Interleukin-dependent modulation of HLA-DR expression on CD4 and CD8 activated T cells

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1 Immunology and Cell Biology (2002) 80, Research Article Interleukin-dependent modulation of HLA-DR expression on CD4 and CD8 activated T cells FRANCISCO J SALGADO, 1 JUAN LOJO, 1 CARMEN M FERNÁNDEZ-ALONSO, 1 JUAN E VIÑUELA, 2 OSCAR J CORDERO 1 and MONTSERRAT NOGUEIRA 1 1 Department of Biochemistry and Molecular Biology, University of Santiago de Compostela and 2 Immunology Service, University Clinical Hospital, Santiago de Compostela, Spain Summary Interleukins (IL) regulate different T-cell surface Ag known as activation markers that have distinct functional roles. In this paper, while studying the influence of some cytokines (IL-12, IL-2 and IL-4) on the expression of several markers [CD69, CD25, CD26, CD3, human leukocyte antigen (HLA-DR), CD45R0] in in vitro activated human T lymphocytes, we observed two groups of donors responding to phytohaemagglutinin (PHA) activation with high or low HLA-DR Ag expression. We also found that CD4 and CD8 populations had different HLA-DR densities under PHA activation (particularly the high HLA-DR-expressing group). Interleukins, in a dose-dependent manner (IL-2 partially), upregulated these HLA-DR levels. In 5 day cultures, IL-12 and IL-2 enhanced the CD8/CD4 ratio of activated T cells, which was responsible, in part, for the IL-dependent HLA-DR upregulation. IL-12 and IL-2 also upregulated the HLA-DR expression at the molecular level on CD8, and IL-12 downregulated it on CD4 cells. It seems that IL-4 upregulated HLA-DR by shortening the mitogen-dependent regulation kinetics. We hypothesize that the different effect of each IL on HLA-DR expression might be related to the regulation of the dose of antigenic peptide presentation and, thus, also influence T H1 /T H2 dominance. Key words: CD4, CD8, HLA-DR, interleukin-2, interleukin-4, interleukin-12. Introduction Within the activation process of T cells, several surface molecules are expressed de novo (CD69, HLA-DR) or are upregulated (CD45R0, CD26). 1 An evaluation of this process can describe, in vivo and in vitro, the activation status of the lymphocyte. Thus, the effects or biological activities of a factor on a T cell are often studied by the expression of these Ag, which are also called activation markers. The network of cytokines exerts a basic control on the immune response status, and the different levels of these markers and the fundamental role that they are playing separately on the immune response could be crucial to the response induced by each IL. The purpose of this study was to test important cytokines for their capacity to regulate some of these surface Ag on T lymphocytes. One example is CD69/VEA, one of the earliest Ag expressed by T cells following activation. The ligand and physiological function of CD69 are currently unknown, but it has costimulatory properties for T-cell activation and proliferation. 2 4 CD69 can also be expressed in an inducible way by B lymphocytes, NK cells, neutrophils, eosinophils, monocytes and immature thymocytes. 3 In NK cells, CD69 can be regulated by cytokines: IL-2, IFN-α or IL-12, but IFN-γ or TNF-α cannot induce its expression, 5 whereas IL-4 inhibits IL-2-generated CD69 expression. 6 Correspondence: Dr OJ Cordero, Departamento de Bioquímica e Bioloxía Molecular, Universidade de Santiago de Compostela, Facultade de Bioloxía, Campus Sur , Santiago de Compostela, Spain. bnojcord@usc.es Received 3 July 2001; accepted 15 July Another well characterized activation Ag is the α subunit of the IL-2 receptor (IL-2R), also called CD25, p55 or Tac antigen. CD25 does not participate in signal transduction resulting from IL-2 interaction, but its non-covalent association with the β (p70-75; CD122) and γ (p64, CD132) subunits gives rise to high affinity IL-2R, which is expressed on activated cells including T, NK and B cells, and monocytes. 7 CD25 has been shown to be positively regulated on T cells by several cytokines, such as IL-2, IL-12, IL-4 and TNF-α At least four isoforms of CD45 are generated from the alternative splicing of the same gene and the role of each one is not totally defined in thymocyte differentiation and T-cell activation. 11 In human T cells, the CD45R0 isoform defines a primed or memory T cell because CD45R0 upregulation with a parallel downregulation of the other isotypes occurs upon activation. 12 Conversion to CD45R0 on T cells and, more rapidly, on NK cells occurs during long-term culture with IL-2, but not with IL-4, IL-6 or IL In contrast, CD26, in addition to its dipeptidylpeptidase IV (DPPIV, EC ) 14 exopeptidase activity, has been implicated in extracellular matrix interactions as well as adenosine deaminase (ADA) binding activity. Moreover, CD26 interacts with CD45 on T-cell surfaces CD26 was originally described as a T-cell activation molecule because its expression is upregulated upon cellular activation. Several in vitro and in vivo findings, including IL-12 and IL-2 upregulation, point to CD26 as a T H1 response marker. With regard to membrane MHC class II molecules (termed HLA-DR, DP and DQ in humans), high levels of these molecules are expressed on professional antigen-presenting cells (APC), such as macrophages/monocytes, B lymphocytes

2 IL-dependent HLA-DR modulation on T cells 139 and dendritic cells, in which they present processed exogenous Ag to CD4 + T cells (APC-T presentation). The upregulation of HLA-DR Ag on APC by IFN-γ, IFN-α, IL-2, IL-4, TNF-α, TNF-β and GM-CSF and their downregulation by IL with fast kinetics have been extensively studied. T cells also express HLA-DR molecules upon activation, although with slower kinetics when compared to professional APC. 24 However, the role of Ag presentation by activated T cells (T T presentation) remains unclear. Accumulating evidence has been obtained supporting, at the same time, a role in increasing the immune response 25 leading to T-cell anergy upon a second encounter with a professional APC, 26 and in partial TCR-transduced signal, which biases the T cell towards a T H2 phenotype. 27 We present data in this paper that points to the implication of cytokines in surface HLA-DR Ag expression. Materials and Methods Cytokines and antibodies Recombinant human IL-12, rhil-2 and rhil-4 were purchased from Peprotech (London, UK), their specific activies being 10 U/ng. Anti-human CD26 mab Ta1-FITC (murine IgG1) was purchased from Coulter (Miami, FL, USA). Pure anti-hla-dr, reacting with a non-polymorphic determinant of HLA-DR antigen, simultest anti- CD3(Leu TM -4)-FITC/anti-HLA-DR-PE, simultest anti-cd4 (Leu TM - 3a)-FITC/anti-CD8 (Leu TM -2a)-PE, simultest CD45-FITC/CD14-PE, anti-cd20-percp, anti-cd4-apc, anti-cd56 (Leu19)-PE, anti-cd16 (Leu-11c) and anti-cd8-percp mab were from Becton Dickinson (San Jose, CA, USA). PE-conjugated antihuman CD45R0 and anti- CD25 mab (anti-il-2 receptor α) were provided by Pharmingen (Becton Dickinson). Ascitic fluid containing IgG2a and IgG1 isotype control mab (UPC10 and MOPC21) and FITC-conjugated polyclonal goat F(ab ) 2 antimouse IgG (GAM-FITC; used at a dilution of 1:100) were purchased from Sigma (Madrid, Spain). Murine mab anti-cd69 (IgG1) was a kind gift from Professor F Sánchez-Madrid (Hospital de la Princesa, Universidad Autónoma, Madrid) and used as hybridoma culture supernatants at a dilution of 1:2. TP1/16 (anti- CD26) from the same origin will be described in detail (FJ Salgado et al., unpubl. data, 2001). Cell isolation and culture Human peripheral blood mononuclear cells (PBMC) were isolated from healthy donors buffy coats provided by the Centro de Transfusiones de Galicia (Santiago, Spain) as previously described Ficoll-Paque (Amersham Pharmacia Biotech, Rainham, Great Britain) purified PBMC were resuspended at 10 6 cell/ml in complete medium (CM): RPMI-1640 medium (Sigma) supplemented with 10% inactivated FBS (56 C, 1 h; Gibco Life Technologies, Grand Island, NY, USA), 100 µg/ml streptomycin (Sigma) and 100 IU/mL penicillin (Sigma). Peripheral blood mononuclear cells were activated with 1 µg/ml of PHA-P (Sigma) in 24 flat-bottom wells plates or 25 cm 2 cell culture flasks in the presence or absence of cytokines for the times and concentrations indicated and with a humidified atmosphere of 5% CO 2 in air. Flow cytometry Peripheral blood mononuclear cells or PHA-activated cells taken from the flasks at the conclusion of the culture period were stained following an indirect or direct immunofluorescence protocol. Briefly, mononuclear cells or lymphoblasts were indirectly labelled for HLA- DR or CD69 Ag in PBS containing 1% BSA and 0.05% sodium azide (FACS buffer), with an optimal dilution of mab (20 µl/10 6 cell), at 4 C for 30 min, and washed three times with cold FACS buffer. Cells were then stained with FITC-conjugated goat antimouse IgG (GAM- FITC; 2 µl/10 6 cell; 1:100). When double, triple or quadruple labelling was performed, PBMC or lymphoblasts were incubated with commercial fluorochrome conjugated mab against CD3, CD4, CD8, CD45R0, CD25, HLA-DR or CD26 Ag at 4 C for 30 min. The cell pellet was washed twice with cold FACS buffer and finally resuspended in the same solution at a concentration of cell/ ml. Background staining of either GAM-FITC or an isotype-specific fluoresceinated Ab were routinely used as negative controls. The expression of each Ag on the cellular samples was quantified on a Becton-Dickinson FACScalibur cytometer and analysed with the WinMDI program, which was kindly provided by J Trotter (Scripps Institute, LaJolla, CA, USA). Viable cells were electronically gated on forward and side scatter para-meters, which are characteristic of activated lymphocytes. In spite of their slightly different distribution, the same region of the cell samples activated in the absence or presence of cytokines was chosen in order to allow comparison. Results Effects of IL-2, IL-12 and IL-4 on the expression of well-known T-cell activation markers The first purpose of our study was to evaluate the effect of IL- 2, IL-12 and IL-4 on the expression (both in percentage of positive T cells and intensity of staining) of some of the wellknown activation markers, such as CD45R0, CD25, HLA-DR and CD69. Table 1 shows a representative experiment analysing PBL or 5 day PHA-activated lymphocytes. IL-12, in addition to CD increased the IL-2R expression. 9 The low extent of CD25 upregulation by IL-2 and IL-4 may be explained by the endogenous PHA-dependent IL secretion. CD45R0 downregulation was also recorded, particularly by IL-12. An analysis of the expression of the early activation marker CD69 at the same incubation time showed that IL-12 upregulated CD69 on PHA-activated T lymphocytes at the same extent as IL-2. As was also described for IL-2, 2 this upregulation occurred early in the activation process, as it was particularly noticeable on the small, less activated cells and tended to disappear on the most activated lymphocytes (data not shown). Table 1 shows the strong positive effect of these IL on the HLA-DR expression with no apparent difference between T H1 (like IL-12) and T H2 (like IL-4) cytokines. It can be deduced from the data in Table 1 that IL are intrinsically affecting the expression of HLA-DR molecules rather than shifting the number of cells into the cycle. First, by observing the values corresponding to the CD3 marker because the three IL were capable of stimulating the proliferation of CD3 cells to the same extent (see the frequencies and intensities of CD3 staining in comparison with the cultures of mitogen alone). Second, by analysing the CD45R0 staining. CD45R0 is considered to be a marker of effector/memory T cells. Apart from the apparent downregulation of this molecule in the presence of IL (FJ Salgado et al., unpubl. data, 2001), the same number of CD45R0 + (i.e. mitogen-experienced) cells can be observed. Third, the IL-dependent upregulation of CD26 and CD25, showing different kinetics to that of

3 140 FJ Salgado et al. Table 1 Cell surface expression, in percentage of T cells, of activation Ag on resting and phytohaemagglutinin (PHA)-activated lymphocytes cultured with or without cytokines CD69 CD26 CD45R0 CD25 DR* CD3 PBL 2 (9) 54 (45) 56 (150) 15 (14) 11 (35) 71 (202) PHA (1 µg/ml) 37 (92) 90 (70) 79 (259) 54 (39) 38 (81) 88 (135) +IL-2 (50 U/mL) 38 (154) 98 (174) 73 (179) 65 (36) 79 (151) 97 (185) +IL-12 (1 ng/ml) 38 (165) 98 (205) 76 (92) 77 (87) 77 (226) 97 (197) +IL-4 (10 ng/ml) ND 96 (122) 73 (183) 70 (32) 71 (212) 97 (198) *Resting B cells of PBL are known to be negative for CD26, CD3, CD45R0, CD69 and CD25, but not for HLA-DR, thus in this column, the HLA-DR + CD3 + percentage is shown. Data for CD45R0 come from the cursor of negative for PE-labelled non-specific Ab and include R0 + RA and R0 + RA + cells. Peripheral blood lymphocytes are physically gated from Ficoll-purified PBMC and staining was performed as described in the Materials and Methods. Mean fluorescence intensity (MFI) data are in parentheses. ND, not done. mitogen, has been documented by us and others, and the results of this study fit well with those kinetics. 9,17,18 In this study, there appeared to be two groups of donors expressing HLA-DR. One group of donors had a weaker response to lectin, 50.5 ± 10.0% positive cells for HLA-DR (n = 6), than the other group, whose T lymphocytes responded strongly to PHA (78.2 ± 6.1%; n = 6; P < between both groups). Table 1 shows an example of the first group. IL-2- and IL-4-dependent upregulation was rarely detected in the strong donors. Kinetics and dose response of IL-12, IL-2 and IL-4 regulation of surface HLA-DR expression in activated T cells We studied the cytokine-dependent HLA-DR regulation in these experiments because it was strong and may have important physiological implications. The kinetics (not shown) of each IL upregulation revealed that, despite slight differences in the peak expression time (IL-4 after 3 4 days and IL-12 after 9 days), the differences among the activation conditions shown in Table 1 were maintained to the 11 day cultures, except in the case of IL-4 where, importantly, upregulation was lost. These data are consistent with published results on the PHA or anti-cd3 dependent HLA-DR peak expression upregulation, reflecting a slow rate of expression. 1,24 Thus, the dose response effect of each cytokine on HLA-DR antigen expression on T lymphocytes was evaluated in 5 day cultures with PHA. HLA-DR expression is a very good activation marker because it is more strongly expressed in the bigger blasts (more complex and activated cells) (Fig. 1a and b), but the regulation was different depending on the cytokine used. Figure 1 shows that low doses of IL-4 and IL-2 were sufficient to upregulate HLA-DR on lymphoblasts in donors with a weak response to PHA, but there was no apparent effect in donors responding with a strong response for HLA-DR expression (data not shown). Nevertheless, anti- HLA-DR mab showed greater staining with increasing doses of IL-4, but not of IL-2. It can be appreciated that HLA-DR expression was also dependent on the cytokine concentration in the case of IL-12 (Fig. 1a and b). At relatively high doses (10 ng/ml; Fig. 1c), IL-12 yielded the highest percentages and intensities in lymphoblast HLA-DR staining. Within the group with a weak response to PHA (related to HLA-DR staining), mean fluorescence intensity (MFI) were enhanced in a range between approximately 20% (like in Fig. 3) and fivefold (like in Fig. 1). We assayed IFN-γ because many IL-12 functions are mediated by this cytokine. Interferon-γ (50 U/ ml) upregulated HLA-DR at a lesser extent (data not shown) than 2 ng/ml of IL-12, so it seems that IL-12, at least in part, directly upregulates HLA-DR. Cytokine-dependent changes on in vitro-activated PBMC populations Therefore, we deduce the existence of differential HLA-DR regulation by cytokines in vitro. However, we have previously reported under these same culture conditions, and contrary to previously described results, 28 that IL-12 preferentially enhanced CD8 + T-cell proliferation. 18 Thus, these patterns of regulation (particularly for IL-4 and IL-12) may be due to: (i) an upregulation of HLA-DR expression at the molecular level and/or (ii) a preferential in vitro proliferation of a particular PBMC population with elevated levels of this marker. Moreover, the effect of IL-12, IL-2 and IL-4 on HLA-DR expression in CD3 APC and CD3 + T cells could be quite different, and these levels of expression on the former populations and their presence might affect the IL effects on T cells. We quadruple labelled for the CD3, CD4, CD8 and HLA- DR antigens, as well as performed CD14/CD20 and CD16/ CD56 double stainings, and evaluated their expression on PHA-activated lymphocytes. In agreement with previous reports 18,29 and as shown in Figs 1(c) and 2, activated lymphoblasts (easily gated as described in Materials and Methods) contained approximately 90% CD3 + T cells. CD14 (monocyte/macrophage) staining was always irrelevant. A consistent CD3 population was not always observed in our culture conditions (see Fig. 1c). If present (Figs 2 and 3) the highest levels for HLA-DR (MFI values) were always present in a fraction of this population. Nevertheless, IL-4 and particularly IL-12 (and IL-2 to a lesser extent), downregulated HLA-DR in this quadrant (Fig. 3). This is due, in part, to a diminution on the B-cell population with respect to PHA blasts (data not shown). However, CD20 (B cells) staining did not achieve the numbers of CD3 cells shown in Fig. 3. In the case of IL-12 and IL-2 this difference is due to an increase in NK cells, which proliferate in the presence of these cytokines 30 and only express HLA-DR at low levels, 31 because we recorded increases in the ratios of CD3 (CD20 ) DR + /CD3 DR (see Fig. 3) and CD16 + CD56 + (NK) cells (data not shown).

4 IL-dependent HLA-DR modulation on T cells 141 Figure 1 The induction of cell surface HLA-DR on phytohaemagglutinin (PHA)-blasts is modulated by cytokines. Isolated human PBMC were incubated for 5 days in flasks with PHA (1 µg/ml) and increasing doses of IL-12 ( ), IL-2 ( ) or IL-4 ( ) (a, b). In (a), HLA-DR levels (y-axis; logarithmic scale) were measured by flow cytometry on activated T cells belonging to the same gated region as non-stimulated lymphocytes (R1 region; data not shown). In (b), HLA-DR antigen expression was analysed on activated T lymphocytes with increased size and cellular complexity (R2 region). Note that the number of cells in both regions could be different in each condition. An experiment representative of three experiments with similar results is shown. Phenotype anlysis with anti-cd3-fitc (x-axis; log scale) and anti-hla-dr- PE (y-axis; log scale) is presented in (c). Mean fluorescence intensity (MFI) for both markers was measured in 5 day PHA (1 µg/ml) activated PBMC costimulated with IL-2 (50 U/mL), IL-12 (10 ng/ml) or IL-4 (10 ng/ml). Cells were gated as described in the Materials and Methods (note that in this case R is the addition of the R1 and R2 regions). Non-specific labelling with irrelevant mab allowed for placement of the HLA-DR-PE mab cursors. The ratio of CD4 : CD8 in fresh PBL T cells did not change with PHA stimulation (Fig. 2). However, simultaneous stimulation with IL-12 strongly modified this ratio 18 (Fig. 2), affecting CD4 + negatively and CD8 + positively. In five experiments IL-2 favoured the CD8 + /CD4 + ratio whereas IL-4 favoured the CD4 + /CD8 + ratio. Different cytokine-dependent regulation of HLA-DR expression on CD4 and CD8 subsets We studied HLA-DR expression in CD8 + and CD4 + T-cell populations and found that HLA-DR expression differed between the T-cell populations in the different cultures. Figure 4 shows a CD4 versus HLA-DR plot [of a previous forward scatter characteristics (FSC) versus CD3 gating] of a donor with a strong response to PHA. It can be appreciated in the PHA panel that the mitogen activated HLA-DR expression at different extents (MFI) on both CD4 + and CD4 (basically CD8 + ) T cells. This effect was reproducible in six experiments, independent of the HLA-DR expression response group, although the effect was weaker in the low response group (Table 2). Table 2, which corresponds to the same blood as Fig. 3, shows that IL (particularly IL-2 and IL-12) were upregulating HLA-DR in the CD8 + subpopulation. As previously noted, only IL-12 maintained the T-cell HLA-DR upregulation in the strong response group (Fig. 4). However, IL-12 upregulated HLA-DR on the CD3 + CD4 cells (both in number of positive cells and, very strongly, in MFI = 38.6 ± 3.2%, n = 3 independent experiments and donors, duplicated samples, P < between PHA and IL-12 groups, using 100% relativization data due to donor-dependent variability in HLA-DR levels), but downregulated this Ag on the CD4 + cells (an impairment of 20.7 ± 4.2% of MFI). In fact, IL-2 had a similar effect (16.3 ± 8.5% of MFI) on the CD3 + CD4 cells. Forward scatter data (not shown) demonstrated that these results were not due to a different cell size between the two cell populations. Finally, data from this group of donors suggest that PHA-activated cells reached the HLA-DR levels found for IL-4 within the other group (Figs 1 and 3 and Table 2). The same experiments were gated for FSC versus side scatter characteristics (SSC) followed by CD3/CD4 or CD3/ CD8 staining before analysing the HLA-DR levels in each

5 142 FJ Salgado et al. Figure 2 Evaluation through flow cytometry of CD4/CD8 percentages present in cytokine costimulated T lymphoblast cultures. Human PBMC were isolated and activated with 1 µg/ml of phytohaemagglutinin (PHA). In parallel cultures, IL-12 (10 ng/ml), IL-2 (50 U/mL) or IL-4 (10 ng/ml) were added at the same time and the cells recovered after 5 days. Direct immunofluorescence was performed with anti-cd4-fitc and anti-cd8-pe mab and measurements obtained with a FACSalibur flow cytometer (Becton Dickinson). Logarithmic mean fluorescence intensity (MFI) for CD4 and CD8 are shown on the x- and y-axis, respectively. The appearance of a CD4CD8 double positive population does not reflect inadequate colour compensation (data not shown). Note that, in the IL dots, the quadrant cursors have been changed in order to achieve simplification for the CD4 and CD8 subsets (due to the dual expression of both markers in the same cell), and the CD8dimCD4 T cells were ascribed to the double negative population. One of several experiments with similar results is presented. subpopulation. Although not shown, we also analysed the HLA-DR levels on CD3 + CD4 CD8 and CD3 + CD4 + CD8 + populations (the former being a cellular group with very important immunological properties). Nevertheless, both populations were not relevant for our studies. Figure 5a shows common histograms that reflect both the different number of cells in each condition and the anti-hla-dr staining. When histograms are normalized (i.e. the same number of events are compared; IL vs PHA control, Fig. 5b), the IL effect can be ascribed to the molecular level, although no numerical data can be obtained through this approach. Both IL-12- and IL-2-dependent HLA-DR upregulation on CD8 + and IL-12 downregulation on CD4 + cells are confirmed at the molecular level (it should be noted that these changes are quantitatively important because MIF data, i.e. number of surface molecules, are high). By this approach, it appears that IL-2 weakly upregulated HLA-DR on the CD4 cells and that IL-4 downregulated HLA-DR in both populations (in two out of three experiments). In conclusion, IL-12 and IL-2 are upregulating HLA-DR expression on CD8 + T cells, traditionally related to CTL responses, and the difference between these IL subpopulations consists of higher dose- and time-dependent proliferative rates of CD8 + T cells (which, after mitogen activation, are subsequently richer in HLA-DR molecules than CD4 + ) in IL-12 cultures. But the results also show a different effect on CD4 + T cells at the molecular level, with IL-12 slightly downregulating HLA-DR. Because there were not big differences between the proliferation of CD4 + and CD8 + T lymphocytes in the presence of IL-4 (Fig. 2), it appears that IL-4- dependent HLA-DR upregulation was at a kinetic level, and occurred faster than with the other factors (or even with PHA alone) in the short term, until mitogen stimulation induced similar levels as those in Fig. 5. Discussion T-cell stimulation leads to the strong and easily measured expression of molecules known as activation markers, which have an important role in the immune response. Usually, activation markers can be directly (in the absence of Ag or mitogen) regulated by many of the known cytokines, due to the constitutive presence of their receptors; in other cases the receptors are expressed de novo upon stimulation (e.g. IL-12R). IL-12, produced by APC, enhances the proliferation and cytolytic activity of NK and T cells and stimulates the appearance of a T H1 phenotype by secretion of cytokines such as IL-2, IL-3, IL-8, TNF-α, GM-CSF and, especially, IFN-γ. 32

6 IL-dependent HLA-DR modulation on T cells 143 Figure 3 HLA-DR expression in a CD3 (non-t) population is reduced when PBMC are activated with phytohaemagglutinin (PHA) plus IL-12. Cultures of PHA blasts, costimulated or not with IL-2 (50 U/mL), IL-12 (10 ng/ml) or IL-4 (10 ng/ml), were established as in Fig. 2. Phenotype analysis was performed with anti-cd3- FITC (x-axis; log scale) and anti- HLA-DR-PE (y-axis; log scale) mab. Non-specific labelling with irrelevant mab was used as a negative control for placing the HLA-DR-PE mab cursor. One experiment representative of several experiments in which a CD3 population can be found (in contrast with Fig. 2c) is presented. Figure 3 HLA-DR expression in a CD3 (non-t) population is reduced when 7(218) PBMC are activated with phytohaemagglutinin (PHA) plus IL-12. Cultures of PHA blasts, costimulated or not with IL-2 (50 U/mL), IL-12 (10 ng/ml) or IL-4 (10 ng/ml), were established as in Fig. 2. Phenotype analysis was performed with anti-cd3- FITC (x-axis; log scale) and anti- HLA-DR-PE (y-axis; log scale) mab. Non-specific labelling with irrelevant mab 6 was used as a negative control for placing the HLA-DR-PE mab cursor. One 45(42) 42 4(228) 5 61(48) 30 experiment representative of several experiments 9(43) in which a 52(55) 5(150) 71(49) CD3 population can be found (in contrast with Fig. 2c) is presented Table 2 HLA-DR expression on phytohaemagglutinin (PHA)- activated CD4 + and CD8 + T-cell subpopulations of a donor from the low and high response to the mitogen groups, and the differential HLA-DR regulation of the cytokines used as costimuli on both populations Low response donor High response donor CD4 CD8 CD4 CD8 PHA (1 µg/ml) 56 (57) 52 (62) 73 (83) 86 (110) +IL-2 (50 U/mL) 57 (44) 77 (76) 74 (80) 87 (117) +IL-12 (10 ng/ml) 57 (44) 76 (67) 72 (65) 90 (153) +IL-4 (10 ng/ml) 77 (44) 82 (76) 74 (75) 87 (103) Five-day PHA (1 µg/ml) blasts (see Fig. 3), cultured in complete medium (CM) supplemented or not with cytokines (IL-12, 10 ng/ml; IL-2, 50 U/mL; IL-4, 10 ng/ml) were simultaneously labelled with anti-cd3-fitc, anti-hla-dr-pe, anti-cd4-apc and anti-cd8- PerCP mab. T lymphocytes were initially gated according to their forward scatter characterisitics (FSC) versus side scatter characteristics (SSC) values and, subsequently, on the basis of their CD3 and CD4/CD8 expression. Data (mean of duplicates) are the percentage of DR positive cells, with mean fluorescence intensity (MFI) data in parentheses. Similar data were obtained in additional experiments (see text). We have recently described CD26 upregulation by IL-12 at low doses and by IL-2, but not by IL-1β, IL-4, IL-15, TNF-α or IFN-γ in human activated T lymphocytes. 17 This upregulation is specific because CD45R0 was not upregulated by IL-12 under the same culture conditions 18 and, in fact, showed differential characteristics to TCR-dependent CD26 upregulation. 18,19 In this paper, we examined the effects of this cytokine on the expression of other well-known activation markers in comparison to CD25, 7 9 and we focused on the observed T-cell HLA-DR upregulation by the three IL used in our in vitro system (Table 1). From early data 24 it is known that HLA-DR expression on T cells requires the recognition of self Ag on the surface of monocytes, as T-cell proliferation is not sufficient for TCRdependent HLA-DR upregulation. The initial results of this paper reveal a strong effect of IL on HLA-DR expression in conditions when activation is not complete (as deduced from the CD25 Ag expression). These results could be ascribed to the IL upregulation of HLA-DR on monocytes or B cells that are present in the conditions we set. This appears to be the case for IL-4, because of the faster kinetics and loss of upregulation when activated cells achieved the highest HLA- DR levels, but not for IL-2 and IL-12 in which the kinetics of upregulation were slower and the differences were maintained with time and APC became undetectable. However, it remains to be determined how CD8 cells recognize HLA-DR on monocytes (if, in fact, they do). The existence of two groups of donors exhibiting weak and strong (>70% positive cells for HLA-DR) responses to PHA have been observed previously

7 144 FJ Salgado et al. Figure 4 HLA-DR expression on phytohaemagglutinin (PHA)- activated CD4 + and CD8 + T-cell subpopulations from a donor in the group showing a high response to the mitogen, and the differential regulation of the cytokines used as costimuli on both populations. Five day PHA (1 µg/ml) blasts, cultured in complete medium (CM) supplemented or not with cytokines (IL-12, 10 ng/ml; IL-2, 50 U/mL; IL-4, 10 ng/ml) were simultaneously labelled with anti- CD3-FITC, anti-hla-dr-pe, anti-cd4-apc and anti-cd8- PerCP mab. T lymphocytes were initially gated according to their forward scatter characterisitics (FSC) versus side scatter characteristics (SSC) values and, subsequently, on the basis of their CD3 and CD4 expression. Dot plots represent HLA-DR levels (y-axis; logarithmic scale) on CD3 + CD4 and CD3 + CD4 + T lymphocytes (x-axis; CD4 mean fluorescence intensity (MFI), logarithmic scale). PHA upregulated HLA-DR preferentially on CD8 T-cell subpopulations. IL-12 or IL-2, when used as costimuli, also upregulated HLA- DR on CD8 T-cell subpopulations. Similar results were obtained in two additional donors (see text for statistical data). by us and others 33,34 for CD25. We deduce that the endogenous IL-2 production upon activation with lectins in the latter group is sufficient to reach the maximum densities of HLA- DR, which explains the weak effect of exogenous IL-2 in this group and the lack of dose-dependency. Another aspect of the initial results, which needed to be unravelled, was the change in CD4/CD8 ratios by the cytokines as changes in HLA-DR staining could be due to differential expression by T-cell populations. Both IL-2 and, particularly, IL-12 favoured the CD8 T-cell proliferation, indicating a T H1 and a CTL response in our conditions. In fact, the accumulation of CD8 in the presence of IL-12 with time is partly responsible for the apparent strong HLA-DR upregulation by this cytokine. It is interesting to note that memory CD8 + T lymphocytes require for proliferation and expansion the presence of a correct class I MHC, but not Ag, 35 thus, this T T cell contact could also be responsible for the expansion/ survival of CD8 + T cells in PHA-activated PBL. 36 Despite the restrictions of our model, that is, the presence of HLA-DR-presenting cells and endogenous production of cytokines, two facts are underlined: (i) in the presence of mitogen alone, CD8 cells presented higher HLA-DR staining than CD4 cells, and this fact might be explained by mechanisms of CD4 inhibition that are dependent on CD8 activation, in which a T T cell contact is required; 36 and (ii) apart from the changes in the CD4/CD8 ratios, these IL showed HLA-DR regulation at the molecular level, which was revealed by flow cytometry when histograms from the different conditions are compared and the number of events normalized. The importance of this finding is emphasized by the fact that each IL showed a different effect. The presence of IL-2 upregulated HLA-DR, particularly on the CD8 population, whereas IL-12 upregulated HLA-DR in the CD8 T cells and downregulated HLA-DR on the CD4 T cells. As both subunits of the IL-12R are expressed at the same extent in almost all T H1 and T C1 cells after 24 h of PHA activation 37,38 it appears that indirect additional effects (cell contacts) are related to these changes. Even IL-4, in PHA-activated cells expressing high HLA-DR, appear to slightly downregulate HLA-DR on both CD4 and CD8 cells, although more polarized T H2 cells should be used to confirm this regulation. In conclusion, the type of immune response and the physiological molecular (presence of different cytokines) and cellular (APC T cell or T T cell contacts) environment appear to be responsible for the levels

8 IL-dependent HLA-DR modulation on T cells 145 Figure 5 HLA-DR levels on CD3 + CD4 + and CD3 + CD8 + T cells activated in different conditions. In the upper rows (a, c) histograms represent gated lymphocytes that were cultured in the absence (black lines) or presence (grey lines) of the same IL concentration as in Fig. 4. In the lower rows of both populations (b, d) the same histograms were normalized (equal number of events for IL-cultured cells and PHA control), so that the IL-12 and IL-2 regulation of HLA- DR expression on CD8 + and CD4 + T cells at the molecular level can be confirmed. It also seems (in this experiment, see text) that IL-2 upregulated HLA-DR on CD4 + T cells, whereas IL-4 downregulated HLA-DR Ag on both populations. of HLA-DR molecules on an activated T cell. In fact, variable levels of HLA-DR expressing T cells are found in different pathological conditions. 1,39 It is now clear that recently activated T cells can present Ag to other T cells 25,26 and this capacity is correlated with the rate of synthesis of class II molecules. In addition to the MHC genotype, the type of costimulator and the TCR affinity, the dose of antigenic peptide presentation (the avidity effect) by APC has an important effect on sorting to T H1 or T H2 immune response against the peptide, with high-ligand density favouring a T H1 response and low-ligand density favouring a T H2 response. 40 Thus, it is reasonable to propose that the different effects of the IL used in this study on HLA-DR synthesis and expression will be related to the density of the presented peptide and, thus, also influence the T H1 /T H2 dominance in the same way as APC. In this sense, it is interesting to consider three recent papers, one study by Yue et al. 41 describes a direct IL-12 (500 U/mL) upregulation of HLA-DR in nonhaematopoietic melanoma cells (which express the IL-12Rβ), while another study by Grohmann et al. 42 reveals that IL-12 enhanced tumour peptide presentation by dendritic cells in a regulated way in vivo, and the third study by Lombardi et al. 27 observed the induction of IL-4 and inhibition of IL-2 production (IL related to the T H1 /T H2 dominance) by T-cell Ag presentation. From our data, different roles for the CD4 and CD8 HLA-DR + populations in this regulation are also deduced. Independent of the molecular mechanism of Ag presentation by T cells, the physiological function of this process is far from clear. Due to the role of many costimulatory molecules and the presence/absence of different cells (B, T, DC or other cells) at different time points of the immune response, the physiological function may vary from an amplification of the immune response to the T-cell anergy state Alternatively, but not mutually exclusively, MHC class II molecules seem to elicit an important function in T T cell interactions in the absence of Ag presentation. In addition, the survival of naive CD8 T cells and the proliferation of memory CD8 T cells requires TCR MHC (class I restricting molecules) interaction, 35 and efficient interactions between CD4 and MHC class II molecules are required for the survival of resting CD4 + T lymphocytes. 43

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