Longitudinal study of intracellular T cell cytokine production in infants compared to adults

Abstract

Intracellular cytokine production in lymphocytes obtained longitudinally from 325 healthy infants aged 2–12 months was compared with adult lymphocytes using four-colour flow cytometry. Peripheral blood samples (180 microlitres) were stimulated with phorbol 12-myristate 13-acetate, ionomycin and brefeldin A to induce production and intracellular accumulation of cytokines. The method was validated by assessing reproducibility, repeatibility, ruggedness (i.e. fresh versus day-old blood samples), precision, linearity and sensitivity. Among infants, the number and percentage of T lymphocytes (helper/inducer T cell subsets and cytotoxic/suppressor T cell subsets) producing IFN-γ (type 1) and IL4 (type 2) increased over the first year of life but remained significantly lower than levels found in adults. In both infants and adults more CD4⁻ T cells than CD4⁺ T cells were induced to make IFN-γ. Infant Th1/Th2 ratios revealed modest Th1-skewed (predominant) profiles compared to adults, which were 5–10 times higher. Infant Tc1/Tc2 ratios revealed Tc1-skewed responses which were equal to adult ratios by age 12 months. At 12 months infant Th2 responses were closer to adult levels than were Th1 cells. Intracellular cytokine detection by flow cytometry is a rapid, sensitive, rugged and precise method to characterize immune status changes over time.

INTRODUCTION

A variety of cytokine assay types have been employed to measure secreted cytokine levels in body fluids and cell culture supernatant. However, these methodologies do not identify specific, cytokine-secreting cell types. Flow cytometric detection of intracellular cytokine production by individual T lymphocytes [1–6] minimizes the influence of extracellular cytokine inhibitors and cytokine depletion by receptor binding. This method permits identification of the cell type synthesizing a given cytokine, and is less time-consuming and labour intensive than ELISPOT assays [7]. Jung et al.[1] and Elson et al.[8] have shown that the frequency of intracellular cytokine staining T lymphocytes correlates positively with the level of secreted cytokine detected by ELISA, although they did not compare the intensity of intracellular cytokine staining with secreted cytokine.

Flow cytometric analysis of intracellular cytokines has been tested on whole blood [4,9–11] and purified peripheral blood mononuclear cells (PBMC) [2]. Suni et al.[6] have shown that intracellular T cell cytokine expression in response to soluble cytomegalovirus antigen is similar in whole blood cultures compared to expression in PBMC cultures. The use of whole blood samples is convenient for large-scale clinical studies to assess immune status.

In this report type 0, type 1 and type 2 intracellular cytokine production [12] in peripheral blood lymphocytes was assessed longitudinally in infants from 2 to 12 months of age, and compared to that of healthy adults. The method was validated by assessing reproducibility, repeatability, ruggedness, precision, linearity and sensitivity. We demonstrate that flow cytometric detection of T cell-derived intracellular cytokines may be used as a sensitive and precise method to characterize immune status changes over time.

MATERIALS AND METHODS

Antibodies

Blood cells were analysed using an extensive phenotyping panel which included monoclonal antibodies (MoAbs) against human CD3 (clone SK7) labelled with allophycocyanin (APC), CD4 (clone SK3) and CD45 (clone 2D1) labelled with peridinin chlorophyll protein (PerCP), and CD14 (clone MφP9) and interleukin-4 (IL4; clone 3010·211) labelled with phycoerythrin (PE) (Becton Dickinson Immunocytometry Systems BDIS], San Jose, CA, USA). Anti-interferon gamma (IFN-γ) MoAb (clone 4S.B3, Pharmingen, San Diego, CA, USA) was labelled with fluorescein isothiocyanate (FITC). All antibodies were titrated to determine optimal staining concentrations.

Samples

Sodium heparinized blood samples from 325 healthy term infants were collected during a blinded, randomized, longitudinal 12-month feeding trial comparing two cow milk formulas (Similac® plus iron with or without supplemented nucleotides, Ross Products Division, Abbott Laboratories, Columbus, OH, USA). A human milk-fed (exclusively for 2 months) reference group was also included. Infants were vaccinated against Haemophilus influenzae b, diphtheria, tetanus, Bordetalla pertussis and oral poliovirus, at 2, 4 and 6 months according to the schedule recommended by the American Academy of Paediatrics (as revised July 1996). Infants enrolled in the study received formula, medical checks and study vaccines for the duration of the feeding trial. Described herein are results of the combined feeding groups in a representative infant population; feeding effects on the development of immune function (as indicated by vaccine responses and immune cell populations) will be described elsewhere.

Sodium heparin anticoagulated infant blood samples, packaged to maintain room temperature, were shipped to the laboratory via overnight express for testing. Blood cells were analysed at 2, 6, 7 and 12 months of life to coincide with the 2-, 4- and 6-month vaccination schedule of infants. The goal was to (1) establish baseline immune responses before the first immunization, (2) capture the peak immune response before and 1 month after the third immunization and (3) provide an assessment of long-term immune responses by 12 months of age. Sodium heparin anticoagulated blood (12 h postcollection) from 22 healthy adult samples was analysed for comparison. Blood samples from an additional four healthy adults were used to validate the intracellular cytokine methodology.

The study was approved by the Institutional Review Board at 18 clinical sites (local hospitals and paediatric practices) from 10 states (AR, CA, FL, GA, IL, LA, NC, NY, OH and PA). Written informed consent was obtained from all study participants, parents or guardians.

Cell preparation and immunofluorescent staining

Cytokine production in stimulated peripheral blood samples was measured according to BDIS [13] with the following modifications: peripheral blood samples collected using sodium heparin anticoagulant were centrifuged at 430 g for 10 min, and the plasma removed and retained for future analyses. Blood cells were reconstituted to their original sample volume using RPMI 1640 medium (Biowhittaker, Walkersville, MD, USA) plus 5% fetal bovine serum (Hyclone, Logan, UT, USA) (RPMI/FBS). Comparison studies testing plasma-free samples and whole blood samples demonstrated equivalent results (data not shown). Briefly, 180 μl of sodium-heparinized plasma-free blood cells were incubated for 4 h at 37°C in a shaking water bath with either 20 μl Hanks’s balanced salt solution (HBSS, Biowhittaker) containing phorbol 12-myristate 13-acetate (PMA: Sigma Chemical, St Louis, MO, USA; 20 ng/ml final concentration), ionomycin (I: Sigma; 1·25 μg/ml final concentration), and an inhibitor of intracellular transport, brefeldin A (BFA: Sigma; 10 μg/ml final concentration), or with 20 μl HBSS containing BFA alone. Samples were surface stained using APC-labelled anti-CD3 (0·25 μg per test) and PerCP-labelled anti-CD4 (0·12 μg per test) for 15 min at room temperature (RT) in the dark. Erythrocytes (RBC) were lysed, and leucocytes were fixed and partially permeabilized by the addition of 2 ml 1× FACS lyse solution (BDIS) for 15 min at RT in the dark. Samples were centrifuged at 430 g for 5 min and supernatants decanted. Pelleted cells were incubated in 2 ml permeabilization buffer (0·1% saponin, 0·1% azide, 1% fetal bovine serum in phosphate buffered saline PBS]) for 5 min at RT in the dark to fully permeabilize leucocytes and lyse residual RBC. Blood samples were vortexed, centrifuged, and supernatants decanted. Permeabilized cells were stained using FITC-labelled anti-IFN-γ (0·2 μg per test) and PE-labelled anti-IL4 (0·25 μg per test) MoAbs for 20 min at RT in the dark. Finally, labelled samples were washed twice in permeabilizing buffer, and resuspended in 1% paraformaldehyde/PBS prior to data acquisition on a FACSort flow cytometer (BDIS). Samples stored for up to 24h in the dark at 4°C were found satisfactory for analysis (data not shown).

Flow cytometric analysis

Four-colour analysis was performed using a FACSort® flow cytometer (BDIS). Data were acquired using CellQuest® software (BDIS) and analysed using Attractors® software (BDIS); approximately 30 000 leucocytes per sample were analysed. FACSort calibration was performed daily using CaliBRITE® beads (BDIS). Four-colour analysis was used to simultaneously identify cytokine producing helper/inducer (CD3⁺CD4⁺) Th cells and putative cytotoxic/suppressor (CD3⁺CD4⁻) Tc cells, as described below. The population of CD3⁺ APC-staining T lymphocytes was discriminated using fluorescence channel 4 (FL4) and laser side-scatter (SSC). Non-T cells (e.g. CD4⁺ monocytes and CD8⁺ NK cells) were excluded from the analysis by gating on CD3⁺ T cells. Subsequently, two-dimensional dot-plot combinations were created using FL1 (IFN-γ), FL2 (IL4) or FL3 (CD4) parameters to show cytokine staining in Th cell subsets designated Th0 CD3⁺ CD4⁺ IFN-γ⁺ IL4⁺], Th1 CD3⁺ CD4⁺ IFN-γ⁺ IL4⁻] and Th2 CD3⁺ CD4⁺ IFN-γ⁻ IL4⁺] or Tc cell subsets designated Tc0 CD3⁺ CD4⁻ IFN-γ⁺ IL4⁺], Tc1 CD3⁺ CD4⁻ IFN-γ⁺ IL4⁻], and Tc2 CD3⁺ CD4⁻ IFN-γ⁻ IL4⁺]. The CD4⁻CD3⁺ T cell subset was termed ‘Tc’ and assumed to represent ‘putative cytotoxic/suppressor CD8⁺ T cells’ but may also include CD4⁻CD8^−/+ gamma/delta T cell receptor positive cells (5–10% of T cells [14]). Stimulated and unstimulated isotype controls were used to confirm antibody specificity, while unstimulated stained samples were used to set quadrants for two-dimensional dot-plot analyses. The manufacturer’s technical data sheets (BD/Pharmingen) also confirmed the specificity of the intracellular cytokine antibodies by preincubating samples with unlabelled anticytokine monoclonal antibodies to inhibit the binding of labelled anticytokine monoclonal antibodies.

Differential white blood count

Total white blood cell counts (WBC) of heparinized whole blood were determined electronically using a Coulter Multisizer II® (Beckman-Coulter Inc., Miami, FL, USA) according to the manufacturer’s instructions. Lymphocytes, monocytes, neutrophils, eosinophils and basophils were identified using light scattering properties, and staining patterns for CD3, CD14 and CD45 using standard flow staining procedures [15]. Absolute cell numbers were calculated from the measured lymphocyte, T cell type and T cell subtype percentages multiplied by the total WBC/μl-value.

Intracellular T cell cytokine method validation criteria

Blood samples obtained from four healthy adult donors were used to determine assay repeatability, reproducibility, precision, ruggedness and linearity.

Assay repeatability was based on the replicate testing of each donor sample. Data are expressed as percentage coefficient of variation (% CV = standard deviation divided by the mean, multiplied by 100). Cell population percentage CVs for each donor’s sample were averaged to obtain a mean percentage CV for each cell population. Repeatability data from two analysts were reported and a combined average calculated.

Reproducibility was determined by comparing results from two analysts, using identical procedures and samples, and is reported as the difference in mean percentage of positive cells from analyst 1 to analyst 2. The differences between the means for all donors were averaged for each cell population.

Precision was determined by calculating the percentage CV for each cell population using duplicate samples from four donors tested by two analysts (n = 16), and subsequently determining the mean percentage CV among the donors for each cell population.

Ruggedness was determined by analysing the ability to obtain valid results from a sample when tested 24-h postsample collection. The mean percentage of positive cells for each cell population and for each donor processed at the time of collection was subtracted from the percent positive cells for each cell population and for each donor processed 24-h postcollection. Differences between the means for all donors were averaged for each cell population.

To address the method’s linearity cells stained with antigen-specific monoclonal reagents were serially diluted twofold with antibody isotype-stained cells, thus changing the measurable concentration of antigen-specific stained cells. The percentage of positive cells from each non-diluted cell population was determined (e.g. 70% of undiluted lymphocytes were CD3⁺); 10 000 lymphocytes were analysed per sample. From this information, the concentration (theoretical value) for each cell population per twofold dilution was calculated. Theoretical values (x) for each cell population per dilution (e.g. 35%, 17·5%, 8·75% CD3⁺ cells, etc.) were plotted against measured values for each population per dilution (experimental values (y); e.g. 38%, 15%, 9·5% CD3⁺ cells, etc.). Linearity was then determined by performing a least squares regression analysis of the plotted data and expressed as R². Sensitivity (i.e. ability to discriminate a cytokine positive cell, as opposed to how much cytokine production constitutes a positive cell) for each cell population was determined from regression line slopes.

Statistical analysis

Intracellular T cell cytokine production among infants aged 2, 6, 7 and 12 months (whether expressed as positive cells per microliter or as a percent) was not normally distributed; therefore, data were analysed using a Wilcoxon signed rank test (a non-parametric comparison method for non-normally distributed differences between paired data). For an experiment-wise (each T cell component) alpha level of 0·05, a Bonferroni criterion (six comparisons) was applied requiring a P-value of 0·05/6 = 0·0083 for significance [16]. Comparisons of intracellular T cell cytokine production between 12-month-old infants and adults were analysed using a Wilcoxon rank sum test; P-values ≤ 0·05 were judged statistically significant. Sample median values of cytokine positive T cells were used to describe group comparisons, as medians are more appropriate than means for non-normally distributed data [16].

RESULTS

Intracellular T cell cytokine method validation

Validation criteria are reported on PMA/I stimulated blood samples because cytokine levels in unstimulated T cells were below the limit of sensitivity of the assay (≥1 cell in 10 000) as reported previously [2,9]. Jung et al.[17] also showed that freshly isolated unstimulated blood lymphocytes do not produce IFN-γ, IL2 or IL4 cytokines as measured by intracellular staining or mRNA analysis. Similar to data from Picker et al.[2], a 4-h incubation with PMA/I at 37°C was optimal for intracellular cytokine expression (data not shown). Furthermore, while PMA/I does reduce the fluorescence intensity of anti-CD4 staining [2], the percentage of CD4 positive helper T cells was not significantly different between unstimulated and stimulated samples (n = 26, mean ± s.e.m.: unstimulated 60·88%± 1·42; stimulated 59·47%± 1·47; P = 0·49) following a 4-h incubation at 37°C.

Because there is no universally accepted standard cell sample for measuring intracellular cytokines, the accuracy of this method was assessed by comparison with other published values. The percentages of cytokine positive T cells from adult samples as determined by the plasma-free peripheral blood method (Table 1) are similar to published values using adult whole peripheral blood [9], or adult peripheral blood mononuclear cells isolated by density-gradient centrifugation [2,3]. In these studies, cytokine synthesis was induced by PMA/I stimulation under similar conditions as described in Materials and methods. There are no published ranges of cytokine production by Tc0 cells for comparison.

Table 1.

Intracellular cytokine method validation results

		Validation measures
Stimulated adult T cell population	% T cell rangea	Repeatability %CV	Reproducibility %	Precision %CV	Ruggedness %	Linearity	Sensitivity
Th	44–60	11·33	±1·27	11·19	4·47
Th0	0·05–0·40	29·14*	±0·04	38·08*	−0·10	0·987	1·019
Th1	4–12	12·35	±0·81	14·94	−1·29	0·967	1·027
Th2	0·44–0·67	22·87*	±0·16	30·48*	−0·08	0·777†	1·019
Tc	40–55	11·74	±1·10	11·77	−4·61
Tc0	0·01–0·06	46·50*	±0·01	81·14*	−0·01	0·365†	1·2
Tc1	7–32	9·40	±1·35	11·91	−3·16	0·962	0·995
Tc2	0·10–0·14	21·65*	±0·01	27·91*	−0·04	0·793†	0·934
Average		11·21	±1·13	12·45	−1·15	0·96	1·01

^aThe proportion of cells producing a particular cytokine is expressed as a percentage of T lymphocytes.

^*Denotes infrequent T lymphocyte subpopulations.

Average values exclude the Th0, Tc0, Th2, Tc2 values. For each adult cell population (n = 4), repeatability data are expressed as percentage CV among the analysts; reproducibility is reported as the difference in mean percentage of positive cells from analyst 2 to analyst 1; precision is expressed as the mean percentage CV among four donors, and ruggedness refers to the difference in mean percentage of positive cells from 0h to 24 h.

^†Denotes low raw data values from the undiluted labelled cells. Calculated linearities are expressed as R² values. Sensitivity data for each cell population were determined from the slope of the regression line where the ideal slope = 1.

Among four healthy adult donors, the mean repeatability, reproducibility, precision and ruggedness for all high frequency populations (the average of the repeatability values in Table 1, excluding Th0, Tc0, Th2 and Tc2 cells which represent low frequency populations) are reported in Table 1. The mean R² linearity value for all high frequency populations (the average of the linearity values in Table 1) was 0·96. The mean sensitivity for all high frequency populations (the average of the sensitivity values in Table 1) was 1 cell, demonstrating that one positive cell per 10 000 cells counted is measurable in this system.

Infant versus adult intracellular T cell cytokine production

Helper/inducer and cytotoxic/suppressor T cell numbers increased significantly in infants from 2 to 6 months of life (P < 0·001), did not change significantly between 6 and 7 months, and decreased significantly from 7 to 12 months (P < 0·001; Fig. 1,Table 2). By 12 months of age Th and Tc cell numbers were significantly higher (P < 0·001) than adult levels (2·3-fold and 1·7-fold, respectively). In contrast, helper/inducer and cytotoxic/suppressor T cell percentages (expressed as a percentage of total T cells) did not change in infants from 2 months to 12 months of life. By 12 months of age percentages of Th cells were significantly higher (1·1-fold, P < 0·001) and Tc cells were significantly lower (1·3-fold, P < 0·001) than adult levels (Fig. 1,Table 3).

Fig. 1.

Total helper/inducer T cells and cytotoxic/suppressor T cells in 2, 6, 7 and 12-month infant (n = 325) and adult (n = 26) T cells. Blood samples were stimulated for 4 h as described in Materials and methods, and stained with MoAb against CD3-labelled APC, CD4-labelled PerCP, IFN-γ-labelled FITC and IL4-labelled PE. 30 000 leucocytes were counted, and the gated CD3⁺versus side scatter population was analysed for cytokine production. Data are presented as median values on a two-dimensional plot. Age groups are shown on the x-axis, cell numbers per μl and percentages are plotted on the y-axes. Th and Tc cells are expressed as a percentage of T lymphocytes. ▪, Th (%); ○, Tc (%); □, Th (no.); ✦, Tc (no.).

Table 2.

Cell number: median and range

		Age
		2 months	6 months	7 months	12 months	Adult
T cell subset				Cell number/μl
Th	(1)	2534b	3183c	3146c	2397a	1032d
		(435–5896)	(657–7903)	(669–7020)	(601–5522)	(647–2091)
Th0		n.d.	n.d.	n.d.	n.d.	0·52
		n.d.	n.d.	n.d.	n.d.	(0–3)
Th1	(2)	5·16a	7·64c	6·80b	15·96d	104e
		(0–41)	(0–77)	(0–85)	(0–108)	(39–320)
Th2	(2)	2·21a	5·58b	5·97b	6·35b	10·13c
		(0–31)	(0–22)	(0–82)	(0–42)	(3–20)
Tc	(1)	994a	1264b	1220b	954a	549c
		(177–3257)	(243–3218)	(323–2876)	(144–2526)	(239–1460)
Tc0		n.d.	n.d.	n.d.	n.d.	0·12
		n.d.	n.d.	n.d.	n.d.	(0–0·74)
Tc1	(3)	18·23a	31·63b	32·21b	43·08c	172d
		(0–274)	(0–445)	(0–338)	(0–316)	(63–393)
Tc2	(3)	0·14a	0·25b	0·31b	0·59c	2·48d
		(0–3)	(0–4)	(0–35)	(0–9)	(0·64–8)
Th/Tc		2·55	2·52	2·58	2·51	1·88

Median values are shown, with ranges in parentheses. Infants n = 325; adults n = 22; n.d. not detectable. Different letters indicate significant differences among T cell populations; differences among infants were judged significant at P-values ≤ 0·0083 and between 12-month infants and adults at P-values ≤ 0·05. Data plotted: (1) Fig. 1, (2) Fig. 2, (3) Fig. 3.

Table 3.

Cell percentage: median and range

		Age
		2 months	6 months	7 months	12 months	Adult
T cell subset				Cell percentage
Th	(1)	72a	72a	72a	71a	63b
		(43–87)	(44–84)	(38–86)	(47–85)	(46–76)
Th0		n.d.	n.d.	n.d.	n.d.	0·06
		n.d.	n.d.	n.d.	n.d.	(0–0·77)
Th1	(2)	0·21a	0·24b	0·22a	0·70c	10·88d
		(0–2)	(0–2)	(0–3)	(0–5)	(5–27)
Th2	(2)	0·09a	0·17b	0·19b	0·28c	1·01d
		(0–1)	(0–1)	(0–4)	(0–3)	(0·26–2)
Tc	(1)	28a	27a	28a	28a	37b
		(9–57)	(11–56)	(14–62)	(15–52)	(24–54)
Tc0		n.d.	n.d.	n.d.	n.d.	0·02
		n.d.	n.d.	n.d.	n.d.	(0–0·15)
Tc1	(3)	1·83a	2·52b	2·65b	4·76c	31·10d
		(0–20)	(0–30)	(0–24)	(0–37)	(13–66)
Tc2	(3)	0·02a	0·02a	0·03b	0·06c	0·39d
		(0–0·21)	(0–0·33)	(0–2)	(0–0·63)	(0·1–1)
Th:Tc		2·58	2·63	2·59	2·55	1·69
Th1/Th2		2·14c	1·55b	1·16a	2·61c	12·01d
Tc1/Tc2		0a	37·28b	48·60b	65·51c	64·41c

Median values are shown, with ranges in parentheses. Percentage of positive T cells are expressed as a percentage of total T cells. Percentage of cytokine positive T cells are expressed as a percentage of the Th or Tc subpopulation, respectively. Infants n = 325; adults n = 26; n.d.: not detectable. Different letters indicate significant differences among T cell populations; differences among infants were judged significant at P-values ≤ 0·0083 and between 12-month infants and adults at P-values ≤ 0·05. Data plotted: (1) Fig. 1, (2) Fig. 2, (3) Fig. 3.

As infants age from 2 to 12 months, the number and percentage (expressed as a percentage of total Th or Tc cells) of intracellular cytokine positive T cells (Th1, Th2, Tc1, Tc2) increased (Figs 2 and 3, Tables 2 and 3). The median number and percentage of Th1 cells increased significantly from 2 to 6 months (number, P < 0·001; percentage, P < 0·001), decreased significantly from 6 to 7 months (number, P = 0·0077; percentage, P = 0·003), and increased significantly from 7 to 12 months (number, P < 0·001; percentage, P < 0·001). By 12 months Th1 numbers and percentages were significantly lower (number, ~sevenfold, P < 0·001; percentage ~16-fold, P < 0·001) than adult levels (Fig. 2). Tc1 cell numbers and percentages increased significantly from 2 to 6 months (number, P < 0·001; percentage, P < 0·001), did not change from 6 to 7 months and increased significantly from 7 to 12 months (number, P < 0·001; percentage, P < 0·001). Infant Tc1 numbers and percentages were significantly lower at 12 months than adult levels (number, ~fourfold, P < 0·001; percentage ~sevenfold, P < 0·001, Fig. 2).

Fig. 2.

Intracellular Th1, Tc1 cytokine production in 2, 6, 7 and 12-month infant (n = 325) and adult (n = 26) T cells. Blood samples were stimulated for 4h as described in Materials and methods, and stained with MoAb against CD3-labelled APC, CD4-labelled PerCP, IFN-γ-labelled FITC and IL4-labelled PE. 30 000 leucocytes were counted, and the gated CD3⁺versus side scatter population was analysed for cytokine production. Data are presented as median values on a two-dimensional plot. Age groups are shown on the x-axis, cell numbers per μl and percentages are plotted on the y-axes. Th1 cells are expressed as a percentage of Th lymphocytes and Tc1 cells are expressed as a percentage of Tc lymphocytes. ▪, Th1 (no.); ○, Tc1 (no.); □, Th1 (%); ✦, Tc1 (%).

Fig. 3.

Intracellular Th2 and Tc2 cytokine production in 2, 6, 7 and 12-month infant (n = 325) and adult (n = 26) T cells. Blood samples were stimulated for 4h as described in Materials and methods, and stained with MoAb against CD3-labelled APC, CD4-labelled PerCP, IFN-γ-labelled FITC, IL4-labelled PE. 30 000 leucocytes were counted, and the gated CD3⁺versus side scatter population was analysed for cytokine production. Data are presented as median values on a two-dimensional plot. Age groups are shown on the x-axis, cell numbers per μl and percentages are plotted on the y-axes. Th2 cells are expressed as a percentage of Th lymphocytes and Tc2 cells are expressed as a percentage of Tc lymphocytes. ▪, Th2 (no.); ○, Tc2 (no.); □, Th2 (%); ✦, Tc2 (%).

Despite the high coefficient of variation associated with Th2 and Tc2 populations (Table 1), all sources of variation were reflected in the distribution of the data, therefore, statistical analyses were performed on these infrequent populations. Th2 cell numbers increased significantly from 2 to 6 months (P < 0·001) and remained unchanged to month 12. Th2 cell percentages increased significantly from 2 to 6 months (P < 0·001), did not change from 6 to 7 months and increased significantly from 7 to 12 months (P < 0·001). (Fig. 3). By 12 months infant Th2 cell numbers and percentages were significantly lower (number, ~twofold, P = 0·004; percentage ~fourfold, P < 0·001) than adult levels. Tc2 cell numbers increased significantly from 2 to 6 months (P < 0·001), did not change significantly between 6 and 7 months and increased significantly from 7 to 12 months (P < 0·001), while Tc2 percentages did not increase from 2 to 6 months, increased significantly from 6 to 7 months (P = 0·006) and increased significantly from 7 to 12 months (P < 0·001). By 12 months infant Tc2 numbers and percentages were significantly lower (number, ~fourfold, P < 0·001; percentage ~sevenfold, P < 0·001) than adult levels (Fig. 3).

In both infants and adults there was a higher number and percentage of Tc1 (IFN-γ⁺) cells than Th1 (IFN-γ⁺) cells (fold increase Tc1 > Th1: 12-month infant number ~threefold, percentage ~sevenfold; adult number ~twofold, percentage ~threefold). In both infants and adults there was a higher number and percentage of Th2 (IL4⁺) cells than Tc2 (IL4⁺) cells (fold increase Th2 > Tc2: 12-month infant number ~11-fold, percentage ~fourfold; adult number ~fivefold, percentage ~threefold).

T cell cytokine ratios were determined to address the balance of type 1 and type 2 cytokine producing cells over time. Th1/Th2 percentage ratios and Tc1/Tc2 percentage ratios were calculated for each subject and the median values reported for each time point. Among infants the level of Th1 cells was slightly higher than Th2 cells, while the level of Th1 cells markedly exceeded Th2 cells in adults. Th1/Th2 ratios were between 1 and 2·6 in infants compared to 12 in adults (Table 3). Among infants Th1/Th2 ratios decreased from 2 to 6 months (P < 0·001), decreased from 6 to 7 months (P < 0·001) and increased from 7 to 12 months (P < 0·001) returning to levels found at 2 months. By 12 months of life, Th1/Th2 ratios were significantly lower (~fivefold, P < 0·001) than adult levels. The level of Tc1 cells markedly exceeded Tc2 cells in both infants and adults. At 2 months the median Tc1/Tc2 ratio was zero. Tc1/Tc2 ratios increased significantly by 6 months (P < 0·001), did not increase from 6 to 7 months, and increased by 12 months (P < 0·001) to that of adult Tc1/Tc2 levels (Table 3).

In 50–60% of infants, Th0 counts (CD3⁺CD4⁺, IFN-γ⁺IL4⁺) were below the limit of assay sensitivity (≥1 cell in 10 000), while 80–90% of Tc0 cells had less than 1 in 10 000 Tc0 cells (CD3⁺CD4⁻, IFN-γ⁺IL4⁺). Th0 cells were detected in adult blood samples at a median level of 0·06% of Th cells, while Tc0 cells were detected at 0·02% of Tc cells (Table 3).

DISCUSSION

Intracellular cytokine detection by flow cytometry is being used increasingly as a human immune status indicator [9,18,19]. This cytometric method was validated to confirm an acceptable level of accuracy and precision for routine testing of clinical blood samples. Presented here is the first report documenting its use in infants in a matched longitudinal study from 2 to 12 months of age.

During the first 12 months of life increases in numbers and percentages of cytokine secreting lymphocytes were observed, possibly due to exposure to a variety of environmental antigens, and infant vaccinations. Immunogen exposure drives T cell differentiation from a naive T cell phenotype (CD45RA⁺, IL2⁺) to a memory/effector T cell phenotype (CD45R0⁺; type 0, type 1 or type 2 T cells) through a stimulation process that includes production of IL2 from naive T cells [12,20]. T cell stimulation with phorbol esters triggers a non-specific activation of T cell cytokine production permitting an assessment of their overall functional capacity. As naive T cells do not generally secrete IFN-γ or IL4, flow cytometric analysis of circulating levels of IFN-γ or IL4 positive T cells (by inference, CD45R0⁺ T cells) might reflect antigen-driven T cell differentiation as the infant immune system matures.

At 2 months of age circulating maternal antibodies are present in infants, while they are virtually absent by 9 months of age [21,22]. As maternal antibody levels diminish, there is a need for de novo antibody synthesis. Cytokine positive T cells were the lowest at 2 months. From 2 to 6 months of age a significant increase in cytokine secreting T cells capable of promoting humoral and cellular immune responses were observed. With the exception of Th1 cells, there were generally few changes in the number and percentage of cytokine positive T cells from 6 to 7 months of age. Th1 cells declined from 6 to 7 months. Although the biological significance of this small but significant decline is unknown, it may be related to the vaccination schedule and exposure to environmental antigens resulting in sequestration of Th1 cells to lymphoid tissue. From 7 to 12 months of age all cytokine positive T cell subsets increased, albeit by 12 months the number and percentage of these cells remained significantly lower than levels determined in adults. In most infants, Th0 and Tc0 cells were below the limit of sensitivity of the assay, whereas these cells were detectable among adults as low frequency populations. Further investigation is required to verify the lower expression of cytokine production (as indicated by fluorescence intensity, data not shown) from infant type 1 and type 2 cells compared to adults, and the biological significance of these differences.

The vaccines administered at 2, 4 and 6 months were against H. influenzae b, diphtheria, tetanus, B. pertussis and oral poliovirus. Although little information is available on vaccine-induced T cell cytokine responses in human infants, recent reports indicate pertussis vaccination induces predominately Th1 responses [23,24], while tetanus induces mixed Th1 and Th2 responses [25]. In the current study, it is difficult to distinguish between the influence of vaccination versus challenge by environmental antigens on cytokine positive T cell levels. Changes in T cell cytokine subsets with time are unlikely to be exclusively vaccine driven. Cytokine producing cells (putative memory/effector T cells) were detected with a non-antigen-specific stimulus (PMA/I), not a vaccine antigen-specific stimulus. Typically, antigen-specific cytokine positive T cells represent infrequent populations in the periphery [6,26]. It is unlikely that infrequent cytokine positive vaccine-specific T cells were detected exclusively using PMA/I. The memory/effector T cell cytokine subsets probably reflect exposure to a plethora of environmental antigens, as well as infant vaccinations.

In both infants and adults more CD4⁻ T cells (Tc1) than CD4⁺ T cells (Th1) expressed IFN-γ, confirming previous reports [27,28]. Infant Th1/Th2 ratios revealed modest Th1 predominant profiles compared to adults, which were five to 10 times higher. However, infant Tc1/Tc2 ratios revealed Tc1-predominant responses, which were equal to adult ratios by age 12 months. These data imply enhanced maturation of IFN-γ⁺ cytotoxic/suppressor T cells in infants compared to IFN-γ⁺ helper/inducer T cells. Infant Th1/Th2 ratios and Tc1/Tc2 ratios were compa-rable to levels previously reported in cord blood (27), but differed from Hagendorens et al.[29] who found Th1/Th2 ratios of 19 and Tc1/Tc2 ratios of 4 in cord blood. One possible explanation for the discrepancy is the higher concentration of PMA used in the latter study. In general, the propensity of infant Th1/Th2 ratios (as defined by IFN-γ and IL4) to be close to 1 might contribute, during the early years of life, to the manifestation of Th2 driven atopy in susceptible individuals.

Interestingly, at 12 months the number and percentage of Th2 cells were closer to adult levels than were Th1 cells, suggesting an earlier maturation of mucosal antigen driven-differentiation towards Th2 cell development. This is an intriguing observation since serum IgA concentrations (Th2 driven) at 12 months are substantially lower than adult levels, while serum IgG levels (Th1 driven) are closer to adult levels [21]. This may reflect poor constitutive B cell memory for IgA production among infants.

The proportions and numbers of T cell cytokine subsets followed similar patterns and continued to increase with age. These data support previous reports [27,28]. However, the helper T (Th) cells and cytotoxic/suppressor T (Tc) cell numbers and proportions were not concordant. The proportion of Th or Tc cells within the T lymphocyte population did not change through the first 12 months of life, hence the Th: Tc ratio remained stable. By adulthood the Th: Tc ratio decreased compared to 12-month infants, due to a decreased proportion of Th cells and an increased proportion of Tc cells. Collectively these data confirm previous reports [30,31] describing T cell changes in the lymphocyte pool. On the other hand, Erkeller et al.[32] reported no change in the proportion of CD4⁺ T cells within the lymphocyte pool and no change in Th: Tc ratios between infants (combined group age 2 days 11 months) and adults (combined group age 18–70 years). The same group later reported a moderate increase in the proportion of CD4⁺ T cells between infants and adults [33].

In contrast to cell proportions, Th and Tc numbers increased ~30% from 2 to 6 months, did not change from 6 to 7 months and declined ~20% from 7 to 12 months to levels more similar to those at 2 months. Transient increases in Th and Tc cells during infant immune maturation have also been described by McCoy et al.[31]. Since changes in Th and Tc cell numbers occurred in parallel, the proportions of Th and Tc were unchanged in the infant. By adulthood, Th and Tc cell numbers continued to decline compared to 12 month infant levels, confirming previous reports [31–33]. (The change in the proportion of Th and Tc by adulthood was driven by a 57% decrease in the number of Th cells and a 42% decrease in Tc cell numbers compared to 12 month infants.)

Changes in the number of helper and cytotoxic/suppressor T cells over time may represent normal immune development. Although data describing T cell development in the absence of vaccination are lacking, the T cell changes described herein may not be exclusively vaccine driven, because antigen-inexperienced naive T cells and antigen-experienced memory/effector T cells followed the same profile (data not shown). Furthermore, vaccination did not increase circulating levels of total immunoglobulin (Ig), but did increase vaccine- specific Ig levels (data not shown). In summary, these T cell changes likely reflect normal immune development, exposure to environmental antigens, and infant vaccinations.

Intracellular cytokine detection by flow cytometry is a precise methodology that allows measurement of new aspects of cytokine production, unapproachable by other techniques. Flow cytometric analysis of intracellular cytokine production by an individual cell identifies the cell source, and with this technique multiple cytokines and cytokine levels may be detected on a per cell basis. This methodology may be a helpful immune status indicator in several cell populations, as well as a prognostic indicator in many diseases, including HIV-1 infection [9], rheumatoid arthritis [34], atopic dermatitis [35] and asthma [19].