Spontaneous internalization of Class I major histocompatibility complex molecules in T lymphoid cells.

A low proportion of T lymphocytes in normal mouse spleen contains small intracytoplasmic vesicles showing Class I MHC molecules. After stimulation in vitro in a mixed lymphocyte reaction or by addition of Con A, the proportion of T cells with such intracytoplasmic vesicles increases progressively and becomes the majority. Labeling with fluorochrome-conjugated antibodies has shown that the vesicles are formed by internalization of molecules from the plasma membrane. The process is spontaneous and does not require cross-linking by antibodies or other ligands; it is selective inasmuch as other molecules (Thy-1 and T200 antigens) are not included and it is specific since it is not performed by other cells such as B lymphoid cells or fibroblasts. On the whole the process shows similarities with the internalization and recycling of other receptors, such as the receptors for different macromolecules of metabolic or informational significance, as seen in other cells. On the other hand, the specificity of Class I MHC mobilization in T lymphoid cells suggest a role for this process which is related to the immune function of these molecules.

Production of autoantibodies by CD5-expressing B lymphocytes from patients with chronic lymphocytic leukemia.

CD5-expressing B lymphocytes from patients with selected chronic lymphoproliferative disorders were used to determine whether monoclonal populations of CD5+ human B cells produce autoantibodies. CD5+ B cells from 19 patients with chronic lymphocytic leukemia (CLL) and one with diffuse well-differentiated lymphocytic lymphoma (DWDL) were cultured, with and without mitogenic stimulation, to obtain Ig from these cells. 17 of the 20 samples produced Ig in vitro. mAb from nine of the 17 patients were reactive with either IgG, ssDNA, or dsDNA. In every instance, the autoantibodies displayed monotypic L chain usage that correlated precisely with the L chain expressed on the CD5+ leukemic B cell surface. These monoclonal autoantibodies varied in their degree of antigenic specificity; some were quite specific, reacting with only one antigen, whereas others were polyspecific, reacting with two or all three autoantigens tested. Three features distinguish these autoantibodies from those observed in prior studies of CD5+ B cells. First, they are clearly the products of monoclonal populations of CD5+ cells; second, several react with dsDNA, a specificity not previously reported and often seen in association with significant autoimmune disorders; and third, two of the monoclonal autoantibodies secreted by the CD5+ clones were of the IgG class. Although not all of the Ig-producing, CD5-expressing clones elaborated mAbs reactive with the autoantigens tested, greater than 50% did. It is possible that with a broader autoantigenic panel or with larger quantities of CLL/DWDL-derived Ig, even more autoantibody-producing clones might be identified. These studies may have important implications for the antigenic specificity of subsets of human B lymphocytes as well as for lymphoproliferative and autoimmune disorders in general.

Intracellular accumulation of T-cell receptor complex molecules in a human T-cell line.

This work was aimed at understanding the mechanisms of T-lymphocyte function by studying the cellular distribution and traffic of molecules of the T-cell receptor complex. The accumulation of specific molecules in intracytoplasmic vesicles is related to the activation of T lymphocytes. Some of these molecules include acid hydrolases, the transferrin receptor, and class I antigens of the major histocompatibility complex. Molecules of the T-cell receptor complex have now also been found in intracytoplasmic vesicles in a human T-cell line derived from a lymphoblastic leukemia. Such vesicles were tightly associated with the cytoplasmic microtubule network. One functional aspect of this association is a cellular pathway by which vesicles traveling to and from the cell surface converge in an area of the cells that is rich in processing enzymes.

Cytokine responses to QuantiFERON® peptides, purified protein derivative and recombinant ESAT-6 in children with tuberculosis.

SETTING: Recent data suggest that interferon-gamma release assays may have reduced sensitivity in children. OBJECTIVE: To explore the cellular responses in children infected with tuberculosis (TB) to different mycobacterial antigens, including the peptides used in the QuantiFERON®-TB Gold In-Tube (QFT) assay. DESIGN: Cytokines were measured by multiplex analyte detection in supernatants after stimulation with peptides in QFT, purified protein derivative (PPD) and recombinant whole protein ESAT-6. Samples from 11 children with active TB, 46 healthy children with latent tuberculosis infection (LTBI), and 35 healthy non-infected children were analyzed. RESULTS: None of the cytokines examined in the QFT peptide stimulation assay distinguished between non-infected children and those aged <5 years with LTBI. Cytokines interleukin-2 and transforming growth factor-beta 1 (TGF-ß1) were shown to distinguish between stages of Mycobacterium tuberculosis infection after blood was stimulated with the QFT peptides. All children had significantly higher Th 1 and 2 cytokine production against PPD than against the other antigens tested. CONCLUSION: Measuring specific cytokine patterns after stimulation with the QFT peptides may not increase sensitivity in diagnosing LTBI in children, but there may be future diagnostic value in determining the stage of infection. PPD-stimulated blood produced a robust and diverse cytokine response in young children, making it an interesting antigen for in vitro diagnostic studies.

CD4-T-cell antigen receptor complexes on human leukemia T cells.

CD4 and T-cell antigen receptor (TCR) comodulate from the surface of human and murine T cells following exposure to monoclonal anti-CD4 or anti-TCR. This comodulation may occur because expression of CD4 and TCR is regulated by similar transmembrane signals or because CD4 and TCR are physically associated. To study multimolecular assemblies on the plasma membrane, we developed a flow cytometric method for detecting singlet-singlet energy transfer between fluorescein isothiocyanate (FITC)- and tetramethylrhodamine isothiocyanate (TRITC)-conjugated monoclonal antibodies as sensitized TRITC emission on intact, single cells. Using this procedure, we detected CD4-TCR complexes on the surface of the transformed human leukemia T cells, HPB-ALL, in the absence of stimulation. More than one CD4 were found in association with one TCR. CD4-TCR complexes were not in rapid equilibrium with free CD4 and free TCR, and they were not induced by the dye-labeled anti-CD4 or anti-TCR.

Effect of CD4 engagement on CD4-T cell receptor complexes.

To detect the presence of CD4-T cell receptor (TCR) complexes, we previously developed a flow cytometric method for measuring singlet-singlet energy transfer on human T cells labeled with fluorescein isothiocyanate-conjugated anti-CD4 and tetramethylrhodamine isothiocyanate-conjugated anti-TCR. Using the same procedure, we have now studied changes in the expression of CD4, TCR, and CD4-TCR complexes following CD4 engagement. Ligation of the D3 domain with OKT4, or the D1 domain with anti-Leu3a, induced CD4 and TCR down-regulation, while ligation of the D1 domain with gp120 did not. OKT4 caused a transient decrease in CD4-TCR association over 1 hr at 37 degrees C, while anti-Leu3a caused a steady-state decrease. In contrast, gp120 decreased CD4-TCR association mainly at 0 degrees C, rather than at 37 degrees C. Such alteration in CD4-TCR assembly may underly anti-Leu3a- and gp120-mediated inhibition of T cell antigen recognition and account for the negative effect of CD4 ligation on TCR-triggered responses.

Acidification of internalized class I major histocompatibility complex antigen by T lymphoblasts.

It has previously been shown that activated murine T lymphocytes express intracellular vesicles containing the class I major histocompatibility complex (MHC) antigen H-2K. Evidence has also been provided that such vesicles may be part of a cellular pathway of spontaneous H-2K antigen internalization and recycling, which is specific to T-lymphoid cells. Dual fluorescence flow cytometry has now been used to establish that H-2K antigen is acidified upon internalization in concanavalin A-stimulated but not lipopolysaccharide-stimulated murine splenocytes, thus providing further support that in T lymphoblasts this class I MHC antigen may travel intracellular routes similar to those reported for other cell surface receptors.

Recycling class I MHC antigens: dynamics of internalization, acidification, and ligand-degradation in murine T lymphoblasts.

We have previously shown that activated T lymphocytes spontaneously internalize their own surface class I MHC antigens and that this phenomenon is specific for these cells since it does not occur in B lymphocytes even after activation. The present work was aimed at defining the quantitative aspects of this phenomenon and, in particular, at the elucidation of the route of the internalized class I MHC antigens. We intended to determine if the internalized molecules are delivered to the lysosomal compartment and digested there or if instead they are brought back to the plasma membrane in a recycling pathway similar to that described for various cell surface proteins known to be engaged in the process of receptor-mediated endocytosis. We have devised a flow cytometric assay based on the use of fluorochrome-labeled monoclonal anti-H-2K antibodies to measure the kinetics of H-2K internalization. Comparison of the time necessary for the internalization of one-half of the surface H-2K molecules in activated T lymphocytes, which is approximately 1 hour, with the half-life of these molecules on the same cells, which is 14 hours, clearly indicates that the internalized molecules are not degraded but are instead recycled. The recycling takes place in an endosomal compartment with an average pH of about 5.6. The monoclonal anti-H-2K antibody used in these studies was not eluted from the H-2K molecules at this pH and is recycled along with them. On the other hand, protein A bound to the Fc of the anti-H-2K antibody was eluted at the low pH of the endosomes, delivered to lysosomes, and digested. We have therefore defined a novel phenomenon, namely the recycling of class I MHC antigens, which occurs selectively in T lymphocytes. The features of this phenomenon are similar to the recycling of surface receptors which mediate the endocytosis of a variety of extracellular ligands in different cells. However, no physiological extracellular ligand is known for class I MHC antigens. It is a reasonable speculation that upon activation T lymphocytes recycle their own surface class I MHC antigens as part of the complex machinery whereby these lymphocytes recognize and respond to non-self moieties on the plasma membranes of presentor or target cells.

Architectural organization in the interphase nucleus of the protozoan Trypanosoma brucei: location of telomeres and mini-chromosomes.

We studied the spatial organization of chromatin in the interphase G1, S and G2 nucleus of the protozoan Trypanosoma brucei, applying in situ hybridization with conventional fluorescence and confocal scanning optical microscopy. The majority of the trypanosome telomere GGGTTA repeats from different chromosomes were found clustered together, either extending in a network through the nuclear interior or localized at the nuclear periphery. The population of one hundred mini-chromosomes was often asymmetrically located: either clustered in a narrow band in close association with the nuclear envelope or distributed into several clusters that segregated into roughly one half of the nucleus. The nuclear organization may undergo modifications during the cell cycle and development. We conclude that non-random spatial positioning of DNA exists in the nucleus of this protozoan. Finding a high level of structural organization in the interphase nucleus of T.brucei is an important first step towards understanding chromosome structure and functioning and its role in the control of gene expression.

In situ activation of helper T cells in the lung.

To better understand the lung and systemic responses of helper T cells mediating memory immunity to Mycobacterium tuberculosis, we used three- and four-color flow cytometry to study the surface phenotype of CD4(+) lymphocytes. Bronchoalveolar lavage (BAL) fluid and peripheral blood (PB) samples were obtained from a total of 25 subjects, including 10 tuberculosis (TB)-infected subjects, 8 purified-protein-derivative-negative subjects, and 7 purified-protein-derivative-positive subjects. In marked contrast to CD4(+) lymphocytes from PB (9% +/- 5% expressing CD45RA and CD29), the majority (55% +/- 16%) of CD4(+) lymphocytes in BAL (ALs) simultaneously expressed CD45RA, a naïve T-cell marker, and CD29, members of the very late activation family. Further evaluation revealed that CD4(+) ALs expressed both CD45RA and CD45RO, a memory T-cell marker. In addition, the proportion of CD4(+) lymphocytes expressing CD69, an early activation marker, was drastically increased in BAL fluid (83% +/- 9%) compared to PB (1% +/- 1%), whereas no significant difference was seen in the expression of CD25, the low-affinity interleukin 2 receptor (34% +/- 15% versus 40% +/- 16%). More importantly, we identified a minor population of CD69(bright) CD25(bright) CD4(+) lymphocytes in BAL (10% +/- 6%) that were consistently absent from PB (1% +/- 1%). Thus, CD4(+) lymphocytes in the lung paradoxically coexpress surface molecules characteristic of naïve and memory helper T cells as well as surface molecules commonly associated with early and late stages of activation. No difference was observed for ALs obtained from TB-infected and uninfected lung segments in this regard. It remains to be determined if these surface molecules are induced by the alveolar environment or if CD4(+) lymphocytes coexpressing this unusual combination of surface molecules are selectively recruited from the circulation. Our data suggest that ex vivo experiments on helper T-cell subsets that display distinctive phenotypes may be pivotal to studies on the human immune response to potential TB vaccines.