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CD4+and CD8+Cell Cytokine Profiles in Neonates, Older Children, and Adults: Increasing T Helper Type 1 and T Cytotoxic Type 1 Cell Populations with Age
CELLULAR IMMUNOLOGY ARTICLE NO.
183, 149–156 (1998)
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CD4/ and CD8/ Cell Cytokine Profiles in Neonates, Older Children, and Adults: Increasing T Helper Type 1 and T Cytotoxic Type 1 Cell Populations with Age James Chipeta, Yoshihiro Komada, Xao-Li Zhang, Takao Deguchi, Kenji Sugiyama, Eiichi Azuma,* and Minoru Sakurai Department of Pediatrics and *Department of Clinical Immunology, Mie University School of Medicine, Mie, Japan Received October 14, 1997
The growing body of evidence suggestive of T helper types 1 and 2 (Th1/Th2) including their counterparts T cytotoxic types 1 and 2 (Tc1/Tc2) cell responses during various human disease states necessitates determination of normal T cell subsets’ cytokine profiles. We show here, using intracellular cytokine staining and flow cytometry, that in healthy subjects interferon (IFN)-g producing CD4/ (Th1) and CD8/ (Tc1) cell populations progressively increase with age with strong correlation to CD45RO surface antigen expression. Meanwhile populations of cells capable of producing IL-4 (Th2 and Tc2) are comparably minimal across all age groups. Collectively, these results may reflect the maturation and expansion of Th1 and Tc1 cell populations from the neonatal period to adulthood, most probably dependent on antigen exposure. q 1998 Academic Press
INTRODUCTION The central regulatory cells of the immune system are the T cells, which mediate much of their function by expression and secretion of cytokines (1). There has been accumulating evidence suggesting that T helper types 1 and 2 (Th1/Th2) cell responses participate in pathological and immunological processes in human disease (2–7). Indeed, previous reports demonstrate gross imbalance in T cell subsets’ cytokine production profiles during various human clinical states. Common examples of these include predominance of Th2 profiles in children with atopy and asthma, Th1 in pathological pregnancy and acute Graft-versus-host disease (acute GVHD), gross cytokine production impairment during infectious diseases such as human immunodeficiency virus (HIV) infection, and inclination to Th1 in autoimmune diseases (8–12). Th1 cells produce high levels of interleukin (IL)-2, interferon (IFN)-g, and tumor necrosis factor (TNF)-b but no IL-4 or IL-5. Th2 cells produce high levels of IL-4, IL-5, and IL-6 but no IL-2 or IFN-
g. Th0 cells produce both types 1 and 2 cytokines. Similarly, their CD8 counterparts, T cytotoxic types 1 and 2 (Tc1/Tc2) have been described and are also involved in immunological responses and disease pathogenesis (13). This is suggestive of the potential importance of determining cytokine production profiles during clinical evaluation of immunological status and disease process. However, literature on normal cytokine production profiles from the neonatal period to adulthood is lacking. This information is vital not only for further elucidation of normal immunological variations among different age groups but may enhance clinical knowledge on immunological maturation. Furthermore, it may shed light on certain clinical issues such as why cord blood stem cell transplantation is infrequently associated with GVHD. Interestingly, several published human studies, to date, reveal marked normal variations in cytokine production patterns among different age groups (14 – 18). However, these studies invariably determined cytokine production from supernatants of stimulated bulk mononuclear cells, making it impossible to delineate responsible T cell subsets. Flow cytometry with the advent of the newly described intracellular cytokine technique has made it possible, now, to determine cytokine production at a single cell level (19, 20). This technique has been evaluated several times, gives comparable results in relation to traditional methods, and is proving to be useful and reliable (21, 22). We thus thought it worthwhile to determine and compare CD4/ and CD8 / cell populations capable of producing each and, simultaneously, two of the three major immunoregulatory cytokines (IL-2, IL-4, and IFN- g) in blood samples from healthy neonates, older children, and adults. Our results reveal, significantly, progressive increase in populations of T cell subsets capable of IFN- g and, simultaneously, IL-2 and IFN- g production from early infancy to adulthood in healthy individuals.
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0008-8749/98 $25.00 Copyright q 1998 by Academic Press All rights of reproduction in any form reserved.
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MATERIALS AND METHODS Subjects and Sample Handling
Cell Stimulation and Determination of Cytokine Producing Cell Populations by Flow Cytometry
Proportions of B cells, T cells, and their subpopulations, NK cells, and monocytes were determined in each sample by immunophenotyping using the lyzed whole blood method as described elsewhere (23) with minor modifications. The following monoclonal antibodies (MoAbs) purchased from Becton Dickinson immunocytometry systems (Mountain View, CA) were used: fluorescein isothiocyanate (FITC)-conjugated Leu-4 (CD3), Leu-3a(CD4), Leu-2a (CD8), anti-HLe-1 (CD45), Leu18 (CD45RA), phycoerythrin (PE)-conjugated Leu-3a (CD4), Leu-2a (CD8), Leu-45RO (CD45RO), Leu-12 (CD19), Leu-M3 (CD14), Leu-11a (CD16), and Leu-19 (CD56). Stained cells were analyzed with FACScan flow cytometer (Becton Dickinson) and results were expressed as percentage of positive cells in sample preparations.
For in vitro cell stimulation, 1 1 106 purified cells (CD4/ and CD8/ separately) from each subject were suspended in RPMI 1640 culture medium supplemented with 10% heat-inactivated fetal calf serum (FCS) in 12 1 75-mm polystyrene round-bottom tubes and incubated with 5 1 1008 M phorbol 12-myristate 13-acetate (PMA; Sigma), 1 mM ionomycin (Sigma) in the presence of 2 mM monensin (Sigma) for 12 h at 377C in an atmosphere of 95% air and 5% CO2 . The following MoAbs were purchased from Pharmingen Company (San Diego, CA): FITC-conjugated anti-human IFN-g (clone 4S.B3), PE-conjugated anti-human IL-2 (clone MQ1-17H12), PE-conjugated anti-human IL-4 (clone 8D4-8), FITC-conjugated isotype-matched control MoAb (IgG1 ; clone R3-34), and PE-conjugated isotypematched control MoAb (IgG2 ; clone R35-95). After stimulation with PMA and ionomycin, cells were double stained in the following combinations: FITC-conjugated anti-human IFN-g/PE-conjugated anti-human IL-2 and FITC anti-human IFN-g/PE-conjugated antihuman IL-4 alongside controls; FITC-conjugated antiIgG1/PE-conjugated anti-IgG2 . The flow cytometry intracellular cytokine staining protocol used was that described by Jung et al. (19) and refined by Prussin et al. (20) with slight modifications. Briefly, stimulated cells were washed once and then fixed and permeabilized by use of ORTHOPermeaFix (Ortho Diagnostic Systems, Raritan, NJ) for 45 min at room temperature followed by washing and nonspecifically blocking with phosphate-buffered saline (PBS) supplemented with 5% FCS for 10 min, again, at room temperature. Then the cells were washed and further blocked with Leu-11b MoAb (CD16; Becton Dickinson) for 30 min at 47C. Next, the cells were washed, resuspended in 5% FCS– PBS, and incubated with the above fluorescein-labeled anti-cytokine MoAbs together with controls for 30 min at 47C. Finally, the stained cells were washed and suspended in 400 ml of FACS flow solution for flow cytometric analysis. Where CD45RO and CD45RA expressions were determined in combination with intracellular cytokine production, cells were stained for surface markers simultaneously at the time of intracellular cytokine staining. The intensity of fluorescence was analyzed by FACScan flow cytometer (Becton Dickinson) having preset instrumentation for uniform analysis of each sample. Ten thousand events of gated viable cells were acquired and analyzed by use of CELLQuest software (Becton Dickinson). Dot plot quadrant statistics were set on the basis of corresponding isotype-matched control antibodies during data analysis such that frequencies of cell populations capable of IL-2, IL-4, IFN-g, IL-2/IFNg, and IL-4/IFN-g production were determined in each sample. Frequencies of cytokine producing cells were
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Inclusion criteria for the study of older children and adults were for strictly healthy individuals with no history of the following: (i) known chronic illness or recent acute illness, (ii) atopy and asthma, and (iii) medication or drug ingestion deemed to affect immune competence. In case of umbilical cord blood, only full term babies of 37 completed gestational weeks, normal birth weights (ú2500 g), and delivered by normal spontaneous vaginal delivery were recruited to the study. In each case heparinized venous blood was collected, under sterile conditions, and analyzed within 12 h of collection. The study was approved by the Human Subjects Committee of Mie University School of Medicine. Informed consent was obtained from all study participants or their parents. Preparation of CD4- and CD8-Rich T-cell Subsets Blood mononuclear cells (BMNC) were separated from umbilical cord, older children, and adult blood samples by Ficoll-Hypaque (Histopaque 1077, Sigma, St. Louis, MO) gradient centrifugation method. CD4and CD8-rich T cell subsets were then obtained from BMNC by positive immunomagnetic separation using DYNABEADS M-450 CD4 and CD8 (Dynal, Skoyon, Norway). In the case of some umbilical cord blood samples, with high concentration of natural killer (NK) cells, the CD30/16/56/ cell populations were sorted out from BMNC using a FACSvantage flow cytometer (Becton Dickinson) prior to separation by DYNABEADS to improve purity. In this way purity of all the purified cells was more than 90% (range 91–99%) except in cord blood CD8/ cells that were slightly lower, an average of 88% (81–98%). Surface Marker Staining
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calculated as follows: (percentage of fluorescent cells stained with anti-cytokine antibody) 0 (percentage of fluorescent cells stained with isotype-matched control antibody) Statistical Analysis Student’s paired t test for mean differences was used to analyze data for levels of statistical significance among the three age groups. Correlations between surface CD45 isoform expression and cytokine production was assessed using the Pearson correlation coefficient. In all statistical applications, P õ 0.05 was considered significant. RESULTS Cytokine Producing Cells in CD4/ and CD8/ T Cell Subsets A total of 47 subjects, 19 cord blood, 16 older children of mean age 11.6 (range, 9–14 years), and 12 adults with mean age of 36.8 (range, 30–53 years) were studied. The CD4/ and CD8/ T cell populations capable of IL-2, IFN-g, and IL-4 production were assessed using double-stained intracellular cytokine flow cytometric technique (Fig. 1). We physically separated and purified cells prior to intracellular staining to determine, with certainty, cytokine producing cell populations only in CD4/ and CD8/ T cells without contamination from monocytes and NK cells, respectively. More importantly, our aim was to check the preexisting cytokine production capability of the respective lymphocyte subpopulations without any accessory help. Irrelevant isotype-matched control antibodies produced less than 1% fluorescent cells. To make sure that only intracellular proteins were being quantified, cells were fixed but not permeabilized, giving less than 1.0% fluorescent cells. In addition, preincubating anti-IFN-g antibody with 20 mg/ml human recombinant IFN-g or anti-IL-2 antibody with 20 mg/ml human recombinant IL-2 for 1 h resulted in more than 98% inhibition of fluorescent cells. For IFN-g, IL-2, and IL-4, the percentage of positive cells for intracellular staining in unstimulated culture was routinely less than 1.0%. In cord blood, a large number of CD4/ T cells could exclusively produce IL-2 with almost no cell population capable of IFN-g production (õ1%). In contrast CD8/ T cell populations contained a small but distinct fraction of IFN-g producing cells besides those capable of IL-2 production. More interestingly, increased cell populations capable of producing IFN-g were clearly identified in both CD4/ and CD8/ cell populations obtained from older children and adults. Although the number of CD8/ cells producing solely IL-2 progressively decreased with age, their fluorescence intensity did not differ as depicted in Fig. 1. It is of note that the fluorescence intensity of intracellular IFN-g staining was sig-
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nificantly higher in older children and adults than that in cord blood. It is also noteworthy that both CD4/ and CD8/ T cells in older children and adults did contain distinct populations producing both IL-2 and IFN-g. Next, mean frequencies of the CD4/ and CD8/ cell populations capable of producing various cytokines were compared among the three age groups (Table 1). A remarkable observed trend is in the pattern of IFNg producing cell populations which significantly and progressively increase with age, particularly so with CD8/ cells of which a greater number are IFN-g producing by adulthood. Conspicuously, CD4/ cell populations capable of simultaneously producing IL-2 and IFN-g are nonexistent in cord blood (õ1%) but significantly increase in older children and adults. Similarly, their CD8/ counterparts progressively increase from less than 2% in early infancy to 5% in adults. Approximately 50 and 10% of the IFN-g producing cells are able to simultaneously produce IL-2 in CD4/ and CD8/ cell populations, respectively. In addition, CD8/ cells capable of producing IL-2 alone distinctly decrease in older children and adults in comparison with cord blood, whereas CD4/ cell populations capable solely of IL-2 production are comparably high in all the three age groups. Cell populations with the ability of IL-4 production and those capable of IL-4 and IFN-g production were comparably minimal across all the ages. Cytokine Production Profiles in CD45RO and CD45RA T Cells Aware of the fact that CD45RA/ and CD45RO/ T cells might produce different cytokines (24), we directly checked the expression of CD45 isoforms in IL-2 and IFN-g producing cells by flow cytometry (Fig. 2). As expected, almost exclusively, IFN-g producing CD4/ cells were CD45RO/. Surprisingly, only about twothirds of CD8/ IFN-g producing cells were CD45RO/ and a distinct fraction of CD45RO0/CD8/ cells could produce IFN-g. However, it should be noted that the fluorescence intensity of intracellular IFN-g staining was clearly lower in CD45RO0/CD8/ cells than in CD45RO//CD8/ cells and there was fluorescence continuity of CD45RO0 and CD45RO/ cells in IFN-g producing cell fraction. IL-2 producing cells, however, have no strong correlation to CD45RA expression. Both CD45RA0 and CD45RA/ cell populations were able to produce approximately equal amounts of IL-2 with a conspicuous fluorescence continuity between CD45RA0 and CD45RA/ cell populations in IL-2 producing CD4/ cells. These findings were further confirmed when individual values for CD45RO expression in 44 subjects’ T cell subsets were plotted on scatter graphs against their respective values of IFN-g production. At birth, invariably, no T-cell subset expresses CD45RO but with increasing age the expression progressively and significantly increases such that by adulthood the ma-
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FIG. 1. Flow cytometric analysis of cytokine producing T cell subsets in cord blood, older children, and adults. CD4/ (A) and CD8/ (B) cell populations capable of IL-2, IL-4, and/or IFN-g production were assessed using dual intracellular cytokine staining and flow cytometry. Representative two-parameter dot plots of IFN-g and IL-2 (IL-4) of each age group are shown in dot plot graphs. Percentages of cytokine producing cells in each quadrant are indicated. Quadrants of irrelevant isotype-matched control antibodies had õ1% fluorescent cells (data not shown).
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The majority of the concepts regarding T helper and T cytotoxic subset development are derived from experiments in murine models, and the maturation of naive human CD4/ and CD8/ T cells has been much less investigated. In the present study we investigated populations of T cells capable of producing IL-2, IL-4, and/ or IFN-g in umbilical cord blood, and older children and adult blood samples using double stained intracellular cytokine flow cytometric analysis. The results demonstrate that T cell subsets capable of IFN-g production progressively increase with age, probably suggestive of a normal physiological increase in Th1 and Tc1 cell populations in peripheral circulation from the neonatal period to adulthood. On the other hand, Th2 and Tc2 cell populations appear to be physiologically kept at minimal levels. The majority of both CD4/ and CD8/ T cells in cord blood express CD45RA antigen indicative of unprimed naive cells. It is now recognized that typical naive CD4/ T cells invariably produce only IL-2 upon stimulation (25, 26). The present study has consistently demonstrated that CD4//CD45RA/ T cells in cord blood contained exclusively a large number of IL-2 producing cells. However, and more interestingly, it is not the case with CD8/ cells. We showed that in cord blood,
CD8/ IFN-g producing T cells could be detected in addition to IL-2 producing cells, although fluorescence intensity of IFN-g staining was relatively low. These data suggest that naive CD8//CD45RA/ T cells could produce an appreciable amount of IFN-g besides the relatively high IL-2. Seemingly, though, the IFN-g producing CD8/ T cells in cord blood may not be truly naive cells as the IL-2 producing cell population could clearly be separated from IFN-g producing cell population in dot plot graphs of the dual staining flow cytometric data. The progressive increase in CD45RO expression with age of T cell subpopulations demonstrated by this study and also documented by several previous reports (27–30) may be inferred as a sign of maturation of naive T cells at birth to the CD45RO/ putative memory/effector subsets in adults. This maturation process most probably is dependent on antigen exposure. We have observed here that IFN-g producing cell populations increase with age among both CD4/ and CD8/ T cells. Moreover, consistent with previous reports is our observation that, among both CD4/ and CD8/ T cells, the CD45RO/ subset accounts for the preponderance of IFN-g production, whereas both CD45RO/ and CD45RA/ subsets appear to be able to produce IL-2 (24). Taken together, it is tempting to hypothesize here that the almost nonexistent IFN-g producing cell populations, observed in normal cord blood, are due to lack of activation in utero such that these increase with bouts of antigenic exposure of childhood and adulthood. Indeed, it has been demonstrated that children with a history of intrauterine exposure to certain antigens do have cytokine profile responses biased to those antigens (31). It may, thus, be further speculated that IFNg producing cell populations, increasing with age, are actually normal physiological Th1 and Tc1 cells in peripheral circulation. They are, most probably, primary reaction effector cells left in long-term memory and a direct reflection of previous antigenic exposures. Simultaneous evaluation of two cytokines within the same cell population revealed the existence of distinct T subsets in older children and adult peripheral blood that manifested synthesis of all possible combinations of the three major immunoregulatory cytokines (IL-2, IL-4, and IFN-g) assessed. Our results indicate that the most common cell populations in CD4/ T cell subset in both these age groups, are those producing IL-2 alone (approximately 40%). We also found that classic Th1 (IL-2//IFN-g//IL-40) and Th1-like (IL-20/IFN-g// IL-40) were displayed by only a fraction (5–6% and 3– 7%, respectively). Meanwhile, classic Th2 (IL-20/IFNg0/IL-4/) cells were minimal. More interestingly, most common in CD8/ T cell population were Tc1-like cells producing IFN-g alone, increasing dramatically with age (16 and 36% in older children and adults, respectively). We also revealed that CD8/ cells producing IL2 alone progressively lose these populations (19, 14, and 8% in cord blood, older children, and adults, respec-
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TABLE 1 Cytokine Production Profiles Cytokine producing cells (mean % { SD) Cord blood (n Å 19)
Cytokine CD4 T cell IL-2 IL-2/IFN-g IFN-g CD8 T cell IL-2 IL-2/IFN-g IFN-g
Older children (n Å 16)
Adult (n Å 12)
56.4 { 11.9a,b 0.77 { 0.53a,b 0.18 { 0.13a,b
41.2 { 8.98 5.58 { 3.47 3.02 { 2.19c
42.4 { 9.89 6.54 { 4.34 7.28 { 5.07
18.7 { 8.77a 1.13 { 0.73a,b 2.84 { 2.29a,b
14.3 { 5.72c 2.97 { 1.24 16.6 { 5.80c
8.55 { 5.11 4.90 { 3.91 36.1 { 13.2
Note. Statistical comparison of cytokine profiles in cord blood and other age groups showed significant difference (P õ 0.05) as follows: a cord blood versus adult, b cord blood versus older children, and c older children versus adult. Under each age group, in parentheses, is the number of samples analyzed.
jority of the cells are positive for CD45RO surface antigen. Significant correlation was demonstrated between CD45RO expression and IFN-g production in both CD4/ and CD8/ T cell populations (Fig. 3). It is of note that the value of correlation coefficient in CD4/ T cell population was slightly higher than that of their CD8/ counterparts. However, no correlation could be found with IL-2 production (data not shown). DISCUSSION
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FIG. 2. CD45RO and CD45RA expression in IL-2 and IFN-g producing cells. CD4/ (A) and CD8/ (B) cells obtained from healthy adults were stained with anti-CD45 isoform antibodies and anti-cytokine antibodies alongside their respective isotype controls as described under Materials and Methods. The experiments were independently performed four times, yielding similar results and representative two-parameter dot plots are shown in dot plot graphs. Percentages of positive cells in each quadrant are indicated.
tively), while classic Tc1 (IL-2//IFN-g//IL-40) patterns are displayed by a fraction (3–5%). However, our descriptions of these observed fluorescent subpopulations that seemingly coexpress IL-2 and IFN-g in either CD4/ or CD8/ cell populations as ‘‘typical’’ Th1 and Tc1, respectively, may not as such be uniquely different from those staining for IFN-g production only (Th1/ Tc1-like). As we have already noted, approximately 50 and 10% of the IFN-g producing cells are invariably able to simultaneously produce IL-2 in CD4/ and CD8/ cell populations, respectively. Thus, the observed increase in the IL-2/IFN-g may well be a function of the increase in IFN-g and not a selective increase in a unique cytokine coexpressing cell population. The variations between CD4/ and CD8/ cytokine
producing cell populations were also demonstrated in the cytokine synthesis capabilities of CD45RO/ and CD45RA/ T cell subsets. Almost exclusively, CD4/ IFN-g producing cells express CD45RO surface antigen, probably suggesting that upon antigen stimulation, CD4/ T cells first gain CD45RO expression followed by the ability to produce IFN-g. On the other hand, only about two-thirds of their CD8/ counterparts express CD45RO antigen. As shown by the continuity of CD45RO0 and CD45RO/ IFN-g producing CD8/ cell fraction, the former, with relatively smaller amount of IFN-g production capability, most probably become CD45RO/ cells capable of producing higher levels of IFN-g. These variations between CD4/ and CD8/ cytokine producing cell populations may be a reflection of
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Finally the increase in Th1 and Tc1 cell populations with age partly explains, as other studies have recently suggested (16, 33), why cord blood stem cell transplantation is infrequently associated with GVHD. The importance of humoral immunity in mediating chronic GVHD is supported by reports of increased IL-4 and IL-10 production in patients with this condition. This has led to the proposal that chronic GVHD is a consequence of Th2 cytokine production by donor CD4/ cells (6). Conversely, cell mediated immunity mediates many of the features of acute GVHD, suggesting that Th1 cytokines may be important in acute GVHD (6). Results presented here indicate that cord blood T cells have the ability to produce adequate IL-2 but lack the capability to produce IFN-g. Hence they may not be able to mount an effective primary reaction. It would be worthwhile to investigate cytokine profiles of cord blood T cells activated with alloantigen stimulation. Studies are now in progress to clarify whether synthesis of Th1 (Tc1) cytokines or Th2 (Tc2) cytokines could be induced by in vitro stimulation of cord blood T cell subsets in comparison with the cytokine producing ability of activated adult T cells. ACKNOWLEDGMENT This work was supported in part by a Scholarship Fund from the Ministry of Education, Science and Culture, Japan (MONBUSHO), under which Dr. James Chipeta is a graduate student in pediatric immunology.
REFERENCES 1. 2. 3. 4. 5. FIG. 3. Correlation of IFN-g production with CD45RO expression. Individual values of CD45RO cells in cord blood (open squares, n Å 19), older children (closed squares, n Å 15), and adults (open triangles, n Å 10) were plotted on scatter graphs against their respective values for IFN-g producing cells in CD4/ (A) and CD8/ (B) T cells. Correlations were assessed using the pearson correlation coefficient and statistical data indicated in each graph.
maturation to respective defined immune functions of these cells, with CD4/ cells acquiring and maintaining their cell-mediated and humoral immunity roles and CD8/ cells inclining to cytotoxicity and suppresser roles as suggested by one clonal study (13). Also, it is possible that the one-third of CD8/ IFN-g producing cells negative for CD45RO are reverted CD45RO/ cells. It has been said that CD45RA/ T cells in adult peripheral circulation and those in cord blood, though phenotypically similar, may be functionally different (33). The former are currently being speculated to be reverted CD45RO/ and not pure CD45RA/ cells (32–34).
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CI981244
CD4/ and CD8/ Cell Cytokine Profiles in Neonates, Older Children, and Adults: Increasing T Helper Type 1 and T Cytotoxic Type 1 Cell Populations with Age James Chipeta, Yoshihiro Komada, Xao-Li Zhang, Takao Deguchi, Kenji Sugiyama, Eiichi Azuma,* and Minoru Sakurai Department of Pediatrics and *Department of Clinical Immunology, Mie University School of Medicine, Mie, Japan Received October 14, 1997
The growing body of evidence suggestive of T helper types 1 and 2 (Th1/Th2) including their counterparts T cytotoxic types 1 and 2 (Tc1/Tc2) cell responses during various human disease states necessitates determination of normal T cell subsets’ cytokine profiles. We show here, using intracellular cytokine staining and flow cytometry, that in healthy subjects interferon (IFN)-g producing CD4/ (Th1) and CD8/ (Tc1) cell populations progressively increase with age with strong correlation to CD45RO surface antigen expression. Meanwhile populations of cells capable of producing IL-4 (Th2 and Tc2) are comparably minimal across all age groups. Collectively, these results may reflect the maturation and expansion of Th1 and Tc1 cell populations from the neonatal period to adulthood, most probably dependent on antigen exposure. q 1998 Academic Press
INTRODUCTION The central regulatory cells of the immune system are the T cells, which mediate much of their function by expression and secretion of cytokines (1). There has been accumulating evidence suggesting that T helper types 1 and 2 (Th1/Th2) cell responses participate in pathological and immunological processes in human disease (2–7). Indeed, previous reports demonstrate gross imbalance in T cell subsets’ cytokine production profiles during various human clinical states. Common examples of these include predominance of Th2 profiles in children with atopy and asthma, Th1 in pathological pregnancy and acute Graft-versus-host disease (acute GVHD), gross cytokine production impairment during infectious diseases such as human immunodeficiency virus (HIV) infection, and inclination to Th1 in autoimmune diseases (8–12). Th1 cells produce high levels of interleukin (IL)-2, interferon (IFN)-g, and tumor necrosis factor (TNF)-b but no IL-4 or IL-5. Th2 cells produce high levels of IL-4, IL-5, and IL-6 but no IL-2 or IFN-
g. Th0 cells produce both types 1 and 2 cytokines. Similarly, their CD8 counterparts, T cytotoxic types 1 and 2 (Tc1/Tc2) have been described and are also involved in immunological responses and disease pathogenesis (13). This is suggestive of the potential importance of determining cytokine production profiles during clinical evaluation of immunological status and disease process. However, literature on normal cytokine production profiles from the neonatal period to adulthood is lacking. This information is vital not only for further elucidation of normal immunological variations among different age groups but may enhance clinical knowledge on immunological maturation. Furthermore, it may shed light on certain clinical issues such as why cord blood stem cell transplantation is infrequently associated with GVHD. Interestingly, several published human studies, to date, reveal marked normal variations in cytokine production patterns among different age groups (14 – 18). However, these studies invariably determined cytokine production from supernatants of stimulated bulk mononuclear cells, making it impossible to delineate responsible T cell subsets. Flow cytometry with the advent of the newly described intracellular cytokine technique has made it possible, now, to determine cytokine production at a single cell level (19, 20). This technique has been evaluated several times, gives comparable results in relation to traditional methods, and is proving to be useful and reliable (21, 22). We thus thought it worthwhile to determine and compare CD4/ and CD8 / cell populations capable of producing each and, simultaneously, two of the three major immunoregulatory cytokines (IL-2, IL-4, and IFN- g) in blood samples from healthy neonates, older children, and adults. Our results reveal, significantly, progressive increase in populations of T cell subsets capable of IFN- g and, simultaneously, IL-2 and IFN- g production from early infancy to adulthood in healthy individuals.
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0008-8749/98 $25.00 Copyright q 1998 by Academic Press All rights of reproduction in any form reserved.
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MATERIALS AND METHODS Subjects and Sample Handling
Cell Stimulation and Determination of Cytokine Producing Cell Populations by Flow Cytometry
Proportions of B cells, T cells, and their subpopulations, NK cells, and monocytes were determined in each sample by immunophenotyping using the lyzed whole blood method as described elsewhere (23) with minor modifications. The following monoclonal antibodies (MoAbs) purchased from Becton Dickinson immunocytometry systems (Mountain View, CA) were used: fluorescein isothiocyanate (FITC)-conjugated Leu-4 (CD3), Leu-3a(CD4), Leu-2a (CD8), anti-HLe-1 (CD45), Leu18 (CD45RA), phycoerythrin (PE)-conjugated Leu-3a (CD4), Leu-2a (CD8), Leu-45RO (CD45RO), Leu-12 (CD19), Leu-M3 (CD14), Leu-11a (CD16), and Leu-19 (CD56). Stained cells were analyzed with FACScan flow cytometer (Becton Dickinson) and results were expressed as percentage of positive cells in sample preparations.
For in vitro cell stimulation, 1 1 106 purified cells (CD4/ and CD8/ separately) from each subject were suspended in RPMI 1640 culture medium supplemented with 10% heat-inactivated fetal calf serum (FCS) in 12 1 75-mm polystyrene round-bottom tubes and incubated with 5 1 1008 M phorbol 12-myristate 13-acetate (PMA; Sigma), 1 mM ionomycin (Sigma) in the presence of 2 mM monensin (Sigma) for 12 h at 377C in an atmosphere of 95% air and 5% CO2 . The following MoAbs were purchased from Pharmingen Company (San Diego, CA): FITC-conjugated anti-human IFN-g (clone 4S.B3), PE-conjugated anti-human IL-2 (clone MQ1-17H12), PE-conjugated anti-human IL-4 (clone 8D4-8), FITC-conjugated isotype-matched control MoAb (IgG1 ; clone R3-34), and PE-conjugated isotypematched control MoAb (IgG2 ; clone R35-95). After stimulation with PMA and ionomycin, cells were double stained in the following combinations: FITC-conjugated anti-human IFN-g/PE-conjugated anti-human IL-2 and FITC anti-human IFN-g/PE-conjugated antihuman IL-4 alongside controls; FITC-conjugated antiIgG1/PE-conjugated anti-IgG2 . The flow cytometry intracellular cytokine staining protocol used was that described by Jung et al. (19) and refined by Prussin et al. (20) with slight modifications. Briefly, stimulated cells were washed once and then fixed and permeabilized by use of ORTHOPermeaFix (Ortho Diagnostic Systems, Raritan, NJ) for 45 min at room temperature followed by washing and nonspecifically blocking with phosphate-buffered saline (PBS) supplemented with 5% FCS for 10 min, again, at room temperature. Then the cells were washed and further blocked with Leu-11b MoAb (CD16; Becton Dickinson) for 30 min at 47C. Next, the cells were washed, resuspended in 5% FCS– PBS, and incubated with the above fluorescein-labeled anti-cytokine MoAbs together with controls for 30 min at 47C. Finally, the stained cells were washed and suspended in 400 ml of FACS flow solution for flow cytometric analysis. Where CD45RO and CD45RA expressions were determined in combination with intracellular cytokine production, cells were stained for surface markers simultaneously at the time of intracellular cytokine staining. The intensity of fluorescence was analyzed by FACScan flow cytometer (Becton Dickinson) having preset instrumentation for uniform analysis of each sample. Ten thousand events of gated viable cells were acquired and analyzed by use of CELLQuest software (Becton Dickinson). Dot plot quadrant statistics were set on the basis of corresponding isotype-matched control antibodies during data analysis such that frequencies of cell populations capable of IL-2, IL-4, IFN-g, IL-2/IFNg, and IL-4/IFN-g production were determined in each sample. Frequencies of cytokine producing cells were
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Inclusion criteria for the study of older children and adults were for strictly healthy individuals with no history of the following: (i) known chronic illness or recent acute illness, (ii) atopy and asthma, and (iii) medication or drug ingestion deemed to affect immune competence. In case of umbilical cord blood, only full term babies of 37 completed gestational weeks, normal birth weights (ú2500 g), and delivered by normal spontaneous vaginal delivery were recruited to the study. In each case heparinized venous blood was collected, under sterile conditions, and analyzed within 12 h of collection. The study was approved by the Human Subjects Committee of Mie University School of Medicine. Informed consent was obtained from all study participants or their parents. Preparation of CD4- and CD8-Rich T-cell Subsets Blood mononuclear cells (BMNC) were separated from umbilical cord, older children, and adult blood samples by Ficoll-Hypaque (Histopaque 1077, Sigma, St. Louis, MO) gradient centrifugation method. CD4and CD8-rich T cell subsets were then obtained from BMNC by positive immunomagnetic separation using DYNABEADS M-450 CD4 and CD8 (Dynal, Skoyon, Norway). In the case of some umbilical cord blood samples, with high concentration of natural killer (NK) cells, the CD30/16/56/ cell populations were sorted out from BMNC using a FACSvantage flow cytometer (Becton Dickinson) prior to separation by DYNABEADS to improve purity. In this way purity of all the purified cells was more than 90% (range 91–99%) except in cord blood CD8/ cells that were slightly lower, an average of 88% (81–98%). Surface Marker Staining
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calculated as follows: (percentage of fluorescent cells stained with anti-cytokine antibody) 0 (percentage of fluorescent cells stained with isotype-matched control antibody) Statistical Analysis Student’s paired t test for mean differences was used to analyze data for levels of statistical significance among the three age groups. Correlations between surface CD45 isoform expression and cytokine production was assessed using the Pearson correlation coefficient. In all statistical applications, P õ 0.05 was considered significant. RESULTS Cytokine Producing Cells in CD4/ and CD8/ T Cell Subsets A total of 47 subjects, 19 cord blood, 16 older children of mean age 11.6 (range, 9–14 years), and 12 adults with mean age of 36.8 (range, 30–53 years) were studied. The CD4/ and CD8/ T cell populations capable of IL-2, IFN-g, and IL-4 production were assessed using double-stained intracellular cytokine flow cytometric technique (Fig. 1). We physically separated and purified cells prior to intracellular staining to determine, with certainty, cytokine producing cell populations only in CD4/ and CD8/ T cells without contamination from monocytes and NK cells, respectively. More importantly, our aim was to check the preexisting cytokine production capability of the respective lymphocyte subpopulations without any accessory help. Irrelevant isotype-matched control antibodies produced less than 1% fluorescent cells. To make sure that only intracellular proteins were being quantified, cells were fixed but not permeabilized, giving less than 1.0% fluorescent cells. In addition, preincubating anti-IFN-g antibody with 20 mg/ml human recombinant IFN-g or anti-IL-2 antibody with 20 mg/ml human recombinant IL-2 for 1 h resulted in more than 98% inhibition of fluorescent cells. For IFN-g, IL-2, and IL-4, the percentage of positive cells for intracellular staining in unstimulated culture was routinely less than 1.0%. In cord blood, a large number of CD4/ T cells could exclusively produce IL-2 with almost no cell population capable of IFN-g production (õ1%). In contrast CD8/ T cell populations contained a small but distinct fraction of IFN-g producing cells besides those capable of IL-2 production. More interestingly, increased cell populations capable of producing IFN-g were clearly identified in both CD4/ and CD8/ cell populations obtained from older children and adults. Although the number of CD8/ cells producing solely IL-2 progressively decreased with age, their fluorescence intensity did not differ as depicted in Fig. 1. It is of note that the fluorescence intensity of intracellular IFN-g staining was sig-
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nificantly higher in older children and adults than that in cord blood. It is also noteworthy that both CD4/ and CD8/ T cells in older children and adults did contain distinct populations producing both IL-2 and IFN-g. Next, mean frequencies of the CD4/ and CD8/ cell populations capable of producing various cytokines were compared among the three age groups (Table 1). A remarkable observed trend is in the pattern of IFNg producing cell populations which significantly and progressively increase with age, particularly so with CD8/ cells of which a greater number are IFN-g producing by adulthood. Conspicuously, CD4/ cell populations capable of simultaneously producing IL-2 and IFN-g are nonexistent in cord blood (õ1%) but significantly increase in older children and adults. Similarly, their CD8/ counterparts progressively increase from less than 2% in early infancy to 5% in adults. Approximately 50 and 10% of the IFN-g producing cells are able to simultaneously produce IL-2 in CD4/ and CD8/ cell populations, respectively. In addition, CD8/ cells capable of producing IL-2 alone distinctly decrease in older children and adults in comparison with cord blood, whereas CD4/ cell populations capable solely of IL-2 production are comparably high in all the three age groups. Cell populations with the ability of IL-4 production and those capable of IL-4 and IFN-g production were comparably minimal across all the ages. Cytokine Production Profiles in CD45RO and CD45RA T Cells Aware of the fact that CD45RA/ and CD45RO/ T cells might produce different cytokines (24), we directly checked the expression of CD45 isoforms in IL-2 and IFN-g producing cells by flow cytometry (Fig. 2). As expected, almost exclusively, IFN-g producing CD4/ cells were CD45RO/. Surprisingly, only about twothirds of CD8/ IFN-g producing cells were CD45RO/ and a distinct fraction of CD45RO0/CD8/ cells could produce IFN-g. However, it should be noted that the fluorescence intensity of intracellular IFN-g staining was clearly lower in CD45RO0/CD8/ cells than in CD45RO//CD8/ cells and there was fluorescence continuity of CD45RO0 and CD45RO/ cells in IFN-g producing cell fraction. IL-2 producing cells, however, have no strong correlation to CD45RA expression. Both CD45RA0 and CD45RA/ cell populations were able to produce approximately equal amounts of IL-2 with a conspicuous fluorescence continuity between CD45RA0 and CD45RA/ cell populations in IL-2 producing CD4/ cells. These findings were further confirmed when individual values for CD45RO expression in 44 subjects’ T cell subsets were plotted on scatter graphs against their respective values of IFN-g production. At birth, invariably, no T-cell subset expresses CD45RO but with increasing age the expression progressively and significantly increases such that by adulthood the ma-
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FIG. 1. Flow cytometric analysis of cytokine producing T cell subsets in cord blood, older children, and adults. CD4/ (A) and CD8/ (B) cell populations capable of IL-2, IL-4, and/or IFN-g production were assessed using dual intracellular cytokine staining and flow cytometry. Representative two-parameter dot plots of IFN-g and IL-2 (IL-4) of each age group are shown in dot plot graphs. Percentages of cytokine producing cells in each quadrant are indicated. Quadrants of irrelevant isotype-matched control antibodies had õ1% fluorescent cells (data not shown).
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The majority of the concepts regarding T helper and T cytotoxic subset development are derived from experiments in murine models, and the maturation of naive human CD4/ and CD8/ T cells has been much less investigated. In the present study we investigated populations of T cells capable of producing IL-2, IL-4, and/ or IFN-g in umbilical cord blood, and older children and adult blood samples using double stained intracellular cytokine flow cytometric analysis. The results demonstrate that T cell subsets capable of IFN-g production progressively increase with age, probably suggestive of a normal physiological increase in Th1 and Tc1 cell populations in peripheral circulation from the neonatal period to adulthood. On the other hand, Th2 and Tc2 cell populations appear to be physiologically kept at minimal levels. The majority of both CD4/ and CD8/ T cells in cord blood express CD45RA antigen indicative of unprimed naive cells. It is now recognized that typical naive CD4/ T cells invariably produce only IL-2 upon stimulation (25, 26). The present study has consistently demonstrated that CD4//CD45RA/ T cells in cord blood contained exclusively a large number of IL-2 producing cells. However, and more interestingly, it is not the case with CD8/ cells. We showed that in cord blood,
CD8/ IFN-g producing T cells could be detected in addition to IL-2 producing cells, although fluorescence intensity of IFN-g staining was relatively low. These data suggest that naive CD8//CD45RA/ T cells could produce an appreciable amount of IFN-g besides the relatively high IL-2. Seemingly, though, the IFN-g producing CD8/ T cells in cord blood may not be truly naive cells as the IL-2 producing cell population could clearly be separated from IFN-g producing cell population in dot plot graphs of the dual staining flow cytometric data. The progressive increase in CD45RO expression with age of T cell subpopulations demonstrated by this study and also documented by several previous reports (27–30) may be inferred as a sign of maturation of naive T cells at birth to the CD45RO/ putative memory/effector subsets in adults. This maturation process most probably is dependent on antigen exposure. We have observed here that IFN-g producing cell populations increase with age among both CD4/ and CD8/ T cells. Moreover, consistent with previous reports is our observation that, among both CD4/ and CD8/ T cells, the CD45RO/ subset accounts for the preponderance of IFN-g production, whereas both CD45RO/ and CD45RA/ subsets appear to be able to produce IL-2 (24). Taken together, it is tempting to hypothesize here that the almost nonexistent IFN-g producing cell populations, observed in normal cord blood, are due to lack of activation in utero such that these increase with bouts of antigenic exposure of childhood and adulthood. Indeed, it has been demonstrated that children with a history of intrauterine exposure to certain antigens do have cytokine profile responses biased to those antigens (31). It may, thus, be further speculated that IFNg producing cell populations, increasing with age, are actually normal physiological Th1 and Tc1 cells in peripheral circulation. They are, most probably, primary reaction effector cells left in long-term memory and a direct reflection of previous antigenic exposures. Simultaneous evaluation of two cytokines within the same cell population revealed the existence of distinct T subsets in older children and adult peripheral blood that manifested synthesis of all possible combinations of the three major immunoregulatory cytokines (IL-2, IL-4, and IFN-g) assessed. Our results indicate that the most common cell populations in CD4/ T cell subset in both these age groups, are those producing IL-2 alone (approximately 40%). We also found that classic Th1 (IL-2//IFN-g//IL-40) and Th1-like (IL-20/IFN-g// IL-40) were displayed by only a fraction (5–6% and 3– 7%, respectively). Meanwhile, classic Th2 (IL-20/IFNg0/IL-4/) cells were minimal. More interestingly, most common in CD8/ T cell population were Tc1-like cells producing IFN-g alone, increasing dramatically with age (16 and 36% in older children and adults, respectively). We also revealed that CD8/ cells producing IL2 alone progressively lose these populations (19, 14, and 8% in cord blood, older children, and adults, respec-
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TABLE 1 Cytokine Production Profiles Cytokine producing cells (mean % { SD) Cord blood (n Å 19)
Cytokine CD4 T cell IL-2 IL-2/IFN-g IFN-g CD8 T cell IL-2 IL-2/IFN-g IFN-g
Older children (n Å 16)
Adult (n Å 12)
56.4 { 11.9a,b 0.77 { 0.53a,b 0.18 { 0.13a,b
41.2 { 8.98 5.58 { 3.47 3.02 { 2.19c
42.4 { 9.89 6.54 { 4.34 7.28 { 5.07
18.7 { 8.77a 1.13 { 0.73a,b 2.84 { 2.29a,b
14.3 { 5.72c 2.97 { 1.24 16.6 { 5.80c
8.55 { 5.11 4.90 { 3.91 36.1 { 13.2
Note. Statistical comparison of cytokine profiles in cord blood and other age groups showed significant difference (P õ 0.05) as follows: a cord blood versus adult, b cord blood versus older children, and c older children versus adult. Under each age group, in parentheses, is the number of samples analyzed.
jority of the cells are positive for CD45RO surface antigen. Significant correlation was demonstrated between CD45RO expression and IFN-g production in both CD4/ and CD8/ T cell populations (Fig. 3). It is of note that the value of correlation coefficient in CD4/ T cell population was slightly higher than that of their CD8/ counterparts. However, no correlation could be found with IL-2 production (data not shown). DISCUSSION
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FIG. 2. CD45RO and CD45RA expression in IL-2 and IFN-g producing cells. CD4/ (A) and CD8/ (B) cells obtained from healthy adults were stained with anti-CD45 isoform antibodies and anti-cytokine antibodies alongside their respective isotype controls as described under Materials and Methods. The experiments were independently performed four times, yielding similar results and representative two-parameter dot plots are shown in dot plot graphs. Percentages of positive cells in each quadrant are indicated.
tively), while classic Tc1 (IL-2//IFN-g//IL-40) patterns are displayed by a fraction (3–5%). However, our descriptions of these observed fluorescent subpopulations that seemingly coexpress IL-2 and IFN-g in either CD4/ or CD8/ cell populations as ‘‘typical’’ Th1 and Tc1, respectively, may not as such be uniquely different from those staining for IFN-g production only (Th1/ Tc1-like). As we have already noted, approximately 50 and 10% of the IFN-g producing cells are invariably able to simultaneously produce IL-2 in CD4/ and CD8/ cell populations, respectively. Thus, the observed increase in the IL-2/IFN-g may well be a function of the increase in IFN-g and not a selective increase in a unique cytokine coexpressing cell population. The variations between CD4/ and CD8/ cytokine
producing cell populations were also demonstrated in the cytokine synthesis capabilities of CD45RO/ and CD45RA/ T cell subsets. Almost exclusively, CD4/ IFN-g producing cells express CD45RO surface antigen, probably suggesting that upon antigen stimulation, CD4/ T cells first gain CD45RO expression followed by the ability to produce IFN-g. On the other hand, only about two-thirds of their CD8/ counterparts express CD45RO antigen. As shown by the continuity of CD45RO0 and CD45RO/ IFN-g producing CD8/ cell fraction, the former, with relatively smaller amount of IFN-g production capability, most probably become CD45RO/ cells capable of producing higher levels of IFN-g. These variations between CD4/ and CD8/ cytokine producing cell populations may be a reflection of
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Finally the increase in Th1 and Tc1 cell populations with age partly explains, as other studies have recently suggested (16, 33), why cord blood stem cell transplantation is infrequently associated with GVHD. The importance of humoral immunity in mediating chronic GVHD is supported by reports of increased IL-4 and IL-10 production in patients with this condition. This has led to the proposal that chronic GVHD is a consequence of Th2 cytokine production by donor CD4/ cells (6). Conversely, cell mediated immunity mediates many of the features of acute GVHD, suggesting that Th1 cytokines may be important in acute GVHD (6). Results presented here indicate that cord blood T cells have the ability to produce adequate IL-2 but lack the capability to produce IFN-g. Hence they may not be able to mount an effective primary reaction. It would be worthwhile to investigate cytokine profiles of cord blood T cells activated with alloantigen stimulation. Studies are now in progress to clarify whether synthesis of Th1 (Tc1) cytokines or Th2 (Tc2) cytokines could be induced by in vitro stimulation of cord blood T cell subsets in comparison with the cytokine producing ability of activated adult T cells. ACKNOWLEDGMENT This work was supported in part by a Scholarship Fund from the Ministry of Education, Science and Culture, Japan (MONBUSHO), under which Dr. James Chipeta is a graduate student in pediatric immunology.
REFERENCES 1. 2. 3. 4. 5. FIG. 3. Correlation of IFN-g production with CD45RO expression. Individual values of CD45RO cells in cord blood (open squares, n Å 19), older children (closed squares, n Å 15), and adults (open triangles, n Å 10) were plotted on scatter graphs against their respective values for IFN-g producing cells in CD4/ (A) and CD8/ (B) T cells. Correlations were assessed using the pearson correlation coefficient and statistical data indicated in each graph.
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