Peripheral Pool of CD8+ T-Cells Contains Lymphocytes with Antigen-Specific Receptors That Recognize Syngeneic MHC Class II Molecules

The capacity of T-lymphocytes to recognize nonself and tolerating self is formed as a result of positive and negative selection in the thymus. While obtaining and testing specificity of T-hybridomas, we demonstrated that the major part of peripheral pool of CD⁸⁺ T-lymphocytes carried receptors specific to self MHC class II molecules. Such an unexpected specificity of receptors has been found in some T-cell hybridomas produced by fusion of activated peripheral CD8⁺ T-lymphocytes with a tumor partner transfected by the coreceptor CD4 gene. The reactivity to self is not an experimental artifact due to an increased avidity of interaction of the hybridoma cells with antigen-presenting cells. Also, it is not an expression of reactivity of T-cells to superantigens, products of endogenous viruses of mouse breast cancer. The formation of a pool of such T-cells involves both cells with double receptor specificity and cells coexpressing two -chains of T-cell receptor. Their appearance in the periphery can be due to the capacity of thymocytes differentiating in the direction of CD4⁺ cells to avoid negative selection via change of expression of coreceptor CD4 to CD8.     

Post-thymic selection of peripheral CD4+ T-lymphocytes on class II major histocompatibility antigen-bearing cells.

Following positive and negative selection in the thymus, mature CD4+ T-cells emigrate into peripheral lymphoid organs. Whether resting T-cells require periodic stimulation to remain viable in the absence of antigen is important for understanding peripheral T-cell homeostasis. A prerequisite for T-cell receptor (TCR)-mediated signals in maintaining peripheral CD4+ T-cell longevity has been demonstrated. Here, we show in mice expressing a mutant I-Abeta transgene on an I-Abeta knockout background that na?ve CD4+ T-cells also require engagement of their CD4 coreceptors by peripheral, class II MHC-bearing cells for their survival. The transgene's product combines with endogenous Aalpha, but this mutant AalphaAbeta heterodimer cannot interact with CD4 molecules, although it efficiently presents antigens to TCRs. Resting CD4+ T-lymphocytes from mutant Abeta transgenic mice die by apoptosis at a much higher rate than do CD4+ T-cells from normal mice. Apoptosis of CD4+ T-cells in mutant Abeta transgenic mice is partially mediated by Fas. Adoptive transfer experiments revealed that the increase in apoptosis is due to a lack of interactions with mutant MHC class II rather than to an intrinsic defect in the CD4+ T-cells selected on mutant Abeta-expressing thymic epithelial cells. Thus, interactions between CD4 and MHC class II molecules contribute to the regulation of homeostasis in the peripheral immune system. Our results further suggest that thymic emigrant cells are continuously retested in the periphery for appropriate coreceptor interactions. Peripheral selection may be important in eliminating potentially autoreactive T-cells.     

Immunocytochemical characterization of S-100 beta-positive human T-lymphocytes by a double immunostaining method

The antigenic properties of S-100 beta-positive human T-lymphocytes (S-100 beta+ T-cells) were investigated by a double immunostaining technique employing an indirect immunoperoxidase method for cytoplasmic S-100 beta subunit and an immunoalkaline phosphatase method for cell surface antigens detected by various monoclonal antibodies to human lymphocytes. S-100 beta+ T-cells recognized by their diffuse intracytoplasmic immunoperoxidase reaction, also expressed CD2, CD3, CD8 antigens demonstrated by surface blue alkaline phosphatase reactivity, but not CD4, CD1, CD25 (interleukin-2 receptor), or HLA-DR antigens. However, they displayed a blastic change to T-cell mitogens, such as Concanavalin A(Con-A) and PHA, followed by the expression of CD25 and HLA-DR antigens. Under normal conditions, S-100 beta+ T-cells comprised approximately 5-22.8% of CD8+ cells amongst human peripheral blood mononuclear cells.     

Beta-adrenoceptor blockade alters thymocyte differentiation in aged mice.

The thymus is the primary site for generation of naive T-lymphocytes in the young animal. With age, the thymus progressively involutes and fewer mature T-cells are produced and migrate to the periphery. With thymic involution, increased density of sympathetic noradrenergic (NA) innervation and concentration of norepinephrine (NE) have been observed. To determine if the age-related changes in thymocyte differentiation are modified by NE signaling through beta-adrenergic receptors, 2-month (mo) and 18-mo old BALB/c mice were implanted subcutaneously with pellets containing the non-selective beta-adrenoceptor antagonist nadolol. Four and one-half weeks later, thymus and peripheral blood were collected to assess changes in thymocyte differentiation and naive T-cell output by flow cytometric analysis of T-cell subpopulations. In old mice, but not in young mice, thymocyte CD4/CD8 co-expression was altered by beta-adrenoceptor blockade. In nadolol-treated old mice, the frequency of the immature CD4-8- population was increased, and the intermediate CD4+8+ population was reduced. A corresponding increase in the frequency of mature CD4-8+, but not CD4+8- cells was observed. The increase in CD4-8+ cells is most likely not mediated by more CD4-8+ cells undergoing positive selection, because CD3hi expression in the CD4+8+ population was not altered by nadolol. The percentage of CD8+44low naive cells in peripheral blood increased in nadolol-treated mice, suggesting that more CD4-8+ cells were exported from the thymus to the periphery. These results indicate that the age-associated increase in sympathetic NA innervation of the thymus modulates thymocyte maturation. Pharmacological manipulation of NA innervation may provide a novel means of increasing naive T-cell output and improving T-cell reactivity to novel antigens with age.     

Transgenic mice to study T-cell receptor gene regulation and repertoire formation

Transgenic mice have been obtained with genes coding for an alpha beta T-cell receptor that recognizes the male-specific antigen H-Y in association with the Db class I major histocompatibility complex molecule. Most if not all of the T-cells express the beta chain encoded by the transgene and show allelic exclusion of endogenous beta genes. In contrast, the expression of the alpha transgene does not completely block rearrangement and formation of functional endogenous alpha genes. In H-2b transgenic female mice the transgenic T-cell receptor is functionally expressed on at least 30% of CD8+ peripheral T-lymphocytes as indicated by their ability to lyse male target cells. Also in transgenic H-2b male mice a large proportion of peripheral T-cells appear to express the transgenic receptor. However, these cells do not react with male target cells because they show only low level or no expression of CD8 cell interaction molecules. Tolerance is established in the male transgenic thymus through deletion of CD4+CD8+ immature thymocytes.     

Major histocompatibility complex class I restricted T-cell autoreactivity in human peripheral blood mononuclear cells

During selection in the thymus or any subsequent response, T-cells recognize peptides bound to major histocompatibility complex (MHC) molecules. Peptides produced by lysosomes or by proteasome/immunoproteasome stimulate CD4+ or CD8+ T-cell, respectively. Inflammation alters components of both antigen-processing pathways resulting in the production of different peptides. The role of such changes in self/non-self discrimination was examined in autologous mixed peripheral blood mononuclear cell cultures. Stimulator cells were incubated in the presence or absence of INF-gamma, with or without lysosome inhibitors (ammonium chloride/chloroquine), cathepsin inhibitor (E-64), or proteasome/immunoproteasome inhibitor (epoxomicin). Responder cells were added and zeta-chain phosphorylated forms were used as read out. INF-gamma did not affect zeta-chain phosphorylated forms, which means that the expected INF-gamma induced alterations in antigen processing machinery do not influence self/non-self discrimination. Surprisingly, the completely phosphorylated 23-kDa zeta-chain was always present except in the case of epoxomicin, indicating the presence of MHC class I restricted autoreactive CD8+ T-cells but not of MHC class II restricted autoreactive CD4+ T-cells, possibly due to more efficient negative selection in the thymus of the latter. Autoimmunity is prevented due to absence of help by CD4+ T-cells. This conclusion was confirmed by the lack of differences in IL-2 levels in cell culture supernatants, as well as, by the absence of differences in cell proliferation under the various conditions described above.     

The relationship between DNA repair after alkylation damage and in vitro aging in human T-lymphocytes

In vitro cultures of human peripheral blood lymphocytes, both unstimulated G0 cells as well as phytohemagglutinin (PHA) stimulated cells, have been used in the investigation of DNA repair following different types of damage, including that induced by ultraviolet light, ionizing radiation, and chemical agents. We report here repair of DNA damage in cultured normal human T-lymphocytes after treatment with the alkylating agents, N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) or methanesulfonate (MMS), as measured by the alkaline elution technique. T-lymphocytes cultured with different sources of T-cell growth factors (TCGFs) were shown to have similar repair levels, as measured by loss of single-strand breaks. However, diminished repair was observed with in vitro culture age when T-cells were cultured with PHA and TCGF for a 3-week period. Cell-cycle analysis performed on asynchronously growing cultures indicated that differential repair with in vitro aging was not cell-cycle-related. Fluorescence Activated Cell Sorting (FACS) was used to determine the percentages of CD4+ and CD8+ T-cell subsets present during the in vitro culture periods. Cultures consisted primarily of CD4+ cells until day 20 when the percentage of CD8+ cells increased to approximately 50% of the T-cell population. Neither the absolute percentages of CD4+ and CD8+ cells not the CD4+/CD8+ ratios correlated with repair rates of cultured T-cells. Therefore, the observed decreases in the repair of alkylating agent-induced damage are not due solely to the change in the CD4+/CD8+ ratio.     

De novo Generation of Cells within Human Nurse Macrophages and Consequences following HIV-1 Infection

Nurse cells are defined as those that provide for the development of other cells. We report here, that in vitro, human monocyte-derived macrophages can behave as nurse cells with functional capabilities that include de novo generation of CD4+ T-lymphocytes and a previously unknown small cell with monocytoid characteristics. We named these novel cells "self-renewing monocytoid cells" (SRMC), because they could develop into nurse macrophages that produced another generation of SRMC. SRMC were not detectable in blood. Their transition to nurse behavior was characterized by expression of CD10, a marker of thymic epithelium and bone marrow stroma, typically absent on macrophages. Bromodeoxyuridine labeling and immunostaining for cdc6 expression confirmed DNA synthesis within nurse macrophages. T-cell excision circles were detected in macrophages, along with expression of pre-T-cell receptor alpha and recombination activating gene 1, suggesting that genetic recombination events associated with generation of the T-cell receptor were occurring in these cells. SRMC expressed CCR5, the coreceptor for R5 HIV-1 isolates, and were highly susceptible to HIV-1 entry leading to productive infection. While expressing HIV-1, SRMC could differentiate into nurse macrophages that produced another generation of HIV-1-expressing SRMC. The infected nurse macrophage/SRMC cycle could continue in vitro for multiple generations, suggesting it might represent a mechanism whereby HIV-1 can maintain persistence in vivo. HIV-1 infection of nurse macrophages led to a decline in CD4+ T-cell production. There was severe, preferential loss of the CCR5+ CD4+ T-cell subpopulation. Confocal microscopy revealed individual HIV-1-expressing nurse macrophages simultaneously producing both HIV-1-expressing SRMC and non-expressing CD3+ cells, suggesting that nurse macrophages might be a source of latently infected CD4+ T-cells. Real-time PCR experiments confirmed this by demonstrating 10-fold more HIV-1-genome-harboring T-cells, than virus-expressing ones. These phenomena have far-reaching implications, and elicit new perspectives regarding HIV pathogenesis and T-cell and hematopoietic cell development.     

Age-related differences in integrin expression in peripheral blood lymphocytes

Alpha integrins play an important role in cell to cell and cell to extra-cellular matrix interactions required for an effective T-lymphocyte-mediated immune response, however little is known about age related differences in expression of alpha integrins on T-cells in humans. We here measured alpha-4 (α4) integrin (CD49d) expression on T-lymphocytes via peripheral blood sampling, comparing parameters between cohorts of young and old adults. No age-related differences were found for the absolute numbers of T-cells, although the percentage of CD4+ T-cells in older adults was significantly greater and the percentage of CD8+ T-cells lower than in younger cohorts. Percentage and absolute numbers of CD3+ T-cells co-expressing CD49d were significantly lower in older adults compared to younger cohorts, and the percentage of gated CD4+ and CD8+ cells that co-labelled positively for CD49d was also reduced in this group. There were no age-related differences in circulating levels of cytokines (Type I interferons) that are known to regulate cell surface integrin expression. Reduced expression of alpha integrins on T-cells may be an early indicator of the loss of homeostatic control that occurs with ageing, contributing to diminished effector T-cell responses during senescence.     

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.     

Organization of the human T-cell receptor genes

T lymphocytes recognize antigens through their membrane bound T-cell receptors. Whereas the conventional T-cell receptors are heterodimers of alpha and beta chains, expressed at the surface of CD3+ CD4+ and CD3+ CD8+ T lymphocytes, the gamma delta T-cell receptors are found at the surface of a subset of T-lymphocytes of phenotype CD3+ CD4- CD8-. The synthesis of the T-cell receptor chains results from the junction (or rearrangement) of DNA segments: Variable (V) gene and joining (J) segment for the alpha and gamma chains, V gene, D (diversity) and J segments for the beta and delta chains. In this review, we summarize the recent findings on the genomic organization of the alpha, beta, gamma and delta T-cell receptor loci in human.     

A role for CD8 in limiting degeneracy of thymocyte selection.

The introduction of a soluble TCR (sTCR) recognizing class I major histocompatibility complex (MHC) in the fetal thymic microenvironment in vitro produces the selection of thymocytes with enhanced avidity for self class I MHC (8). The sTCR was supposed to impose enhanced avidity for self MHC at an early degenerate phase of TCR-driven selection. This could determine increased reactivity to self at later stages of differentiation when specificity of TCR-ligand interaction augments and the effect of sTCR vanishes. This hypothesis was based on the observed deletion of CD4+8+ thymocytes upon upregulation of TCR and the increase in cell size of some CD8+ cells which are expanded in long-term fetal thymus organ cultures (FTOC) as well as in the periphery of adoptively transferred nude mice. Here we show that the developing alphabeta thymocyte which does not express CD8 at the cell surface has a selective advantage in FTOC with sTCR, thus suggesting that participation of CD8 in self peptide/MHC recognition confers specificity to T-cell selection and results in excessive signaling in thymocytes in spite of the presence of sTCR.     

Quantification of T-cell dynamics during latent cytomegalovirus infection in humans

Cytomegalovirus (CMV) infection has a major impact on the T-cell pool, which is thought to be associated with ageing of the immune system. The effect on the T-cell pool has been interpreted as an effect of CMV on non-CMV specific T-cells. However, it remains unclear whether the effect of CMV could simply be explained by the presence of large, immunodominant, CMV-specific memory CD8⁺ T-cell populations. These have been suggested to establish through gradual accumulation of long-lived cells. However, little is known about their maintenance. We investigated the effect of CMV infection on T-cell dynamics in healthy older adults, and aimed to unravel the mechanisms of maintenance of large numbers of CMV-specific CD8⁺ T-cells. We studied the expression of senescence, proliferation, and apoptosis markers and quantified the in vivo dynamics of CMV-specific and other memory T-cell populations using in vivo deuterium labelling. Increased expression of late-stage differentiation markers by CD8⁺ T-cells of CMV+ versus CMV- individuals was not solely explained by the presence of large, immunodominant CMV-specific CD8⁺ T-cell populations. The lifespans of circulating CMV-specific CD8⁺ T-cells did not differ significantly from those of bulk memory CD8⁺ T-cells, and the lifespans of bulk memory CD8⁺ T-cells did not differ significantly between CMV- and CMV+ individuals. Memory CD4⁺ T-cells of CMV+ individuals showed increased expression of late-stage differentiation markers and decreased Ki-67 expression. Overall, the expression of senescence markers on T-cell populations correlated positively with their expected in vivo lifespan. Together, this work suggests that i) large, immunodominant CMV-specific CD8⁺ T-cell populations do not explain the phenotypical differences between CMV+ and CMV- individuals, ii) CMV infection hardly affects the dynamics of the T-cell pool, and iii) large numbers of CMV-specific CD8⁺ T-cells are not due to longer lifespans of these cells.     

Non-regulatory CD8+CD45RO+CD25+ T-lymphocytes may compensate for the loss of antigen-inexperienced CD8+CD45RA+ T-cells in old age

The age-related decline in immune system functions is responsible for the increased prevalence of infectious diseases and the low efficacy of vaccination in elderly individuals. In particular, the number of peripheral naive T-cells declines throughout life and they exhibit severe functional defects at advanced age. However, we have recently identified a non-regulatory CD8+CD45RO+ CD25+ T-cell subset that occurs in a subgroup of healthy elderly individuals, who still exhibit an intact humoral immune response following influenza vaccination. Here, we demonstrate that CD8+CD45RO+CD25+ T-cells share phenotypic and functional characteristics with naive CD8+CD45RA+CD28+ T-cells from young individuals, despite their expression of CD45RO. CD8+CD45RO+ CD25+ T-cells also have long telomeres and upon antigenic challenge, they efficiently expand in vitro and differentiate into functional effector cells. The expanded population also maintains a diverse T-cell receptor repertoire. In conclusion, CD8+CD45RO+CD25+ T-cells from elderly individuals compensate for the loss of functional naive T-cells and may therefore be used as a marker of immunological competence in old age.     

Internalization of CD4 molecules in human T-cells demonstrated by immuno-electron microscopy.

Internalization of CD4 molecules on human CD4-enriched T-cells was demonstrated by immunocytochemical electron microscopy. CD4+ T-cell subclones were obtained from normal human peripheral blood, followed by one-way MLC screening and co-culturing with IL-2. Fixed and non-fixed T-cell samples were indirectly immunolabeled with mouse anti-human CD4 monoclonal antibody and goat anti-mouse IgG conjugated with peroxidase. Unfixed T-cells were immunolabeled at 4 degrees C and then re-incubated for 5-45 min at 37 degrees C. The selected CD4+ T-cell subclones showed strong CD4 binding on the cell surface after IL-2 incubation. However, fresh T-cells, monocytes, bone marrow cells and CD8+ T-cells all stained negative for CD4. The distribution of CD4 molecules on the fixed cell surface showed a homogeneous pattern. Capping and internalization of CD4-antibody-peroxidase complexes from the cell surfaces were observed follow a pathway of receptor-mediated endocytosis in unfixed T cells. Endocytotic vesicles, vacuoles of diverse sizes and shapes near the cell membrane or deep in the cell center were found to contain CD4 molecules. Negatively stained Golgi saccules were observed up to 45 min after re-incubation. These results suggest that increased CD4 molecules can be induced on the surface of normal human T-cells in vitro. Internalization and accumulation of CD4 molecules occurred in CD4-enriched T-cells with IL-2 pretreatment.     

The 5'-flanking region of the human CGL-1/granzyme B gene targets expression of a reporter gene to activated T-lymphocytes in transgenic mice.

The human CSP-B/CGL-1 gene is the homologue of the mouse granzyme B/CCPI gene and encodes a cytotoxic T-lymphocyte-specific serine protease. We have used regulatory sequences upstream from the CSP-B gene to drive human growth hormone gene expression in transgenic mice. Eleven founder mice were screened for transgene expression in activated T-cells. Expression was detected in 10 mice; levels of expression were integration site-dependent. The transgene was not expressed in resting lymphocytes but could be activated by treatment with concanavalin A or interleukin-2, indicating that CSP-B regulatory sequences are responsive to signals originating at either the T-cell receptor or the interleukin-2 receptor. Transgene expression was detected at the whole organ level only in lymph nodes and small intestine, where endogenous mouse CCPI mRNA was also present. The time course of transgene activation in T-lymphocytes was similar to that of the mouse CCPI gene. No differences in levels of expression of the transgene were observed in activated lymphocyte populations that had been depleted of either CD4+ or CD8+ cells; in contrast, the mouse CCPI gene was expressed primarily in CD8+ cells. Six CD4+ T-cell clones with Th0, Th1, or Th2 phenotypes were generated from a transgenic animal. All clones expressed moderate to high levels of the transgene, but only three clones expressed mouse CCPI, indicating that the transgene is disregulated in CD4+ T-cell subsets. The CSP-B regulatory unit represents a novel reagent for targeting gene expression to activated T-lymphocytes.     

PD-1 blockage reverses immune dysfunction and hepatitis B viral persistence in a mouse animal model

Persistent hepatitis B viral (HBV) infection results in chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma (HCC). Recent studies in animal models of viral infection indicate that the interaction between the inhibitory receptor, programmed death (PD)-1, on lymphocytes and its ligand (PD-L1) play a critical role in T-cell exhaustion by inducing T-cell inactivation. High PD-1 expression levels by peripheral T-lymphocytes and the possibility of improving T-cell function by blocking PD-1-mediated signaling confirm the importance of this inhibitory pathway in inducing T-cell exhaustion. We studied T-cell exhaustion and the effects of PD-1 and PD-L1 blockade on intrahepatic infiltrating T-cells in our recently developed mouse model of HBV persistence. In this mouse animal model, we demonstrated that there were increased intrahepatic PD-1-expressing CD8+ and CD4+ T cells in mice with HBV persistence, but PD-1 upregulation was resolved in mice which had cleared HBV. The Intrahepatic CD8+ T-cells expressed higher levels of PD-1 and lower levels of CD127 in mice with HBV persistence. Blockade of PD-1/PD-L1 interactions increased HBcAg-specific interferon (IFN)-γ production in intrahepatic T lymphocytes. Furthermore, blocking the interaction of PD-1 with PD-L1 by an anti-PD-1 monoclonal antibody (mAb) reversed the exhausted phenotype in intrahepatic T lymphocytes and viral persistence to clearance of HBV in vivo. Our results indicated that PD-1 blockage reverses immune dysfunction and viral persistence of HBV infection in a mouse animal model, suggesting that the anti-PD-1 mAb might be a good therapeutic candidate for chronic HBV infection.     

OK T-positivity in stimulated T-lymphocytes

T-lymphocytes express different antigenic determinants which can be recognized using specific anti-T monoclonal antibodies. OK T3 (Ortho Diagnostics, Raritan, N.J.) detects 95% circulating T-lymphocytes, while OK T4 reacts with helper/inducer T-lymphocytes and OK T8 with suppressor/cytotoxic T-lymphocytes. In normal peripheral blood the proportion of mononuclear cells (after "Lymphoprep' separation) positive with the various anti-T monoclonal antibodies is, according to our standards, as follows: OK T3 77 +/- 9.4%, OK T4 51 +/- 7.8%, OK T8 27+/- 6.4%. In this study we have evaluated the positivity with OK T MoAb after activation and proliferation of the T-cell population in a double layer T-lymphocyte colony assay. After 4-5 days of incubation, the proportion of OK T3 + cells had increased to 94 +/- 4.6%, while that of OK T4+ and OK T8+ had raised to 62 +/- 14.1% and 65 +/- 7.1% respectively. These data suggest that T-colony formation gives rise to an increased expression of OK T8 positivity, possibly through a mechanism of T-cell activation (shown also by the 'Ia' positivity), and/or of proliferation of T-cells with a double antigenic phenotype.     

Activation of CD4+ T-lymphocytes in asymptomatic HIV infected patients induce the program action of lymphocyte death by apoptosis]

Activation-induced programmed cell death, or apoptosis, is a physiological cell suicide process involved in the negative thymic selection of the T-cell repertoire. We have proposed that the inappropriate re-emergence in the mature CD4+ T-cell population of such a death program could explain both the early dysfunction and the late depletion of CD4+ T-cells from human immunodeficiency virus (HIV)-infected individuals. We present evidence showing that the selective failure of T-cells from 10 HIV-infected asymptomatic individuals (with normal CD4+ T-cell counts) to proliferate in vitro to pokeweed mitogen and to self-major histocompatibility complex class-II T-cell receptor mobilization by superantigens is due to the induction by these stimuli of CD4+ T-cell death. This death process has characteristic features of apoptosis, including DNA fragmentation into multiples of a 200 base pair unit, and the preventive effect of the protein synthesis inhibitor cycloheximide. These findings suggest that in vivo CD4+ T-cell suicide upon activation might account, independently of any HIV-mediated cytopathic effect, for the progressive depletion of CD4+ T-cells that leads to AIDS.