Molecular characterization of L-type calcium channel splice variants expressed in human T lymphocytes

Calcium (Ca²⁺) influx is a fundamental intracellular signal that is required to initiate and sustain T lymphocyte activation. Dihydropyridine-sensitive, L-type Ca²⁺ channels appear to play a significant role in Ca²⁺ mobilization during T cell activation, but very little is known about the molecular structure of these channels in T lymphocytes. Here we identify two novel splice variants of the Ca_V1.4 (_1F) L-type Ca²⁺ channel that are expressed in human T lymphocytes, and also demonstrate expression of the Ca_V1.4 protein in the human Jurkat T cell leukemia line and human peripheral blood T lymphocytes (PBTs). The carboxy-termini of both Ca_V1.4 splice isoforms contain unique exon usages distinct from the Ca_V1.4 channel isolated from human retina that may render these channel variants insensitive to changes in membrane depolarization. Additional evidence of the importance of these new splice variants comes from the demonstration that the mRNA expression of the Ca_V1.4 splice isoforms is regulated by TCR-induced activation in Jurkat T cells, and to a lesser extent in human PBTs. Overall these results provide the first evidence that structurally unique L-type Ca²⁺ channels exist in T lymphocytes, which can contribute to a Ca²⁺ influx during T lymphocyte activation.     

Seasonal Variation and Sex Differences of Circulating Macrophages, Immunoglobulins and Lymphocytes in Healthy School Children

Subpopulations of T and B lymphocytes and levels of serum immunoglobulins G, A, M, E and subclasses G1, G2 and G3 were studied in 45 healthy school children aged 8–16 years during four seasons of the year. There were significant increases in CD4⁺ T helper cells, total T lymphocytes and CD4⁺/CD8⁺ (helper/cytotoxic) T-cell ratio during the spring season. While the levels of CD8⁺ T cells and total B lymphocytes remained statistically unchanged during all four seasons, the levels of natural (HNK-1) killer cells and macrophages increased significantly during the autumn and summer seasons respectively. The levels of immunoglobulins G, A, M and E remained statistically unchanged during all four seasons. Girls had higher levels of CD4⁺ T cells and a higher CD4⁺/CD8⁺ T-cell ratio than boys. Girls also had slightly higher levels of immunoglobulin G and M. These observations suggest that seasonal variations of some immunological parameters occur in healthy children. This may be an adaptive response to variable climatic and other environmental factors. These natural variations due to seasonal changes should be taken into account when immunological tests are used in clinical investigations.     

Human Th0-type T Helper-Cell Clone Supports Antigen-Specific Immunoglobulin Production in Scid/beige-hu Mice

Tetanus toxoid-specific T cells have been generated from human splenic lymphocytes by an initial 6-day stimulation period with antigen, followed by a proliferation period with recombinant IL-2 and human feeder cells. Proliferating T cells were subsequently cloned by limiting dilution. A human T-cell clone that was functionally characterized showed: (i) a specific proliferative response to tetanus toxoid in the presence of autotogous Epstein—Barr virus (EBV)-transformed lymphoblastoid cells; (ii) a phenotype characteristic for the helper/inducer CD4⁺/CD8⁻/CD45RO⁺ T cells, and (iii) a lymphokine profile, as determined by mRNA analysis, representative of Th0-like human CD4⁺ T helper cells. This tetanus toxoid-specific T-cell clone which showed antigen-dependent helper activity for antibody production by autologous B cells in vitro could also provide T-cell help to antigen-specific human B cells transplanted into severe combined immunodeficiency (scid/beige) mice.     

Activated Human γδ T Lymphocytes Express Functional Lactoferrin Receptors

Lactoferrin (Lf), an iron-binding protein in milk, mucosal secretions and neutrophil granules has bactericidal properties and is a source of iron for breast-fed infants. In this paper the authors show that most in vivo activated lymphocytes, i.e. freshly isolated lymphocytes from first trimester human decidua, and most in vitro activated human blood lymphocytes, express lactoferrin receptors (Lf-R), while unstimulated blood lymphocytes do not. All major lymphocyte subsets, i.e. αβ T cells, γδ T cells, CD8⁺ T cells, CD4⁺ T cells, B cells and NK cells, express Lf-R after activation. The proportion of Lf-R expressing activated γδ T cells is significantly larger than that of activated αβ T cells. Lf-R and transferrin receptors (Tr-R/CD71) show the same kinetics of appearance on activated blood lymphocytes and are, to a large extent, expressed on the same cells. However, 35% of decidual lymphocytes and 15% of activated blood lymphocytes express Lf-R only. Addition of Lf to cultures containing an optimal concentration of Tr augments the proliferative response to polyclonal T cell activators and alloantigens, suggesting that presently used standard culture conditions for in vitro activation are suboptimal in particular for γδ T cells. Lf-R on decidual lymphocytes contain bound Lf, which probably is produced locally. The results suggest that Lf is a growth-supporting factor, especially important in local immune responses in the mucosa.     

Interleukin-2 and Serum Thymic Factor Enable Autologous Rosette-forming T Lymphocytes to Generate Helper and Cytotoxic Functions

The autologous rosette-forming T cells (Tar cells) isolated by means of their ability to form rosettes with autologous erythrocytes were characterized by the use of OKT monoclonal anti-human T-cell subset antibodies and a monoclonal anti-HLA-DR antibody. We found that the phenotype of Tar cells was OKT 3⁺4⁺8⁺Dr⁻ as determined by both indirect imnrunofluorescence microscopy and complement-mediated killing of ⁵¹Cr-labelled Tar cells. In addition, we found that Tar lymphocytes were able to develop cytotoxicity against allogeneic and trinitrophenol (TNP)-conjugated autologous target cells in the presence of interleukin-2 (IL-2) or serum thymic factor. However, these cells showed little or no cytotoxicity in the absence of interleukin-2 or serum thymic factor. Tar lymphocytes generated helper function for B lymphocytes in the presence of interleukin-2 in both pokeweed mitogen (PWM)- and purified protein derivative (PPD)-stimulated cultures. Nevertheless, non-IL-2-treated Tar cells did not exhibit any helper activity on B cells. Finally, pretreatment of Tar cells with 1000–1500 rad of X ray made these cells unable to develop helper function for B lymphocytes. It is concluded that: (1) OKT 3⁺4⁺8⁺Dr⁻ Tar cells are able to generate cytotoxicity against alloantigens and TNP-labelled self structures provided they are stimulated by IL-2 or serum thymic factor; (2) these cells need both to proliferate and to receive help from IL-2 to develop helper cells capable of assisting B-lymphocyte differentiation into plasma cells in both PWM- and PPD-stimulated cultures.     

A Short Incubation with PGE2 Affects the Proliferative Response of T Lymphocytes to PWM

In this study we have investigated the role of PGE2 in the activation of human T lymphocytes by PWM. A preincubation of these cells with molar concentrations of the prostaglandin ranging from 10^-9 M to 10^-4 M is able to reduce the expression of IL-2R and CD71 on T lymphocyte membrane during the first days of culture, while the DR molecule which is expressed later in the same experimental conditions is not affected by the treatment of T lymphocytes with PGE2. The PGE2-induced inhibition of IL-2R and CD71 well correlates with the reduction of ³H-thymidine incorporation by T cells, indicating that a preincubation of T lymphocytes with PGE2 profoundly affects the proliferative apparatus of these cells when they are stimulated by PWM.     

Cryopreservation of human lymphocytes for assessment of lymphocyte subsets and natural killer cytotoxicity

Cryopreservation of lymphocytes has become increasingly important, especially when the cells are to be used in retrospective studies of selected and dwindling populations, such as A-bomb survivors. This report describes an efficient method for cryopreservation of human lymphocytes which does not significantly alter various immunological characteristics of these cells. The proportions of Leu-1⁺ cells (T cells), Leu-2a⁺ cells (suppressor-cytotoxic T cells), Leu-3a⁺ cells (helper-inducer T cells), HLA-DR⁺ cells, Mo2⁺ cells (monocytes), B1⁺ cells (B cells), and Leu-7⁺ cells (natural killer (NK) cells), as determined by monoclonal antibodies, were found to be stable following cryopreservation. NK cell activity against K-562 target cells showed a 40–60% decrease immediately after thawing, but recovered to approximate pre-freezing levels after preincubation for 18 h. Neither lymphocyte subsets nor cell viability significantly changed following preincubation after cryopreservation. However, the ratio of cells binding to K-562 cells increased after this preincubation and may account for the observed recovery of NK cell activity. NK cell activity remained relatively stable up to 14 months of storage which confirms that freezing damage depends on the freezing process rather than on the duration of cryopreservation.     

Effect of Prostaglandin E2 on Pokeweed Mitogen-Activated Human Lymphocyte Cultures

It is shown that a short incubation of peripheral human lymphocytes with PGE₂ is able to reduce the B cell differentiation induced by PWM. The target of PGE₂ action appears to belong to T lymphocytes, since the treatment of non-T cells is uneffective in reducing the immune response. Both OKT4⁺ and OKT8⁺ subsets are sensitive to PGE₂. Data concerning the role of endogenous as well as exogenous PGE₂ either on unfractionated or fractionated OKT4⁺, OKT8⁺ and non-T lymphocytes are also discussed. The PGE₂ -inhibition on immunoglobulin synthesis in PWM-stimulated cultures seems to be mediated by a complex effect on both the T cell subsets.     

Enrichment of nucleofected primary human CD4+ T cells: a novel and efficient method for studying gene function and role in human primary T helper cell differentiation

Identification of key factors mediating the differentiation of naïve CD4⁺ T helper cells into Th1 and Th2 subsets is important for understanding the molecular mechanisms of the development of autoimmune diseases as well as asthma and allergy. Functional importance of a given gene in the initiation of human T helper cell differentiation has been hard to study due to the difficulty in transfecting primary resting human T lymphocytes. In this study we have successfully transfected human primary CD4⁺ T helper cells using Amaxa's Nucleofection technology. To overcome the background caused by untransfected cells, we have developed a system for enriching nucleofected unstimulated human primary T helper cells that express the gene of interest. This is achieved by introducing a plasmid construct containing a bicistronic unit coding for a truncated mouse MHC class l H-2K^k cell surface marker followed by selection of H-2K^k positive cells using antibody coated beads. We demonstrate that the nucleofected and enriched H-2K^k positive T helper cells differentiate into Th1 and Th2 cells as well as the non-transfected control cells. We also show that by using this novel method, introduction of an shRNA targeting Stat6, a key molecule driving the Th2 cell development, results in impaired Th2 cell differentiation, as expected. The method described here, enables fast and feasible preparation of highly pure transfected primary CD4⁺ T cell cultures ideal for studying the influence of overexpression or knockdown of a given gene on T helper cell differentiation and other primary human T cell functions.     

Differentiation of T Lymphocytes in the Human Adenoid as Measured by the Expression of CD45 Isoforms

Encounter of antigen by T lymphocyte on antigen-presenting cells results in changes in the expression of several cell surface molecules, including the abundant cell surface glycoprotein CD45. We have characterized the expression of the CD45 isoforms CD45RA and CD45RO in CD4⁺ and CD8⁺ T lymphocytes in the adenoids and peripheral blood of young children. We found that the relative proportions of CD45RA⁻,CD45RO⁺ antigen-experienced T cells was higher in the adenoids than in peripheral blood, and that the proportion of naive or resting CD45RA⁺,CD45RO⁻ T cells was lower in the adenoids than in peripheral blood. The frequency of bright double-positive CD45RA⁺,CD45RO⁺ T cells, which represent cells in transition from the CD45RA⁺ to CD45RO⁺ phenotype, was higher in the adenoids than in peripheral blood. The frequency of another double-positive cell population, but with unknown ontogeny, expressing both CD45RA and CD45RO at a low level, was higher in peripheral blood than in adenoidal T cells. It was found that the frequency of adenoidal antigen-experienced CD45RA⁻,CD45RO⁺ T lymphocytes increased with increasing age of the child. These results are consistent with the model that the adenoids serve as a site for conversion of CD45RA⁺ to CD45RO⁺ T lymphocytes, and that the maturation of the immune system in young children is associated with phenotypic changes in T lymphocytes residing in secondary lymphoid organs.     

Analysis of Human Peripheral Blood T Lymphocytes Proliferating in Response to Sensitizing Antigens: Effect of Interleukin-2 (Il-2)-Responsive Bystander Cells

The contribution of interleukin-2 (IL-2)-responsive bystander cells to the proliferative responses of human peripheral blood T lymphocytes to antigens used for sensitization such as Purified Protein Derivative (PPD), Tetanus toxoid (TT) and Influenza virus was investigated. Marked proliferation of the unfractionated peripheral blood mononuclear cells (PBMC) was observed following stimulation with these antigens to which the individuals were known to have been sensitized previously. Depletion of large granular lymphocytes (LGL) from PBMC resulted in substantial reduction in the response of the lymphoid cells in proliferating to the antigens. Proliferation of the T4⁺T8^- (helper)-enriched population, or T4⁺T8^- subset depleted of any IL-2 receptor (IL-2R)-bearing lymphoid cells to these antigens was comparable to that of LGL-depleted PBMC cultures. Cell titration experiments of the blast cells generated from these cultures revealed that PBMC-derived population contained fewer antigen-reactive lymphocytes. These results, therefore, suggested that IL-2-responsive LGL through expansion affected the concentration of antigen-proliferating T cells in the antigen-stimulated PBMC cultures.     

RT-PCR Based Analysis of T-Cell Receptor B Variable Region Gene Usage in Normal Human Breast Skin Resident T Lymphocytes (SRT)

The skin interfaces directly with the external environment that contains innumerable infectious agents. Therefore, an appropriate and rapid immunologic response is required to preserve internal homeostasis. An essential feature of the 'skin immuno system' (SIS) is the presence of substantial numbers of T cells in normal skin. The T-cell receptor repertoire from normal human breast skin was analysed quantitatively and qualitatively by using PCR amplification of reverse transcribed RNA, T-cell receptor BV3 and BV14 gene usage was increased in skin T lymphocytes in all individuals tested ( n  = 8) compared to peripheral blood CD4⁺ and CD8⁺ T lymphocytes from the same individuals. The T-cell receptor junctional diversity analysed by high resolution gel electrophoresis showed skin T-cell BV3 and BV14 gene usage to be predominantly polyclonal. Superantigen stimulation of T cells in human skin is considered a likely explanation of the present finding.     

Peripheral Immune Response in Pulmonary Tuberculosis

Cellular immune response and delayed-type hypersensitivity reactions are considered to play a major role in the immunopathogenesis of pulmonary tuberculosis (PTB). But the exact mechanism is still to be clarified. Th1 cells are mainly involved in cellular immune responses in PTB and provide a normal healing process with minimal or no sequela whereas Th2 cell and CD8⁺ T lymphocyte responses may lead to more severe type of disease. In this study, we investigated the peripheral blood immune responses in PTB. The study group consisted of acid fast positive young male soldiers with PTB and a negative HIV serology. The control group included healthy young volunteer male soldiers without a history of PTB. Intracytoplasmic cytokine content of CD8⁺ T cells and lymphocytes, including IL-2, IL-4, IL-5, IL-10 and IFN-γ were determined by flow cytometry, and IL-2, IL-4, IL-5, IL-10, IFN-γ and TNF-α serum levels were measured by cytometric bead array (CBA). No difference was observed between the percentages of T, B, NK cells and HLA-DR expression in both groups, however, the number of CD3⁺HLA-DR⁺ activated T cell percentages was higher in PTB group as compared to healthy subjects. IL-2, IL-4, IL-5, IL-10 contents of lymphocytes and IFN-γ⁺CD8⁺ T cells were found to be significantly lower in PTB patients when compared with healthy subjects, and in parallel, serum IL-2, IL-4, IL-5 and TNF-α levels were also significantly lower in PTB patients. In conclusion we suggest that, CD8⁺ T cells producing both Th1 and Th2 type cytokines, may play important role in the peripheral immune response to mycobacteria.     

Human platelet alloantigens Br/Br are expressed on the very late activation antigen 2 (VLA-2) of T lymphocytes

We report the molecular localization of the human platelet alloantigens Br^a/Br^b on activated T lymphocytes. By radioimmunoprecipitation anti-Br^a precipitated two proteins from T lymphocytes of Br(a+) donors after long-term activation (24 days), but not from resting or short-term-activated T lymphocytes (4 days). No bands could be precipitated with anti-Br^a from T lymphocytes of Br(a−) donors, but the same two bands were seen with anti-Br^b. The apparent molecular weights of unreduced molecules were 155 and 130 kDa, respectively. Monoclonal antibody Gi14 directed against an epitope on the platelet membrane glycoprotein Ia/IIa complex precipitated the same two bands with long-term-activated T lymphocytes, whereas MoAb TS2/7 directed against very late antigen (VLA)-1 molecules precipitated two bands with M_r 180 and 130 kDa (₁β heterodimer). The physicochemical properties of these two bands precipitated by anti-Br^a, anti-Br^b or monoclonal antibody Gi14 with molecular weights of 155 and 130 kDa, respectively, correspond to VLA ₂ and β chains and fit defined criteria for the VLA-2 antigen. These results were corroborated by an assay employing monoclonal antibody for antigen immobilization showing that Br^a and Br^b antigens were present only on long-term activated T lymphocytes. Our results provide evidence for the expression of the Br^a/Br^b platelet alloantigen system on VLA-2 antigens of activated T lymphocytes and demonstrate a genetic polymorphism of VLA-2 molecules on both cell lines.     

Enhanced expression of HLA-class II molecules on activated human T lymphocytes following treatment with tumor necrosis factor alpha

Many factors induce or enhance expression of major histocompatibility complex class I and class II molecules on various cell types. Human T lymphocytes are class II negative in the resting state but show expression of class II molecules following activation. We analyzed the modulating capacity of the lymphokines recombinant interferon gamma (rIFN-γ), interleukin-4 (IL-4), and recombinant tumor necrosis factor alpha (rTNF-) on class II expression in subsets of alloactivated human T lymphocytes. The activated CD4⁺ T cells expressed all three class II isotypes (DR, DQ, and DP), whereas the cytotoxic CD8⁺ T-cell lines expressed DR and DP molecules but failed to bind DQ-specific monoclonal antibodies significantly. Treatment with rIFN-γ and IL-4 had no effect on class II expression on any of the T-cell lines or clones, whereas rTFN- enhanced class II expression in both subsets. rTNF- could modulate expression of all three class II isotypes but, in principle, it appears only to affect ongoing class II synthesis as de novo synthesis of class II molecules with a resultant change in the class II phenotype from DR⁺DQ⁻DP⁺ to DR⁺DQ⁺DP⁺ in the CD8⁺ T lymphocytes was not observed. No synergic effects of rINF-γ and rTNF-γ were observed; this results from the fact that activated T cells express few, if any, receptors for rIFN-γ.     

Characterization by monoclonal antibodies of lymphocyte subsets present in B-enriched suspensions

B lymphocyte enriched suspensions isolated by E rosette depletion (E⁻ cells) or by nylon fiber adherence (adherent cells) were identified by their cellular composition using different T and B cell markers (SI_g, E receptor, T3, T4, T8 and Ia-like antigens). The cells were isolated from peripheral blood both of healthy donors and uremic dialysis patients. A variable proportion of non-B cells was found in some preparations. This contaminant was represented mainly by Null cells in E⁻ lymphocyte suspensions and by T cells in the adherent population. Contaminating T lymphocytes were most frequently found in adherent cell preparations from uremic individuals and appeared to be an heterogeneous population including variable proportions of T4, T8 and Ia positive T cells. A significant increase of T8⁺ cells and a decrease of the ratio T4/T8 was seen among adherent T cells as compared to the normal distribution among T peripheral lymphocytes.     

T-cell receptor triggering differentially regulates bim expression in human lymphocytes from healthy individuals and patients with infectious mononucleosis

Bim, a proapoptotic member of the Bcl-2 protein family, is a major regulator of central and peripheral T-cell deletion. Regulation of Bim activity by T-cell receptor (TCR) triggering is not well understood. We investigated expression of Bim in different subpopulations of ex vivo isolated human T cells from healthy donors and patients with infectious mononucleosis (IM). Upregulation of Bim expression in response to TCR-triggering was observed only in a small proportion of analyzed samples of peripheral blood lymphocytes (PBLs) from healthy donors and only occasionally upon longitudinal analysis of cells isolated from the same individuals. Populations of naive or memory T cells enriched on the basis of CD45RO or CD45RA expression showed only slight and comparable Bim upregulation. In contrast, ex vivo isolated PBLs from IM patients in the acute stage of the disease with significant expansions of CD8⁺ cells expressed increased levels of Bim, and lymphocytes from the majority of analyzed IM patients exhibited significant upregulation of all major Bim isoforms in response to TCR triggering. These results demonstrate that at least some antigen-induced expansions of human CD8⁺ T cells are associated with increased levels of Bim, and TCR triggering in effector T lymphocytes may increase Bim activity by upregulation of its expression.     

Rapid expression of the CD69 antigen on T cells and natural killer cells upon antigenic stimulation of peripheral blood mononuclear cell suspensions

The CD69 antigen has been identified as the earliest activation marker on the surface of cytokine- or mitogen-activated lymphocytes. The expression of this molecule may be a useful early marker of antigen- or allergen-specific activation of lymphocytes in vitro. We evaluated the expression of the CD69 and CD25 antigens on antigen- or allergen-stimulated lymphocytes and the proliferative responses as detected by thymidine incorporation. Peripheral blood mononuclear cells (PBMC) of allergic patients sensitized to Dermatophagoides pteronyssinus , bovine casein, or nickel sulfate were cultured in the absence or presence of clinically relevant allergens, tetanus toxoid, or recombinant interleukin (IL)-2. The respective binding of CD69 or CD25 antibodies to PBMC and thymidine incorporation were measured. An early expression of CD69, but not of CD25, antigen was detectable after 24-72 h of stimulation on up to 80% of natural killer (NK) cells and up to 10% of CD4⁺ T cells in PBMC cultures. Anti-IL-2 antibodies inhibited these increases of CD69 on NK cells and T cells by up to 60%. After 6 days of antigenic stimulation, the rates of both CD25⁺ and CD69⁺ lymphocytes were higher. Seventy-four percent of the CD25⁺ PBMC but only 55% of the CD69⁺ cells were CD3⁺ T lymphocytes at this time. No qualitative differences were detectable in allergen- or tetanus-toxoid-stimulated PBMC from allergic patients. The high expression of CD69 on NK cells in antigen-stimulated cultures suggests that these cells are easily activated by cytokines from antigen-stimulated T cells. CD69⁺ NK cells may serve as early-indicator cells in cultures with antigen- or allergen-stimulated mononuclear cells.     

T-Lymphocyte Functions in Acute Leukaemia Patients with Severe Chemotherapy-Induced Cytopenia: Characterization of Clonogenic T-Cell Proliferation

Intensive chemotherapy for acute leukaemia is followed by a period of severe chemotherapy-induced leukopenia. We used a limiting dilution assay to investigate whether remaining CD4⁺ and CD8⁺ T lymphocytes derived from such leukopenic patients could be activated and undergo clonogenic proliferation. The activation signal in our model was accessory cells (irradiated normal peripheral blood mononuclear cells) + phytohaemagglutinin (PHA) + interleukin-2 (IL-2). During severe leukopenia a majority of circulating lymphocytes were CD4⁺ T cells. Clonogenic proliferating T lymphocytes were detected for all patients. Higher frequencies of clonogenic cells were detected in the CD8⁺ subset as compared to the CD4⁺ subset. However, for both subsets frequencies of proliferating cells were decreased compared with healthy individuals. The CD4⁺ and CD8⁺ lymphocytes were also capable of proliferation in response to alloactivation, and accessory cells mainly containing acute myelogenous leukaemia blast were efficient as accessory cells for activation. For the CD4⁺ cells, increased proliferation was detected in the presence of acute myelogenous leukaemia (AML) blasts compared with normal accessory cells. Based on our results we conclude that: (1) although acute leukaemia patients with therapy-induced leukopenia have both a quantitative and a qualitative T-cell defect, (2) the remaining T-cell population includes a subset capable of clonogenic proliferation. However, (3) proliferation of the clonogenic CD4⁺ cells can be modulated by AML blasts.     

GD2 Ganglioside-Specific Monoclonal Antibody Reacts with Murine Cytotoxic T Lymphocytes Reactive with FBL-3N Erythroleukaemia

Ganglioside expression on cytotoxic T lymphocytes induced against the mouse erythroleukaemia line FBL-3N, was analysed, compared with that of naive T lymphocytes in the thymus, lymph nodes and spleen. Although normal uncultured and cultured T lymphocytes expressed no GD2, GD3 or GM2 gangliosides, cytotoxic T cells with CD4⁺ CD8⁻, CD4⁻CD8⁺, and CD4⁻CD8⁻ phenotypes reacted with anti-GD2 monoclonal antibody with various intensities. The reactivity with anti-GD2 was associated with the intensity of cytotoxic activity as analysed using cytotoxic T lymphocyte (CTL) clones established from the bulk CTLs of each phenotype. An increase of the mRNA expression of GM2/GD2 synthase gene was demonstrated by Northern blot hybridization using RNA extracted from thymocytes, spleen cells, Con A-treated spleen cells and various types of CTL cells. These results indicated that GD2 ganglioside expression might be associated with the functional differentiation of murine T lymphocytes.