Suppression of adult B cell differentiation in pokeweed mitogen-stimulated cultures by Fc(IgG) receptor-negative T cells from cord blood.

Unfractionated T lymphocytes from cord blood suppressed adult B cell differentiation into immunoglobulin-producing cells in pokeweed mitogen-stimulated co-culture system. Cord blood T cells were fractionated into T cells bearing Fc receptors for IgG (Tgamma cells) and T cells lacking Fc receptors for IgG(Tnon-gamma cells) by rosette formation with ox erythrocytes coated by the IgG fraction of rabbit antisera followed by Ficoll-Hypaque gradient sedimentation. T gamma cells from cord blood, even though isolated after the interaction with immune complexes, showed no suppressor activity on adult B cell differentiation, whereas Tnon-gamma cells exerted strong suppression to a similar extent to that by unfractionated cord T cells. The suppressor activity on B cell differentiation by Tnon-gamma cell as well as by unfractioned T cells from cord blood was completely abrogated by irradiation with 2000 rads. These results indicated that, contrary to suppressor function found in adult T cells, the suppressor activity in cord T cells might be exerted by a T cell subset lacking Fc receptors for IgG(Tnon-gamma cells).     

Separation of human lymphocyte subpopulations by density gradient electrophoresis. I. Different mobilities of T (T mu, T alpha) and B lymphocytes from human tonsils

T and B lymphocytes from human tonsils were separated by density gradient electrophoresis on the basis of their surface charge. The high-mobility cell fractions were found to be highly enriched in T lymphocytes with only very small proportions of B cells. In contrast, the low-mobility fractions were predominantly B lymphocytes, and had only 10 to 30% contamination of T cells. The intermediate-mobility fractions contained both T and B lymphocytes in approximately equal proportions. IgM-bearing lymphocytes, as well as cells with receptors for mouse erythrocytes, the Fc portion of IgG, and complement were found in the intermediate- and low-mobility fractions. T lymphocytes, prepared by E rosetting, were also electrophoresed by this method and found to be of higher mobility as compared with peripheral blood T lymphocytes. T cells with Fc receptors for IgM (Tμ) or IgA (Tα) were found to be considerably heterodisperse with regard to surface charge and were present in all fractions. The separated cell fractions were treated in vitro with various concentrations of concanavalin A and thereafter examined for Tμ, Tγ, and Tα phenotypes. Low concentrations of Con A (2.5 μg/ml) had no effect on cell surface phenotypes. However, higher concentrations of Con A (20μg/ml) significantly reduced the numbers of T cells having IgM receptors (Tμ), but failed to alter the expression of the Tγ phenotype. The latter finding contrasts to that observed with T cells from the peripheral blood where high concentrations of Con A increase the proportions of the Tγ cells. This study demonstrates that density gradient electrophoresis can be used for the separation and study of lymphocyte subpopulations from human tonsils.     

Daily variation in circulating lymphocyte counts, T and B proportions and responsiveness to phytohemagglutinin.

Peripheral blood was obtained from six healthy individuals over five consecutive days under uniform conditions and the total lymphocyte counts, T and B proportions, and response to phythohemagglutinin (PHA) were determined. The daily variation in T lymphocytes as measured by the spontaneous sheep erythrocyte (SRBC) assay was much greater when total T concentrations rather than T percentages were compared. There was considerable daily variation in PHA responsiveness and in the percentages of cells bearing Fc and C3 receptors and surface immunoglobulin (SIg). Cryopreservation did not affect the proportions of T and B lymphocytes although it resulted in a significant enhancement of PHA responsiveness following the freeze-thaw procedure. The significance of these results is discussed.     

Active and late rosette-forming cells: immunological and cytochemical characterization

In order to characterize T-cell subpopulations with different affinities for sheep red blood cells, a possible correlation between active and late rosette-forming cells (RFC), expression of Fc receptors, and the presence of Ia antigens was investigated. Further analysis of their spontaneous in vitro DNA synthesis, and their cytochemical pattern was also performed. Most of the active T cells lacked Fc receptors and expressed Ia antigens, whereas the large majority of cells with Fc-IgG and -IgM receptors were included as late T cells, but lacked Ia determinants. Analysis of spontaneous blastogenesis showed that late RFC, but not active RFC, synthesize DNA in unstimulated cultures. Cytochemical analysis suggested that Active RFC display a more immature phenotype compared to late RFC, which have enzymatic markers present in mature T cells. Possible interpretations of these findings are discussed.     

Insoluble immune complexes suppress mitogen-induced proliferation of human T lymphocytes bearing Fc gamma receptors.

Human peripheral blood lymphocytes were mixed with erythrocyte-antibody (EA) complexes and separated into EA-rosette forming cell (EA-RFC)-enriched and EA-RFC-depleted suspensions. Thymidine incorporation of EA-RFC-enriched population in the presence of T cell mitogens (PHA, Con A, PWM) was about half of that of EA-RFC-depleted or of unseparated cells. The dose-response curves and kinetics of proliferation were found to be very similar in the three populations. Proliferative response of EA-RFC-enriched lymphocytes was strictly T cell dependent, although non-T cells were later recruited to incorporate thymidine. The interaction of T lymphocytes bearing surface receptors for IgG (T_G) with insoluble complexes followed by a post-binding temperature sensitive event, resulted in the modulation of Fc receptors associated with an impaired proliferative response to PHA, Con A, and PWM, without significant change in metabolic cell activity as shown by cell viability, sponaneous leucine incorporation, or β₂ microglobulin release.     

The effects of corticosteroids on immunoregulation in sarcoidosis.

The effects of in vivo hydrocortisone administration on the kinetics and functional capabilities of cells involved in the immune response in sarcoidosis were examined. Untreated sarcoidosis patients have a decrease in the absolute numbers of circulating T lymphocytes (P < 0.05). However, with regard to the proportions of T lymphocyte subpopulations, there is an increase in the relative proportions of IgG Fc receptor positive T cells (T_G) (P < 0.01), which have suppressor capabilities in certain in vitro systems of mitogen-induced antibody production, and a relative decrease in IgM Fc receptor positive T lymphocytes (T^M) which have helper effects in this system (P < 0.05). Additionally, sarcoidosis patients have circulating “suppressor” monocytes capable of suppressing anti-sheep red blood cell (SRBC) plaque-forming cell (PFC) responses by pokeweed mitogen (PWM)-stimulated lymphocytes. The in vitro removal of this cell abrogated this depressed response (P < 0.01). Intravenous administration of hydrocortisone produced a transient absolute T lymphocytopenia (P < 0.01) accompanied by a relative increase in T^G cells (P < 0.01) and a relative decrease in T_M cells (P < 0.02). Four hours after hydrocortisone therapy, at the point of maximal hydrocortisone-induced monocytopenia (P < 0.01), the suppressed ability of sarcoidosis lymphocytes to synthesize and secrete in vitro anti-SRBC antibody after polyclonal activation was corrected (P < 0.01), and PFC responses comparable to those seen in untreated normal subjects were obtained. These studies demonstrate that corticosteroid administration has profound effects on certain in vitro demonstrable immunoregulatory abnormalities in sarcoidosis.     

Immunosuppression by cobra factor: distribution, antigen-induced blast transformation and trapping of lymphocytes during in vivo complement depletion.

In vivo administration of cobra factor (CoF), the C3-activating protein of cobra venom, suppresses thymus-dependent antibody production. In a study of possible mechanisms for this effect binding of CoF to murine spleen cells in vitro was not detected, nor was there any effect on C3 or Fc receptors. The numbers of spleen cells bearing C3 receptors, Fc receptors, θ antigen or surface immunoglobulin were not altered by in vivo complement depletion of mice with CoF. The distribution and antigen-induced trapping of transferred ⁵¹Cr-labelled syngeneic spleen cells were unaffected by treatment of either donors or recipients with CoF. Furthermore, the antigen-induced generation, trapping and specific retention of immunospecific blast cells were normal in CoF-treated mice, despite profound suppression in these animals of IgG antibody production. The majority of these blast cells 3 days after immunisation were T cells, suggesting that complement depletion interferes with the process of T-dependent antibody production at a later stage than the activation of T cells by antigen.     

Subpopulations of human T lymphocytes. III. Distribution and quantitation in peripheral blood, cord blood, tonsils, bone marrow, thymus, lymph nodes, and spleen.

Human T cell subpopulations (Tμ and Tγ) were examined for their distribution in the peripheral blood, cord blood, bone marrow, tonsils, thymus, lymph nodes and spleen. The proportions of Tμ and Tγ cells are comparable in the peripheral blood, tonsils and bone marrow. The proportions of Tγ cells in cord blood are significantly higher than those in the peripheral blood. Almost complete lack of Tγ cells was observed in lymph nodes. Spleen has very high proportions of Tγ cells. Thymuses have very low proportions of both Tμ and Tγ cells when compared with peripheral blood, cord blood, tonsils, and bone marrow. The receptors for IgM on Tμ cells appear to be masked by passively absorbed IgM and require prior in vitro incubation in medium containing fetal calf serum for the full expression of this marker.     

Immature T lymphocytes in human cord blood identified by monoclonal antibodies: a model for the study of the differentiation pathway of T cells in humans

The reactivity of human cord blood lymphocytes was assessed against a panel of monoclonal antibodies (MoAb). The mean proportion of OKT3+ cells (pan-T) was significantly lower in cord blood (52 +/- 13.8%; mean +/- SD) compared with that of adult blood (75 +/- 8.9%) and paralleled well with the E-rosette-forming capacity (50 +/- 16.3%). Both the proportions of OKT4+ cells (helper/inducer phenotype) and of OKT8+ cells (suppressor/cytotoxic phenotype) were significantly reduced in cord blood (43 +/- 11.8% vs 50.3 +/- 7.4% and 20 +/- 10.3% vs 25.6 +/- 6.0%, respectively), while the overall OKT4/OKT8 ratio was increased compared with adult blood (2.87 +/- 1.83 vs 2.04 +/- 0.61). Unlike adult blood, in 30 of the 35 samples of cord blood an overlap was observed between the total proportion of OKT4+ and OKT8+ cells (65 +/- 15.2%) and that of OKT3+ cells (52 +/- 14.3%). Although small numbers of cells coexpressing both antigens were occasionally found, double-staining analysis showed that the overlap in cord blood was mostly due to an expanded proportion of OKT3 (Leu-4)-/OKT8 (Leu-2)+ cells. Relevant proportions of OKT6+ (common thymocyte antigen) and OKT10+ (thymocytes, activated T cells, precursor cells) cells were found in cord blood as opposed to adult blood (10.8 +/- 8.6% vs 0.6 +/- 0.6% and 67 +/- 18.0% vs 8 +/- 2.1%, respectively), while terminal deoxynucleotidyl transferase-positive cells were observed only in two samples of cord blood. A small proportion of T cells (E-rosette+) reacted with the MoAb OKIa1 (HLA-DR). Finally, the proportion of cord blood cells recognized by the MoAb Leu-7 (HNK-1 clone) was almost negligible compared with adult blood (2.8 +/- 2.4% vs 15 +/- 7.5%). These data confirm the immaturity and heterogeneity of cord blood lymphocytes and demonstrate the presence at birth of circulating lymphocytes which express a surface phenotype reminiscent of that found in the late stages of intrathymic differentiation and in some human T-cell leukemias. Human cord blood may thus represent a suitable model for the study of the differentiation pathway of normal and pathological T-cells in humans.     

Countercurrent distribution of lymphocytes from human peripheral blood in an aqueous two-phase system. I. Separation into subsets of lymphocytes bearing distinctive markers

Lymphocytes from human peripheral blood have been separated by countercurrent distribution in a charged aqueous two-phase system composed of Dextran T 500 and polyethylene glycol 6000 with a cell yield of 59–88% and viability above 90%. A highly reproducible partition pattern was seen with four distinct peaks. Lymphocytes with surface membrane immunoglobulin (SmIg) were located in the first part of the distribution corresponding mainly to peak I. T lymphocytes as detected by E rosetting and α-naphthyl acetate esterase (ANAE) staining showed a broad distribution with a maximum in peaks II and III. ANAE-negative lymphocytes were seen in both extremes of the distribution, corresponding to B cells in the first part and to a population of E^? and SmIg^? lymphocytes in the last part. Monocytes were present in all fractions with some enrichment in peaks II–IV. Lymphocytes with low-affinity Fc receptors were found in B-cell-containing fractions in the first part of the distribution, but also in the last part. Lymphocytes with high-affinity Fc receptors were detected mainly in peak IV. It is thus demonstrated that peripheral blood lymphocytes can be fractionated into subpopulations enriched in cells with characteristic markers.     

Structure and biological function of human IgD: XV. Biological potential of IgD-bearing cells from cord blood,and normal human adults

Specifically purified intact IgG anti-human δ or F(ab′)₂ anti-δ stimulated the in vitro incorporation of [³H]thymidine by cord blood and adult human peripheral blood lymphocytes. The magnitude of stimulation was unrelated to the increased frequency of IgD-bearing cells observed in cord blood over that seen in adult blood. Purified T cells lacked the capacity to undergo mitogenesis when incubated with anti-δ antibody. Of special interest was the lack of anti-δ enhancement of phytohemagglutinin (PHA) responsiveness of cord blood lymphocytes that is often observed when adult peripheral blood lymphocytes are treated with anti-δ, then PHA. This observation could be interpreted as indicating: (a) that the anti-δ-activated neonatal B cells, or their lymphokines, were preferentially activating a population of suppressor cells, which in turn prevented the PHA-augmentation usually seen with adult cells, (b) that the neonatal B cells do not possess the T cell-augmenting capacity or, (c) that the neonate lacks a subpopulation of T cells that can be acted upon by the B-cell or B-cell factors.     

Fractionation of mouse T and B lymphocytes by preparative cell electrophoresis. Efficiency of the method

Mouse lymphocytes have been fractionated in preparative cell electrophoresis into two functionally viable populations, a high mobility cell (HMC) and a low mobility cell (LMC) population. The distribution of HMC in CBA spleen, blood, and lymph node corresponds to known proportions of θ-positive cells in these organs. The HMC carry the θ-isoantigen, respond to phytohemagglutinin in vitro, and induce a graft-versus-host reaction in newborn F₁ hybrid mice. Nearly all spleen LMC have complement receptors on their surface. About 70% of spleen LMC are sensitive to anti-MBLA serum and form “caps” when incubated with FITC-conjugated anti-Ig. Only LMC respond to E. coli lipopolysaccharide. Thus, T cells localize in the HMC population and B cells in the LMC population. There is no detectable contamination of T lymphocytes among the LMC, nor of B lymphocytes among the HMC.     

Macrophage--T cell interactions. I. The uptake by T cells of Fc receptors released from macrophages.

Using a rosette assay for the detection of cells carrying Fc receptors (Fc+) we have been able to show that in nylon wool (NWC), separated spleen cells from different strains of mice 12 to 18% are Fc+.Within 4 hr of culture in vitro at 37 °C, 75 to 85% of the Fc+ cells lose their Fc receptors and remain Fc receptor negative even after culture for 24 hr. However the addition of 5 to 10% syngeneic (but not allogeneic) peritoneal macrophages in the NWC, resulted in the preservation of the Fc receptors on 75 to 85% of the Fc+ cells originally present.Brief exposure of NWC which have been cultured in vitro for 4 hr (lost their Fc receptors) to supernates from 3 hr cultures of peritoneal macrophages reconstituted the fc+ cells by 75 to 85%. Only the supernates from syngeneic, but not allogeneic macrophages are active. Evidence is presented which indicates that these supernates contain Fc receptor molecules of small molecular weight. These molecules can be removed by antisera directed against the I region of the major histocompatibility complex.     

DNA methylation of cord blood cell types: Applications for mixed cell birth studies

Epigenome-wide association studies of disease widely use DNA methylation measured in blood as a surrogate tissue. Cell proportions can vary between people and confound associations of exposure or outcome. An adequate reference panel for estimating cell proportions from adult whole blood for DNA methylation studies is available, but an analogous cord blood cell reference panel is not yet available. Cord blood has unique cell types and the epigenetic signatures of standard cell types may not be consistent throughout the life course. Using magnetic bead sorting, we isolated cord blood cell types (nucleated red blood cells, granulocytes, monocytes, natural killer cells, B cells, CD4⁺T cells, and CD8⁺T cells) from 17 live births at Johns Hopkins Hospital. We confirmed enrichment of the cell types using fluorescence assisted cell sorting and ran DNA from the separated cell types on the Illumina Infinium HumanMethylation450 BeadChip array. After filtering, the final analysis was on 104 samples at 429,794 probes. We compared cell type specific signatures in cord to each other and methylation at 49.2% of CpG sites on the array differed by cell type (F-test P < 10^?8). Differences between nucleated red blood cells and the remainder of the cell types were most pronounced (36.9% of CpG sites at P < 10^?8) and 99.5% of these sites were hypomethylated relative to the other cell types. We also compared the mean-centered sorted cord profiles to the available adult reference panel and observed high correlation between the overlapping cell types for granulocytes and monocytes (both r=0.74), and poor correlation for CD8⁺T cells and NK cells (both r=0.08). We further provide an algorithm for estimating cell proportions in cord blood using the newly developed cord reference panel, which estimates biologically plausible cell proportions in whole cord blood samples.     

Activation of Fc receptor-bearing lymphocytes by immune complexes. I. Stimulation of lymphokine production by nonadherent human peripheral blood lymphocytes

Activation of human peripheral blood lymphocytes by incubation with particulate immune complexes or aggregated human gamma-globulin was studied by measuring the release of leukocyte migration inhibitory factor (LIF) activity. LIF-active supernatants were consistently produced when nonadherent lymphocytes containing less than 1% surface immunoglobulin-bearing cells and less than 0.2% nonspecific esterase-positive monocytes were incubated in the presence of RBC sensitized with rabbit or human antibodies or with pooled heat-aggregated human gamma-globulin. This immune complex-induced lymphokine production (ICLP) was dependent on the presence of cells bearing receptors for the Fc portion of IgG (Fc gamma). ICLP could not be demonstrated with lymphocyte preparations enriched for B cells even though the latter showed vigorous LIF production in the presence of complement-sensitized erythrocytes. ICLP was dependent on the concentration of lymphocytes and of stimulant as well as on the duration of coincubation, and it required active metabolic processes and RNA and protein synthesis but not DNA synthesis. Ca++ but not Mg++ was obligatory. ICLP by non-B Fc gamma receptor-bearing lymphocytes may play a role in antibody-dependent protective inflammation and immunologic injury phenomena, which is similar to that of lymphokine release by antigen-activated T cells in delayed hypersensitivity responses.     

Characterization of human T lymphocytes that express the C3b receptor

The presence of the C3b receptor (C3bR) on human peripheral blood T lymphocytes was recognized by the capacity of rabbit F(ab')2 anti-C3bR and tetramethylrhodamine isothiocyanate (TRITC)-conjugated goat F(ab')2 anti-rabbit F(ab')2 to stain 14.5 +/- 3.7% (mean +/- SEM; n = 5) of lymphocytes forming rosettes with sheep erythrocytes (E). The F(ab')2 anti-C3bR also blocked the capacity of peripheral blood lymphocytes stained with OKT11 to form rosettes with bovine E bearing C3b and immunoprecipitated a single membrane protein having a m.w. of approximately 250,000 from detergent lysates of 125I-labeled, purified T cells. Measurement by fluorescent flow cytometry of the quantitative expression of the C3bR indicated that T cells had slightly more antigenic sites/cell than did E and approximately 10-fold fewer sites than were present on B cells. The surface constituents of the peripheral blood T cells expressing the C3bR were assessed in an assay that employed simultaneously three markers: rosette formation with sheep E, TRITC staining with anti-C3bR and fluorescein isothiocyanate (FITC)-staining with a panel of monoclonal antibodies or with aggregated IgG. Among lymphocytes forming rosettes with sheep E and expressing the C3bR, 99.6 +/- 0.4%, 65.0 +/- 5.8%, 17.2 +/- 6.2%, and 15.3 +/- 5.0% of the cells expressed antigens detected by OKT3, OKT4, OKT8, and OKM1 monoclonal antibodies, respectively. Ninety-seven per cent of the C3bR-bearing T cells were also capable of specifically binding aggregated IgG, indicating the presence of Fc receptors for IgG (Fc gamma R) on these cells. The T cells expressing the C3bR had large nuclei, thin rims of basophilic cytoplasm and no azurophilic granules. Thus, the C3bR is present on some T cells, all of which have a typical lymphocyte morphology, the T3 antigen and the Fc gamma R.     

Studies on T cells in newborns. Higher reactivity of umbilical cord blood lymphocytes to PHA as measured by whole blood microtechnique.

The capacity of human foetal lymphocytes to respond to PHA and to form E-rosettes have been compared with data from adult individuals. For this purpose a microculture system that uses whole blood and avoids the problems of lymphocyte separation, has been developed. Foetal lymphocytes reached optimal stimulation with lower dosis of PHA (31,2 microgram/ml) as compared with adult cells (125-252 microgram/ml). However their quantitative response (measured by 14C-thymidine uptake) was equal in both groups. In addition, peripheral T cells (E-rosetting cells) reached values of 36.47 +/- 9% in newborn and 49.6 +/- 10% in normal adult controls. These results are discussed as to the status and development of cellular inmunity in human foetus.     

Acute lymphoblastic leukaemia: a heterogenous disease.

By using several techniques to detect surface markers on T and B lymphocytes, 11 cases of acute lymphoblastic leukaemia (A.L.L.) were studied. In four cases an insignificant number of markers were detected on the lymphoblast populations. In one case a significant number of blasts formed both sheep red blood cell rosettes and Fc rosettes, suggesting a T-cell origin for the neoplastic cells, and in another case the presence of Fc and C3 receptors on the lymphoblast population indicated a B-cell origin. In a further five cases 14-43% of the blasts had detectable surface immunoglobulin. It is concluded that A.L.L. is a heterogeneous disorder, some cases failing to express surface markers and others having either a T-or a B-lymphocyte origin or both.     

Comparative analysis of the heterogeneity of mononuclear cells present in adult and cord blood by simultaneous examination of multiple phenotypic characteristics

In an attempt to better relate specific membrane characteristics of human adult and cord blood lymphocytes to specific functional activities, the phenotypic differences that exist in these two populations have been examined. Cord blood cells have considerably more spontaneous suppressor cell activity than adult cells. A technique that allows cells to be examined simultaneously for their ability to ingest latex beads, react with specific monoclonal antisera, bind sheep erythrocytes, or react with the Fc portion of IgG was used. As well as assessing fresh populations, phenotypic changes that occur when such cells are held in culture or stimulated with phytohemagglutinin for 3 days were sought. Many differences were found when comparing these mononuclear populations. These included the observations that 12% of adult and 9% of cord blood E-rosette-forming cells ingest latex beads and that 9% of OKT3 reactive cells in both populations did not form E rosettes. In cord blood 58% of T cells that bind OKT8 do not form E rosettes. A similar percentage of cord blood T8-positive cells express a receptor for Fc gamma, such cells being very uncommon in adult blood. Four "monocyte" subpopulations were identified in both samples. One such population (an OKM1- and Fc gamma-positive, nonphagocytic cell) was three times more common in cord blood. In cord blood some OKM1-positive cells also appeared to be simultaneously OKT8 positive. These phenotypic variations forward populations that may be candidates responsible for the functional differences noted in vitro.     

Epstein-Barr virus (EBV) receptors, complement receptors, and EBV infectibility of different lymphocyte fractions of human peripheral blood. I. Complement receptor distribution and complement binding by separated lymphocyte subpopulations.

Human peripheral lymphocytes were fractionated into a variety of B-, T-, and O-cell fractions and were characterized with regard to several surface receptors. There was a strong correlation between the frequency of EAC receptor-positive cells and the percentage of complement membrane fluorescence (CMF)-stained cells following exposure to fresh human serum and subsequent staining with an anti-C3 conjugate. CMF staining did not diminish in C4-deficient or hypogammaglobulinemic serum, or in the presence of EDTA or EGTA-Mg²⁺, but was completely negative with C3-depleted normal human serum. In all likelihood, the staining is therefore due to the direct binding of C3 to preformed receptors on the lymphocyte surface. In addition to the surface Ig-positive B-cell fractions, C3 receptors were also detected on part of the O-cell population and on a proportion of the Fc receptor-positive T cells.