In vitro differentiation of B lymphocytes from pre-B cells.

Adult bone marrow contains both B lymphocytes and their immediate precursors, pre-B cells. These two cells differ in size and can be separated by velocity sedimentation; B cells are enriched in the subpopulation of cells sedimenting at between 2.0 and 3.5 mm/hr and pre-B cells in the subpopulation between 5.0 and 7.0 mm/hr. Incubation of pre-B cells in vitro for 4 or 5 days leads to their differentiation into functional B lymphocytes. The transition form pre-B to B appears to occur in two steps. The first step gives mitogen responsive B cells with an intermediate sedimentation velocity and the second step produces typical small, slowly sedimenting B cells. Pre-B cells can be quantified by using a limiting dilution assay and occur at a frequency of 1/60 in the subpopulation of rapidly sedimenting bone marrow cells.     

B lymphocyte subpopulations separated by velocity sedimentation. II. Characterization of tolerance susceptibility.

Neonatal and adult splenic B lymphocyte subpopulations, separated by velocity sedimentation, were tested in an in vitro splenic focus assay for their susceptibility to tolerance induction with hapten-protein conjugates. At least two tolerizable B cell subsets have been defined in the neonatal spleen; one comprising the slowly sedimenting, small lymphocytes and the other comprising the very rapidly sedimenting, large lymphocytes. In addition, a rapidly sedimenting large B cell subset in the adult spleen was found to be highly susceptible to tolerance induction. It was suggested that the inability to detect this susceptibility in unfractionated adult spleen was due to the low proportion of these very large lymphocytes in the total spleen cell population. The tolerizable B cell subsets presently defined probably represent the least mature B lymphocytes detectable in the splenic focus assay.     

Ecto-5'-nucleotidase expression during human B cell development. An explanation for the heterogeneity in B lymphocyte ecto-5'-nucleotidase activity in patients with hypogammaglobulinemia

Ecto-5'-nucleotidase (ecto-5'-NT) activity was measured in human B cells at different stages of development. Ecto-5'-NT activity of B cell preparations from fetal spleen and cord blood was 5.08 and 5.59 +/- 2.8 nmol/hr/10(6) cells, respectively; that of B cell preparations from adult peripheral blood, spleen, or lymph node was fivefold to sixfold higher (27.9 +/- 12, 29.2 and 33.8 nmol/hr/10(6) cells, respectively). The increased enzyme activity in B cell preparations from adult peripheral blood as compared with cord blood paralleled increased percentages of 5'-NT+ cells (69 +/- 12% vs 32 +/- 17%) and an average of twice as much enzyme activity per positive cell. Small, resting B cells that cannot synthesize Ig in vitro in response to pokeweed mitogen (PWM) were isolated from adult peripheral blood by mouse erythrocyte rosetting. Total ecto-5'-NT activity and the percentage of 5'-NT+ cells were equivalent in total B cells and the mouse erythrocyte rosette-positive subpopulation. Thus, ecto-5'-NT activity is acquired before B cells gain the ability to differentiate into Ig-secreting plasma cells in response to PWM. Ecto-5'-NT activity was also measured in B cell preparations from eight patients with common variable immunodeficiency. Six had reduced ecto-5'-NT activity (2.83 to 15.4 nmol/hr/10(6) cells), and two had normal activity (34.7 and 58.2 nmol/hr/10(6) cells). B cells from all six patients with low ecto-5'-NT activity failed to synthesize Ig when cultured with PWM and normal irradiated T cells. Of the two patients with normal B cell ecto-5'-NT activity, one also had B cells unresponsive to PWM, but B cells from the other patient appeared to more normal, in that they synthesized IgM and IgG when cultured with PWM plus irradiated allogeneic T cells. Thus, measurement of B cell ecto-5'-NT activity allows the subclassification of patients who have a common inability to synthesize immunoglobulin in vitro response to PWM. B cells with low ecto-5'-NT activity are presumably blocked at an earlier stage in development than B cells with normal ecto-5'-NT activity. Evaluation of ecto-5'-NT activity along with the expression of other B cell surface antigens should aid in the definition of discrete stages of B cell development.     

Lymphocytes binding polyriboadenylic acid and synthesizing antibodies to nucleic acids in autoimmune and normal mice.

Splenic lymphocytes from both normal and autoimmune mice bind significant quantities of polyriboadenylic acid (poly rA) when incubated with radiolabeled poly rA for 40 min at 37 degrees C. This poly rA binding is specifically inhibited by an excess of nonradioactive poly rA and by anti-mouse immunoglobulin. Poly rA binding is decreased by exposing spleen cells to Pronase and is restored by subsequent culture for 18 to 72 hr. Poly rA-binding activity is associated more with bone marrow-derived than with thymus-derived lymphocytes. These results suggest the presence of autoantigen-binding lymphocytes in normal as well as autoimmune mice. Furthermore, spleen cells from normal and autoimmune mice cultured for 72 hr synthesize and secrete antibodies to poly rA and DNA. These antibodies can be recovered from the culture supernatants by a solid immunoadsorbent technique and antibody immunoprecipitation. The synthesis of antibodies to nucleic acids by normal spleen cells suggests that autoreactive lymphocytes may be released from normal immunoregulatory control during in vitro culture conditions.     

Characterization of rat bone marrow lymphoid cells. I. A study of the distribution parameters of sedimentation velocity, volume and electrophoretic mobility

Various cell populations in rat bone marrow were characterized by means of a two dimensional separation using velocity sedimentation and free flow electrophoresis and by electrical sizing of the separated cells. Up to 4.5 mm/hr five different populations with discrete distributions in volume (coefficient of variation 10% to 13%) and sedimentation velocity (coefficient of variation 6% to 10%) were observed. Three of the small sized populations represented lymphocytes and small normoblasts and two of the larger sized populations represented myeloid cells. Almost all of these cells were in the G0/G1 cycle phase. In the faster sedimenting fractions which contained immature myeloid, erythroid and undefined blast cells and two S phase populations, discrete volume distributions were not evaluated. The cell populations with homogeneous volume (particularly the small lymphocytes) showed high density variations which condiserably impair the separation resolution. The cells sedimenting slower than 3.5 mm/hr were further separated by means of free flow electrophoresis into three peaks differing in electrophoretic mobility (EPM). The peaks of low and high EPM contained two populations and the peak of medium EPM contained three populations all characterized by normal volume distributions of uniform coefficient of variation between 11% and 14%. The small cells in the peaks of high and medium EPM were normolblasts and the other cells were lymphocytes. The biological significance of these results is discussed.     

Cellular characteristics and specificity of alloactivated regulatory T cells modulating T-cell-mediated responses

The characteristics of human alloactivated regulatory T (T_r) cells influencing the proliferative responses of mixed lymphocyte reactions (MLR) were analyzed based on relative major histocompatibility complex (MHC) specificity, proliferative requirements, Fc receptor expression, and buoyant density. Optimal T_r-mediated suppression required MHC homology with the responder, but not the stimulator populations. In addition, T_r suppression consisted of both proliferative and nonproliferative components. By either Fc receptors for immunoglobulin G (IgG) (Tγ) or buoyant density, no precursor populations predestined to differentiate into specific T_r subpopulations could be delineated. However, fractionation of the T_r populations by buoyant density demonstrated slowly sedimenting suppressor subpopulations and rapidly sedimenting amplifier cells with the net effect representing a balance among these differing functional subpopulations. Finally, the T_r suppressive populations failed to demonstrate FcIgG surface receptors therefore indicating that both T-γ and T non-γ subpopulations are capable of differentiating into alloactivated T non-γ suppressor cells.     

SHORT-TERM CULTURES OF MOUSE MARROW CELLS SEPARATED BY VELOCITY SEDIMENTATION

Our experiments were designed to identify the source of the increased number of cells forming colonies in culture (CFU-C) detected after short-term culture of mouse marrow cells over 'feeders' of renal tubules. Accordingly, marrow cell suspensions were fractionated by velocity sedimentation and aliquots of each fraction cultured for 2 days over tubule 'feeders'. We found a greater increase of CFU-C in suspensions of slowly sedimenting cells as compared to rapidly sedimenting cells. Colcemid and vinblastine were used to alter the peak sedimentation velocity of marrow suspensions by changing the distribution of cells in the cell cycle. After such manipulations, the peak increase in CFU-C continued to be associated with fractions containing slowly sedimenting cells. Such fractions also contained most of the pluripotent stem cells identified by the spleen colony technique.     

Ability of chicken B cells from different compartments to respond to TNP-Ficoll

The responsiveness of chicken B cells from various compartments to T-independent antigens was studied by immune transfers of spleen and bursa cells into immunosuppressed recipients. Bursa cells from 8- to 10-wk-old donors failed to respond to trinitrophenylated Ficoll (TNP-F) even when thymus cells or splenic T cells were added. Spleen cells from the same donors transferred responses, as judged both by anti-TNP plaque-forming cells (PFC) per spleen and serum anti-TNP titers. In contrast, responses to TNP-Brucella abortus (TNP-BA) were transferred at least as well as by bursa as by spleen cells. Rabbit anti-chicken T cell serum plus complement treatment of the spleen cells reduced their ability to transfer responses to sheep erythrocytes, but either did not affect or enhanced serum antibody responses to TNP-BA and TNP-F. In intact animals, responsiveness to i.v. injected TNP-F was found to develop slowly after hatching in the chicken. At the age of 2 and 3 mo, PFC/spleen on day 4 after TNP-F injection were only 20% and 40%, respectively, of the adult response. Thymectomy at hatching further delayed this development, resulting in 12% and 45% of the adult control response at ages of 3 and 4 mo. It is concluded that responsiveness to the TI-2 antigen, TNP-F, develops slower than that to the TI-1 antigen, TNP-BA, and is restricted to the splenic B cell compartment. In addition, this development appears to be faster in the presence rather than in the absence of the thymus. In view of the previously shown effect of thymus on bursa development, these data suggest that the maturation of TI-1 antigen (TNP-F)-respondent chicken B cells requires residence in both the bursa and spleen before the development of responsiveness to such antigens.     

Cellular Origin of a Mouse Leukemia Viral Ribonucleic Acid

Mouse erythroblastosis virus, a member of the mouse leukemia virus group, was obtained from chronically infected C(3)H mouse embryo cells and purified on sucrose gradients. The ribonucleic acid (RNA) extracted from ribonuclease-treated virus consisted of a rapidly sedimenting (72S) species and a more slowly sedimenting component (4 to 30S). The 72S RNA did not contain base sequences homologous to deoxyribonucleic acid (DNA) from infected cells as determined by hybridization studies. In contrast, the slowly sedimenting RNA enclosed within the virus had base sequences homologous to DNA from infected and uninfected C(3)H mouse embryo cells.     

Pronase treatment of lymphocytes reduces the time required for onset of S phase in response to anti-immunoglobulin

Murine B lymphocytes in the presence of antibody specific for surface membrane immunoglobulin begin to synthesize DNA at about the 36th hr of culture, although the onset of synthesis in response to other B cell-reactive mitogens occurs at approximately 18 hr. In contrast, the onset of DNA synthesis by Pronase-treated cells in response to anti-immunoglobulin required only 18 hr. This earlier onset of S phase was not observed when Pronase treatment was performed in the presence of ovalbumin or the protease inhibitors phenylmethylsulfonyl fluoride or aprotinin. It is unlikely that simple carryover of Pronase from the treatment procedure to the cell culture process was involved, because Pronase treatment for 1 hr at 3 degrees C rather than at 37 degrees C did not result in early onset of DNA synthesis. Cells treated with Pronase and then with mitomycin C to irreversibly inhibit their capacity to synthesize DNA were incapable of inducing early onset of S phase on co-culture with untreated cells, suggesting that Pronase may act directly on B cells rather than indirectly via other cells in the splenocyte population.     

Polyclonal activation of CR+ and CR- B lymphocytes: the kinetics of initiation of DNA and immunoglobulin synthesis by lipopolysaccharide.

B cells that carry the complement receptor (CR+) were separated from B cells that lack the complement receptor (CR-) by velocity sedimentation or by passage through C-coated Sephadex columns. The kinetics of responses to bacterial lipopolysaccharide (LPS) in both B cell subpopulations were determined in three assay procedures: 1) incorporation of radioactive thymidine into DNA; 2) incorporation of radioactive leucine into immunoglobulin; 3) enumeration of cells forming polyclonal antibody to the 2,4,6-trinitrophenyl hapten. Although both subpopulations of B cells responded to LPS, they differed in the time course. CR- B cells responded with a delay of approximately 24 hr as compared with the response of CR+ B cells. The implications to the ontogenetic status of CR+ and CR- B subpopulations are discussed.     

Velocity sedimentation profiles of normal and regenerating primitive erythroid burst-forming units: relation to phase of cell cycle and growth in vitro

Abstract. The primitive burst-forming unit-erythroid (BFU-e) derived from normal and regenerating murine bone marrow was examined by velocity sedimentation at unit gravity. An increase in the modal sedimentation velocity and the percentage of rapidly sedimenting BFU-e was found in regenerating marrow as compared to normal marrow. Neither hypertransfusion-induced plethora nor administration of erythropoietin (Ep) during regeneration altered the changes from normal in the velocity sedimentation profile observed during regeneration. Separated marrow cells were pooled as rapidly sedimenting and slowly sedimenting and then examined for percentage of BFU-e in DNA synthesis and growth response in vitro to increasing concentrations of a partially purified Ep preparation. The percentage of BFU-e in DNA synthesis as determined by tritiated thymidine killing does not correspond to the BFU-e growth response to Ep in vitro . No difference in growth was noted between BFU-e from rapidly and slowly sedimenting normal marrow cells despite an increased percentage in DNA synthesis of normal BFU-e which sedimented rapidly. No significant difference in the percentage of BFU-e in DNA synthesis was found between the rapidly and slowly sedimenting subpopulations of regenerating BFU-e, but the latter had a reduced growth response to low concentrations of Ep.     

A role for Lys-His-Gly-NH2 in avian and murine B cell development

Lys-His-Gly-NH2 has been claimed to selectively induce B cell precursors to differentiate into mature B lymphocytes. In the present study, the effects of this tripeptide and a control compound having the reverse sequence (Gly-His-Lys-NH2) on growth and differentiation of chicken and mouse B cell precursors were investigated. When chicken bone marrow (BM) cells from 15-day-old embryos were treated for 18 hr with either of the tripeptides, the frequency of Bu-1 antigen-bearing cells increased. Moreover, when embryonic bursa cells were stimulated in vitro with phorbol myristate acetate, which induces them to proliferate and undergo terminal differentiation into immunoglobulin (Ig)-secreting cells, these compounds caused a 10-fold increase in the number of Ig-secreting cells but did not increase cell proliferation. They had no effect on neonatal or adult bursa cells. Embryonic bursa cells were cultured in the presence of either of the tripeptides and metabolically labeled with [35S]methionine. When immunoprecipitated Ig was analyzed by two-dimensional gel electrophoresis, no differences in mu heavy or lambda light chain diversity patterns could be detected, indicating that neither of these compounds enhances Ig diversification. The effect of these tripeptides on murine B cell precursors was assayed in cultures of BM cells depleted of mature B cells by 5-fluorouracil. When precursor cells were incubated without adherent BM stromal cells, they did not respond to the tripeptides. However, after incubation of precursors with adherent stromal BM cells for 2 days, followed by treatment with either of the two tripeptides, differentiation into lipopolysaccharide-reactive mature B cells took place. Incubation of precursors with adherent stromal BM cells in the absence of tripeptides was not sufficient to allow the precursors to complete differentiation. In addition, both tripeptides acted synergistically with interleukin 1 or interleukin 3. In conclusion, these tripeptides seem to enhance precursor B cell differentiation in a lineage-nonspecific manner rather than to function as lineage-specific differentiation hormones.     

Subpopulations of physiological and ovarian follicular fluid peptide induced apoptotic cells

Ovarian follicular fluid peptide (OFFP) purified from sheep ovaries enhances apoptotic changes in ovarian granulosa cells of mice. To get an insight into the cell subpopulations responding to OFFP, the heterogeneity of granulosa cells was resolved. Subpopulations of granulosa cells were obtained from ovaries of immature mice treated with PMSG alone and autopsied 48 hr (control) and 72 hr after injection (atretic) and from animals injected OFFP 24 hr after PMSG injection and autopsied 24 hr later (OFFP treated) by separation on discontinuous Percoll gradient. Four fractions were collected and studied for their relative distributions and percent apoptotic cells measured by acridine orange staining. FSH binding to granulosa cell (sedimenting as a major) fraction was studied by radio receptor assay. There is a difference in densities in subpopulations of apoptotic cells induced by OFFP and those generated during the physiological process of atresia. This difference may be a reflection of different granulosa cell subpopulations involved in peptide response or differences in phases as the cells transit from normal to apoptotic phenotype. FSH binding to granulosa cells from OFFP treated animals was significantly less than those from control and atretic group.     

Splenic B cells from CBA/N mice acquire responsiveness to anti-immunoglobulin after a brief treatment with pronase

Although splenic B cells of CBA/N mice do not synthesize DNA in response to anti-mouse IgM (mu-chain specific), the cells respond readily to Sepharose linked anti-mu. Subsequent to a brief treatment with pronase, CBA/N splenocytes exhibited anti-mu-mediated DNA synthesis at 40 to 100% of the DNA synthetic capacity detected with Sepharose linked anti-mu. Furthermore, spleen cell populations treated with anti-Thy-1.2 and complement or populations purified on anti-immunoglobulin-coated Petri plates (greater than 90% surface immunoglobulin positive) acquired responsiveness to anti-mu after pronase treatment.     

Three Size-Classes of Intracellular Adenovirus Deoxyribonucleic Acid

When human adenovirus type 2 or 12 infects cells, either productively or non-productively, three classes of viral deoxyribonucleic acid (DNA) are found within the cells: (i) viral DNA which cosediments with DNA extracted from infectious adenovirions at 31.3S for adenovirus type 2 and at 29.0S for adenovirus type 12, (ii) viral DNA which sediments at about 18S, and (iii) viral DNA which sediments at >45S and is apparently integrated into the cellular DNA. A precursor-product relationship is suggested as a working hypothesis; the intact viral DNA is hydrolyzed to slowly sedimenting DNA and the slowly sedimenting DNA is integrated into the cellular DNA. Both the parental and the newly synthesized viral DNA are altered by this route. The intact viral DNA within the cells apparently is cleaved into the slowly sedimenting DNA by a preformed enzyme.     

Immune reconstitution in the immunodeficient chicken by transfer of embryonic lymphoid cells

Embryonic chickens were rendered immunodeficient by in ovo injection of homologous IgM on the 10th embryonic day. The immunodeficient embryos were intravenously given lymphoid cells taken from normal embryonic bursa, spleen or thymus on the 15th embryonic day. Gain of splenic or bursal weight, natural antibodies to sheep red blood cells (SRBC), frequencies of rosette forming cells (RFC) binding to SRBC or dinitrophenyl-SRBC (DNP-SRBC) and of immunoglobulin bearing cells (IBC) in the bursa and the spleen were investigated to assess the effect of transferred cells during the embryonic stage. Transferred bursal and splenic cells showed an ability to restore the frequency of RFC or IBC in the recipients. However, reversion from the immunodeficient state was not observed in the thymic cell transfer. These findings suggested that the cells derived from embryonic bursa and spleen contained stem cells which developed into RFC and also into precursors of IBC.     

Kinetics of CTL induction by concanavalin A.

Induction of maximal CTL activity was achieved within 12 hr of exposure to Con A in vitro in various mouse lymphoid cell populations. These included spleen cells from normal unsensitized mice, spleen cells from mice previously immunized with alloantigen, and mouse spleen cells exposed to alloantigen in long-term mixed leukocyte culture (LTMLC). Although induction of maximal incorporation of tritiated thymidine was accomplished within this same period in the cells obtained from LTMLC, a much longer period of Con A exposure (greater than 24 hr) was required for freshly prepared spleen cells from normal or previously immunized mice. These findings indicate that the increased tritiated thymidine uptake induced in freshly prepared spleen cells on continued exposure to Con A beyond 12 hr is not associated with the development of cytolytic activity, and that it probably represents stimulation of subpopulations no longer present in the LTMLC population where positive selection for cells responsive to cellular alloantigens has taken place.     

Relationship between the expression of class I antigen and reactivity of chicken thymocytes

The expression of major histocompatibility complex (MHC) class I antigens in ontogenesis and the distribution of B-F⁺ cells, defined by means of a monoclonal antibody, were studied by indirect membrane immunofluorescence tests on suspensions of thymus, bursa, spleen, peripheral blood lymphocytes (PBL) and red blood cells (RBC) from 18-day-old chicken embryos and chickens from 1–90 days after hatching. At 18 days of incubation and at the first day after hatching, RBC, PBL, and the cells from bursa and thymus are negative. The percentage of positive PBL and bursal cells increases up to 9 days after hatching. By 2 weeks after hatching almost 100 % of the RBC, PBL, bursa, and spleen cells were positive whereas the thymus showed only 20% positive cells. Analysis on 4-m-thick, frozen acetone-fixed tissue sections of thymus showed that medullary cells are positive, while the cortical area is negative. The graft-versus-host (GvH) competence of these thymus subpopulations was compared after sorting by the fluorescence-activated cell sorter and injection into MHC incompatible embryos. GvH reactivity was associated primarily with the B-F⁺ population. Double staining studies with peanut agglutinin (PNA)-fluorescein isothiocyanate and a rabbit-anti-Ig tetramethyl isothiocyanate-conjugate proved that the PNA^– thymocytes are identical with B-F⁺ thymocytes.Abbreviations used in this paper: FACS fluorescence-activated cell sorter - FCS fetal calf serum - FITC-Ig fluorescein isothiocyanate-conjugated immunoglobulin - GvH graft-versus-host - HAT hypoxanthineaminopterin-thymidine - HBSS Hanks' balanced salt solution - IIF indirect immunofluorescence - MCA monoclonal antibody - MHC major histocompatibility complex - NWL normal white Leghorn - OS Obese strain - PBL peripheral blood lymphocytes - PBS phosphate-buffered saline - PNA peanut agglutinin - RBC red blood cells - TRITC-Ig tetramethyl isothiocyanate-conjugated immunoglobulin     

Immunogenicity of allograft components: II. Relative immunogenicity of rat kidney parenchymal versus “passenger” cells

Well-perfused adult DA kidneys were enzymatically dispersed under conditions which do not affect the expression of cell surface major histocompatibility antigens. The kidney cell suspensions were separated via sedimentation at unit gravity into three fractions: I, rapidly sedimenting (>6.5 mm/hr) enriched for kidney tubular and glomerular cells and depleted of passenger leukocytes (76 and 8%, respectively); II, intermediate (5.1–6.0 mm/hr) mixed population equivalent to the unseparated kidney cell suspension (52% tubular and glomerular cells, 20% endothelial cells, and 28% passenger leukocytes); and III, slow sedimenting (<5.0 mm/hr) enriched for passenger leukocytes (63%). The three isolated fractions were analyzed for their ability to accelerate allograft rejection in the “primed heart rejection assay.” The cells in fraction I were unable to reduce heart allograft survival, while the cells from fraction III reduced it significantly. Cells from fraction II were intermediary effective. The results are in agreement with the hypothesis that the urine-producing apparatus of rat kidney is relatively nonimmunogenic, while the main stimulus for graft rejection is provided by the “passenger” cell component.