface markers, IgM for B cells and θ antigen for T cells. A direct binding of ¹²⁵I-labelled anti-IgM detected natural surface IgM, while an indirect binding following a prior exposure to anti-θ antibody detected θ antigen. Cells remaining unlabelled with the latter procedure were considered to lack both markers (double negative). While the incidence of IgM+ve small lymphocytes within the tumour declined, their absolute numbers increased with tumour growth. Low levels of antiglobulin binding shown by these cells were considered to reflect low levels of maturation, because (1) our previous studies indicated that they were newly formed, and (2) the extent of antiglobulin binding by B lymphocytes in the marrow is known to increase with increasing post-mitotic age. The proportions and the absolute numbers of θ+ve as well as the double negative small lymphocytes increased within the growing tumours. Within the host spleen, the incidence of IgM+ve small lymphocytes remained unchanged but their absolute numbers increased because of splenomegaly. The degree of antiglobulin binding by these cells was comparable to that of the normal splenic population. The incidence of θ+ve cells dropped but their absolute numbers remained unchanged in the spleen during tumour growth. In contrast, the incidence as well as the absolute numbers of double negative cells increased markedly. This cell category increased also in the blood, possibly in transit to the tumour site and other lymphoid organs from the bone marrow, where they were most prevalent. Their bone marrow origin was further suggested by a preponderance of marrow derived small lymphocytes at the tumour site as well as in the host spleens found in our earlier studies. Double negative population in the spleen showed a paucity of C′3 and Fc receptors on the cell surface and included cells capable of producing B lymphoid colonies in vitro.     

Immunophenotypic and genotypic characterisation of multiple myelomas with adverse prognosis characterised by immunohistological expression of the T cell related antigen CD45RO (UCHL-1).

AIMS: To investigate whether plasma cell expression of early B cell, late B cell/preplasma cell, T cell, and myelomonocytic antigens or myeloma associated lymphocytic infiltrates correlated with prognosis in bone marrow biopsy specimens of patients with multiple myeloma. METHODS: Bone marrow biopsy specimens of 23 patients with multiple myeloma were investigated for plasma cell expression and interstitial lymphocyte expression of T cell related antigen CD45RO (UCHL-1). RESULTS: Eight patients showed plasma cell expression of CD45RO and 16 showed increased tumour infiltrating CD45RO positive lymphocytes, which were correlated with poor survival by multivariate analyses (p = 0.005 and p = 0.04, respectively). B cell antigens (MB2, CD20) but no T cell specific antigens (CD3) or T cell receptor gene rearrangements were expressed by plasma cells in CD45RO positive myelomas. Of 16 patients with myeloma who had increased tumour infiltrating CD45RO positive lymphocytes, four had interstitial lymphocyte expression of B cell antigens and two had interstitial lymphocyte expression of the T cell specific antigen CD3. CONCLUSIONS: The recognition of plasma cell expression of CD45RO and increased interstitial CD45RO lymphocytes in bone marrow biopsy specimens of patients with multiple myeloma is an adverse prognostic finding not indicative of an aberrant T cell phenotype or genotype; it is consistent with B cell/pre-plasma cell antigen expression by myeloma cells and their lymphocytic precursors.     

Lectin-binding patterns of small lymphocytes in bone marrow,thymus and spleen: demonstration of lymphocyte subsets by quantitative radioautography

Cells from mouse bone marrow, thymus and spleen were exposed to ¹²⁵I-labeled concanavalin A (Con A), Lens culinaris lectin (LCL), soybean agglutinin (SBA), Helix pomatia agglutinin (HPA), phytohemagglutinin-P (PHA-P), peanut agglutinin (PNA), or wheat germ agglutinin (WGA) in a range of concentrations and examined radioautographically. Small lymphocytes in the three organs differed in the minimal concentration of each lectin which gave detectable surface labeling, while at optimal lectin concentrations, their labeling intensity profiles differed markedly. Inhibition by sugars demonstrated the labeling specificity. Major populations of bone marrow small lymphocytes bound WGA strongly, while Con A, SBA, HPA, PHA-P and LCL were bound only weakly, and PNA binding was lacking. Most thymus cells bound Con A, SBA, HPA, PHA-P and PNA strongly, WGA and LCL weakly. Subsets of bone marrow and thymus small lymphocytes differed from the major populations in their lectin-binding intensities. Spleen small lymphocytes were heterogeneous in the binding of each lectin. However, a major population bound LCL exceptionally strongly, while few cells bound PNA. Using a panel of lectins under standardized conditions, these studies show distinctive lectin-binding patterns for small lymphocytes in the bone marrow, thymus and spleen, respectively. Major and minor cell populations are distinguishable in each organ, providing an approach to discriminating lymphocyte lineages, subtypes and differentiation stages.     

Antigen-binding small lymphocytes in the guinea-pig: I. The immunological response to human thyroglobulin

The time course of the relative distribution of small lymphocytes binding ¹²⁵I-labelled human thyroglobulin (HTg) in cell suspensions from the peripheral blood and various lymphoid organs was studied in guinea-pigs at progressive intervals up to 28 days after immunization with an emulsion of HTg and BCG in Freund's incomplete adjuvant (FIA). Small lymphocytes binding ¹²⁵I-labelled HTg were first detected in peripheral blood, popliteal (draining) lymph node, spleen and bone marrow preparations on the 10th day, and in mesenteric (distant) lymph node and thymus preparations on the 14th day after primary immunization. In general, the percentage of these cells increased progressively thereafter until the end of the period of study. Blocking experiments with unlabelled antigens indicated that the binding of ¹²⁵I-labelled HTg by small lymphocytes was specific. An anti-HTg antibody cytophilic for guinea-pig small lymphocytes was demonstrated by the passive transfer of antigen-binding capacity to lymphocytes of unimmunized animals with hyperimmune guinea-pig serum. It is proposed that, in these experiments, anti-HTg cytophilic antibody was bound first to small lymphocytes in the tissues participating actively in the immune response (popliteal node, spleen and bone marrow) before spilling over into the general circulation to coat lymphocytes at other sites (mesenteric node and thymus).     

T cells with chimeric antigen receptors have potent antitumor effects and can establish memory in patients with advanced leukemia

Tumor immunotherapy with T lymphocytes, which can recognize and destroy malignant cells, has been limited by the ability to isolate and expand T cells restricted to tumor-associated antigens. Chimeric antigen receptors (CARs) composed of antibody binding domains connected to domains that activate T cells could overcome tolerance by allowing T cells to respond to cell surface antigens; however, to date, lymphocytes engineered to express CARs have demonstrated minimal in vivo expansion and antitumor effects in clinical trials. We report that CAR T cells that target CD19 and contain a costimulatory domain from CD137 and the T cell receptor ζ chain have potent non-cross-resistant clinical activity after infusion in three of three patients treated with advanced chronic lymphocytic leukemia (CLL). The engineered T cells expanded >1000-fold in vivo, trafficked to bone marrow, and continued to express functional CARs at high levels for at least 6 months. Evidence for on-target toxicity included B cell aplasia as well as decreased numbers of plasma cells and hypogammaglobulinemia. On average, each infused CAR-expressing T cell was calculated to eradicate at least 1000 CLL cells. Furthermore, a CD19-specific immune response was demonstrated in the blood and bone marrow, accompanied by complete remission, in two of three patients. Moreover, a portion of these cells persisted as memory CAR(+) T cells and retained anti-CD19 effector functionality, indicating the potential of this major histocompatibility complex-independent approach for the effective treatment of B cell malignancies.     

Immunocytochemical characterization of monocyte colonies of human bone marrow: a clue to the origin of Langerhans cells and interdigitating reticulum cells

Human bone marrow was cultured and cells from individual monocyte colonies stained with a variety of polyclonal and monoclonal antibodies. Erythroid colonies and peripheral blood monocytes were used as controls. Both bone marrow cultured and peripheral blood monocytes stained with antibodies to lysozyme, non-specific cross reacting antigen (NCA) and alpha-1-antitrypsin and the cells stained variably for HLA-DR. A small number of cells in each cultured bone marrow colony stained with antibody to human thymic antigen (HTA T6) and most of the cells stained with variable intensity using anti-S-100 protein. No cells reactive with these two antibodies were detected in peripheral blood monocyte preparations and erythroid colony cells were negative for all antigens studied. The presence of cells within individual bone marrow colonies with phenotypic properties of both phagocytic macrophages (lysozyme positive, NCA positive, alpha-1-antitrypsin positive) and Langerhans cells/interdigitating reticulum cells (HTA(T6) positive, S-100 protein positive) suggests that these cells share a common stem cell and are both components of the mononuclear phagocyte system.     

Monoclonal antibodies define the characteristics and cellular distribution of an Ia-associated protein

In a recent report we described the identification of physical associations between Major Histocompatibility Complex (MHC) Class II (Ia) antigens and other structures of Mr 67,000, which were significantly enhanced following brief T-B cell co-culture (1). To further investigate this 67K Ia-associated product, monoclonal antibodies (MAb) were produced against isolated 67K material and their reactivity examined. Cell surface binding by these MAb was detected only after perturbation of the membrane by cellular adherence or following aldehyde fixation, which indicates that the determinant recognized by these mAb is retained in the plasma membrane in a covert fashion. All lymphoid cells tested showed reactivity with the MAb as determined by immunofluorescence and by ELISA, but no binding was detected on bone marrow or peritoneal macrophages. Expression of the antigen reactive with these antibodies followed a similar pattern with established murine cell lines, with T and B cell lines and a pre-B cell line showing reactivity, while no antigen was detected on macrophage-like and fibroblast cell lines. The intensity of antigen expression by normal lymphoid cells was ordered: thymocytes greater than splenic T cells greater than or equal to bone marrow lymphocytes greater than splenic B cells. No correlation was observed between expression of Ia antigens by non-lymphoid cells and expression of the 67K molecule. These observations suggest that this antigen is primarily a marker of lymphoid cells, with the highest expression on cells of the T lymphocyte lineage. Finally, inhibition of antigen-specific, MHC-restricted T-cell activation by the MAb directed against the 67K structure suggests an important functional role for this interesting molecule originally identified by its physical association with Ia following T-B cell interactions.     

Polyreactive antigen-binding B cells in the peripheral circulation are IgD+ and B7−

Polyreactive antibodies are naturally occurring antibodies, primarily of the IgM isotype, that are capable of reacting with a wide variety of different self and non-self antigens. Previously, we reported that a B cell capable of making polyreactive antibody has Ig receptors on its surface that can bind different antigens. The present investigation was initiated to characterize these polyreactive antigen-binding B cells further. A panel of fluorescein isothiocyanate-labeled antigens (insulin, IgG Fc fragment or β-galactosidase) served as probes to select polyreactive antigen-binding B cells by cell sorting. Our experiment revealed that these polyreactive antigen-binding B cells were mainly of the IgD isotype. They expressed high levels of CD40 and major histocompatibility complex class II molecules, but little or no B7-1, B7-2, or Fas. In contrast to the binding of antigens to monoreactive receptors (usually high affinity), the binding of antigens to polyreactive receptors (usually moderate or low affinity) did not up-regulate the expression of B7-1 or B7-2. Antigens that bound to polyreactive receptors, however, were internalized and degraded, although not as efficiently as antigens that bound to monoreactive receptors. Despite the ability of these B7⁻ cells to process antigens, they were not able to activate T cells in a mixed leukocyte reaction. It is concluded that polyreactive antigen-binding B cells have properties that are consistent with the ability to induce immunological tolerance.     

Dendritic cells derived from bone marrow cells fail to acquire and present major histocompatibility complex antigens from other dendritic cells

Dendritic cells stimulate primary T-cell responses and a major activation route is via presentation of antigens pre-processed by other dendritic cells. This presentation of pre-processed antigens most likely proceeds through transfer of functional major histocompatibility complex (MHC) antigens through exosomes, 'live nibbling' or apoptotic vesicles. We hypothesized that not all dendritic cell populations may both donate MHC antigen to dendritic cells and present antigens acquired from other dendritic cells. All populations tested, including those derived from bone marrow precursor cells stimulated primary, allogeneic T-cell responses and acted as accessory cells for mitogen stimulation. Populations of responder type, splenic dendritic cells promoted allogeneic responses indirectly but those derived from bone marrow cells blocked rather than promoted T-cell proliferation. To identify mechanisms underlying this difference we studied transfer of I-A antigens between cells. Active, two-way transfer of allogeneic I-A occurred between splenic primary antigen presenting cells including CD8alpha+ lymphoid dendritic cells, CD8alpha- myeloid dendritic cells and B220+ cells; all these cell types donated as well as acquired MHC molecules. By contrast, the bone marrow-derived dendritic cells donated I-A antigens but acquired negligible amounts. Thus, dendritic cells derived directly from bone marrow cells may stimulate primary T-cell responses through transferring functional MHC to other dendritic cells but may not be able to acquire and present antigens from other dendritic cells. The evidence suggests that T-cell activation may be blocked by the presence of dendritic cells that have not matured through lymphoid tissues which are unable to acquire and present antigens pre-processed by other dendritic cells.     

Sequential expression of B lymphocyte surface antigens in vitro

Serological techniques were used to examine the process of sequential surface antigen expression on differentiating B cells in vitro. A 4-day culture system is described in which bone marrow lymphocytes from neonatal mice acquire in sequence the ability to express Lyb-2, IgM, Ia and IgD in response to a 3-h-induction with E. coli lipopoly-saccharide (LPS). Lyb-2 can be induced on day 1, IgM can be induced after 24 h, Ia after 48 h and IgD only after 96 h in culture. This sequence mimics the order of appearance of B cell surface antigens during ontogeny. When DNA synthesis is blocked from 0–24 h with hydroxyurea (HU), all surface antigens can be induced simultaneously by LPS. Immunoselection of one antigen-bearing population results in the loss of cells bearing other B cell antigens indicating that the surface antigens are induced on the same cells. When both HU and LPS were added to the cultures from the start, IgM appears after 11–14 h, Ia after 14–15 h and IgD only after 19 h. Induction of antigen was demonstrated by the cytotoxicity assay, quantitative absorption and the protein A sheep red blood cell resetting assay. The results obtained show that there is a population of surface IgM-negative precursor B cells in young bone marrow which, when grown in vitro, become sequentially inducible for expression of B surface antigens. Inhibition of DNA synthesis promotes acquisition of the inducible state, and the sequence of antigen expression is correlated with specific time intervals after DNA synthesis has stopped.     

The peritoneal Ly-1 (CD5) B cell repertoire is unique among murine B cell repertoires.

Ly-1 (CD5) B cells and conventional B cells represent two distinct lineages of murine B cells which are distinguishable by expression of surface molecules, organ location, ontogeny and development and antibody production in vivo. In order to assess whether the different developmental pathways of Ly-1 B cells and conventional B cells result in different antibody repertoires, we have used limiting dilution analyses to determine frequencies of B cells making antibodies capable of binding to a range of antigens including haptens, proteins, bacterial polysaccharides and bromelain-treated mouse red blood cells. Starting populations of B cells were purified from spleen, peritoneum and bone marrow of adult BALB/c mice or from spleens of newborn mice by use of the fluorescence-activated cell sorter. The peritoneal Ly-1 B cell repertoire was found to be different from that of conventional B cells, with between 5- and 100-fold higher frequencies of clones producing IgM antibodies capable of binding to the antigens tested. However, when tested, the majority of Ly-1 B cell anti-haptenic antibodies did not show the high affinity binding or fine specificity characteristics of specific antibodies elicited in immune responses in vivo. The high frequencies of antigen-reactive antibodies within the Ly-1 B repertoire are most likely explained by the presence of clones secreting low-affinity or multireactive antibodies. The Ly-1 B cell repertoire is not mirrored in repertoires from either newborn B cells or virgin B cells in adult bone marrow. Therefore, either Ly-1 B cells develop from distinct precursors with intrinsically different mechanisms of V gene usage and recombination, or newly formed Ly-1 B are heavily selected on specificity for entry into this peritoneal lineage. If the second alternative is true, bacterial antigens in the gut are not required for selection of this unique repertoire, as Ly-1 B cells in germ-free mice also show the multireactive repertoire characteristic of this B cell lineage in normal mice.     

Antigen-binding human T suppressor cells and their association with the HLA-DR locus

The ability of human lymphocytes to bind antigen was studied by direct binding of ¹²⁵I-labeled streptococcal protein antigen, followed by autoradiography. T-enriched lymphocytes depleted of adherent cells and B cells showed specific binding of ¹²⁵I-labeled streptococcal antigen (SA) at 4°C and in the presence of sodium azide. Further depletions of the T-enriched population by the monoclonal T4 or T8 antiserum and complement revealed that the antigen-binding T cell is T4^?, T8⁺. This was confirmed by positive selection of T8 cells, by rosetting with ox red blood cells and by the binding of SA by in vitro induced suppressor but not helper cells. Antigen specificity of binding to the suppressor cells was established by complete inhibition with the SA but no inhibition with keyhole limpet hemocyanin. A characteristic dose-response of binding 1 or 10 ng SA to HLA-DRw6 lymphocytes and 1000 ng SA to DR4,1,2,3 or 5 lymphocytes was found. A comparison of the dose-responses of antigen-binding T⁺ suppressor cells with those of helper and suppressor functions showed that the dose of SA which binds to suppressor cells is similar to the dose required to induce helper but not suppressor function. A plausible interpretation of these observations is that the T8⁺ antigen-binding suppressor cells might function as “contrasuppressor cells” which compete successfully for the membrane receptors of helper cells, thereby preventing suppression by the major subset of suppressor cells.     

The Proliferation of Plasma Cells from Mouse Bone Marrow in Vitro III. Primary and Secondary Immune Responses Associated with Thymic RNA

In vitro primary and secondary immune responses involving the proliferation of IgG-forming plasma cells from cultures of normal or antigen-primed mouse bone marrow cells have been investigated in order to clarify the relationships between the bone marrow cells and thymic RNA derived from each animal. By an administration of soluble and insoluble antigens with thymic RNA to bone marrow cultures, 54 ± 13% of IgG-forming plasma cells were found to produce specific immunoglobulin following stimulation with antigen. Thymus independent antigen polyvinylpyrrolidone (PVP) induced the proliferation of anti-PVP specific antibody forming cells in the abcence of thymic RNA, although more of these cells were generated when PVP-primed thymic RNA was administered with PVP to normal bone marrow cultures. Other antigens such as keyhole limpet hemocyanin (KLH), flagellar antigen of Salmonella, apofferitin and SRBC were investigated using the same bone marrow culture system. These antigens induced various numbers of antibody forming cells from the cultures. Antigen-primed thymic RNA derived from mice inoculated with antigen caused secondary immune response-patterns of plasma cell proliferation from normal bone marrow cultures. Bone marrow cells, however, derived from antigen primed animals proliferated with patterns of primary immune response when cultured with normal thymic RNA and the same antigen. On the basis of these observations, it is concluded that anamnestic immune memory is mainly associated with thymic RNA and not with immunoblasts in bone marrow.