The epidemiological analysis of monitoring of the immune status in liquidators of consequences of the Chernobyl accident for early identification of risk groups and diagnostics of oncological diseases. Report 2. Dependence of frequency and changes in the immune status on risk factors of radiation accident

Malignant neoplasms (MN) have been found to develop most frequently in the liquidators of entry into the ChNPP zones in 1986 (43.75%), as well as among the liquidators who worked for long, one quarter of whom participated in liquidation of the consequences of failure (LCF) in 1986. Specific features of the immune status depending on the timing of participation in LCF and the year of entry into the ChN PP zone have been established. Changes in the immune system in the persons with a confirmed diagnosis of MN who took both a non-permanent and permanent part in liquidating the consequences of the ChNPP failure in 1986 had the same character of deviations and differed in the magnitudes of deviations of immunological parameters. Continuous participation in the period of extreme conditions and a greater exposure to the radiation factor led to the increased content of CD8(+)-T-cells, CD16(+)-lymphocytes and activated T-lymphocytes, as well as to the reduced index of immune regulation, decreased content ofCD3-16/56+(NK)-cells (%) and the total IgE and to a greater deficiency of B-lymphocytes. Distinctions in the groups of liquidators who participated in LCF in 1986 and 1987 have been revealed. The greatest deviations in the IS indicators were found in liquidators-87. A similar effect came to light in case of a continuance in the ChNPP zones in 1986 and 1987; however, the degree of deviation of the content of CD4(+)-T-lymphocytes (41), CD8(+)-T-lymphocytes (1) and the immune regulation index (41) were remarkably higher in liquidators-87. A continuous stay in the ChNPP zones in 1987 led to the deficiency of CD4(+)-T-lymphocytes, increased values of CD8(+)-T-lymphocytes, a decreased index of CD4+/CD8+, as well as to the change in the ratio between NK-T and NK cells, increased numbers of CD95+, HLA-DR+ and activated T-lymphocytes, and a lower level of the total IgE. Long-term participation in LCF didn't cause any enhanced expression of cellular activation markers in liquidators-86. Specific features of changes in IS depending on a dose of external gamma-irradiation have been established. Increase in the frequency of MN among liquidators, in relation to the number of examinees in each age group, with age has been revealed. Distinctions in the age dynamics of IS in liquidators in the presence and in the absence of MN manifested themselves in a stable level of values of CD3+, CD4+, CD8(+)-T-lymphocytes, immune regulation index, CD95+, serum IgA at the age between 40 and 70 years old with a subsequent reduction in indicators and increase in the content of CD8(+)-T-lymphocytes with age in the absence of MN; continuous increase of CD3-16/56(+)-NK-cells in the presence of MN and decrease in the values after 70 in the absence of MN. Also revealed in IS of the both age groups of liquidators over 70 with and without MN was the deficiency of the T-cell component (CD3+, CD4(+)-T-lymphocytes, CD4+/CD8+ index) and the increase in absolute values of CD8(+)-T-lymphocytes. The growing deficiency of CD4(+)-T-lymphocytes during monitoring against the background of ever rising values of CD8(+)-T-lymphocytes leading to the weakening of the immune regulation due to progressing disorders of the T-lymphocyte regulatory subpopulation distribution can serve an indicator for the adverse prognosis of the life expectancy in the presence of MN.     

Expression of T cell receptor-alpha and -beta subunits in human thymocytes. An immunocytologic study

The expression of the T cell receptor (Ti)-alpha and -beta subunits in human thymocytes was studied with the use of two rabbit antisera directed at constant regions of human Ti-alpha- and Ti-beta-chains (H36 and H38, respectively). Immunoperoxidase techniques were employed to count by light and electron microscopy the cells, in the various thymocyte subsets, bearing Ti-alpha and Ti-beta subunits. Of the unfractionated thymocytes, 88% +/- 5 SD and 56% +/- 8 SD were labeled by H38 and H36, respectively. More than 90% of cells in cluster of differentiation (CD)-1+ (mainly cortical) and CD1-CD3+ (mainly medullary) subsets were stained with H38. When tested by H36, 51% +/- 6 SD of CD1+ and 81% +/- 8 SD of CD1-CD3+ thymocytes were positive. In the immature CD3-CD1- subpopulation, less than 3% of cells reacted with H36, whereas 15% +/- 3 SD were stained by H38. Flow cytometry revealed that CD1+ (mainly cortical) thymocytes expressed CD3 surface antigen in a percentage similar to that of CD1+ cells positive for Ti-alpha subunits. Indirect double labeling procedures with immunogold- and peroxidase-conjugated second antibodies demonstrated that almost all CD1+/Ti-alpha + cells expressed the surface CD3 antigen, whereas a large proportion of CD1+/Ti-beta + cells did not. These results indicate a sequential expression of Ti-beta and Ti-alpha subunits during intrathymic T cell differentiation. They also suggest that assembly and surface expression of the CD3-Ti complex are linked to the production of Ti-alpha-chains in addition to Ti-beta subunits. Last, the expression of Ti-alpha and Ti-beta subunits was studied in peanut agglutinin (PNA)+, CD1+ blasts representing the main, spontaneously proliferating intrathymic pool. The lack of Ti-alpha and Ti-beta subunits and the absence of surface CD3 antigen on most of these blasts suggest that immature T cells are compelled to proliferate in the thymus in a CD3-Ti complex independent manner.     

Expression of variable exon A-, B-, and C-specific CD45 determinants on peripheral and thymic T cell populations.

A mAb (I/24) has been generated that is specific for a determinant on mouse CD45 molecules. Reactivity of this mAb with a panel of CD45 transfected cell lines demonstrated that the determinant recognized is dependent upon expression of one or more CD45 variable exons and that exon C is sufficient for its expression. The exon C-specific epitope detected by I/24 is expressed at high density on essentially all B lymphocytes and at an intermediate density on the vast majority of CD8+ splenic T cells. Two distinct subpopulations of CD4+ splenic T cells were detected, a minor subpopulation that expresses this exon determinant at high density and a major subpopulation that expresses it at a much lower density. This first identification of a CD45RC-specific reagent allowed a comparison of the expression of exon A-, exon B-, and exon C-specific determinants on peripheral and thymic lymphoid populations. When splenic lymphocytes were analyzed for expression of CD45RA (reactive with mAb 14.8), CD45RB (reactive with mAb 23G2 or mAb 16.A), and CD45RC (reactive with mAb I/24) determinants, it was found that each of these CD45 determinants had a distinct pattern of expression on CD4+ and CD8+ T cells and B cells. CD45RB and RC epitopes were also detected at high density on a small proportion (0.7 to 4.1%) of thymocytes. Both CD45RB and RC epitopes were found predominantly on CD4-CD8- and CD4-CD8+ thymocytes but were also found on small numbers of CD4+CD8+ and CD4+CD8- cells. The population of thymocytes that expressed CD45RB and CD45RC determinants displayed a novel TCR CD3 phenotype characterized by a level of expression that was intermediate between that seen in the larger CD3 bright and CD3 dull populations of thymocytes.     

Natural killer cells in patients with pulmonary tuberculosis]

In the existent literature the number of works devoted to the subpopulation of natural killer cells (NK) is not significant. The purpose of the present study was to determine the NK content in the blood of pulmonary tuberculosis patients; to establish correlation of the level of their content with the content of the previously studied T-lymphocyte subpopulations; to determine the intensity of the fluorescence of NK, CD3+, CD4+, CD8+ lymphocytes. The data were obtained on the significant increase in the NK mean level in tuberculosis patients (20.37 +/- 1.74) as compared with that in healthy subjects (12.77 +/- 2.56). The NK fluorescence intensity (56.33 +/- 2.28) conditioned by the Fc-receptor expression intensity is significantly lower than the analogous index in healthy volunteers (82.4 +/- 7.69). The NK level in the blood of tuberculosis patients correlates with the content of CD3+, CD8+ lymphocytes as well as with the CD4+/CD8+ index.     

The subpopulation structure of human T-lymphocytes studied by using a monoclonal antibody (MCA) to CD27]

The obtained murine mAb LT27 (IgG2a) assigned to the cluster of differentiation CD27 was used to study the distribution of antigen CD27 among human lymphocytes scbpopulations in normal state and immunopathology. In normal donors the antigen CD27 was found to be expressed most frequently on CD4+ cells (90 +/- 8% of which coexpressed antigen CD27) and to the lesser extent on- CD8+ cells (only 77 +/- 28% of CD8+ cells carried antigen CD27). 79 +/- 12% of double negative lymphocytes (CD3+CD4-CD8-) expressed antigen CD27. In patients with hypogammaglobulinemia the proportion of CD4+CD27+/CD4+ and CD8+CD27+/CD8+ was significantly reduced to 80 +/- 11% (p < 0.01) and 45 +/- 19% (p < 0.001), respectively. The ratio CD4+CD27+/CD4+ varied insignificantly with the increase of CD4+ population, but the increase of the CD8+ population was accompanied by the definite tendency to a decrease of the ratio CD8+CD27+/CD8+. The distribution of CD27 antigen inside CD4+ and CD8+ subpopulations was found to be different from the distribution of CD29 and CD45RA antigens.     

Clonal analysis of human CD4-CD8-CD3- thymocytes highly purified from postnatal thymus.

Human triple-negative (CD4-CD8-CD3-) thymocytes purified from postnatal thymus by the use of magnetic bead columns and cell sorting were cultured in bulk or cloned with a feeder cell mixture of irradiated PBL, irradiated JY cells, and PHA. Triple-negative thymocytes proliferated well under these culture conditions, and after 12 days in bulk culture they remained triple negative. Limiting dilution experiments revealed that the frequency of clonogenic cells in fresh triple-negative thymocytes was less than 1%. Of 40 clones obtained in a representative experiment, 37 were triple negative and 3 were CD4+ TCR-alpha beta+. No TCR-gamma delta+ clones were isolated. Some of the triple-negative clones expressed CD16 and were apparently NK cells. Seven representative CD16-triple-negative clones were expanded and characterized in detail. These clones shared the common cell surface phenotype of CD1-CD2+CD3-CD4--CD8-CD5-CD7+CD16-CD56+. One of them expressed cytoplasmic CD3 delta and CD3 epsilon Ag, but these Ag were not detected in any peripheral blood-derived CD16- NK clones examined for comparison. The seven CD16- thymus-derived clones exhibited significant cytolytic activity against K562. The clone that expressed cytoplasmic CD3 Ag was shown to have the germ-line configuration of the TCR-beta and TCR-gamma genes. Thus, it is suggested that in vitro culture of triple-negative thymocytes can give rise to NK-like cells, including those that express cytoplasmic CD3 Ag. In contrast to previous reports, our results gave no evidence of differentiation of triple-negative thymocytes into TCR-alpha beta+ or TCR-gamma delta+ T cells.     

Critical role for IL-4 in the development of transplant arteriosclerosis in the absence of CD40-CD154 costimulation.

Blockade of the CD40-CD154 pathway can inhibit CD4(+) T cell activation but is unable to prevent immune responses mediated by CD8(+) T cells. However, even in the absence of CD8(+) T cells, inhibition of the CD40-CD154 pathway is insufficient to prevent the development of transplant arteriosclerosis. This study investigated the mechanisms of transplant arteriosclerosis in the absence of the CD40 pathway. C57BL/6 CD40(-/-) (H2(b)) recipients were transplanted with MHC-mismatched BALB/c (H2(d)) aortas. Transplant arteriosclerosis was evident in both CD40(-/-) and CD40(+/-) mice (intimal proliferation was 59 +/- 5% for CD40(-/-) mice vs 58 +/- 4% for CD40(+/-) mice) in the presence or absence of CD8(+) T cells (intimal proliferation was 46 +/- 7% for CD40(-/-) anti-CD8-treated mice vs 50 +/- 10% for CD40(+/-) anti-CD8-treated mice), confirming that CD8(+) T cells are not essential effector cells for the development of this disease. In CD40(-/-) recipients depleted of CD8(+) T cells, the number of eosinophils infiltrating the graft was markedly increased (109 +/- 24 eosinophils/grid for CD40(-/-) anti-CD8-treated mice vs 28 +/- 7 for CD40(+/-) anti-CD8-treated mice). The increased presence of eosinophils correlated with augmented intragraft production of IL-4. To test the hypothesis that IL-4 was responsible for the intimal proliferation, CD8 T cell-depleted CD40(-/-) recipients were treated with anti-IL-4 mAb. This resulted in significantly reduced eosinophil infiltration into the graft (12 +/- 5 eosinophils/grid for CD40(-/-) anti-CD8(+), anti-IL-4-treated mice vs 109 +/- 24 for CD40(-/-) anti-CD8-treated mice), intragraft eotaxin, CCR3 mRNA production, and the level of intimal proliferation (18 +/- 5% for CD40(-/-) anti-CD8(+)-, anti-IL-4-treated mice vs 46 +/- 7% for CD40(-/-) anti-CD8-treated mice). In conclusion, elevated intragraft IL-4 production results in an eosinophil infiltrate and is an important mechanism for CD8(+) T cell-independent transplant arteriosclerosis in the absence of CD40-CD154 costimulation.     

Analysis of the functional capabilities of CD3+CD4-CD8- and CD3+CD4+CD8+ human T cell clones

The functional capabilities of human peripheral blood CD3+CD4-CD8- and CD3+CD4+CD8+ T cell clones were examined. The clones were generated by culturing purified populations of CD3+CD4-CD8- and CD3+CD4+CD8+ T cells at limiting dilution (0.3 cell/well) in the presence of PHA, rIL-2, and irradiated PBMC as feeders. Twelve CD3+CD4-CD8- and 5 CD3+CD4+CD8+ clones were generated. Clonality was documented by analyzing TCR gamma- and beta-chain rearrangement patterns. All CD3+CD4-CD8- clones were stained by the TCR-delta 1 mAb that identifies a framework epitope of the TCR delta-chain, but not by mAb WT31 that identifies the TCR-alpha beta on mature T cells. In contrast, the CD3+CD4+CD8+ clones were all stained by WT31 and not by TCR-delta 1. All 17 clones were screened for various functional activities. Each secreted IL-2, IFN-gamma, and lymphotoxin/TNF-like factors when stimulated with immobilized mAb to CD3 (64.1), albeit in varying quantities. These clones secreted far less IL-2 and IFN-gamma than CD3+CD4+CD8- or CD3+CD4-CD8+ alpha beta expressing clones, but comparable amounts of lymphotoxin/TNF. All clones also functioned as MHC-unrestricted cytotoxic cells. This activity was comparable to that mediated by the CD3+CD4+CD8- or CD3+CD4-CD8+ alpha beta clones. Nine of 12 CD3+CD4-CD8- and 4 of 5 CD3+CD4+CD8+ clones were able to support B cell differentiation when activated by immobilized anti-CD3, but usually not as effectively as the CD3+CD4+CD8- or CD3+CD4-CD8+ alpha beta clones. The differences in the functional capabilities of the various clones could not be accounted for by alterations in the signaling capacity of the CD3 molecular complex as mAb to CD3 induced comparable increases in intracellular free calcium in each clone examined. When clones were stimulated with PWM, each suppressed B cell differentiation supported by mitomycin C-treated fresh CD4+ T lymphocytes. Suppression was dependent on the number of clone cells added to culture, but could be observed with as few as 12,500 cells per microtiter well. Phenotypic analysis of the clones revealed that all expressed CD29, CD11b, and the NKH1 surface Ag. These results demonstrate that the CD3+CD4-CD8- and CD3+CD4+CD8+ T cell clones exhibit many of the functional characteristics of mature T cells, although they produce IL-2 and IFN-gamma and provide help for B cell differentiation less effectively than CD3+CD4+CD8- and CD3+CD4-CD8+ alpha beta T cell clones.     

Leucoagglutinin induces differential proliferation of lymphocyte subsets

In this study we have established culture conditions that allow the preferential and rapid expansion of either T cell receptor (TCR)+/CD3+16- T lymphocytes or TCR-/CD3-16+ natural killer (NK) cells, or the non-selective outgrowth of both subsets. Optimal proliferation of lymphocytes was obtained using a combination of irradiated allogeneic peripheral blood lymphocytes (PBL) and irradiated Epstein Barr virus (EBV) transformed lymphoblastoid B cell lines (B-LCL). Addition of 1 microgram/ml leucoagglutinin to the culture medium induced a preferential outgrowth of TCR+/CD3+16- T lymphocytes. The proportion of TCR-/CD3-16+ NK cells was decreased to 5% or less, although still a 2000-fold multiplication of TCR-/CD3-16+ NK cells was obtained at day 13. Without leucoagglutinin a 1000-fold increase of about 70% pure TCR-/CD3-16+ NK cells was obtained at day 13. Intermediate concentrations of leucoagglutinin (0.1-0.3 micrograms/ml) resulted in a non-selective expansion of both NK cells and T cells. Irrespective whether leucoagglutinin was added or not, the number of TCR+/CD3+8+ lymphocytes increased more rapidly relative to the TCR+/CD3+4+ lymphocytes resulting in an increased TCR+/CD3+8+ population size. Also under limiting dilution conditions leucoagglutinin increased the frequency of proliferating cells. In contrast to the preferential outgrowth of TCR+/CD3+8+ lymphocytes in bulk cultures, approximately 80% of the clones generated was TCR+/CD3+4+, demonstrating a growth promoting effect of TCR+/CD3+4+ lymphocytes on TCR+/CD3+8+ lymphocytes in PBL bulk cultures.     

Characterization of porcine T lymphocytes and their immune response against viral antigens.

T lymphocytes play a central role in the antigen-specific immune response against various pathogens. To detect and to characterize porcine T lymphocytes, monoclonal antibodies (mAb) against leukocyte differentiation antigens had been raised and classified for their specificity. Analyses of porcine T lymphocytes with specific mAb against CD4 and CD8 differentiation antigens revealed differences in the composition of the porcine T-lymphocyte population compared to other species. In addition to the known subpopulations, CD4+CD8- T helper cells and CD4-CD8+ cytolytic T lymphocytes, extra-thymic CD4+CD8+ T lymphocytes and a substantial proportion of CD2-CD4-CD8- T cell receptor (TcR)-gamma delta+ T cells could be detected in swine. Functional analyses of porcine T-lymphocyte subpopulations revealed the existence of two T-helper cell fractions with the phenotype CD4+CD8- and CD4+CD8+. Both were reactive in primary immune responses in vitro, whereas only cells derived from the CD4+CD8+ T-helper-cell subpopulation were able to respond to recall antigen in a secondary immune response. With regard to T lymphocytes with cytolytic activities, two subsets within the CD4-CD8+ T-cell subpopulation could be defined by the expression of CD6 differentiation antigens: CD6- cells which showed spontaneous cytolytic activity and CD6+ MHC I-restricted cytolytic T lymphocytes including virus-specific cytolytic T lymphocytes. These results enable now a detailed view into the porcine T-cell population and the reactivity of specific T cells involved in the porcine immune response against pathogens. Furthermore this knowledge offers the possibility to investigate specific interactions of porcine T lymphocytes with virus-specific epitopes during vaccination and viral infections.     

Role of a thymic stromal cell clone in inducing the stage-specific differentiation of various subpopulations of double negative thymocytes

The monolayer of a thymic stromal cell clone termed MRL104.8a induced the differentiation of adult double negative (DN) thymocytes (CD3-4-8-) through a CD3-4-8+ intermediate into CD3- (or dull) 4+8+ stages. DN thymocytes were separated into three subpopulations depending on their cell-surface expression of Pgp-1 and IL-2R, namely, Pgp-1+IL-2R-, Pgp-1-IL-2R+, and Pgp-1-IL-2R-. The present study investigated the requirements of the MRL104.8a monolayer for inducing the differentiation of these DN thymocyte subpopulations. The following were revealed: i) the MRL104.8a monolayer failed to induce the differentiation of a Pgp-1+IL-2R- subpopulation; ii) whereas a Pgp-1-IL-2R+ subpopulation did not express either CD4 or CD8 Ag when cultured in medium, culturing this subpopulation on the thymic stromal cell monolayers resulted in the expression of CD8 but not CD4 Ag; and iii) a Pgp-1-IL-2R- DN subpopulation obtained through less extensive treatments with anti-CD4 and anti-CD8 antibodies in the presence of C before sorting procedures spontaneously differentiated into double positive cells in medium. In contrast, most of DN cells with the same phenotype obtained through extensive anti-CD4 and -CD8 treatments before sorting failed to express CD4 and/or CD8 Ag in medium but could differentiate through a CD3-4-8+ into more mature stages only when they were cultured on the thymic stromal monolayer. These results indicate differential requirements of thymic stromal cells for the differentiation of various DN subpopulations with qualitatively distinct phenotypes and different magnitudes (very low vs almost zero levels) of CD4/CD8 expression.     

Tumor-selective cytolysis is executed exclusively by CD45R+ CTL whereas allo-specific cytotoxicity can be executed also by CD45R- CTL

Naive and memory CD4+ T helper cells can be distinguished on the basis of expression of the CD45R molecule. Whether this dichotomy applies also to CD8+ T cells has not yet been established. In the present investigation the cytolytic activity of peritoneal CD8+CD45R+ and CD8+CD45R- T cells from tumor- and allo-immunized rats has been studied. More than 90% of the CD8+ peripheral blood T lymphocytes expressed the CD45R molecule, whereas in the peritoneal cavity about 60% of the CD8+ T cells displayed the CD45R+ phenotype. Analysis of cytotoxicity of sorted peritoneal cells of W439 tumor-immunized donors demonstrated selective cytolytic activity of the CD5+CD4-CD8+CD45R+ subpopulation to W439 lymphoma target cells but no effect of CD5+CD4-CD8+CD45R- lymphocytes. None of these lymphocyte populations exhibited cytolytic activity to the NK-sensitive cell line YAC-1, whereas the CD5-CD45R+ population showed strong cytotoxicity to YAC-1 cells. In allo-immunized rats both CD5+CD4- CD8+CD45R+ and CD5+CD4-CD8+CD45R- peritoneal cells exhibited strong allo-specific cytolytic activity, but no activity to YAC-1 cells. Both CD5+CD4-CD8+CD45R+ and CD5+CD4-CD8+CD45R- cells from tumor-immunized rats proliferated in response to Con A and rIL-2. This is the first study demonstrating that tumor-selective cytolytic CD8+ T cells express the CD45R molecule and that allo-specific cytolytic CD8+ T cells are found in both the CD45R+ and CD45R- populations.     

A model for the origin of TCR-alphabeta+ CD4-CD8- B220+ cells based on high affinity TCR signals

The origin of TCR-alphabeta+ CD4-CD8- cells is unclear, yet accumulating evidence suggests that they do not represent merely a default pathway of unselected thymocytes. Rather, they arise by active selection as evidenced by their absence in mice lacking expression of class I MHC. TCR-alphabeta+ CD4-CD8- cells also preferentially accumulate in mice lacking expression of Fas/APO-1/CD95 (lpr) or Fas-ligand (gld), suggesting that this subset might represent a subpopulation destined for apoptosis in normal mice. Findings from mice bearing a self-reactive TCR transgene support this view. In the current study we observe that in normal mice, TCR-alphabeta+ CD4-CD8- thymocytes contain a high proportion of cells undergoing apoptosis. The apoptotic subpopulation is further identified by its expression of B220 and IL2Rbeta and the absence of surface CD2. The CD4-CD8- B220+ phenotype is also enriched in T cells that recognize endogenous retroviral superantigens, and can be induced in TCR transgenic mice using peptide/MHC complexes that bear high affinity, but not low affinity, for TCR. A model is presented whereby the TCR-alphabeta+ CD2- CD4-CD8- B220+ phenotype arises from high intensity TCR signals. This model is broadly applicable to developing thymocytes as well as mature peripheral T cells and may represent the phenotype of self-reactive T cells that are increased in certain autoimmune conditions.     

CD4-CD8- human T cells: phenotypic heterogeneity and activation requirements of freshly isolated "double-negative" T cells

In the present study we have analyzed the in vitro activation requirements of freshly isolated CD4-CD8- "double-negative" (DN) human peripheral blood T cells. DN cells were isolated from E+ cells by removal of CD4+, CD8+, and CD16+ cells through consecutive steps of C'-mediated lysis and panning. While the majority (79.0 +/- 12.0%) of DN cells were TCR gamma delta+ as shown by staining with mAb TCR delta-1, a minor fraction (6.7 +/- 4.7%) expressed TCR alpha beta as revealed by staining with mAb BMA031. Within the gamma delta+ DN fraction, most cells reacted with mAb Ti gamma A which delineates a V gamma 9JPC gamma 1 epitope, whereas a minor fraction stained with mAb delta TCS-1 which identifies a V delta 1J delta 1 epitope. Functional studies performed at low cell number (1000) per microculture indicated that DN cells can be activated by anti-CD3 mAb, PHA and allogeneic stimulator cells, provided that exogenous growth factors are supplied. Both rIl-2 and rIl-4 acted as efficient growth factors for DN cells, and a synergistic stimulatory effect of rIl-2 and rIl-4 was observed when DN cells were cocultured with allogeneic LCL stimulator cells. As compared to unseparated E+ cells, isolated DN responder cells had a reduced capacity to secrete Il-2 upon PHA stimulation in the presence of LCL feeder cells. The majority of DN cells maintained their CD3+ CD4-CD8- phenotype upon coculture with allogeneic LCL stimulator cells. These data demonstrate that CD3+ DN cells in human peripheral blood are heterogeneous with respect to TCR expression. In addition, they show that freshly isolated DN cells are deficient in Il-2 production but may be normally stimulated by anti-CD3, PHA, or alloantigen if exogenous growth factors (rIL-2 and/or rIl-4) are provided.     

Phenotypic and functional characterization of c-kit expression during intrathymic T cell development.

We have studied the expression and function of c-kit on subsets of mouse thymocytes. c-kit was primarily expressed on subpopulations of CD4-CD8-CD3- triple negative (TN) cells. The strongest c-kit expression was associated with subsets that represent the least mature TN cells, including CD44+CD25- TN, and a subpopulation of CD25+ TN. These cells were also Thy-1lo, H-2Khi TSA-1hi, HSAlo, B220-, Mac-1-, and Gr-1-. Additionally, the recently described pre-TN thymocyte population (CD4loCD3-CD8-) was also c-kit+. CD25+ TN thymocytes proliferated in the presence of IL-7 and stem cell factor (the ligand for c-kit), and this proliferation was completely inhibited in the presence of anti-c-kit. Furthermore, the addition of anti-c-kit to 2-deoxyguanosine-treated fetal thymic lobes undergoing reconstitution with fetal liver-derived precursor cells inhibited their T cell differentiation potential. These observations indicate an important role for c-kit/stem cell factor interactions during early thymocyte development.     

Reactivity of new adhesion molecules on lymphocytes from patients with chronic graft versus host disease

Reaction patterns of the 7th Human Leukocyte Differentiation Antigen Workshop blind panel adhesion molecules were studied on CD3/CD4, CD3/CD8, CD3/TCR gamma delta double positive T cells from peripheral blood of patients with chronic graft versus host disease (n = 8) and healthy controls (n = 4). Reactivity of 14 adhesion antibodies was tested by three-colour immunophenotyping. The mean proportion of CD3+ T cells (69 +/- 19%). CD3/CD8++ (31 +/- 13%) and CD3/TCR gamma delta++ (4 +/- 2%) T sub-populations of patients were comparable with the healthy controls. However, the mean percentage of CD3/CD4++ T cell subset in patients (14 +/- 12%) proved to be significantly decreased in comparison with the normal control value (34 +/- 16%) presumably due to secondary immunodeficiency. The workshop antibodies proved to be reactive with three T cell subsets expressing the examined antigens. Based on the results of the adhesion molecule workshop new CD categories have been introduced: CD156b as a transmembrane protein, CD167a as an epithelial tyrosin kinase receptor, CD168 as a receptor for hyaluronan mediated motility (RHAMM) and CD171 as a co-stimulatory adhesion molecule. There were significant differences in the expression of the CD167a and CD156b antigens on the CD3/CD4++ subset between the samples of patients compared with the controls characterizing the CD4+ T lymphocyte subpopulation in chronic graft versus host disease.     

Evidence for the existence of two parallel differentiation pathways in the thymus of MRL lpr/lpr mice.

MRL mice homozygous for the lpr/lpr gene develop a massive lymphadenopathy caused by the accumulation of CD4-CD8-, Thy-1-positive T cells that express B220. This phenotypically unusual T cell population coexists with normal, B220- T cells in lpr/lpr animals. To investigate the origin and differentiation pathway of B220+ T cells, the expression of a panel of developmentally regulated cell surface markers including TCR, CD4, CD8, Thy-1, and B220 was examined. Thymocytes and peripheral T lymphocytes from lpr/lpr mice were analyzed by four-color flow cytometry. The results showed that both B220+ and B220- thymocytes contained all of CD4-CD8-, CD4+CD8+, and CD4 or CD8 single positive T cell subpopulation in the lpr thymus. Expression of the V beta 11 TCR, measured by flow cytometry and reverse polymerase chain reaction, was demonstrated in lpr thymus. However, the number of T cells expressing V beta 11 was greatly reduced in both the B220+ and B220- T cell populations in lymph node, spleen, and liver. Taken together, the data provide evidence for maturation and selection of a distinct population of B220+ T cells in the thymus of MRL lpr/lpr mice.     

Human lymphokine-activated killer (LAK) cells: identification of two types of effector cells

We analyzed the antigenic phenotype of lymphokine-activated killer (LAK) effector cells. Human blood lymphocytes were cultured for 3 days with 100 U/ml recombinant interleukin 2 (rIL 2), subpopulations isolated with monoclonal antibodies and a fluorescence-activated cell sorter (FACS) and assayed for cytotoxic activity against 51chromium labeled noncultured melanoma tumor cells. Initial experiments compared the LAK effector function of CD5+ T lymphocytes vs CD5- cells (predominantly CD16+ NK cells). The mean percent specific release at a 10:1 effector:target (E:T) ratio was 25% +/- 16 for CD5- cells, 10% +/- 6 for CD5+ cells, and 22% +/- 9 for unsorted cells. In contrast, when lymphocyte subpopulations were isolated before rIL 2 culture (LAK precursors), CD5- cells but not CD5+ cells developed LAK activity (28% +/- 12 vs 1% +/- 1, mean percent specific release, 10:1 E:T ratio), confirming our previous results showing that only CD16+ cells were LAK precursors. The discrepancy between LAK effector and precursor phenotypes suggested that LAK precursors acquired CD5 determinants during rIL 2 culture; however, double label immunofluorescence of rIL 2 cultured CD16+ cells showed that this was not the case. The data suggested that in the presence of other cell types, some T lymphocytes may develop LAK activity, but purified blood T lymphocytes do not develop LAK function when cultured with rIL 2 alone. We also analyzed LAK effector function in lymphocyte subpopulations defined by CD4 and CD8 antigens. The data showed that lymphocytes with a low density expression of CD8 and no expression of CD4 were enriched for LAK effector cells, whereas CD4+ and CD8- had less activity than unsorted cells. Lymphocytes with a high density expression of CD8 had activity similar to unsorted cells. We also assessed the contribution of Leu-7 (HNK-1) granular lymphocytes to LAK effector function. After culture with IL 2, lymphocytes were depleted of Leu-7+ cells by antibody and complement treatment and then were sorted into CD5+ and CD5- fractions. The cytotoxic activity of Leu-7-CD5+ cells was a mean 5% +/- 5 vs a mean 14% +/- 8 for the total CD5+ population (20:1 E:T ratio). The activity of Leu-7- CD5- was slightly less than the total CD5- fraction (21% +/- 9 vs 28% +/- 14, 10:1 E:T ratio). In conclusion, LAK effector function was highest in non-T cell (CD5- CD16+) populations and some activity was also present in T cell populations (CD5+ and predominantly Leu-7+).     

Subpopulations of mature murine thymocytes: properties of CD4-CD8+ and CD4+CD8- thymocytes lacking the heat-stable antigen

The heat-stable antigen (HSA), recognized by the monoclonal antibodies M1/69, B2A2, and J11d, is low or absent on the surface of most murine peripheral T cells but present on all but 3% of thymocytes. The CD4-CD8+ and CD4+CD8- or "single positive" thymic populations may be divided into further subgroups based on surface HSA expression. One group, CD4-CD8+ and expressing very high levels of HSA (HSA++), is an immature, T cell antigen receptor (TcR) negative, outer cortical blast cell. However, a further subdivision of CD4-CD8+ and CD4+CD8- single positives may be made, into those negative to low for HSA (HSA-) and those expressing moderate amounts of HSA (HSA+). The proportion of HSA- single positives is low in the thymus of young mice, whereas the proportion of HSA+ single positives is similar to that of the adult. Both the HSA- and the HSA+ subsets of single positive thymocytes from adult mice are CD3+ and express the normal peripheral T cell incidence of V beta 8 determinants on the TcR. On stimulation with concanavalin A in limit-dilution culture both HSA- and HSA+ subsets of single positive thymocytes give a high frequency of proliferating clones, and the clones from both HSA- and HSA+ subsets of CD4-CD8+ thymocytes are cytotoxic. Thus both HSA- and HSA+ single positive thymocytes are functionally mature. The HSA- subsets of single positive thymocytes differ from the HSA+ subsets in being slightly larger in size, in expressing higher levels of MEL-14, in binding more peanut agglutinin, and in including a proportion of cells expressing high levels of the Pgp-1 glycoprotein. It is suggested that HSA- CD4-CD8+ and HSA- CD4+CD8- thymocytes are more mature than their HSA+ counterparts, and might represent a previously activated or "memory" thymic subpopulation.