Activation State Of Lymphocytes Research Articles

Hepatitis C virus screening and clinical monitoring of biomarkers in patients undergoing hemodialysis

In this study, 395 volunteers were enrolled to investigate the seroprevalence of hepatitis C virus, the immunological and the alanine aminotransferase (ALT) biomarkers amongst hemodialysis patients, living in Manaus, Brazil. An overall seroprevalence of 13.9% was found in the hemodialysis patients. Analysis of seroconversion patterns demonstrated that most patients with HCV seroconverted up to 10 years following the first hemodialysis session. Anti-NS5 antibody was detectable in 60.4% of patients with HCV. A lower percentage of circulating CD3(+) and CD4(+) T-cells was found in patients seronegative for HCV, whereas a higher frequency of CD8(+) T-cells was the hallmark of patients with HCV. An overall low activation state of monocytes and eosinophils were observed in hemodialysis patients. In contrast, a higher frequency of activated neutrophils was observed in patients with HCV, selectively in the NS5+ subgroup. All hemodialysis patients had a higher percentage of activated lymphocytes, with the higher activation state in patients with NS5- reactivity. Higher ALT levels were observed in patients with HCV, especially in the NS5+ subgroup. Interestingly, the ALT levels were correlated negatively with the lymphocyte activation state, selectively in the NS5- subgroup, suggesting a protective role of these activated lymphocytes in patients with HCV. These findings reinforce the importance of the transmission of HCV among hemodialysis patients, suggesting that apart from the HCV screening, the serological and ALT biomarkers may represent important predictors of morbidity and/or mortality among patients undergoing hemodialysis.

The Role of Apoptosis in the Ameliorating Effects of a CDR1-Based Peptide on Lupus Manifestations in a Mouse Model

Experimental systemic lupus erythematosus (SLE) can be induced in mice following immunization with an anti-DNA mAb expressing a major Id, 16/6Id. Treatment with a peptide, designated human CDR1 (hCDR1; Edratide), that is based on the sequence of CDR1 of the 16/6Id ameliorated disease manifestations. In the present study, we investigated the roles of apoptosis and related molecules in BALB/c mice with induced experimental SLE following treatment with hCDR1. A higher state of activation and increased rate of apoptosis were found in lymphocytes of SLE-afflicted mice as compared with healthy controls. The latter effects were associated with up-regulated caspase-8 and caspase-3, and down-regulated Bcl-x(L). The ameliorative effects of hCDR1 were associated with down-regulation of caspase-8 and caspase-3, up-regulation of Bcl-x(L), and a reduced rate of apoptosis. Treatment of diseased mice with an apoptosis-reducing compound that inhibited caspases down-regulated the secretion of the pathogenic cytokine IFN-gamma and lowered the intensity of glomerular immune complex deposits and the levels of proteinuria. Furthermore, coincubation of Bcl-x(L) inhibitors with hCDR1-treated cells abrogated the ability of hCDR1 to reduce the activation state of lymphocytes and to down-regulate the secretion of IL-10 and IFN-gamma. Moreover, the Bcl-x(L)-expressing CD4(+)CD25(+) cells from hCDR1-treated mice induced the expression of Bcl-x(L) in CFSE-labeled CD4(+)CD25(-) cells of the SLE-afflicted mice. Thus, the reduction of apoptosis and the up-regulation of Bcl-x(L), which plays an apparent role in tolerance induction, contribute to at least part of the beneficial effects of hCDR1 on lupus manifestations.

Identification of Urop11, a novel leptin-modulated gene that is upregulated in the hypothalamus of mice with virus-induced obesity

Obesity results from disturbances of tightly regulated interactions between the nervous, endocrine, and metabolic systems that can be caused by external factors, such as viral infections. A mouse model of obesity induced by brain infection with a morbillivirus, canine distemper virus, allowed us to identify obesity-related genes. Using a subtractive library for the hypothalamus, the main brain structure regulating energy homeostasis, we identified a new gene on mouse chromosome 19 which we named upregulated obese product (Urop) 11 and, which has no homology with any known mRNA. A step-by-step molecular approach allowed us to isolate the full-length mRNA, predict the protein sequence, and identify consensus sites. Urop11 was mainly detected in the hypothalamus and adipocytes, and was dramatically upregulated in these central and peripheral structures in obese mice. Urop11 was also expressed in human neural and lymphoid samples and its expression seemed to be regulated by the state of lymphocyte activation. Interestingly, Urop11 expression was strongly upregulated both in vivo in mouse hypothalamus and in vitro in mouse neural cell lines, after leptin treatment. Taken together, our data show that Urop11 is a target of leptin, the satiety factor produced by adipocytes, in physiological and pathological conditions, including obesity. This new gene can be considered a key molecule in the hypothalamic integration pathway and demonstrates the importance of Urop11 as a target of leptin action.

Kv1.3 channel redistribution in the immunological synapse varies according to the T lymphocyte activation state

Kv1.3 channel redistribution in the immunological synapse varies according to the T lymphocyte activation state

Expression of DHX32 in lymphoid tissues

DHX32 is a novel putative RNA helicase identified based on its downregulation in acute lymphoblastic leukemia. DHX32 gene has 12 exons, alternative usage of exons 1 and 2 results in the expression of two transcripts that differ in their 5′ untranslated region (UTR), consistent with the involvement of two different promoters. In addition, exon 5 skipping results in the production of a full-length and alternatively spliced transcripts. We used transcript-specific primers in reverse transcriptase (RT)-PCR to show that the thymus and spleen express all of DHX32 transcripts. We also used immunohistochemistry to study the expression of DHX32 protein. The expression pattern of DHX32 protein in normal lymphoid tissues is variable but suggests association with state of lymphocyte activation and/or differentiation. Dysregulated expression of DHX32 is noted in some types of lymphoma compared to their normal counterpart.

Lymphocytes from bipolar and schizophrenic patients share common biochemical markers related to histone synthesis and histone cell membrane localization characteristic of an activated state

In a previous communication, based on the total histone and histone variants’ synthesis rates, biochemical parameters used for the characterization of the activation state of lymphocytes, we showed that a portion of the lymphocyte population obtained from peripheral blood of patients with bipolar disorder in the manic and/or depressed phases of the illness were in an activated state as opposed to normothymic patients and control subjects whose lymphocytes are in a resting, Go, state. In light of these previous findings, in the present investigation, we have analyzed total histone synthesis rates and the H2A and H3 histone variants’ synthesis pattern of acid-extracted histones from the lymphocytes’ nuclear fraction obtained from control subjects, patients with bipolar disorder in all phases of the illness, and patients with schizophrenia. Additional biochemical parameters, such as total cellular protein and DNA synthesis rates, were also studied. Moreover, recent findings from other investigators showed the association of histones on the plasma membrane fraction of PHA-activated, but not Go resting lymphocytes. Based on these results, acid-extracted proteins from the plasma membrane fraction obtained from control, bipolar patients in all phases of the illness, and schizophrenic patients were analyzed by immunoblotting using a polyclonal histone antibody, anti-H2B. All biochemical parameters tested show that a portion of the lymphocyte population from bipolar, i.e. manic and depressive, as well as schizophrenic patients are in an activated state and clearly indicate that the unusual for lymphocytes cell cycle-related histone biochemical properties are common to both disorders.

Fas expression on peripheral blood lymphocytes in systemic lupus erythematosus (SLE): relation to lymphocyte activation and disease activity.

Levels of apoptotic lymphocytes have been found to be increased in SLE and persistence of apoptotic cells has been associated with autoantibody production. Increased lymphocyte Fas (CD95) expression due to lymphocyte activation may account for increased susceptibility to Fas-mediated apoptosis in SLE. Flowcytometry was performed to evaluate membrane expression of Fas in combination with the activation markers CD25, HLA-DR and CD38 on, respectively, CD4+, CD8+ and CD19+ lymphocytes of SLE patients with inactive (n = 20) and with active disease (n = 13). SLEDAI-scores were calculated. Healthy volunteers (n = 14) served as controls. Percentages of CD4+ T-cells expressing CD25 and CD19+ B-cells expressing CD38 were increased in patients with active disease compared to controls (P = 0.03, P = 0.04, respectively). In contrast to CD4+ and CD8+ cells, percentages of CD19+ cells expressing Fas were increased in SLE patients with active disease (P = 0.0002 vs controls). In these patients percentages of cells double positive for both CD38 and Fas were increased compared to patients with inactive disease (P = 0.006) and controls (P = 0.0007). Percentages of CD19+ cells expressing Fas correlated with SLEDAI-scores. In SLE patients, percentages of Fas-expressing B-lymphocytes are increased, are related to the state of lymphocyte activation, and correlate to disease activity. Increased Fas expression results in a higher susceptibility for Fas-mediated apoptosis, which might contribute to the increased levels of apoptotic lymphocytes in SLE patients.

Lymphocyte homing to allografts.

A complex system of lymphocyte trafficking has evolved to provide immune surveillance and recognition of foreign antigens (1). When the regulation of this process is disrupted leukocyte recruitment continues inappropriately resulting in pathological inflammation (2, 3). During graft rejection activa

Measles virus nucleocapsid protein binds to FcgammaRII and inhibits human B cell antibody production.

Despite the development of an efficient specific immune response during measles virus (MV) infection, an immunosuppression occurs contributing to secondary infections. To study the role of nucleocapsid protein (NP) in MV-induced immunosuppression, we produced recombinant MV NP. Purified recombinant NP exhibited biochemical, antigenic, and tridimensional structure similar to viral NP. By flow cytometry, we showed that viral or recombinant NP bound to human and murine B lymphocytes, but not to T lymphocytes. This binding was specific, independent of MHC class II expression, and dependent of the B lymphocyte activation state. The murine IIA1. 6 B cell line, deficient in the Fc receptor for IgG (FcgammaRII) expression, did not bind NP efficiently. Transfected IIA1.6 cells expressing either murine FcgammaRIIb1 or b2, or human FcgammaRIIa, b1*, or b2 isoforms efficiently bound NP. Furthermore, this binding was inhibited up to 90% by monoclonal antibodies 2.4G2 or KB61 specific for murine and human FcgammaRII, respectively. Finally, the in vitro Ig synthesis of CD40- or Ig-activated human B lymphocytes in the presence of interleukin (IL)-2 and IL-10 was reduced by 50% in the presence of recombinant NP. These data demonstrate that MV NP binds to human and murine FcgammaRII and inhibits in vitro antibody production, and therefore suggests a role for NP in MV-induced immunosuppression.

An assay for the analysis of lymphocyte migration across cerebral endothelium in vitro

We describe a recently developed assay for the analysis of leukocyte migration across cerebral endothelium in vitro. The endothelium is grown as monolayers on Goretex or Cyclopore membranes coated with extracellular matrix proteins and supported on inserts. This system permits the recovery and phenotyping of cells which migrate down through the endothelium. Using labelled lymphocytes we were able to differentiate four populations of cells, with differing degrees of mobility in the migration assay. We have compared the results from this system with those from conventional adhesion assays. Binding of cells to the endothelium is rapid, but is confined to a particular subpopulation of the applied lymphocytes. We have followed cell migration over 24 h in the system using normal and cytokine-activated endothelium and have found that whereas adhesion depends both on the state of lymphocyte activation and on the condition of the endothelium, the level of migration of stimulated lymphocytes is largely independent of endothelial activation. Moreover, whereas CD8 + cells bind well to the endothelium, it is the CD4 + cells which migrate most effectively. Comparison of brain and epididymal fat endothelium showed similar migration levels over 2 h, but migration was greater across epididymal fat endothelium at 24 h.

Lymphocyte activation status, expression of adhesion molecules and adhesion to human endothelium in rheumatoid arthritis — relationship to disease activity

The synovial tissue of patients with rheumatoid arthritis (RA) is characterized by infiltration with inflammatory cells, mainly memory helper cells (CD4+CD29+). An important initiating step in tissue infiltration is the adhesion of peripheral blood lymphocytes to the vascular endothelium. Therefore, we studied lymphocyte-endothelium adhesion in 40 RA patients and in 19 controls by a sensitive fluorimetric assay, using human umbilical vascular endothelial cells. Furthermore, expression of adhesion molecules VLA (CD29) and LFA-1 (CD11a) on CD4+ and CD8+ T cells was determined. In order to evaluate the activation state of lymphocytes, the soluble interleukin-2 receptor (sIL2R) was measured. The relationship to disease activity was evaluated using the Ritchie articular index. RA patients had a higher percentage of CD4+ cells (p < 0.005) and a lower percentage of CD8+ cells (p < 0.001) than controls did. The CD4+CD29+/CD4+CD29- ratio and the CD8+CD29+/CD8+CD29- ratio were increased in patients with active RA (p < 0.01 and p < 0.05, respectively) and in patients with inactive disease (p = 0.09 and p < 0.005, respectively) compared with controls. LFA-1 (CD11a) was present on almost all T lymphocytes and its density did not differ between patients and controls. Serum levels of sIL2R were significantly higher in both patient groups compared with controls (p < 0.0005); patients with active disease showed significantly higher levels than patients with inactive disease (p < 0.05). Lymphocyte-endothelium adhesion was not increased in patients, although the expression of the adhesion molecule CD29 on T lymphocytes of RA patients was higher.(ABSTRACT TRUNCATED AT 250 WORDS)

Lymphocyte migration into the CNS modelled in vitro

We report on a series of experiments which examines the factors controlling lymphocyte adhesion to brain endothelium in vitro and the factors which control cell migration across the endothelium, using a new migration assay. Although lymphocyte adhesion preceded migration across the brain endothelium, the two processes are not identical. We noted that activated CD4 + T cells were particularly good at migrating across endothelia. CD8 + T cells and B cells did not migrate but adhered well to endothelia. Moreover, the endothelium maintained high levels of cell traffic without being disrupted and without exhausting the molecular systems which allowed migration. From the viewpoint of migration of dividing cells, the state of lymphocyte activation appeared to be the most important controlling factor — these cells migrated equally well across endothelium activated with cytokines or untreated endothelium. The kinetics of adhesion suggested that the LFA-1/ICAM-1 and VLA-4/VCAM combinations of adhesion molecules were important in controlling migration. With antibody blocking studies, the role of the LFA-1/ICAM-1 system was equivocal. While anti-LFA-1 blocked lymphocyte adhesion, anti-ICAM-1 did not, suggesting that the level of ICAM-1 was not critical.

Cell numbers and in vitro responses of leucocytes and lymphocyte subpopulations following maximal exercise and interval training sessions of different intensities.

In vitro lymphocyte function and the mobilisation of peripheral blood leucocytes was examined in eight trained subjects who undertook an incremental exercise test to exhaustion and a series of interval training sessions. Venous blood samples were obtained before the incremental test, immediately after, and 30, 60, and 120 min after the test. Interval training sessions were undertaken on separate days and the exercise intensities for each of the different sessions were 30%, 60%, 90% and 120% of their maximal work capacity respectively, as determined from the incremental exercise test. There were 15 exercise periods of 1-min duration separated by recovery intervals of 2 min in each session. Venous blood samples were obtained immediately after each training session. Significant increases in lymphocyte subpopulations (CD3+, CD4+, CD8+, CD20+, and CD56+) occurred following both maximal and supramaximal exercise. This was accompanied by a significant decrease in the response of cultures of peripheral blood lymphocytes to Concanavalin A (ConA), a T-cell mitogen. The state of lymphocyte activation in vivo as measured by CD25+ surface antigen was not, however, affected by acute exercise. The total number of lymphocytes, distribution of lymphocyte subpopulations and in vitro lymphocyte response to ConA had returned to pre-exercise levels within half an hour of termination of exercise but serum cortisol concentrations had not begun to fall at this time. There was a significant decrease in the CD4+:CD8+ cell ratio following exercise; this was more the result of increases in CD3-CD8+ cells (CD8+ natural killer cells) than to CD3+CD8+ cells (CD8+ T-lymphocytes). Decreased responsiveness of T-cells to T-cell mitogens, postexercise, may have been the result of decreases in the percentage of T-cells in postexercise mixed lymphocyte cultures rather than depressed cell function. The cause of this was an increase in the percentage of natural killer cells which did not respond to the T-cell mitogen. The results indicated that while a substantial immediate in vitro "immunomodulation" occurred with acute exercise, this did not reflect an immunosuppression but was rather the result of changes in the proportions of reactive cells in mononuclear cell cultures. We have also demonstrated that the degree of the change in distribution of lymphocyte subpopulation numbers and responsiveness of peripheral blood mononuclear cells in in vitro mitogen reactions increased with increasing exercise intensity. Plasma volume changes may have contributed to some of the changes seen in leucocyte population and subpopulation numbers during and following exercise.

Identification of monoclonal antibodies reactive with the rat homolog of ICAM-1, and evidence for a differential involvement of ICAM-1 in the adherence of resting versus activated lymphocytes to high endothelial cells

We have produced four mAbs reactive with the rat homolog of ICAM-1 (intercellular adhesion molecule-1), and presented evidence to indicate that the ICAM-1 molecule plays a differential role in the binding between high endothelial cells and lymphocytes, depending on the state of lymphocyte activation. The conclusion that the four antibodies (1A29, 6A22, 10A25, 10A56) specifically recognize the rat ICAM-1 homolog was based on: (i) their ability to inhibit homotypic aggregation of PHA-blasts; (ii) SDS-PAGE analysis of the antigen recognized; (iii) antigen distribution as assessed by immunoperoxidase staining of frozen sections; and (iv) cytokine-induced up-regulation of the antigen. Involvement of the ICAM-1 molecule in lymphocyte binding to high endothelial cells, the vital step in lymphocyte extravasation in lymph nodes, was examined by the use of one of the newly developed antibodies, and it was found that binding of activated but not resting lymphocytes to cultured high endothelial cells was significantly blocked by the anti-ICAM-1, implying that ICAM-1 has differential involvement in the adherence of lymphocytes to high endothelial cells and that it may play an important role in dissemination of memory lymphocytes to systemic circulation by facilitating or strengthening the binding to the specialized postcapillary high endothelial venules (HEV).

Regulation of T lymphocyte proliferation. Interleukin 2-mediated induction of c-myb gene expression is dependent on T lymphocyte activation state.

We previously reported that with time, after antigenic stimulation of antigen-regulated murine T lymphocyte clones, total IL-2-R expression decayed 10-50-fold, commensurate with a decline in the ability of the cells to proliferate to IL-2. However, late after antigenic stimulation, when the cells were refractory to the IL-2-proliferative stimulus, high levels of high affinity IL-2-R remained. In this report we further explore the basis of unresponsiveness to IL-2 in the quiescent clones. We show that the proto-oncogene c-myc is induced in the late cell population by IL-2 to comparable levels observed early after antigen stimulation. IL-2-dependent c-myb induction, however, is seen only early after activation but not in the late-activated population. Analysis of the IL-2-dependent expression of c-myb mRNA with time after antigenic stimulation showed that steadystate c-myb expression declines dramatically with kinetics closely paralleling a decay in IL-2-dependent proliferative ability. In contrast, steadystate c-myc expression remains high throughout this period. Expression of c-myb is critical for proliferation of these cells since antisense oligodeoxy-nucleotide to c-myb can inhibit their IL-2-dependent proliferation. We present evidence for a pathway of c-myb induction via the TCR that is independent of the IL-2/IL-2-R interaction. In addition, the inhibition of IL-2-R-induced c-myb expression by 2-aminopurine and enhanced induction of c-myb via the TCR demonstrate that TCR activation and IL-2-R activation lead to induction of c-myb by different mechanisms.

T lymphocyte activation in euthyroid Graves' ophthalmopathy: soluble interleukin 2 receptor release, cellular interleukin 2 receptor expression and interleukin 2 production.

The present study was undertaken to examine the cellular control arm of the immune response with regard to T lymphocyte proliferation in euthyroid Graves' ophthalmopathy. Twenty patients with euthyroid Graves' ophthalmopathy (7 on antithyroid drugs and 13 on no treatment) and 18 healthy controls were studied in an infection-free period. Mitogen-stimulated cellular interleukin 2 (IL2) receptor expression, soluble interleukin 2 receptor release, and interleukin 2 production, were studied in peripheral blood mononuclear cells cultured for 24 h. The cellular IL2 receptor expression and soluble IL2 receptor release did not differ between the patients and healthy controls. In contrast, IL2 production in response to pokeweed mitogen stimulation was increased in lymphocytes from patients with Graves' ophthalmopathy. The IL2 release did not correlate with the quantities of cellular and soluble IL2 receptor. The mitogen-stimulated cellular IL2 receptor expression, IL2 receptor release, and IL2 production did not differ between patients with or without carbimazole therapy. Despite a suggested role of autoreactive T cells in mediating the development and propagation of autoimmune thyroid disease, this study fails to demonstrate a defective T lymphocyte activation state in patients with Graves' ophthalmopathy during an euthyroid state.

An immunoperoxidase-autoradiography double labeling method for analysis of lymphocyte activation markers and DNA synthesis

An immunoperoxidase-autoradiography double labeling method for analysis of lymphocyte activation markers and DNA synthesis is described. For this study expression of MHC locus II coded Ia antigen, interleukin-2 receptor, transferrin receptor and gp 40/80 glycoprotein was analyzed using monoclonal antibodies in avidin-biotin-peroxidase complex staining combined with visualization of [ 3H]thymidine incorporation by autoradiography. Compared to spontaneous [ 3H]thymidine incorporation assay information is obtained at single cell level. In contrast to blast indexes calculated from MGG stained preparates, information on the expression of various functional cell surface structures as well as DNA synthesis is also obtained. Double-assay for lymphocyte phenotype and DNA synthesis by flow cytometry might be preferred to light microscopy, but the widespread use of immunoperoxidase staining and autoradiography may make this new kind of approach more easily available. Other advantages worth considering are the possibility of transporting, staining and counterstaining as microscope slides and the permanent nature of the documentation and morphological information obtained. In our experience, this method seems to be useful for studying resting peripheral blood lymphocytes as well as mitogen and antigen induced changes in the lymphocyte activation state.

T lymphocyte activation state in the minor salivary glands of patients with Sjögren's syndrome.

Local lymphoplasmacytoid infiltration of the diseased exocrine glands is a cardinal sign of Sjögren's syndrome (SS). The state of T lymphocyte activation present in these local infiltrations was studied by three different techniques: determination of interleukin 2 (IL2) receptor (Tac) on cell surface membrane; autoradiography combined with immunoperoxidase staining of T cell epitopes; and electron microscopic analysis of the lymphoblast subclasses. Although 64 (SEM 4)% of the local inflammatory cells expressed Ia antigen, only 4 (SEM 1)% of them displayed the T cell activation antigen Tac. Autoradiography-immunoperoxidase double labelling showed that less than 1% of all T cells in situ were [3H]thymidine incorporating blasts. This finding suggests that although T lymphocyte is the dominant cell in situ, only a few of these cells have passed the G0/G1 interphase, and even fewer have been pushed to the S phase of the cell cycle by IL2. Transmission electron microscopy showed that few T blasts were present, even though there were many plasma cells. This result further confirms the impression that only a minor T cell subpopulation in situ is blast transformed despite the fact that many of the local T lymphocytes in the diseased salivary glands in SS are Ia positive.

Lymphocyte Subpopulations, Activation Phenotypes, and Spontaneous Proliferation in Tuberculous Pleural Effusions

The state of lymphocyte activation in tuberculous pleural effusions was studied. The proportion of cells at and beyond the G1 phase of the cell cycle displaying interleukin-2 receptor, transferrin receptor or gp 40/80 glycoprotein in avidin-biotin-peroxidase complex (ABC) staining was 6 +/- 2 percent, 8 +/- 3 percent, and 14 +/- 4 percent of all pleural fluid mononuclear cells, respectively. These findings imply that only a fraction of pleural fluid lymphocytes is activated in tuberculosis. The proportion of autoradiographically visualized 3H-thymidine incorporating blasts at the S phase of the cell cycle was 1.2 +/- 0.3 percent. This phenomenon further confirms the impression that, in spite of activation, most of the pleural fluid mononuclear cells are resting cells. The T4/T8 ratio in the functionally active blast cell population determined by a double labeling method combining ABC staining with autoradiography was similar to the total T4/T8 ratio (2.3 +/- 0.7 vs 2.7 +/- 0.8, p greater than 0.05) calculated for all pleural fluid mononuclear cells.

Expression of MHC class II antigen, interleukin-2 receptor, transferrin receptor and gp 40/80 glycoprotein during different phases of a normal PHA-driven lymphocyte activation in vitro.

This study characterizes the temporal profile of MHC class II antigen, interleukin-2 receptor, transferrin receptor and gp 40/80 glycoprotein lymphocyte activation markers in relation to each other during different phases of PHA-dependent cellular activation in vitro. Binding of these monoclonal lymphocyte activation-probes was visualized by the avidin-biotin-peroxidase complex method. Maximum PHA-dependent MHC class II antigen expression of 27 +/- 3% was observed on culture day 1, but later no significant differences were observed in MHC class II antigen expression between PHA-driven or culture media-containing control cultures. On the contrary, interleukin-2 receptor (78 +/- 6%) and transferrin receptor (75 +/- 5%) expression reached a maximum on culture day 3, coinciding with a maximum proliferative response. On culture day 5, when 3H-thymidine incorporation was already on the decline, gp 40/80 glycoprotein reached a maximum PHA-dependent expression of 78 +/- 2%, which differed significantly from interleukin-2 receptor (60 +/- 8%, p less than 0.05) and transferrin receptor (51 +/- 8%, p less than 0.01) expression. This study suggests that MHC class II antigen, interleukin-2 receptor, transferrin receptor and gp 40/80 glycoprotein, although all of them are lymphocyte-activation markers, differ as to the chronological sequence of their appearance and disappearance. Their combined use in lymphocyte-activation marker profile assay therefore gives valuable information about the lymphocyte activation state.