Subpopulations of human peripheral blood lymphocytes.

Multiple staining protocols have been developed for the classification of subpopulations of human peripheral blood lymphocytes. Of the non-T (E^?) cells, roughly half (10–20% PBL) have receptors for complement components as detected with complement-coated zymosan particles, but do not show Fc receptors as detected with Ripley IgG-coated human RBC. The other half are C^?, Fc⁺, with a small percentage possessing both receptors. The C⁺, Fc^? cells can be subdivided into cells which are IgM⁺ (75%) or IgM^?. Cells with Fc receptors detected with aggregated IgG were IgM⁺.     

Fc receptors on human T lymphocytes. II. Cytotoxic capabilities of human T gamma, T mu, B, and L cells.

Subpopulations of human peripheral blood lymphocytes were prepared by rosetting techniques employing neuraminidase-treated sheep erythrocytes (SRBC_n), sheep erythrocytes coated with IgM and murine complement (EAC′), and bovine erythrocytes coated with IgG and IgM. The isolated subpopulations were tested in assays of natural cytotoxicity (NC), antibody-dependent cellular cytotoxicity (ADCC), and mitogen-induced cellular cytotoxicity (MICC). B cells (SRBC_n?, EAC′+) did not mediate cytotoxicity. L cells (SRBC_n?, EAC′?) mediated NC and ADCC but not MICC. T cells (SRBC_n+) mediated NC, ADCC, and MICC. Separation of T cells into Fc-IgG (Tγ) and Fc-IgM (Tμ) subsets revealed that Tγ cells mediated NC, ADCC, and MICC while Tμ cells mediated only MICC. Thus MICC but not NC or ADCC was solely T-cell mediated. Tγ and L cells were functionally distinguishable in that Tγ cells but not L cells mediated MICC. Tγ cells and Tμ cells differed with regard to NC and ADCC effector function while both subsets mediated MICC.     

Subpopulations of human T lymphocytes. VII. Cellular basis of concanavalin A-induced T cell-mediated suppression of immunoglobulin production by B lymphocytes from normal humans.

Purified peripheral blood T lymphocytes from normal subjects were pretreated with varying concentrations of concanavalin A (Con A) for a period of 18 or 48 hr. Following treatment, these T lymphocytes were examined for the proportions of Tμ and Tγ cells and their regulatory effect on immunoglobulin production by normal allogeneic B cells in presence of pokeweed mitogen. A significant decrease in the proportions of Tμ cells and increase in Tγ cells were observed when concentration of Con A, 40 μg/ml, was used to treat purified T cells for either 18 or 48 hr. Significant suppression of in vitro immunoglobulin synthesis was observed at a similar concentration in mixing experiments. The mechanism of Con A-induced T cell-mediated suppression of immunoglobulin secretion by allogeneic B cells is discussed.     

Subpopulations of chicken B lymphocytes.

Immunoglobulin-synthesizing cells from the spleen and bursa were fractionated by the 1 X G sedimentation velocity technique and characterized by their ability to synthesize immunoglobulin and by staining with fluorescent anti-light chain chain. Four subpopulations of immunoglobulin-synthesizing cells were identified. In the bursa, slowly sedimenting (S 2.3 mm/hr) and rapidly sedimenting (S greater than 3.5 mm/hr) subpopulations with surface immunoglobulin were present; in the spleen, a slowly sedimenting (S 2.3 mm/hr) subpopulation with surface immunoglobulin and plasma cells (S greater than 3.5 mm/hr) with large concentrations of intracellular immunoglobulin existed. The subpopulations differed most markedly in their ability to synthesize immunoglobulin (cpm Ig synthesized/10(6) Ig-positive cells); the rates of immunoglobulin synthesis were in the ratio 1:2:1:900. The slowly sedimenting B cells from the spleen and both subpopulations of B cells from the bursa released small amounts of immunoglobulin into the culture media, whereas, the plasma cells released immunoglobulin at a rate as much as 3700 times greater. Bursal B cells could be further distinguished from splenic B cells by a greater amount of DNA synthesis.     

IgD-receptor-positive human T lymphocytes. II. Identification and partial characterization of human IgD-binding factor.

Membrane receptors specific for IgD (IgD-R) have been identified on murine CD4+ and human CD4+ and CD8+ T lymphocytes. Up-regulation of these IgD-specific receptors can be achieved by exposure of such T cells to various stimuli, including oligomeric or Ag cross-linked IgD, IL-2, IL-4, and T cell mitogens, such as PHA. Previous studies with murine IgD-R+ splenic T cells and IgD-R+ T hybridoma cells have demonstrated the existence of soluble IgD-binding factors (IgD-BF) that are shed or released into the medium in which these cells are grown. In our study, human peripheral blood T cells and IgD-R+ T hybridoma cells were examined for their ability to produce human IgD-BF. PHA stimulation of peripheral blood T cells results in their release of an IgD-specific factor with an apparent Mr of 70 kDa. IgD- Sepharose-purified IgD-BF was able to competitively inhibit rosetting of IgD-R+ T cells with IgD-coated RBC. Immunoblot assays in which alkaline phosphatase-conjugated human IgD myeloma protein was used as a probe, confirmed the IgD specificity of IgD-BF. An IgD-BF-specific mAb (LTB9) that also reacts with membrane IgD-R was produced after immunization of BALB/c mice with this factor. LTB9 was able to detect IgD-BF in the supernatants derived from human IgD-R+, tetanus toxoid-specific T hybridoma cells, H9-CEM1, and to stain membrane IgD-R by indirect immunofluorescence. Stimulation of H9-CEM1 cells with immobilized IgD resulted in up-regulation of membrane IgD-R expression, as measured cytofluorometrically with LTB9-stained cells, and potentiated release of IgD-BF from these cells. Finally, LTB9 as well as IgD-Sepharose, immunoprecipitated a 70-kDa protein from the lysates of biosynthetically labeled H9-CEM1 cells. Similar immunoprecipitation results were obtained with H9-CEM1-derived supernatants containing IgD-BF. Taken together, these results support the hypothesis that human T cell membrane IgD-R are released as soluble IgD-BF.     

Ultraviolet-induced DNA excision repair in human B and T lymphocytes. II. Effect of inhibitors and DNA precursors.

Despite their great sensitivity to ultraviolet light purified human B and T lymphocytes are capable of complete repair provided that the ultraviolet dose does not exceed 0.5 Jm-2. Their capacity to repair, as measured by the restoration of DNA supercoiling in preparations of nucleoids, and their survival are significantly increased in the presence of deoxyribonucleosides. Certain agents which inhibit semi-conservative DNA synthesis (hydroxyurea, 1-beta-D-arabinofuranosylcytosine (arafCyt) either stop or delay the repair process in lymphocytes. The effect of hydroxyurea is eventually overcome spontaneously, but changes in the sedimentation behaviour of ultraviolet-irradiated nucleoids caused by arafCyt can only be neutralized by addition of deoxycytidine. The effective inhibition of repair by arafCyt permits the detection of extremely small amounts of ultraviolet damage and also the estimation of when repair is complete.     

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.     

1.5 MHz ultrasound irradiation of human lymphocytes.

Human lymphocyte cultures are exposed to 1.5 MHz continuous wave ultrasound, and it is demonstrated that cell death, as monitored by pyknosis, follows immediately on sonication at intensities within the usual therapeutic range (less than 1.7 W/cm2,spatial average). The number of cells affected is determined by the ultrasound intensity only, but the rate at which they proceed through their pyknosis cycle is modified by both the intensity and the duration of exposure. There is a clear indication of an intensity threshold for the effect approximately 1.1 W/cm2. Pulsed 1.5 MHz ultrasound (70 mus, 1 :1 pulses, 1.7 W/cm2 space-time average) results in a 15-20 hour delay in the measurable response to sonication. It is shown that the intracellular presence of a lysosomal-enzyme inhibitor strongly modifies the course of the ultrasound action. Evidence is presented to suggest that the basic interaction mechanism is via a cavitation process, but there are some difficulties with this interpretation, which are also discussed.     

The amino acid sequence of thymopoietin II.

The amino acid sequence of bovine thymopoietin II is presented. This T cell differentiating hormone of the thymus is a single 49 amino acid polypeptide chain of 5562 daltons. There is microheterogeneity at the C terminus with approximately two thirds of the molecules lacking the C terminal arginine found on the remaining molecules. Determination of the primary structure of thymopoietin II was facilitated by a long automated sequenator run on thymopoietin II coupled to 2-isothiocyanonaphthalene-4,8-disulfonic acid (NITC), tryptic cleavage of maleated thymopoietin II to yield the overlapping C terminal peptide, and efficient manual sequencing of this peptide using benzene extractions to minimize extractive losses of peptide.     

Subpopulations of human T lymphocytes. X. Alterations in T, B, third population cells, and T cells with receptors for immunoglobulin M (Tmu) or G (Tgamma) in aging humans.

Peripheral blood from 120 healthy subjects, of whom 59 were young (35.5 +/- 9.6 years) and 61 aging (69.2 +/- 4.2 years), was examined for the proportions and numbers of lymphocyte populations, with a battery of surface markers. Absolute lymphocyte count and T,B and third population cells were comparable in both groups. Tgamma cell proportions were significantly (p less than 0.001) increased in aging subjects when compared with the young subjects. However, this difference was more significant (p less than 0.001) when aged females were compared with the young females as compared to aged males vs young males (p less than 0.05). When data were anlayzed for absolute numbers of Tmu and Tgamma cells, a similar significant decrease in Tmu, and increase in Tgamma cells were observed. Interestingly, when these data were anlayzed according to gender, significant differences in Tmu and Tgamma cell number were observed between young and old females but not between young and old men. Implications of these results are discussed.     

Radiosensitivity of T and B lymphocytes. IV. Effect of whole body irradiation upon various lymphoid tissues and numbers of recirculating lymphocytes.

Groups of 10-week-old female CBA/J mice were exposed in whole body fashion to 0,5,50, and 500 rads and sacrificed in serial fashion 1,3,5,7,9,15, and 30 days after irradiation for morphologic evaluation of thymus, spleen, lymph node, and Peyer's patch, and assessment of the relative numbers of thymus-derived (T) and bone marrow-derived (B) cells in these tissues. The absolute and relative numbers of recirculating T and B cells mobilizable by thoracic duct cannulation were also determined and compared with similar determinations with respect to peripheral blood lymphocytes. B cell depletion occurred more quickly and was more pronounced in spleen and lymph node than T cell depletion at all three exposure doses. Depletion of T and B cells was roughly equal in peripheral blood and thoracic duct lymph. When present, regeneration of the T cell component occurred more rapidly than did B cell restoration. The latter often was incomplete at the time of the final sacrifice (day 30). PHA-responsive and Con A-responsive cells also appeared to differ with respect to the kinetics of cell death after whole body irradiation.     

Human B cell development. II. Subpopulations in the human fetus

In man, during fetal development the B cell populations show distinct phenotypes at different tissue sites. The pre-B and B lymphocytes of the fetal liver and bone marrow express IgM and B cell markers, B1 (CD20) and BA-1 (CD24). These "early" cells are negative with a number of other reagents, anti-IgD, RFB4 (CD22), RFB6 (CD21), and RFA-2, which on the other hand recognize peripheral B cells. These peripheral B lymphocytes in the developing fetus are heterogeneous. The diffusely distributed B cells in the earliest lymph node samples, 16 to 17 wk of gestational age, and from 16 to 21 wk in the spleen, are strongly IgM+ (IgD+,RFB4+,RFB6+, and RFA-2+) but lack T cell-associated markers such as T1 (CD5, p 67,000 dalton equivalent of murine Ly-1) and Tü-33. In fetal lymph nodes, primary nodules develop around the follicular dendritic (FD) cells from 17 wk onward, and contain a virtually pure population of B cells; B1+,BA1+,RFB4+,RFB6+,RFA-2+, which simultaneously express IgM,IgD together with T1 (CD5), a T cell-associated antigen. A sizeable subpopulation of these IgM+,T1+ cells are also positive for Tü-33, another T cell-associated marker. In the spleen, the B cells of the IgM+,IgD+,T1+ type appear in smaller numbers and only relatively late around wk 22. These cells are diffusely distributed at first, and start accumulating around the small FD cell clusters as soon as these emerge about the 23rd gestational wk. At that time, the IgM+,T1+B cells can also be washed out from the peritoneal and pleural cavities. The T1+,IgM+B cells may represent the normal equivalent cells of B chronic lymphoid leukemia and centrocytic lymphoma, and appear to be the counterpart of Ly-1+,IgM+B cells in the mouse.     

Class II MHC self-antigen presentation in human B and T lymphocytes

Human CD4(+) T cells process and present functional class II MHC-peptide complexes, but the endogenous peptide repertoire of these non-classical antigen presenting cells remains unknown. We eluted and sequenced HLA-DR-bound self-peptides presented by CD4(+) T cells in order to compare the T cell-derived peptide repertoire to sequences derived from genetically identical B cells. We identified several novel epitopes derived from the T cell-specific proteome, including fragments of CD4 and IL-2. While these data confirm that T cells can present peptides derived from the T-cell specific proteome, the vast majority of peptides sequenced after elution from MHC were derived from the common proteome. From this pool, we identified several identical peptide epitopes in the T and B cell repertoire derived from common endogenous proteins as well as novel endogenous epitopes with promiscuous binding. These findings indicate that the endogenous HLA-DR-bound peptide repertoire, regardless of APC type and across MHC isotype, is largely derived from the same pool of self-protein.     

Subpopulations of splenic T and B lymphocytes producing and regulating leukocyte adherence inhibition factor

Picryl chloride induces contact hypersensitivity in mice, accompanied by spleen cell sensitization that is demonstrable in vitro by specific antigen-induced formation of leukocyte adherence inhibition factor (LAIF). This cellular activity was detected only up to 7 days after sensitization; thereafter the spleen cells appeared to be unreactive with the antigen. The cells were still normally reactive with the mitogen concanavalin A. Antigen reactivity of such “late” cells was restored by passage through a glass-bead column (provided resulting nonadherent cells were reconstituted with normal macrophages), and the restored reactivity was again suppressed by the eluted glass-bead-adherent cells. Suppression was antigen specific. Separation of T and B lymphocytes by affinity chromatography, after glass-bead treatment of sensitized spleen cells, showed that two subpopulations of B cells—those responsible for producing LAIF as well as those suppressing LAIF production by T cells—were glassbead adherent. This was extended by showing directly with anti-Thy-1.2 serum that B cells producing LAIF and suppressor T cells were glass adherent. Thus two suppressive cell populations, and the B cell producing LAIF, were glass adherent while the T-cell LAIF producer was not. Tests for adoptive transfer of cutaneous hypersensitivity in vivo demonstrated the relevance of many of the above observations to conditions in the whole animal. “Late” spleen cells from sensitized mice could not transfer hypersensitivity but this property was restored by glass-bead passage. The eluted adherent cells suppressed transfer. Both adoptive transfer and its suppression were antigen specific.     

Distinctive functional properties of human blood L lymphocytes: a comparison with T lymphocytes, B lymphocytes, and monocytes.

Human blood lymphocytes with high affinity Fc receptors have been operationally named L lymphocytes because of membrane-labile IgG markers. L lymphocytes lack membrane-incorporated immunoglobulin and do not form rosettes with sheep red blood cells coated with IgM antibody and mouse complement. These lymphocytes are capable of binding IgG in normal human serum at 4 degrees C and will form rosettes with human lymphocytes coated with Ripley IgG. In this study, functional in vitro properties of isolated L lymphocytes were compared with T lymphocytes, B lymphocytes, and monocytes. To obtain these mononuclear populations, first, plastic adherent monocytes were harvested. T lymphocytes were then isolated by centrifugation of E rosette-forming cells, and other rosetting techniques were employed to isolate L and B lymphocytes by negative selection. The functional properties of L lumphocytes were completely unlike those of T cells, B cells, or monocytes. L lymphocytes did not proliferate in response to mitogens, soluble antigens, or cell surface antigens. Moreover, this population could not replace monocytes in helping T lymphocytes respond to concanavalin A and pokeweed mitogen. Once T cells were supplemented with monocytes, however, the addition of L lymphocytes to the culture greatly enhanced the T lymphocytes proliferative response to phytohemagglutinin, concanavalinA, purified protein derivative (PPD), and streptokinase/streptodornase. L lymphocytes were not a subset of B cells. They did not spontaneously develop surface Ig in culture, and pokeweek mitogen could not induce them to transform and generate cytoplasmic Ig detectable by immunofluorescence. Mixtures of B cells and T cells responded to pokeweed mitogen better than do T cells alone. In contrast, enhanced reactivity with L and T cell combinations was not observed. Another sharp difference between these two populations was the stimulator capacity of each in mixed lymphocyte culture. When B and L lymphocytes were carefully monocyte-depleted, only B cells were effective stimulators of autologous and allogeneic lymphocytes. In comparison with T cells, B cells, and monocytes, L lymphocytes were the only effective killers of human blood lymphocytes sensitized with IgG. L lymphocytes, then, have cytotoxic potential, but cannot proliferate in response to various stimulants or become antibody-producing cells. These findings suggest that L lymphocytes comprise a third lymphocyte population.     

Serologically defined subpopulations of human T lymphocytes.

Several rabbit antisera to T cells obtained from various sources (thymus, peripheral blood, brain, T-derived leukemias) were studied with the aim to obtain reagents specific for a subset of T cells. Sera were first absorbed on human tissues and B cells; thereafter these T cell-specific sera were additionally absorbed with T cells of different origin and especially with leukemic T cells, which are likely to represent a clonal expnasion of a subset of T cells with potentially unique antigenic markers. Three antigenically distinct subpopulations of T cells were delineated. The relationship of these subsets with previously defined human T cell subpopulations (T subsets with a receptor for the Fc or IgG or IgM or with a receptor for a lectin from wheat germ agglutinin) was investigated.     

Chromosomal proteins in human B and T lymphocytes.

Histones and non-histone chromosomal proteins were characterized in B and T human lymphocytes by means of polyacrylamide disc gel electrophoresis. It was found that while histones do not present appreciable differences in the two examined populations, non-histone chromosomal proteins exhibit distinct electrophoretic profiles. Low molecular weight proteins predominate in B lymphocytes whereas high and intermediate proteins are largely represented in T lymphocytes. The latter proteins may be related to the capability of these resting cells to proliferate under appropriate antigenic stimuli.     

Activated and memory T lymphocytes in human milk.

Since activated macrophages and cytokines are found in human milk (HM), a flow cytometry study was conducted to determine whether T cells in HM display phenotypic markers of recent or previous activation. HM was collected during the first 3 d of lactation. The Paint-a-Gate program was used to optimize gating on the lymphocyte population. A mean +/- 1 SD of 4 +/- 3% of total HM leukocytes were lymphocytes and 96 +/- 3% were macrophages and granulocytes (N = 33 subjects). HM lymphocyte populations were further analyzed in five subjects. T cells (CD3+) represented 83 +/- 11% and B cells (CD19+) were 6 +/- 4% of HM lymphocytes. The mean CD4/CD8 ratio of T cells in HM was 0.88 (range 0.40-1.25). This ratio was significantly decreased compared to the peripheral blood (PB) of control adults (P less than 0.02) and postpartum women (P less than 0.02), due mostly to a significant increase in CD8+ CD3+ cells in HM compared to the PB of control adults (P less than 0.002) and postpartum women (P less than 0.05). T cells bearing markers of recent activation were significantly increased in HM compared to the PB of control adults: 85 +/- 7% of CD3+ cells in HM were HLA-DR+ (controls, 10 +/- 4%; P less than 0.001), and 15 +/- 6% of CD3+ cells in HM were IL-2R+ (controls, 6 +/- 2%; P less than 0.001).(ABSTRACT TRUNCATED AT 250 WORDS)