SEARCH

SEARCH BY CITATION

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

Aside from an intermediate stage in thymic T-cell development, the expression of CD4 and CD8 is generally thought to be mutually exclusive, associated with helper or cytotoxic T-cell functions, respectively. Stimulation of CD8+ T cells, however, induces the de novo expression of CD4. We demonstrate that while superantigen (staphylococcal enterotoxin B, SEB) and anti-CD3/CD28 costimulation of purified CD8+ T cells induced the expression of CD4 on CD8+ T cells by 30 and 17%, respectively, phytohaemagglutinin (PHA) stimulation did not induce CD4 expression on purified CD8+ T cells but significantly induced the expression of both CD4 on CD8 (CD4dimCD8bright) and CD8 on CD4 (CD4brightCD8dim) T cells in unfractionated peripheral blood mononuclear cells (PBMC). The level of the PHA-mediated induction of CD4dimCD8bright and CD4brightCD8dim was at 27 and 17%, respectively. Depletion of CD4+ T cells from PBMC abrogated this PHA-mediated effect. Autologous CD4+ and CD8+ T-cell co-cultures in the presence of PHA induced this CD4dimCD8bright T-cell expression by 33%, demonstrating a role for CD4 cells in the PHA-mediated induction of the double positive cells. The induction of CD4dimCD8bright was independent of a soluble factor(s). Phenotypic analysis of CD4dimCD8bright T cells indicated significantly higher levels of CD95, CD25, CD38, CD69, CD28, and CD45RO expression than their CD8+CD4 counterparts. CD4dimCD8bright T cells were also negative for CD1a expression and were predominately T-cell receptor (TCR) αβ cells. Our data demonstrate that CD4dimCD8bright T cells are an activated phenotype of CD8+ T cells and suggest that CD4 upregulation on CD8+ T cells may function as an additional marker to identify activated CD8+ T cells.


Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

Maturation of T cells in the thymus leads to the expression of CD4 or CD8 molecules, which are associated with T helper or cytotoxic T cells, respectively. Although during thymic T-cell development, a CD4+CD8+ immature T-cell stage exists, the expression of CD4 and CD8 cell surface molecules on mature T cells in the periphery is thought to be mutually exclusive. On the contrary, approximately 3–5% of peripheral blood lymphocytes express both CD4 and CD8 molecules on their surface.1–4 The expression of CD4+CD8+ T cells in the periphery was previously believed to be a result of the premature release of double positive (CD4+CD8+) thymocytes from the thymic medulla into the periphery. Substantial evidence, however, indicates that mature CD8+ T cells can be induced to express CD4, generating a CD4dimCD8bright phenotype.5–8 CD4 cells can also be induced to express CD89 (reviewed in 10), generating a CD4brightCD8dim phenotype. The CD4brightCD8dim phenotype is especially prominent in a number of animal models, including swine, rodent, chicken, and monkeys (reviewed in 10). Additionally, the expression of these double-positive T cells is enhanced in a number of clinical conditions including human T lymphotropic retrovirus type 1 infection,11 chronic T lymphoid leukaemia,12 Sjögren's syndrome,13 myasthenia gravis,2–4,14 multiple sclerosis,15 idiopathic thrombocytopenic purpura,16 and Behçet's syndrome.17 The significance of this induction, however, remains to be elucidated.

To understand the role of CD4dimCD8bright T cells in immunity, we initiated studies to define the conditions leading to CD4 induction on CD8+ T cells. Previously, we and others demonstrated that purified CD8+ T cells can be induced to express CD4 on their surface in response to superantigen (staphylococcal enterotoxin, SEB), anti-CD3/CD28 costimulation, or allogeneic dendritic cell interaction.5–8 While superantigen and anti-CD3/CD28 costimulation have consistently demonstrated the induction of CD4 on CD8+ T cells, the effect of phytohaemagglutinin (PHA) on mediating CD4 upregulation on CD8+ T cells is discordant. In one study, PHA was reported to induce the upregulation of CD4 on purified CD8+ T cells,6 while another study reported that this was not the case.5 We therefore evaluated the impact of PHA on the induction of CD4 on CD8+ T cells. We also evaluated the phenotypic profile of CD4dimCD8bright T cells in comparison to CD8+ T cells that do not express CD4 (CD8+CD4). To phenotypically define these CD4dimCD8bright T cells, we evaluated a number of markers that are associated with T-cell activation, naïve/primed cells, and functional status. The activation markers included CD25 (interleukin (IL)-2 receptor α chain), CD69 (early activation antigen), CD38 (activation marker), human leucocyte antigen (HLA)-DR (major histocompatibility complex (MHC)-II) and CD95 (the Fas receptor). CD25 and CD69 define early cellular activation while HLA-DR is associated with late events in cell activation. We also analysed the expression of the isoforms of CD45RA and CD45RO, which classically define naïve (CD45RA+CD45RO) and memory (CD45RACD45RO+) phenotypes but nonetheless have a number of drawbacks in classifying naïve, memory/primed T cells.18–21 CD28 is a costimulatory molecule that binds to B7 on antigen presenting cells. The expression of CD28 defines a functional phenotype of cells. We also examined CD1a expression, which is associated with immature thymocytes as well as T-cell receptor (TCR) αβ expression. Collectively, these markers were utilized to distinguish the phenotype of CD4dimCD8bright T cells from CD4CD8+ T cells.

Materials and methods

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

Isolation of peripheral blood mononuclear cells and T-cell subsets

Peripheral blood mononuclear cells (PBMC) were isolated by Ficoll–Hypaque density gradient centrifugation from venous blood collected from healthy donors as previously described.22 Purified CD4+ T cells were isolated from PBMC by positive immunuoselection using magnetic beads (Dynal, Lake Success, NY). The purity of the isolated CD3+CD4+ T cells was > 98%, as determined by flow cytometric analysis. Purified CD8+ T cells were isolated by negative immunoselection using magnetic beads (Dynal), depletion of CD4 in these cultures was consistently > 99%. CD19-depleted (B-depleted) PBMC, and CD16, 56-depleted (natural killer (NK) cell-depleted) PBMC were generated by the removal of CD19+ B cells and CD16,56+ NK cells, respectively, by positive immunoselection using magnetic beads (Dynal). Isolated cells were suspended in RPMI-1640 medium (Biowhittaker, Walkersville, MD) supplemented with 10% human AB serum (Gemini Bio Products, Inc., Calabasas, CA), 1% penicillin/streptomycin (Sigma, St. Louis, MO), and IL-2 (20 U/ml). Cultures were subsequently stimulated with 1 µg/ml SEB (Sigma), 1 µg/ml soluble anti-CD3 and anti-CD28 antibodies (PharMingen; San Diego, CA), or 4 µg/ml PHA (Sigma) and maintained for 6 days, unless otherwise indicated.

Transwell and neutralization assays

PHA-stimulated CD4+ and CD8+ T cells (1 × 106 cells/ml) were added to the transwell chambers (0·4 µm transwell; Costar, Corning, NY). CD4+ T cells and CD8+ T cells were both added either to the top or bottom of the chamber in multiple experiments. Six days post-PHA stimulation, the cells were monitored for CD4dimCD8bright and CD4brightCD8dim expression by flow cytometry. Anti-leucocyte function-associated antigen-1 (LFA-1), anti-MHC-II, anti-intracellular adhesion molecule-1 (ICAM-1), and anti-MHC-I (1 µg/ml; PharMingen) antibodies were separately added to PHA-stimulated cultures and CD4dimCD8bright expression was monitored by flow cytometry 6 days post-PHA stimulation.

Immunofluorescence staining and flow cytometric analysis

Cell surface staining of the lymphocyte subsets was preformed at day 0 and 6 days following stimulation. Briefly, cells were incubated at room temperature for 20 min with the appropriate antibody panel conjugated to fluorescein isothiocyanate (FITC), phycoerythrin (PE), peridinin chlorophyll protein (PerCP), or allophycocyanin (APC). The phenotypic panel used in these experiments consisted of: (1) immunoglobulin G1 (IgG1) FITC: IgG1 PE: IgG1 PerCP: IgG1 APC; (2) CD3 FITC: CD4 PE: CD8 PerCP: CD19 APC; (3) IgG1 FITC: CD4 PE: CD8 PerCP: IgG1 APC; (4) CD45RA FITC: CD4 PE: CD8 PerCP: CD45RO APC; (5) CD28 FITC: CD4 PE: CD8 PerCP: CD95 APC; (6) CD25 FITC: CD4 PE: CD8 PerCP: CD69 APC; (7) anti-HLA-DR FITC: CD4 PE: CD8 PerCP: CD38 APC; (8) TCR αβ-1 FITC: CD4 PE: CD8 PerCP; (9) TCR γδ-1 FITC: CD4 PE: CD8 PerCP; and (10) CD1a FITC: CD4 PE: CD8 PerCP. Immunofluorescent-labelled cells were then washed with phosphate-buffered saline (PBS) and fixed in 1% formaldehyde. All antibodies were purchased from either Becton Dickinson (San Jose, CA) or PharMingen. Four-colour flow cytometric analysis was preformed using a FACSCalibur flow cytometer utilizing CELLQuest software (Becton Dickinson). Dual expression of CD4 and CD8 was determined by gating on CD3+ lymphocytes. In some experiments, the percentage of CD4dimCD8bright expression was determined by gating on CD3+CD8bright lymphocytes. Inversely, the percent of CD4brightCD8dim expression was evaluated by gating on CD3+CD4bright lymphocytes.

Statistical analysis

The data was analysed using SAS and S-Plus statistical software packages. Paired data signed rank test was applied to compare the differences between the phenotypic profile of CD4dimCD8bright and CD8+CD4 cells. A P-value < 0·05 is indicative of significant difference. Descriptive statistics such as mean, standard deviation, median, minimum, and maximum were also generated to analyse the data.

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

Induction of CD4 on CD8+ T cells

To examine the impact of PHA on the induction of CD4 on CD8+ T cells, CD8+ T cells were isolated by negative immunoselection, which depleted > 99% of the CD4+ T cells. The highly purified CD8+ T cells were treated with superantigen (SEB at 1 µg/ml), soluble anti-CD3/CD28 antibodies (1 µg/ml), or PHA (4 µg/ml). Six days post-stimulation, the cultures were stained using several fluorescent antibodies (CD3 FITC: CD4 PE: CD8 PerCP) and subjected to three-colour flow cytometric analysis by gating on CD3+ lymphocytes. While unstimulated CD8+ T cells expressed approximately 3% CD4 on their surface, SEB and anti-CD3/CD28 costimulation induced CD4 expression on CD8+ T cells by approximately 30 and 17%, respectively (Fig. 1a,b). This CD4 induction exhibited a dim phenotype, while the CD8 expression appears brighter on these cells (Fig. 1b). The level of CD4 expression on PHA-stimulated CD8+ T cells (3·7%) was equivalent to that detected in unstimulated culture (3%, Fig. 1a), indicating that PHA stimulation of purified CD8+ T cells does not induce CD4 expression.

image

Figure 1. CD4 upregulation on CD8+ T cells. (a) CD8+ T cells were isolated from PBMC and stimulated with Staphylococcal enterotoxin B (SEB; 1 µg/ml), anti-CD3/CD28 antibodies (1 µg/ml), or phytohaemagglutinin (PHA, 4 µg/ml). On day 6 post-stimulation, the cells were stained with CD3 FITC: CD4 PE: CD8 PerCP-conjugated antibodies and analysed using flow cytometry. Asterisk denotes P < 0·05. Data is representative of at least three independent experiments. (b) Flow cytometric representation of above experiment gating on CD3+ lymphocytes. (i) Unstimulated culture. (ii) SEB-stimulated culture. (iii) Anti-CD3/CD28 costimulated culture. Flow cytometric representation of the PHA effect on CD4 expression on purified CD8+ T cells was similar to unstimulated culture and is not shown.

Download figure to PowerPoint

PHA induces CD4dimCD8bright and CD8dim CD4bright T cells in total PBMC

To determine if PHA can mediate the upregulation of CD4 on unfractionated cells, total PBMC were isolated and stimulated with PHA for 6 days. Although PHA stimulation of purified CD8+ T cells did not induce CD4 on the surface of CD8+ T cells, PHA stimulation of total PBMC induced not only CD4 on CD8+ T cells (CD4dimCD8bright) but also CD8 on CD4+ T cells (CD4brightCD8dim; Fig. 2a). This double-positive (CD4dimCD8bright+CD4brightCD8bright+CD4brightCD8dim) expression was at a mean of 28% (Fig. 2b). To distinguish between the level of CD4dim induction on CD8bright T cells and CD8dim induction on CD4bright T cells, we performed additional flow cytometric analysis by gating on the CD8bright and CD4bright T cells, as illustrated in one representative example shown in Fig. 2(a). We demonstrate that a mean of 27% of the CD8bright T cells expressed CD4dim while a mean of 17·0% of CD4bright cells expressed CD8dim (Fig. 2b).

image

Figure 2. Effect of PHA on the induction of CD4dimCD8bright and CD4brightCD8dim expression in PBMC. (a) Isolated PBMC and CD8+ T cells were stimulated with PHA (4 µg/ml) for 6 days. Stimulated CD8+ T cells and PBMC were stained with CD3 FITC: CD4 PE: CD8 PerCP-conjugated antibodies and analysed by flow cytometry. Top panel represents unstimulated CD8+ (i) and PBMC (ii). Bottom panel represents PHA-stimulated CD8+ T cells (iii) and PHA-stimulated PBMC (iv). The percent of CD4brightCD8dim and CD4dimCD8bright are shown as histogram of gates a and b, respectively. Results shown in (a) are representative of one experiment. (b) The percentage of CD8bright T cells expressing CD4dim on their surface in PHA-stimulated PBMC cultures was obtained by gating on CD3+CD8bright lymphocytes. Similarly, the percentage of CD4bright T cells expressing CD8dim on their surface was obtained by gating on CD3+CD4bright lymphocytes. CD4+CD8+ cells are indicative of CD4brightCD8dim+ CD4brightCD8dim+ CD4brightCD8bright cells. Results shown in B are the mean of at least three independent experiments.

Download figure to PowerPoint

To examine the mechanism leading to the PHA-mediated induction of CD4dimCD8bright and CD4brightCD8dim cells, PBMC were depleted of CD19 (B cells), CD16 and CD56 (NK cells), and CD4 (T helper cells) and subsequently stimulated with PHA. As seen in Fig. 3, depletion of CD4 abrogated the PHA-mediated effect to levels comparable to PHA stimulation of purified CD8+ cells, which was approximately 5%. Depletion of either B cells or NK cells, however, did not diminish this effect. This data suggests that CD4+ T cells are essential for the PHA-mediated induction of CD4dimCD8bright phenotypic expression.

image

Figure 3. CD4 is essential for PHA-mediated induction of CD4dim CD8bright cells. PBMC, CD4-depleted PBMC, B-depleted PBMC, NK-depleted PBMC, and purified CD8+ T cells were stimulated with PHA (4 µg/ml). The samples were stained for flow cytometry at day 6 with CD3 FITC: CD4 PE: CD8 PerCP-conjugated antibodies. Percent expression of CD4dimCD8bright T cells was obtained by gating on CD3+CD8bright lymphocytes. Results shown are the mean of at least two independent experiments performed in duplicates.

Download figure to PowerPoint

To further verify the role of CD4+ T cells in the PHA-mediated induction of CD4dimCD8bright phenotype, highly purified CD4+ and CD8+ T cells were isolated by immunoselection and co-cultured at 1 : 1 ratio with or without PHA. Co-cultures of PHA-stimulated CD4+ and CD8+ T cells induced the expression of CD4dimCD8bright cells to a mean of 33% (Fig. 4a,b). Notably, a mean of 8·8% of PHA-treated CD4+ T cells, without co-culturing with CD8+ T cells, induced CD8 expression on their surface (Fig. 4b). This is in contrast to the inability of purified CD8+ T cells to induce CD4 expression post-PHA stimulation. Examining the kinetics of the PHA-mediated induction of CD4dimCD8bright T cells after co-culturing CD4 and CD8 T cells at 1 : 1 ratio indicated that maximum induction (37%) in the co-cultures occurred by day 3 then gradually declined to 12% by day 12 (Fig. 4c). The media cultures, however, began to spontaneously express CD4dimCD8bright by day 9, reaching 16% by day 12, this level however, never reached the 37% induction observed with PHA stimulation (Fig. 4c).

image

Figure 4. The effect of CD4+ : CD8+ T-cell co-cultures on the PHA-mediated induction of CD4dimCD8bright T cells. (a) CD8+ T cells, PBMC, or a co-culture of autologous CD4+ : CD8+ T cells at a 1 : 1 ratio were stimulated with PHA (4 µg/ml) then stained for flow cytometric analysis at day 6 using CD3 FITC: CD4 PE: CD8 PerCP-conjugated antibodies. Percentage CD4dimCD8bright expression was obtained by gating on CD3+CD8bright lymphocytes. Results shown in (a) are the mean of at least three independent experiments. (b) Flow cytometric representation of unstimulated and PHA-stimulated cultures of CD8+ T cells (i and v), CD4+ T cells (ii and vi), CD4+ and CD8+ autologous T cell co-cultures (iii and vii), and PBMC (iv and viii). Top panel represents unstimulated cultures while bottom panel represents PHA-stimulated cultures. Gates a1–a3 represent CD4bright cells expressing CD8dim at 6, 25 and 27%, respectively. Gates b1–b2 represent CD8bright cells expressing CD4dim at 8 and 12%, respectively. The total percentage of CD4+CD8+ (CD4brightCD8dim+ CD4dimCD8bright+ CD4brightCD8bright) are indicated within each figure as 10, 24 and 19% of CD4+ T cells, CD4+ and CD8+ T-cell co-culture, and PBMC, respectively. (c) Kinetic analysis of CD4dimCD8bright T-cell expression post-PHA stimulation. CD4 and CD8 T cells were cultured as described previously and the percentage expression of CD4dimCD8bright T cells was analysed by flow cytometry for 12 days. Results shown are the mean of at least three independent experiments.

Download figure to PowerPoint

Given that the presence of CD4+ cells appears to be essential for the induction of CD4dimCD8bright T cells, we performed titration studies, whereby CD4+ T cells were added to CD8+ T cells at decreasing ratios ranging from 1 : 1 to 1 : 64. As seen in Fig. 5, decreasing ratios of CD4 : CD8 T cells reduced the mean percentage expression of the CD4dim CD8bright cells post-PHA stimulation. Specifically, at a 1 : 1 ratio of CD4 : CD8 T cells, CD4dimCD8bright expression post-PHA stimulation was at approximately 34%. However, at 1 : 4 ratio of CD4 : CD8 T cells, the level of CD4dimCD8bright T cells was decreased to 13·0%, and at 1 : 32 ratio of CD4 : CD8 T cells, CD4dim CD8bright T-cell expression was reduced to background levels (4·2%). These data demonstrate that the induction of CD4dimCD8bright T cells is dependent on the presence of CD4+ T cells (Fig. 5).

image

Figure 5. Dose–response curve of the role of CD4 cells in the PHA-mediated induction of CD4dimCD8bright cells. Autologous CD4+ and CD8+ T cells were co-cultured at ratios from 1 : 1 to 1 : 64 and stimulated with PHA (4 µg/ml). The co-cultures were stained for flow cytometric analysis on day 6 using CD3 FITC: CD4 PE: CD8 PerCP-conjugated antibodies. Percentage CD4dimCD8bright cells were obtained by gating on CD3+CD8bright lymphocytes. Results shown are the mean of at least three independent experiments.

Download figure to PowerPoint

Induction of CD4dimCD8bright T cells is not mediated by a soluble factor

To examine whether the induction of double-positive cells, specifically the induction of CD4dimCD8bright T cells, was mediated by a soluble factor, supernatants from PHA-stimulated PBMC and CD4+ : CD8+ T-cell co-cultures were added to unstimulated and PHA-stimulated CD8+ T cells. These supernatants did not mediate the induction of CD4dimCD8bright expression (data not shown). Purified CD4+ and CD8+ T cells were also cultured in a transwell system for 6 days with PHA stimulation. At least three independent experiments were conducted where CD4+ T cells were placed in the top or bottom wells. Co-culturing of CD4+ and CD8+ T cells in the presence of PHA with physical separation by a transwell membrane also did not induce CD4 expression on CD8+ T cells (Fig. 6) nor CD8 expression on CD4+ T cells (data not shown). Collectively, these experiments indicate that the induction of CD4dimCD8bright T cells is not mediated by a soluble factor and also suggest that direct cell-to-cell-contact between CD4+ and CD8+ T cells leads to the induction of double-positive cells.

image

Figure 6. Induction of CD4dimCD8bright cells is not mediated by a soluble factor(s). Purified CD8+ T cells or CD4+ T cells separated by transwell were stimulated with PHA (4 µg/ml) and cultured in this transwell system for 6 days as described in Materials and methods. Cells in the top (CD8+) and bottom (CD4+) wells were stained with CD3 FITC: CD4 PE: CD8 PerCP-conjugated antibodies and expression of CD4dimCD8bright lymphocytes was analyzed by flow cytometry. Data shown is the mean of at least three independent experiments.

Download figure to PowerPoint

Kinetics of CD4dimCD8bright T-cell expression

To determine the stability of CD4 expression on CD8+ T cells post-stimulation, purified CD8+ T cells were isolated as described previously and at day 0 stimulated with anti-CD3/CD28 or SEB. The cultures were maintained for at least 12 days in medium supplemented with 10% human AB serum and IL-2 (20 U/ml) and fed as necessary without additional stimulation. The SEB-mediated CD4 induction on CD8+ T cells reached its peak on day 6 (40%), which transiently declined to 22% at day 9 but peaked to 36% by day 12 (Fig. 7). The CD4dimCD8bright T-cell expression was stable for up to 24 days post SEB stimulation (data not shown). Anti-CD3/CD28 costimulation resulted in peak CD4dimCD8bright T-cell expression at day 6 (18%), which declined to 7% by day 9 and remained at that level for 12 days post-anti-CD3/CD28 costimulation (Fig. 7).

image

Figure 7. Kinetics of CD4 induction on CD8+ T cells. Purified CD8+ T cells were stimulated with SEB (1 µg/ml) or anti-CD3/CD28 antibodies (1 µg/ml) for at least 12 days. IL-2-supplemented medium was replaced as needed. Samples were stained for flow cytometry every 3 days with CD3 FITC: CD4 PE: CD8 PerCP-conjugated antibodies and analysed by flow cytometry. Percentage CD4dimCD8bright expression was obtained by gating on CD3+ lymphocytes. In SEB-stimulated culture, CD4dimCD8bright percentage expression was not statistically different (P > 0·05) between days 6 and 9. Results shown are the mean of at least three independent experiments.

Download figure to PowerPoint

Phenotypic characterization of CD4dimCD8bright T cells

To distinguish between CD8+ T cells that are induced to express CD4 (CD4dimCD8bright) and CD8+ T cells that do not express CD4 (CD8+CD4) under the same experimental conditions, purified CD8+ T cells were stimulated with SEB and CD4dimCD8bright and CD8+CD4 cells were analysed for the expression of functional (CD25, CD28, CD95, CD1a), activation (HLA-DR, CD38, CD69), naïve/primed (CD45RA, CD45RO), and TCR (αβ, γδ) phenotype. CD4dimCD8bright T cells are phenotypically distinct from their single-positive (CD8+CD4) counterpart (Fig. 8). The paired signed rank test demonstrates that SEB-stimulated CD4dimCD8bright cells significantly express higher levels of CD95 (P = 0·03), CD28 (P = 0·03), CD69 (P = 0·03), CD25 (P = 0·03), CD38 (P = 0·03), HLA-DR (P = 0·03), and CD45RA+CD45RO+ (P = 0·03) than SEB-stimulated CD8+ that did not express the CD4dim phenotype (Fig. 8a,c). Median percentage expression of these phenotypic markers is shown in Table 1. Although HLA-DR expression is statistically elevated on CD4dimCD8bright T cells, this level of induction is not remarkable and is within the normal range of HLA-DR expression on CD8+ T cells from healthy donors. Additionally, CD95 (98·3 versus 49·2%), CD25 (99·6 versus 44·1%), CD38 (98·2 versus 41·7%), and CD45RACD45RO+ (61·4 versus 37·3%) median percentage inductions were more strikingly elevated in CD4dimCD8bright than CD8+CD4 cells. Expression of CD45RA+CD45RO (P = 0·03) was also significantly less on the CD4dimCD8bright population than on CD8+CD4 cells (Fig. 8b,c). Unstimulated CD8+ cells (medium control) and SEB-stimulated CD8+CD4 cells demonstrated a phenotypically similar profile (Fig. 8c). Collectively, these phenotypic markers indicate that CD4dimCD8bright cells are an activated phenotype of CD8+ T-cell stimulation.

image

Figure 8. Phenotypic analysis of CD4dimCD8bright and CD8+CD4 cells. CD8+ T cells were stimulated with SEB (1 µg/ml) for 6 days. The stimulated CD8+ T cells were stained with a variety of phenotypic markers as indicated in Materials and Methods. The expression of the cell surface markers was evaluated by gating on CD4dimCD8bright versus CD8+CD4 lymphocytes. Unstimulated cultures were also evaluated. Paired data signed rank test, generated from six donors, for activation (a), functional (a), and primed/naïve phenotypes (b) are shown. Asterisk (*) denotes P-value < 0·05. White lines within the bar denote median difference. The black bars represent the 75% confidence interval. Error bars denote the maximum and minimum levels of the difference. Flow cytometric representations of the phenotypic panel of unstimulated CD8+ T cells (medium, CD8+CD4), SEB-stimulated CD8+ T cells that did not induce CD4 expression (SEB, CD8+CD4) and SEB-stimulated CD8+ T cells that induced CD4 expression (SEB, CD4dimCD8bright) are shown in (c).

Download figure to PowerPoint

Table 1.  Minimum, median, and maximum values * of the percentage of CD4 dimCD8bright cells post SEB-stimulation
Surface markerCD8+CD4CD8+CD4CD4dimCD8bright
  • *

    Values are minimum on the left, median in bold and maximum on the right.

CD9511·2, 25·8, 93·924·2, 49·2, 98·085·7, 98·3, 99·9
CD282·8, 57·1, 92·553·9, 85·3, 92·074·4, 97·0, 99·2
CD691·2, 7·0, 14·53·6, 6·7, 19·26·0, 15·3, 37·5
CD250·3, 6·5, 42·917·9, 44·1, 89·193·1, 99·6, 99·8
CD383·5, 10·5, 33·319·7, 41·7, 93·084·8, 98·2, 99·7
HLA-DR0·6, 9·0, 25·30·3, 0·6, 7·31·7, 5·3, 24·6
CD45RA+CD45RO38·6, 57·2, 74·05·9, 39·4, 65·70·2, 0·6, 0·8
CD45RA+CD45RO+2·1, 10·5, 11·83·4, 15·4, 25·29·1, 31·5, 88·1
CD45RACD45RO+15·1, 32·3, 47·513·4, 37·3, 73·111·2, 61·4, 84·9
CD1a0·3, 0·5, 0·50·1, 0·2, 0·70·9, 3·1, 8·0
TCR αβ86·8, 95·2, 99·196·1, 97·8, 98·797·6, 99·4, 100

To confirm that these CD4dimCD8bright cells are not prematurely released from the thymus, CD1a, a thymic marker, was evaluated. Neither CD4dimCD8bright nor CD8+CD4 cells expressed CD1a (data not shown). TCR γδ+ cells may coexpress CD4 and CD8; therefore, to confirm that these CD4dimCD8bright cells were not an expanded phenotype of TCR γδ+ T cells, we analysed TCR γδ and TCR αβ expression of both CD4dimCD8bright and CD8+CD4 T cells. Both cell populations predominately expressed TCR αβ at 99·4 and 97·8%, respectively (Table 1).

Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

The expression of CD4 can be induced on CD8+ T cells well after thymic selection. Limited studies have shown that SEB and anti-CD3/CD28 costimulation upregulate both protein and mRNA expression of CD4 on purified CD8+ T cells.5–8 However, the impact of PHA on mediating the induction of CD4 on CD8+ T cells has been discordant.5,6 In our studies, PHA did not mediate the induction of CD4 on purified CD8+ T cells. However, we demonstrate that PHA mediated the induction of CD4 on total/unfractionated PBMC. This PHA-mediated induction included both CD8 T cells expressing CD4 (CD4dimCD8bright) and CD4 T cells expressing CD8 (CD4brightCD8dim). Cell subset depletion experiments of PBMC and CD4 : CD8 co-culture experiments indicated that CD4 cells are required for the PHA-mediated induction of CD4 on CD8+ T cells. Based on our data, the previous discordant finding of the PHA effect on the upregulation of CD4 on CD8+ T cells may be explained by a minimum contamination of CD4+ T cells in the isolated CD8+ T-cell fraction, leading to CD4 mediation of the induction of the CD4dimCD8bright phenotype post-PHA stimulation. This effect was independent of a soluble factor, as demonstrated by transwell experiments, suggesting a direct cell-to-cell contact mechanism between CD4+ and CD8+ T cells, in the presence of PHA, to upregulate CD4 on CD8+ T cells.

It is critical to exclude the generation of doublets post-stimulation to account for CD4dimCD8bright T cells. Several lines of evidence demonstrate that these CD4dimCD8bright are not doublets but represent a genuine T-cell population: (i) The ‘dim’ expression of CD4 on CD8+ T cells indicates that these cells are not artefacts of doublets or antigen transfer; (ii) CD4 mRNA was shown to be 20-fold higher in CD4dim CD8bright T cells than CD8+CD4 cells;5 (iii) Highly purified CD8+ T cells (> 99% depletion of CD4+ cells) still responded to SEB and anti-CD3/CD28 costimulation by inducing CD4dimCD8bright; (iv) the phenotype of CD4dimCD8bright cells is distinct from CD8+CD4 cells; and (v) we were able to sort these CD4dimCD8bright by flow cytometry (data not shown). Collectively, these data indicate that these cells are not doublets but represent a distinct CD8 cell population that is induced to express CD4.

We have shown that CD4 cells are required for the PHA-mediated induction of CD4dimCD8bright cells, a requirement that is independent of a CD4 soluble factor, but may involve cell-to-cell contact. We hypothesized that this cell-to-cell contact may be mediated by CD4 binding to the MHC class-II molecule and conversely CD8 binding to the MHC class-I molecule. Antibodies to both MHC class-II and MHC class-I, however, did not abrogate this effect, nor did antibodies to LFA-1 or ICAM-1 (data not shown). It is possible, however, that these antibodies did not specifically block the binding epitopes involved in this interaction. Soluble CD4 also did not induce the CD4dimCD8bright phenotype (data not shown). PHA and to some extent co-culturing CD4 and CD8 T cells over time will induce CD4dimCD8bright T cells; we cannot exclude the possibility of antigen transfer or microvesicle formation as a possible mechanism for the induction of this phenotype. Nonetheless, this phenotype spontaneously generated or generated post-PHA stimulation may still be biologically relevant. In one study, microvesicles transferred from CCR5+ to a CCR5-negative cell rendered the recipient cell susceptible to M-tropic human immunodeficiency virus (HIV) infection.23 Therefore, although the exact mechanism of PHA-mediated induction of CD4 on CD8 T cells, aside from the requirement for the presence of CD4 T cells, remains to be elucidated, the presence of these cells may still be biologically significant.

Activation of CD8+ T cells appears to be critical in the induction of CD4dimCD8bright T cells. Phenotypic analysis of these cells demonstrated that they represent an activated phenotype. These CD4dimCD8bright T cells were more activated than CD8+CD4 cells, as indicated by elevated expression of CD25 (α chain of IL-2 receptor), CD69 (early activation marker), CD95 (Fas receptor) and CD38 (activation marker). Although CD95 is the Fas receptor, this molecule may not be associated with apoptosis but rather may be a marker for primed/memory cells. In fact, CD95hi in conjunction with CD27 (tumour necrosis factor receptor) and CD45RO+ is often used as a marker to identify primed/memory cells (CD45RO+CD27CD95hi). CD4dimCD8bright T cells are also larger and more granular than CD8+CD4 cells (data not shown), which is an indicator of activated cells. Conventional naïve (CD45RA+CD45RO)/primed (CD45RACD45RO+) phenotypic analysis of CD4dimCD8bright T cells also demonstrated a switch from naïve to activated phenotype. CD4dimCD8bright cells expressed higher levels of CD45RA+CD45RO+ than CD8+CD4 T cells, which maybe indicative of a naïve/primed transitional state. CD45RA/CD45RO markers to distinguish between naïve and memory/primed T cells, respectively, may not always be an adequate distinction when used alone; the additional markers of T-cell priming (CD25, CD69, CD38, HLA-DR, CD95), which we have evaluated collectively, indicate that these CD4dimCD8bright T cells have an activated phenotype.

CD4dimCD8bright also expressed higher levels of CD28 (costimulatory molecule) than CD8+CD4 cells. Unlike the phenotype of CD4dimCD8bright T cells, the phenotype of SEB-stimulated CD8+ T cells that did not express CD4 (CD8+CD4) was similar to that of unstimulated CD8+ T cells. A similar phenotypic profile was also observed post-PHA stimulation of PBMC and gating on CD4dimCD8bright T cells (data not shown). The phenotypic analysis of CD4dimCD8bright cells indicates that these cells constitute a distinct activated cell population from stimulated CD8+ T cells that do not upregulate CD4 expression. Lack of CD1a expression on CD4dimCD8bright cells demonstrate that these cells are not prematurely released from the thymus. Low-level expression of TCR γδ indicate that these double positive cells are not preferentially expanded gut-associated TCR γδ cells (reviewed in 24), which are known to express double-positive T cells, but are rather predominately TCR αβ cells.

Although these CD4dimCD8bright cells are an activated phenotype of CD8+ cells, whether these populations constitute an effector CD8+ population remains to be elucidated. High expression of CD28 on these cells may promote their interaction with antigen-presenting cells via CD80/CD86 engagement. This interaction may be critical in mounting a cytotoxic T lymphocyte response. Effector CD8+ populations have been linked to the downregulation of CD28.25–28 CD28 expression on CD4dimCD8bright cells may be downregulated post-antigen presentation. CD4 upregulation on CD8+ T cells may also be an additional marker to distinguish between activated and non-activated CD8+ T cells, possibly an indicator of early activation. The CD4 molecule induced on CD8+ T cells is thought to be functional, as indicated by its linkage to the Src family protein tyrosine kinase p56lck (Lck)6 and its ability to transduce an appropriate chemotactic signal following ligation by IL-16 (data not shown).

CD4 expression on CD8+ T cells may have a critical impact on the pathogenesis of the HIV or any other CD4-utilizing viruses such as human herpes virus-6. Our data demonstrate that CD4dimCD8bright cells are generated in response to CD8+ T-cell activation. HIV infection is associated with hyperactivation of the immune system,29–31 which may induce CD4 on CD8+ T cells. This induction may provide a mechanism for HIV entry into CD8+ T cells, especially in advanced HIV disease where CD4+ T cells are progressively depleted and CD8+ T cells are increased. CD4dimCD8bright T cells in this scenario may support the replication of HIV. CD8+ T cells from HIV seropositive patients have been shown to harbour HIV in vivo;6 whether these same cells also express CD4 remains to be elucidated and may be complicated by the fact that CD4 expression on these CD8+ T cells may be down-modulated post-HIV infection.

While the induction of CD8 on CD4+ T cells has been reported extensively in the literature (reviewed in 10), we provide evidence here in support of limited studies documenting the expression of CD4 on CD8+ T cells.5–8 This expression is induced by different mechanisms mediated by superantigen, anti-CD3/CD28 costimulatory signals, allogeneic dendritic cell interaction, or by mitogen stimulation. Understanding the biological significance of this induction is critical into gaining better insights into T-cell biology, HIV pathogenesis, and other clinical conditions leading to the increased expression of these mature double-positive T cells.

Acknowledgments

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

This work is submitted in partial fulfilment of the requirements for the degree of Doctor of Philosophy for Y.B.S. at Rush University (Chicago, IL). J.A.Z. is an Elizabeth Glaser Scientist supported by the Elizabeth Glaser Pediatric AIDS Foundation. We thank Dr Shande Chen (Department of Preventative Medicine, Section of Biostatistics, Rush-Presbyterian-St. Luke's Medical Center, Chicago, IL), for performing the statistical analysis and Dr Joan Siegel (Department of Immunology and Microbiology, Rush University, Chicago, IL) for helpful discussion. This work was partially supported by NIH grant AI48392 (J.A.Z.).

References

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References
  • 1
    Blue ML, Daley JF, Levine H, Schlossman SF. Coexpression of T4 and T8 on peripheral blood T cells demonstrated by two-color fluorescence flow cytometry. J Immunol 1985; 134:2281.
  • 2
    Schlesinger I, Rabinowitz R, Brenner T, Abramsky O, Schlesinger M. Changes in lymphocyte subsets in myasthenia gravis: correlation with level of antibodies to acetylcholine receptor and age of patient. Neurology 1992; 42:2153.
  • 3
    Ortolani C, Forti E, Radin E, Cibin R, Cossarizza A. Cytofluorimetric identification of two populations of double positive (CD4+,CD8+) T lymphocytes in human peripheral blood. Biochem Biophys Res Commun 1993; 191:601.DOI: 10.1006/bbrc.1993.1260
  • 4
    Sala P, Tonutti E, Feruglio C, Florian F, Colombatti A. Persistent expansions of CD4+ CD8+ peripheral blood T cells. Blood 1993; 82:1546.
  • 5
    Kitchen SG, Korin YD, Roth MD, Landay A, Zack JA. Costimulation of naive CD8 (+) lymphocytes induces CD4 expression and allows human immunodeficiency virus type 1 infection. J Virol 1998; 72:9054.
  • 6
    Flamand L, Crowley RW, Lusso P, Colombini-Hatch S, Margolis DM, Gallo RC. Activation of CD8+ T lymphocytes through the T cell receptor turns on CD4 gene expression: implications for HIV pathogenesis. Proc Natl Acad Sci USA 1998; 95:3111.DOI: 10.1073/pnas.95.6.3111
  • 7
    Yang LP, Riley JL, Carroll RG et al. Productive infection of neonatal CD8+ T lymphocytes by HIV-1. J Exp Med 1998; 187:1139.
  • 8
    Laux I, Khoshnan A, Tindell C, Bae D, Zhu X, June CH, Effros RB, Nel A. Response differences between human CD4 (+) and CD8 (+) T-cells during CD28 costimulation: implications for immune cell-based therapies and studies related to the expansion of double-positive T-cells during aging. Clin Immunol 2000; 96:187.DOI: 10.1006/clim.2000.4902
  • 9
    Blue ML, Daley JF, Levine H, Craig KA, Schlossman SF. Biosynthesis and surface expression of T8 by peripheral blood T4+ cells in vitro. J Immunol 1986; 137:1202.
  • 10
    Zuckermann FA. Extrathymic CD4/CD8 double positive T cells. Vet Immunol Immunopathol 1999; 72:55.DOI: 10.1016/s0165-2427(99)00118-x
  • 11
    Macchi B, Graziani G, Zhang J, Mastino A. Emergence of double-positive CD4/CD8 cells from adult peripheral blood mononuclear cells infected with human T cell leukemia virus type I (HTLV-I). Cell Immunol 1993; 149:376.DOI: 10.1006/cimm.1993.1163
  • 12
    Mizuki M, Tagawa S, Machii T et al. Phenotypical heterogeneity of CD4+ CD8+ double-positive chronic T lymphoid leukemia. Leukemia 1998; 12:499.
  • 13
    Aziz KE, McCluskey PJ, Wakefield D. Phenotypic and functional abnormalities in the peripheral blood T-cells of patients with primary Sjögren's syndrome. Cytometry 1994; 18:35.
  • 14
    Matsui M, Fukuyama H, Akiguchi I, Kameyama M. Circulating CD4+ CD8+ cells in myasthenia gravis: supplementary immunological parameter for long-term prognosis. J Neurol 1989; 236:329.
  • 15
    Munschauer FE, Stewart C, Jacobs L, Kaba S, Ghorishi Z, Greenberg SJ, Cookfair D. Circulating CD3+ CD4+ CD8+ T lymphocytes in multiple sclerosis. J Clin Immunol 1993; 13:113.
  • 16
    Mizutani H, Katagiri S, Uejima K et al. T-cell abnormalities in patients with idiopathic thrombocytopenic purpura: the presence of OKT4+8+ cells. Scand J Haematol 1985; 35:233.
  • 17
    Valesini G, Pivetti-Pezzi P, Mastrandrea F, Moncada A, Cuomo M, Natali PG. Evaluation of T cell subsets in Behcet's syndrome using anti-T cell monoclonal antibodies. Clin Exp Immunol 1985; 60:55.
  • 18
    Summers KL, JLOD, Hart DN. Co-expression of the CD45RA and CD45RO antigens on T lymphocytes in chronic arthritis. Clin Exp Immunol 1994; 97:39.
  • 19
    Peakman M, Mahalingam M, Leslie RD, Vergani D. Co-expression of CD45RA (naive) and CD45R0 (memory) T-cell markers. Lancet 1994; 343:424.
  • 20
    LaSalle JM & Hafler DA. The coexpression of CD45RA and CD45RO isoforms on T cells during the S/G2/M stages of cell cycle. Cell Immunol 1991; 138:197.
  • 21
    Murali-Krishna K & Ahmed R. Cutting edge: naive T cells masquerading as memory cells. J Immunol 2000; 165:1733.
  • 22
    Coligan JE, Kruesbeek AM, Margulies DH, Shevach EM, Strober W. Current Protocols in Immunology New York: John Wiley and Sons, Inc., 1994.
  • 23
    Mack M, Kleinschmidt A, Bruhl H et al. Transfer of the chemokine receptor CCR5 between cells by membrane-derived microparticles: a mechanism for cellular human immunodeficiency virus 1 infection. Nature Med 2000; 6:769.
  • 24
    Takahashi I & Kiyono H. Gut as the largest immunologic tissue. J Parenter Enteral Nutr 1999; 23:S7.
  • 25
    Trimble LA & Lieberman J. Circulating CD8 T lymphocytes in human immunodeficiency virus-infected individuals have impaired function and downmodulate CD3 zeta, the signaling chain of the T-cell receptor complex. Blood 1998; 91:585.
  • 26
    Weekes MP, Wills MR, Mynard K, Hicks R, Sissons JG, Carmichael AJ. Large clonal expansions of human virus-specific memory cytotoxic T lymphocytes within the CD57+CD28CD8+ T-cell population. Immunology 1999; 98:443.DOI: 10.1046/j.1365-2567.1999.00901.x
  • 27
    Weekes MP, Carmichael AJ, Wills MR, Mynard K, Sissons JG. Human CD28 CD8+ T cells contain greatly expanded functional virus-specific memory CTL clones. J Immunol 1999; 162:7569.
  • 28
    Hamann D, Baars PA, Rep MH, Hooibrink B, Kerkhof-Garde SR, Klein MR, Van Lier RA. Phenotypic and functional separation of memory and effector human CD8+ T cells. J Exp Med 1997; 186:1407.
  • 29
    Al-Harthi L, Siegel J, Spritzler J, Pottage J, Agnoli M, Landay A. Maximum suppression of HIV replication leads to the restoration of HIV-specific responses in early HIV disease. AIDS 2000; 14:761.
  • 30
    Valdez H, Al-Harthi L, Landay A, Lederman MM. Rationale for immune-based therapies for HIV-1 infection. J Lab Clin Med 1998; 131:197.
  • 31
    Kelleher AD, Al-Harthi L, Landay AL. Immunological effects of antiretroviral and immune therapies for HIV. AIDS 1997; 11 (Suppl. A):S149.