0031-3998/04/5606-0891
PEDIATRIC RESEARCH
Copyright © 2004 International Pediatric Research Foundation, Inc.

Vol. 56, No. 6, 2004
Printed in U.S.A.

Effect of Dietary Ribonucleotides on Infant Immune
Status. Part 2: Immune Cell Development
RACHAEL H. BUCK, DEBRA L. THOMAS, TIMOTHY R. WINSHIP, CHRISTOPHER T. CORDLE,
MATTHEW J. KUCHAN, GERALDINE E. BAGGS, JOSEPH P. SCHALLER, J. GARY WHEELER
Abbott Laboratories [R.H.B., D.L.T., T.R.W., C.T.C., M.J.K., G.E.B., J.P.S.], Ross Products Division,
Columbus, Ohio 43219, Arkansas Children’s Hospital Research Institute, Inc. [J.G.W.], Little Rock,Arkansas 72204

ABSTRACTThe objective of this study was to determine whether dietaryribonucleotides alter immune cell phenotypes or function in the firstyear of life. Newborn term infants in a double-blind, 12-mo, multicenter trial were randomized to cow milk formula groups with
(FN, n ⫽ 138) or without (F, n ⫽ 147) 72 mg/L supplemental
ribonucleotides. A nonrandomized HMF cohort ( n ⫽ 192) was
concurrently enrolled. Eighty-eight immune blood cell types werecharacterized by flow cytometry. Data were analyzed by multivariate ANOVA (MANOVA), ANOVA, and repeated measures analysis (RMA), with adjustments made for multiple comparisons.
Ribonucleotide feeding changed subpopulations of T and naturalkiller (NK) cells. FN had higher numbers and percentages ofmemory/effector (M/E) cytotoxic/suppressor (CD45R0⫹CD8⫹,
RMA) T, Fas⫹ M/E (CD45R0⫹CD95⫹CD3⫹, 6 mo) T, andCD56⫹CD16⫺ NK cells (CD56⫹CD16⫺CD3⫺CD8⫺, 12 mo), andhigher percentages of M/E helper (CD45R0⫹CD4⫹, RMA) T,
Tc1 (IFN␥⫹CD4⫺CD3⫹, RMA), total interferon (IFN)␥T(IFN␥⫹CD4⫹/⫺CD3⫹, RMA), Th2 (IL-4⫹CD4⫹CD3⫹, 7 mo),and CD57⫹ NK-T cells (CD57⫹CD56⫺CD3⫹, 6 mo, 7 mo) compared with F. Percentages of naive helper T (CD45RA⫹CD4⫹, 12mo) and numbers and percentages of CD56⫹ NK-T cells(CD56⫹CD16⫺CD3⫹CD8⫺, 2 mo, 6 mo) were lower in FN than F.
Percentages of M/E cytotoxic/suppressor, Th2, and CD56⫹CD16⫺
NK cells in FN were significantly higher than F but were notdifferent from HMF, whereas F was significantly lower than HMF.
Ribonucleotide supplementation of infant formula supported increased T-cell maturation and affected immunoregulatory NK cellsubsets. These FN-associated immune cell profiles either did not

Immaturity of the infant immune system may place infants at
risk for infectious disease–induced morbidity and mortality (1).
Further, mode of feeding may affect susceptibility inasmuch as
infants fed human milk appear to have fewer infections thanformula-fed infants (1). Maternal secretory antibodies against
Received June 14, 2004; accepted June 26, 2004.
Correspondence: Rachael H. Buck, Ph.D., Abbott Laboratories, Ross Products Division, 3300 Stelzer Rd., Columbus, OH 43219; e-mail: rachael.buck@abbott.com
Supported by grants from the Ross Products Division of Abbott Laboratories.
DOI: 10.1203/01.PDR.0000145577.03287.FA

differ from those infants fed HMF or tended to be more like thosefed HMF than those fed F. (Pediatr Res 56: 891–900, 2004)

Abbreviations
APC, allophycocyanin
CD, cluster of differentiation
ExHMF, exclusive human milk-fed
F, formula without nucleotides
FBS, fetal bovine serum
FN, formula with supplemented nucleotides
FSC, forward light scatter
HBSS, Hanks’ balanced salt solution
Hib PRP, Hib capsular polysaccharide (polyribosylribitol
phosphate)
Hib-TITER, Haemophilus influenzae type b vaccine
HMF, human milk-fed
IFN, interferon
M/E cell, memory/effector cell
NK cell, natural killer cell
PerCP, peridinin chlorophyll protein
RMA, repeated measures analysis
SSC, side light scatter
Staph, Staphylococcus aureus
SWI, Similac with ironTc cell, cytotoxic T cellTh cell, helper T cellWBC, white blood cell

environmental pathogens largely mediate the diseasepreventive effect of human milk. However, other potentiallyimmunoenhancing and anti-infective substances in human milk
(growth factors, cytokines, oligosaccharides, complement, enzymes, lactoferrin) may also provide protective effects (2).
Ribonucleotide 5' monophosphates (nucleotides) exhibit immunopotentiating and gastrointestinal developmental effects(3–5). The nucleotide content of human milk is approximately
72 mg/L, comprising nucleosides, nucleotides, mono-, di-, andtriphosphates, nucleotide adducts (nucleotide-containing com-

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BUCK ET AL.

pounds such as coenzymes), and free bases (6). In a priorstudy, cow milk– based infant formula supplemented with 72
mg/L of dietary nucleotides enhanced Haemophilus influenzae
type b and diphtheria toxoid immunization responses comparedwith an unsupplemented control formula (7). In a recent study
in Taiwan (8), nucleotide supplementation of formula increased serum IgA levels and reduced diarrhea incidence inhealthy, term infants.
Dietary-induced changes in immune status may also bereflected in peripheral blood immune cell populations. Previous reports comparing immune cells from cow milk formula–or human milk–fed infants identified changes mainly among
lymphocyte subsets (T, B, and NK cells), and lymphocytemediated functions, such as antibody production, lymphocyte
proliferation, and cytokine production (2,9 –11). Kuchan et al.
(3) observed that nucleotide supplementation of cow milk
formula tended to increase some lymphocyte populations compared with a control formula.
Immune development of infants fed unsupplemented or
nucleotide-supplemented cow milk formula was further evaluated in this longitudinal, masked, 12-mo, parallel feeding
trial. A nonrandomized, initially HMF group was concurrently
enrolled. Changes in immune cell development may be reflected by differences in cell numbers and/or percentages,
maturation, or functional state. Responses to childhood immunizations during the first year of life are reported elsewhere(12).
METHODS
Ethical considerations. Only infants whose mothers had
decided not to breast-feed before enrollment were recruited forthe formula-fed groups. Infants received cow milk– based formula, medical checks, and study vaccines during the trial. Eachparticipating site’s Institutional Review Board approved thestudy. Written informed consent was obtained from parent(s)or legal guardian(s).
Subjects, study design, and formula. This 12-mo, prospective, randomized, double-blind, parallel, longitudinal study ofhealthy, term, formula-fed infants was conducted at 18 sites(local hospitals and pediatric practices) in 10 states (AR, CA,
FL, GA, IL, LA, NC, NY, OH, and PA) from fall 1996 tospring 1998. Formula-fed infants were enrolled before 8 d ofage, and randomized to the control formula (F, n ⫽ 147) or thesame formula supplemented with 72 mg/L ribonucleotide 5'
monophosphates (FN, n ⫽ 138). Subjects were fed the assigned formula ad libitum from enrollment to 12 mo of age.
Infants in the HMF reference group (n ⫽ 192) were enrolled
before 8 d of age, fed human milk exclusively for at least 2 mo,and followed the study visit schedule for 12 mo. After 2 mo,infants whose parent(s) chose to wean were fed commerciallyavailable Similac with Iron (SWI, Ross Products Division,
Abbott Laboratories, Columbus, OH), which at that time was
not supplemented with ribonucleotides. Other foods were permitted for all infants after 4 mo of age. SWI was comparable
to F except for a higher level of linoleic acid and a different
protein blend (12). The proportion of HMF subjects whose
milk source was exclusively human milk, or human milk in

combination with formula, respectively, was 54% and 73% at
3 mo, 35% and 60% at 5 mo, 22% and 34% at 8 mo, and 15%
and 30% at 12 mo.
Each formula batch (manufactured by Ross Products Division, Abbott Laboratories) met or exceeded nutrient levels
recommended by the Committee on Nutrition of the American
Academy for Pediatrics (13), as required by the Infant Formula
Act of 1980 and subsequent amendments. The nucleotide
formula was supplemented with 72 mg/L of yeast-derived,
food-grade ribonucleotide 5' monophosphate monomers. Formula compositions and ribonucleotide concentrations for eachof the formula batches are reported elsewhere (12).
Subjects were immunized as previously described (12) with
Hib-TITER (Haemophilus influenzae b), Tripedia DTaP (diphtheria toxoid, tetanus toxoid, acellular pertussis) pediatric vaccine (Connaught, Swiftwater, PA), and Orimune (oral poliovirus, Orimune, Lederle, Pearl River, NY) at 2, 4, and 6 mo ofage, consistent with the 1996 and 1998 recommendations of
the American Academy of Pediatrics.
Blood collection and immune cell analysis. Sodium heparinized, coded blood samples were collected at 2, 6, 7, and 12
mo, packaged to maintain room temperature (RT), and transported to Ross Products Division where they were tested within
24 h of collection.
One milliliter of blood from each sample was used for
granulocyte phagocytosis assays (see below), and 100 ␮L
removed for a total WBC count. The remaining blood volume
was marked on the collection tube and centrifuged for 10 min
at 430 ⫻ g. The plasma fraction was then removed with asterile transfer pipette and stored at ⫺80°C for serologicaltesting. The remaining cell fraction, referred to as plasma-freeblood, was reconstituted to its original volume with RPMI
1640 culture medium (BioWhittaker, Walkersville, MD) plus
5% FBS (HyClone Laboratories, Logan, UT).
Immunophenotyping. Total WBC numbers were determined electronically using a Coulter Multisizer II (Beckman
Coulter, Inc., Fullerton, CA). Cell processing, staining procedures, and flow cytometric analyses were performed using
standard methods for flow cytometry (13). Plasma-free blood
samples were stained using MAb labeled with FITC, phycoerythrin, PerCP, or APC fluorochromes [Becton Dickinson
Immunocytometry Systems (BDIS), San Jose, CA] mixed in 3
or 4 fluor combinations. Appropriate isotype control antisera
were used with each assay. Samples were evaluated using
BDIS FACScan, BDIS FACSCalibur, or BDIS FACSort flow
cytometers and BDIS Attractors software, as described previously (13). For the discrimination of lymphocytes, monocytes,
neutrophils, eosinophils, and basophils, 10,000 leukocytes
were counted, and results expressed as a percentage of the total
WBC count. The absolute numbers of cells per microliter ofblood were derived by multiplying percentages of leukocyte
subset populations of interest by the WBC count per microliter.
Approximately 10,000 lymphocytes, discriminated by forward(FSC) and side light scatter (SSC) characteristics, werecounted from each of the immunophenotyping tubes. Lymphocyte subset results were expressed as percentages of total
lymphocytes. Absolute lymphocyte cell numbers were derived

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NUCLEOTIDES AND IMMUNE CELL DEVELOPMENT

by multiplying the lymphocyte count per microliter by thepercentage of the subset of interest.
Intracellular cytokine expression. T-cell-derived IFN-␥ andIL-4 intracellular cytokines were induced using phorbol 12myristate 13-acetate and ionomycin (14), and T-cell subsets
were identified by surface staining with anti-CD4 PerCP andanti-CD3 APC MAb. Intracellular cytokine staining and flow
cytometric analysis were performed as described previously(14).
CD40 ligand expression. Plasma-free blood was stimulated
for 4 h at 37°C in a shaking waterbath with 0.02 ␮g/mL
phorbol 12-myristate 13-acetate and 1.25 ␮g/mL ionomycin, or
HBSS (unstimulated control). Cells were stained for 15 min at
RT with anti-CD40L FITC, anti-CD69 PE, anti-CD3 PerCP,
and anti-CD19 APC MAb. Erythrocytes were then lysed using
0.15M ammonium chloride, leukocytes were washed with PBS(BioWhittaker) plus 1% FBS, and fixed with 1% paraformaldehyde. Cells were analyzed by flow cytometry as above.
Granulocyte extravasation/migratory potential. Plasmafree blood was incubated for 10 min with 10⫺8 M N-formylmet-leu-phe (Sigma Chemical Co., St. Louis, MO) or HBSS
(unstimulated control) in a 37°C shaking waterbath. Cells were
washed with PBS and stained with anti-CD11b FITC andanti-CD62L (L-selectin) phycoerythrin MAbs for 15 min at RT.
Erythrocytes were lysed with BDIS FACS lysing solution andleukocytes were washed once with PBS containing 0.2% sodium azide, and then fixed in 1% paraformaldehyde. Cells wereanalyzed by flow cytometry. Approximately 10,000 granulocytes, determined by FSC and SSC characteristics, were analyzed for CD11b and CD62L expression.
Granulocyte phagocytosis. One milliliter of whole blood
was treated with 0.15M ammonium chloride to lyse erythrocytes. Remaining leukocytes were washed twice with Dulbecco’s PBS (BioWhittaker) plus 5% FBS. Cells were resuspended in HBSS and incubated for 10 min in a 37°C shaking
waterbath with either heat-killed, plasma-opsonized Staph(Calbiochem-Novabiochem, La Jolla, CA), propidium iodide(PI)-stained Staph (100 ␮g/mL PI in HBSS; Molecular Probes,
Inc., Eugene, OR), or HBSS alone. Approximately 10,000
granulocytes, determined by FSC and SSC, were analyzed byflow cytometry. The median fluorescence channel of granulocytes ingesting fluorescent PI-labeled bacteria determined
phagocytosis; the greater the amount of PI-stained bacteria
ingested, the higher the median fluorescence channel.
Statistical methods. Formula groups were randomized
within each site stratified by gender. Random permuted blocks
of size 2, designed to achieve balance in the feeding allocation
after every four subjects, were used. Although the HMF group
was not randomized at enrollment it was included as a reference group, as is commonly reported in infant feedingcomparisons.
Intent-to-treat analyses were performed on cellular outcomes. The majority of cell populations were not normally
distributed, therefore, data from these populations were log10transformed to normalize the data before statistical evaluation.
To reduce the burden of testing 88 individual cell populations,data were analyzed in 24 clusters of immune function/typerelated cells (e.g. type 1 T-cell cluster contained two compo-

nents: Th1 and Tc1 cells; Table 1) using a per protocol
two-group (FN versus F) multivariate ANOVA (MANOVA),and a three-group posthoc MANOVA (FN versus F versus
HMF), using site as a blocking factor. Sites with low enrollment were combined in these assessments. The rationale for
MANOVA was that, if clusters of related cell populations did
not show differences among the treatment groups, then the
differences between individual cluster components might not
be statistically significant. In essence, time point MANOVA onclusters of immunologically related cells were performed followed by time point ANOVA on all cluster component cell
types if a cluster was found to be significantly different. For
simplicity, MANOVA-triggered ANOVA analyses are referredto throughout as being identified by MANOVA. Multiple
pairwise comparisons of cell types by three-group MANOVA(FN versus F versus HMF) were implemented using Holm’s
step-down procedure to adjust significance levels. The smallest
p value was tested at 0.05/3 ⫽ 0.0167; if this was not foundsignificant, then testing stopped and the groups were declared
not different, otherwise the second lowest p value was tested at
0.05/2 ⫽ 0.025, continuing only when significance was detected; the largest p value was tested at 0.05. In addition, twoand three-group RMA (to indicate change over time) of selectcell types (selected by visual inspection of the data) wereanalyzed using SAS PROC MIXED (SAS Institute, Cary, NC).
Multiple pairwise comparisons by three-group RMA were
implemented using Holm’s step-down procedure, as describedabove. The smallest p value was tested at 0.05/12 ⫽ 0.0042,
Table 1. Immunological/biological cell clusters for MANOVA
analysis
No. of clustercomponent celltypes
Leukocyte differential clusters
Lymphocyte
Granulocyte
Granulocyte function cluster
Monocyte
T cell clusters
␥␦ T cell
T cell differentiation
Helper cell
Cytotoxic/suppressor cell
Memory/effector cell
Naive cell
Apoptosis capable cell
Type 1 T cell
Type 2 T cell
CD40L⫹ T cell
CD40L⫺ T cell
B cell clusters
Auto antibody B cell
Primary B cell
Secondary B cell
NK/NK-T cell clusters
CD8⫹ NK
CD16⫹ ADCC NK
CD57⫹ NK
CD56⫹/CD57⫹ NK-T
CD8⫹ NK & NK-T
CD16⫹ADCC NK & NK-T

43
41
CD markers(␥␦⫹ CD3⫹)(CD4⫹CD8⫹)(CD4⫹)(CD8⫹)(CD45R0⫹)(CD45RA⫹)(CD95⫹)(IFN␥⫹)(IL-4⫹)(CD40L⫹ CD3⫹)(CD40L⫺CD3⫹)(CD5⫹)(CD23⫹)(CD23⫺)

3
3
6
7
11
7
3
2
2
3
3
3
1
2
2
4
1
7
4
7

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BUCK ET AL.

and the second lowest at 0.05/11 ⫽ 0.0045, etc., to account for
the three feeding groups and four time points. Finally, exploratory post hoc statistical analyses were performed to expand
the MANOVA observations: two- and three-group time point
ANOVA (unadjusted for site as a blocking factor), and twoand three-group RMA were performed on all cell populations.
Due to the large proportion of individuals who failed togenerate cytokine positive Th0 cells, a categorical variable(responders/nonresponders) was defined and a CochranMantel-Haenszel test was performed comparing the proportionof Th0 responders among the feeding groups.
Null hypothesis testing of feeding group differences for eachcell population was two-sided and considered statistically significant at ␣ ⫽ 0.05. Because of the large number of cellcomparisons, and because many comparisons should be considered not truly independent, a p value correction for multiplicity of endpoints (cell types) was not performed.
Percentage changes in the number or proportion of lymphocytes between FN and F were calculated as follows: ([FN ⫺ F]⫼ F) ⫻ 100%. Percentage changes in the number or proportionof lymphocytes between either formula and HMF were calculated as follows: ([F or FN] ⫺ [HMF] ⫼ HMF) ⫻ 100%.

RESULTS
Significant cell type differences between FN and F formulas
were judged at p values ⱕ0.05 (two-group analyses), whereas
significant cell type differences among HMF and formulagroups (three-group analyses) were judged with the lowest pvalue ⱕ 0.0167 (0.05/3), the second lowest at p ⱕ 0.025(0.05/2), and the largest at p ⱕ 0.05, using Holm’s stepdown
procedure, as described above, to account for multiple groupcomparisons. Consequently, differences between formulagroups identified by two-group statistical analyses did notalways reach significance by three-group analyses; in these
instances p values ⬎ 0.0167 but ⱕ 0.05 were regarded assuggestive of trends in the data. The most likely explanation
for the loss of significance by three-group analysis is that thevariability among the groups was increased by the addition of
HMF, therefore, some differences between formulas were nolonger statistically significant due to higher variability andmultiple comparison adjustments.
With the exception of the leukocyte differential, granulocyte
function, total T, B, and NK cell populations, and because of
the large number of populations identified (88 cell types), only
those populations that demonstrated differences between FN
and F are reported.
Nucleotides did not affect WBC and leukocyte differential.
There were no significant differences in WBC counts or in thenumber or percentage of lymphocytes, monocytes, neutrophils,
basophils, or eosinophils between FN and F (two-group
MANOVA or ANOVA) during the course of the study. Furthermore, there were no significant differences in these leukocyte populations between either formula and HMF (threegroup MANOVA or ANOVA), except at 6 mo, wherelymphocyte numbers were higher in F compared with HMF (p⫽ 0.01).

Nucleotides did not affect granulocyte function. There wereno significant differences in granulocyte phagocytosis or granulocyte extravasation potential between FN and F (two-group
MANOVA or ANOVA). In addition, neither formula wassignificantly different from HMF (three-group MANOVA).
However, by three-group ANOVA both formula groups at 2
mo (FN ⬍ HMF, p ⫽ 0.003; F ⬍ HMF, p ⫽ 0.002) had aslightly lower increase in the density of CD11b (a marker ofextravasation affinity) on activated cells compared with HMF;
no differences in CD62L levels were noted (data not shown).
Upon activation, granulocytes typically shed CD62L and increase the expression of cell surface CD11b (15).
Nucleotides did not affect total T cells, B cells, or NK cells;
MANOVA and RMA. Figure 1, a– c, depicts the profiles of
total T (CD3 ⫹ ), total B (CD19&20 ⫹ ), and total NK(CD56⫹CD57⫹/⫺CD16⫹/⫺CD3⫺) lymphocyte percentages
and numbers over time. Statistical analyses comparing thechange from time point to time point, either within or among
the three feeding groups were not performed, however, these
profiles appeared to be similar among the three groups. Figure
1, a– c, reveals no significant differences between FN and F forthese cell populations, but does show significant differences
between formula-fed and breast-fed infants, as described
below.
There were no significant differences in T (CD3⫹), Th(CD3⫹CD4⫹), and Tc (CD3⫹CD8⫹) cell numbers or percentages between FN and F groups (two- or three-group
MANOVA). However, both formula groups had significantly
higher (up to 10%) numbers of T cells at 2 and 6 mo, andhigher proportions (up to 18%) of T cells at 2, 6, and 7 mocompared with HMF (Fig. 1-a and Fig. 3-group MANOVA).
These T-cell differences were driven by naive Th cells, and not
M/E T cells, as described below. At 7 mo, T cell numbers in
FN were not different from HMF, whereas F was significantly
higher than HMF (p ⫽ 0.005).
There were no significant differences in B cell (CD19&20⫹)
numbers or percentages between formula groups. However,both formula groups had significantly lower proportions (up to17%) of B cells at 2, 6, and 7 mo compared with HMF. FN hadsignificantly lower numbers of B cells at 7 mo compared with
HMF (p ⫽ 0.001), whereas F was not different from HMF (Fig.
1b).
There were no significant differences in NK cell
(CD56⫹CD57⫹/⫺CD16⫹/⫺CD3⫺) numbers or percentages between FN and F. However, NK cell proportions were significantly lower (14%) in both formula groups at 6 mo comparedwith HMF (Fig. 1c).
Nucleotides affected T-cell subsets; MANOVA and RMA.
Figures 2–7 describe significant differences in T-cell subsets
between FN and F, and compare each formula group to breastfed infants. The proportion of naive helper (CD45RA⫹CD4⫹)
T cells was significantly lower (5%) in FN compared with F at
12 mo (two-group MANOVA; Fig. 2). Both formula groups
had significantly higher proportions (5–14%) of these cells at
2, 6, and 7 mo compared with HMF (three-group MANOVA).
At 12 mo, naive helper T-cell percentages in FN were notsignificantly different from HMF, however, F was significantly

NUCLEOTIDES AND IMMUNE CELL DEVELOPMENT

Figure 1. Panels a– c reveal no significant differences between FN and F but
do show differences between formula-fed and breast-fed infants. (a) Bothformula groups (⫹) had significantly higher numbers of T cells (CD3⫹)compared with HMF at 2 mo (FN ⬎ HMF, p ⫽ 0.016; F ⬎ HMF, p ⫽ 0.0003)
and 6 mo (FN ⬎ HMF, p ⫽ 0.022; F ⬎ HMF, p ⫽ 0.0001), and higher
percentages of T cells at 2, 6, and 7 mo (FN, F ⬎ HMF, p ⫽ 0.0001). FN
numbers were not different from HMF at 7 mo, whereas F was significantly
higher than HMF (p ⫽ 0.005). Data represent mean ⫾ SEM. (b) Both formulagroups (⫹) had significantly lower percentages of B cells (CD19&20⫹)compared with HMF at 2 mo (FN ⬍ HMF, p ⫽ 0.0005; F ⬍ HMF, p ⫽0.0001), 6 mo (FN ⬍ HMF, p ⫽ 0.0001; F ⬍ HMF, p ⫽ 0.0005), and 7 mo
(FN ⬍ HMF, p ⫽ 0.0001; F ⬍ HMF, p ⫽ 0.0004). FN had significantly lower
numbers of B cells at 7 mo compared with HMF (p ⫽ 0.001), whereas F was
not different from HMF. Data represent mean ⫾ SEM. (c) Both formula groups(⫹) had lower percentages of NK cells (CD56⫹CD57⫹/⫺CD16⫹/⫺CD3⫺) at 6
mo (FN ⬍ HMF, p ⫽ 0.006, F ⬍ HMF, p ⫽ 0.004,) compared with HMF. Data
represent geometric mean ⫾ SEM.

higher than HMF, whereas FN was not different from F(three-group MANOVA).
Two-group RMA (indicating change across all time points)
revealed higher percentages of M/E helper (CD45R0⫹CD4⫹)
T cells in FN compared with F (Fig. 3). Neither formula groupwas significantly different from HMF (three-group RMA);

895

however, the percentage differences in M/E helper T cells
between FN and F were maintained. Figure 3 describes the
MANOVA-identified time point differences between FN and F
and comparisons with HMF.
Two-group RMA showed higher percentages and numbersof M/E cytotoxic/suppressor (CD45R0⫹CD8⫹) T cells in FN
compared with F (Fig. 4). FN was not different from HMF ineither percentages or numbers (three-group RMA), whereas F
was significantly lower than HMF (percentages only) and FN(percentages and numbers). Figure 4 describes the MANOVAidentified time point differences between FN and F, and comparisons with HMF.
M/E Fas⫹ (apoptosis competent, CD45R0⫹CD95⫹CD3⫹)
T-cell proportions (Fig. 5) were significantly higher (22%) in
FN compared with F at 6 mo (two-group MANOVA). Neither
formula group was different from HMF nor each other by
three-group MANOVA.
Two-group RMA showed higher percentages of Tc1(IFN␥⫹CD4⫺CD3⫹) cells in FN compared with F (Fig. 6), andhigher percentages of total IFN␥⫹ (Th1 and Tc1) T cells in FN
compared with F (p ⫽ 0.026, data not shown). Three-group
RMA showed significantly higher percentages of Tc1 cells (p⫽ 0.007) and total IFN␥⫹ T cells (p ⫽ 0.0002, data not shown)
in HMF compared with F. No pairwise differences were seen
comparing FN to HMF or F. Tc1 cell proportions (Fig. 6) weresignificantly higher (33%) in FN compared with F at 12 mo(two-group MANOVA). F was lower than HMF at 7 mo by
three-group MANOVA.
Th2 (IL-4⫹CD4⫹CD3⫹) cell proportions (Fig. 7) were significantly higher (27%) in FN compared with F at 7 mo
(two-group MANOVA). At 7 mo, Th2 cell percentages in FN
were not significantly different from HMF (three-group
MANOVA), whereas F was significantly lower than HMF and
FN.
Subjects were defined as Th0 cell (IFN ␥ ⫹ IL⫹
4 CD4⫹CD3⫹) responders or Th0 cell nonresponders basedon the sensitivity of the intracellular cytokine assay (16).

Figure 2. Naive helper (CD45RA⫹CD4⫹) T-cell percentages (*) were lower in
FN vs F at 12 mo (two-group MANOVA). Both formula groups (⫹) had higher
percentages of these cells at 2, 6, and 7 mo vs HMF (p ⬍ 0.005). At 12 mo, naive
helper T-cell percentages (three-group MANOVA) in F were significantly higher
than HMF (p ⫽ 0.0001), whereas FN was not different from HMF (p ⫽ 0.028) or
F (p ⫽ 0.066). Data represent geometric mean ⫾ SEM.

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BUCK ET AL.

Figure 3. Two-group (p ⫽ 0.006) and three-group (p ⫽ 0.009) RMA revealed
higher percentages of M/E helper T cells (CD45R0⫹CD4⫹) in FN vs F; neither
formula group differed from HMF. M/E helper T-cell percentages weresignificantly higher in FN compared with F at *2 (two-group MANOVA), **6(two-group MANOVA), and ***7 mo (two-group ANOVA). Neither formulagroup differed from HMF by three-group MANOVA, and the percentage
differences between formulas at 2 (p ⫽ 0.045) and 7 mo (p ⬎ 0.05) did notreach significance, whereas at 6 mo (p ⫽ 0.01) percentages of these cells in FN
were greater compared with F. Data represent mean ⫾ SEM.

Figure 4. Two- and three-group RMA showed higher percentages andnumbers of M/E cytotoxic/suppressor (CD45R0⫹CD8⫹) T cells in FN vs F
(two-group, p ⫽ 0.002 and 0.006, respectively; three-group, p ⫽ 0.006 and p⫽ 0.012, respectively). FN was not different from HMF in either percentages
or numbers (three-group RMA), whereas F was significantly lower than HMF(F ⬍ HMF, p ⫽ 0.009, percentages only) and FN (F⬍FN p ⫽ 0.002
percentages; p ⫽ 0.006 numbers). Percentages of M/E cytotoxic/suppressor T(Tc) cells were significantly higher in FN vs F (two-group MANOVA) at *2,**
6, ***7, and ***12 mo. Percentages of M/E Tc cells in FN were notsignificantly different from HMF (three-group MANOVA) at 2 or 7 mo,
although F was significantly different from HMF at 2 mo (F ⬍ HMF, p ⫽0.007) and tended to be different from HMF at 7 mo (p ⫽ 0.037). Neither
formula significantly differed from HMF at 6 and 12 mo. Three-group
MANOVA supported the formula differences in M/E Tc cells at 6 (FN ⬎ F, p
⫽ 0.006) and 12 mo (FN ⬎ F, p ⫽ 0.005), although the differences inpercentages between FN and F at 2 and 7 mo did not reach significance (p ⫽0.048 and 0.026, respectively). Numbers of M/E Tc cells were higher at a6 moand b12 mo in FN compared with F (two-group ANOVA); neither formulagroup differed from HMF. Data represent geometric mean ⫾ SEM.

Although Th0 cells represent an infrequent T-cell cytokine
population, two-group Cochran-Mantel-Haenszel analysis revealed significantly more Th0-positive subjects in FN (54%)
compared with F (36%) at 6 mo (p ⫽ 0.005). No differenceswere seen among the feeding groups in a three-group analysis.

Figure 5. *Percentages (two-group MANOVA) and anumbers (two-group
ANOVA) of M/E Fas⫹ (apoptosis competent; CD45R0⫹CD95⫹CD3⫹) T cells
were higher in FN vs F at 6 mo. By three-group analyses, neither formulagroup was different from HMF. Data represent geometric mean ⫾ SEM.

Figure 6. Two-group RMA showed higher percentages of Tc1(IFN␥⫹CD4⫺CD3⫹) cells in FN vs F (p ⫽ 0.044). Three-group RMA showed
lower percentages of Tc1 cells (p ⫽ 0.007) in F compared with HMF. No
pairwise differences were seen comparing FN to HMF or F. Percentages of Tc1
cells were higher in FN vs F (p ⫽ 0.011, two-group MANOVA) at 12 mo (*).
Tc1 percentages in F were lower than HMF at 7 mo (⫹) by three-group
MANOVA (p ⫽ 0.006). Data represent geometric mean ⫾ SEM.

Proportions of naive CD4⫹CD8⫹ T cells were significantly
lower (12.5%) in FN compared with F at 12 mo (two-group
MANOVA). However, there were no significant feeding differences by three-group MANOVA (data not shown). Proportions of M/E CD4⫹CD8⫹ T cells were significantly higher(20%) in FN compared with F at 6 mo (two-group MANOVA).
However, there were no significant feeding differences by
three-group MANOVA (data not shown).
Exploratory RMA on all cell populations supported theinitial RMA results and did not reveal additional nucleotide
effects.
Nucleotides affected NK and NK-T cells: exploratory twogroup and three-group ANOVA. Two- and three-group
ANOVA time point analyses for each cell type supported theresults of the two- and three-group MANOVA. However,
several lymphocyte populations were not seen by MANOVA
cluster analysis but were identified by time point ANOVA ofindividual cell types, generally because the MANOVA cluster
was not significant at a given time point. ANOVA revealed
additional changes in M/E T cells and NK subsets. FN had up

NUCLEOTIDES AND IMMUNE CELL DEVELOPMENT

Figure 7. Percentages of Th2 (IL-4⫹CD4⫹CD3⫹) cells were higher in FN vsF (p ⫽ 0.017, two-group MANOVA) at 7 mo (*). By three-group MANOVA,
Th2 cell percentages in FN were not significantly different from HMF at 7 mo,
whereas F was significantly lower than HMF and FN (F ⬍ HMF, p ⫽ 0.001;
F ⬍ FN, p ⫽ 0.018). Data represent geometric mean ⫾ SEM.

to 43% higher numbers of M/E Tc cells (6 mo, 12 mo), 9%
higher proportions of M/E Th cells (7 mo), 15% higher numbers of M/E Fas⫹ cells (6 mo), 11% higher proportions of M/EFas⫺ T cells (12 mo), and 22% higher proportions of ␥⫹␦⫹ Th
cells (6 mo) compared with F. With respect to NK subsets,
FN had 17–18% higher numbers and proportions of
CD56⫹CD16⫺ NK cells, 30 – 42% higher proportions of
CD57⫹ NK-T cells, and 20 –29% lower numbers and proportions of CD56⫹ NK-T cells compared with F (Table 2).
Exclusive breast-feeding (ExHMF) versus mixed breastfeeding (HMF) and nucleotide effects. After 2 mo of exclusive breast-feeding, infants weaned to HMF were comparedwith infants whose milk source was exclusively human milk
(ExHMF) at 6 mo and 7 mo (n value insufficient at 12 mo).
Because ribonucleotide feeding affected eight lymphocyte
populations (M/E Th, M/E Tc, IFN␥, Tc1, Th2, CD56⫹NK,
CD56⫹NK-T, CD57⫹NK-T) at these time points, only thosecell types were analyzed comparing HMF or ExHMF. There
were no significant differences in these cell populations at 6
mo and 7 mo between ExHMF and HMF. Three-group
analyses using ExHMF, FN, and F supported the nucleotide
effects seen using HMF as a reference group. Similar to theresults with HMF, FN immune cell types either did not differ
from those infants fed ExHMF, or tended to be more likethose fed ExHMF than those fed F (except for CD56⫹ NK-T
cells at 6 mo, FN⬍ExHMF, although no significant differences by RMA).
DISCUSSION
This large study of 477 infants represents an extensive,
longitudinal assessment of infant immune development during
the first year of life. Several statistical methods were applied toelucidate the possible effects of nucleotides on immune cell
development. These techniques included multivariate time
point ANOVA (MANOVA) of clusters of functionally related
cell types, and exploratory time point ANOVA of all cell types,
which led to similar but not identical conclusions. ANOVA
analysis of all cell types supported the nucleotide effects
identified by MANOVA. However, ANOVA also revealed
nucleotide effects not identified by MANOVA, including

897changes in M/E Tc cell numbers and NK cell subsets (Table 2).
MANOVA analysis did reduce the burden of testing individual
cell types but it did not identify all nucleotide-associated cellchanges. Some of the nucleotide-associated changes affected
cell percentages but not absolute cell numbers. In these instances, nucleotide effects on cell percentages may have been
driven by changes in the target cell of interest (i.e. numerator)or the parent lymphocyte population (i.e. denominator), or
both. Although the absolute cell numbers may not have beensignificantly different, relationships among cells (expressed asa percentage) were significantly different.
Analysis of immune cell populations revealed significant
differences between the two formula feeding groups for several
lymphocyte types associated with immune maturation. Immune maturation of T cells is associated with a decrease innaive T lymphocytes and an increase in M/E T lymphocytes,
both of which may be identified by the presence or absence ofcell surface markers, and/or the production of various cytokines. Naive T cells (CD45RA⫹), which predominate in theinfant immune system, often have not previously encounteredantigen and mediate primary immune responses via IL-2 cytokine production. M/E T cells (CD45R0⫹) are produced as aresult of primary antigen exposure, and their numbers expand
after subsequent antigen challenges (booster immunization,
environmental pathogens). During this process, most T cells
differentiate into effector cells to facilitate antibody productionor act as cytotoxic cells, and then undergo apoptosis. Remaining T cells become memory cells capable of mediating a morepotent secondary, or memory immune response (17). Thesecells produce type 1 cytokines (e.g. IL-2, IFN-␥), and/or type2 cytokines (e.g. IL-4, IL-5).
The relative proportion of several M/E T lymphocyte populations increased up to 44% in the nucleotide group comparedwith the control group [M/E Th cells, M/E Tc cells, apoptosis
competent (Fas⫹) M/E T cells, Fas⫺ M/E T cells, and M/E T
cells with both helper (CD4) and cytotoxic/suppressor (CD8)markers]. The number of M/E Tc cells and Fas⫹ T cells also
increased up to 43% in the nucleotide group compared with thecontrol group. M/E T-cell populations in the nucleotide groupwere comparable to those in the HMF group, whereas M/E Tc
cells (RMA) in F were lower than HMF. The HMF grouprepresents an appropriate reference group for breast-fed infants
inasmuch as there were no differences in M/E T cellpopulations between exclusively breast-fed and HMF infants.
Consistent with changes in M/E T cells, percentages of T
lymphocytes positive for intracellular IFN-␥ (Tc1) and for IL-4
(Th2) were also higher in the nucleotide group compared withthe control group; Tc1 and Th2 cells represent subsets of M/E
T cells. These cytokine-positive T cells in the nucleotide groupwere comparable to those in the HMF group, whereas total
IFN␥ (RMA), Tc1 (RMA), and Th2 (7 mo) cells in the control
group were lower than HMF. The increase in M/E T cells was
associated with a concomitant decrease in the proportion ofnaive T lymphocytes (naive helper T cells and naive T cellswith both helper and cytotoxic/suppressor markers) in the
nucleotide group compared with the control group.
Decreases in naive T-cell populations and increases in memory T-cell populations characterize normal immune matura-

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BUCK ET AL.
Table 2. Effect of nucleotides on NK-T cells and NK cells (exploratory ANOVA)
Age
Cell population
NK-T cells
CD57⫹ NK-T (% of lymphocytes)(CD57⫹ CD56⫺CD3⫹)

Feeding
FN
F
HMF

CD56⫹ NK-T (% of lymphocytes)(CD56⫹CD16⫺CD8⫺CD3⫹)

FN
F
HMF

CD56⫹ NK-T (#)(CD56⫹CD16⫺CD8⫺CD3⫹)

FN
F
HMF

NK cells
CD56⫹ NK (% of lymphocytes)(CD56⫹CD16⫺CD8⫺CD3⫺)

FN
F
HMF

CD56⫹ NK (#)(CD56⫹CD16⫺CD8⫺CD3⫺)

FN
F
HMF

2 mo

6 mo

7 mo

12 mo

0.05
(0.04 – 0.06)
0.05(0.04 – 0.06)
0.06(0.05– 0.07)
0.04(0.04 – 0.05)
0.05(0.05– 0.07)
0.04(0.04 – 0.05)
2.2(1.8 –2.8)
3.1(2.5–3.8)
2.1(1.7–2.5)

0.13(0.11– 0.16)
0.10(0.08 – 0.12)
0.14(0.11– 0.17)
0.06(0.05– 0.07)
0.08(0.06 – 0.09)
0.08(0.07– 0.10)
3.8(3.1– 4.6)
5.1
(4.3– 6.1)
5.1
(4.4 –5.9)

0.17(0.14 – 0.19)
0.12(0.10 – 0.15)
0.18(0.14 – 0.21)
0.08(0.07– 0.10)
0.08(0.07– 0.10)
0.07(0.06 – 0.08)
4.6(3.7–5.8)
5.3(4.4 – 6.5)
4.1
(3.5– 4.8)

0.33
(0.27– 0.41)
0.28(0.23– 0.33)
0.42(0.35– 0.50)
0.14(0.12– 0.16)
0.12(0.11– 0.14)
0.15(0.13– 0.17)
7.3(6.4 – 8.3)
6.3
(5.5–7.3)
7.4(6.6 – 8.3)

0.44
(0.41– 0.48)
0.43(0.39 – 0.46)
0.59(0.56 – 0.64)
24(22–27)25(23–27)32(30 –35)0.44(0.40 – 0.47)
0.41(0.38 – 0.45)
0.52(0.49 – 0.56)
28(25–31)28(25–31)32(30 –35)0.45(0.42– 0.48)
0.44(0.40 – 0.47)
0.48(0.45– 0.51)
27(24 –29)28(26 –31)29(27–31)0.49(0.45– 0.54)
0.42(0.39 – 0.47)
0.52(0.49 – 0.56)
26(23–28)22(20 –24)26(24 –28)

Data represent geometric means with 95% confidence intervals in parentheses. Bold items reflect significant differences between formulas (two-group analyses,p ⬍ 0.05). NK-T and NK subset populations are expressed as proportions (%) or numbers (#).

tion/development and normal responses to vaccinations andinfections (18,19). Increases in memory cells in nucleotide-fed
infants compared with control-fed infants may be beneficial, as
increases in memory cells correlate with higher vaccine antibody titers and improved cell-mediated immune responses.
The inability to generate adequate levels of memory cells is
associated with some types of immunodeficiency and lessrobust cell-mediated responses (20 –22). Increased levels of
Tc1 and total IFN␥-producing cells in nucleotide-fed infants
compared with control-fed infants may also be beneficial, as
increases in IFN␥ correlate with proliferative responses, anindicator of cell-mediated immunity, and inversely correlatewith the severity of respiratory syncytial disease; that is, thegreater the amount of IFN␥, the less severe the disease (23,24). The increased proportion of Th2 cells in the nucleotidegroup may enhance mucosal and systemic antibody responses.
Finally, increased proportions of Th0 responders in the nucleotide group compared with the control group may also support
T-cell maturation, because naive Th cells transition to Th0
after encountering antigen and subsequently differentiate into
Th1 or Th2 cells (25).
Interestingly, other nutritional factors have been shown tomodulate T-cell immune maturation. Specifically, supplementation of preterm formula with docosahexanoic acid and ara-

chidonic acid increased immune maturation at 42 d of age
compared with unsupplemented formula, as indicated by a
ⵑ25% increase in the proportion of circulating M/E helper T
cells, and a decrease in the proportion of naive helper T cells
to levels consistent with those in human milk (26).
Nucleotide feeding was associated with a significantly elevated neutralizing antibody response to poliovirus type 1, atransient increase in Hib protection levels at 6 mo (⬎1 ␮g/mL),and trends favoring nucleotide supplementation for antibody
responses to Hib and diphtheria antigens (12). In a previous
study (7), nucleotide supplementation was associated withsignificantly higher Hib and diphtheria antibody responses (FN⬎ F, FN ⬎ HMF). Although M/E T cells were not measured
by Pickering et al. (7), Cordle et al. (16) observed a 29%
increase in the number of M/E Th cells in infants fed Isomil (asoy-based formula with inherent nucleotide levels ⵑ300 mg/L)
supplemented with nucleotides (ⵑ74 mg/L) compared withthose fed HMF. The same group (27) also reported an increased antibody response to the PRP component of HibTITER vaccine in the nucleotide-fed group compared with
HMF. Enhanced levels of M/E T cells may be responsible fordriving the significantly improved vaccine antibody responses
seen in these studies (7,12,27).

NUCLEOTIDES AND IMMUNE CELL DEVELOPMENT

With regard to nucleotides and animal research, Jyonouchi
et al. (28) reported nucleotide-mediated increases in type1–associated antibodies (anti-keyhole limpet hemocyanin
IgG2a and IgG2b) in type 1– dominant mice, indicating that
dietary nucleotides shift a type 1–predominant immune systemfurther in the same direction. Consistent with this observation,
Jyonouchi et al. (29) also showed dietary nucleotides result ina type 1 shift in anti-ovalbumin antibody responses in type2–predominant mice. In addition, lymphocytes isolated frommice fed nucleotides expressed more IFN␥ mRNA, and produced more IFN␥ and more IFN␥⫹ T cells after restimulation
with antigen in culture (28 –30). These changes indicate that
nucleotide supplementation results in shifts toward type 1
responses. Thus, the current clinical finding that nucleotides
enhance the proportion of type 1 T cells (IFN␥⫹) supports
Jyonouchi’s observations in mice fed nucleotides. The current
study also demonstrated nucleotides support a type 1/type 2
cytokine balance consistent with breast-fed infants.
Nucleotide-associated changes were not restricted to T cells.
Nucleotide supplementation enhanced the numbers and proportions of CD56⫹CD16⫺ NK cells, enhanced the proportionsof CD57⫹ NK-T cells (NK-T cells express CD3 and CD56
and/or CD57), and lowered the numbers and proportions of
CD56⫹ NK-T cells compared with the control-fed infants. Thechanges in NK and NK-T cells were consistent with the HMF
group, whereas the control group significantly differed from
HMF. HMF represents an appropriate reference group forbreast-fed infants because there were no differences in theseNK or NK-T-cell populations between exclusively breast-fed
and HMF infants. CD56⫹CD16⫺ NK cells represent ⵑ10% ofCD56⫹ NK cells (supported by the current study), they express
high levels of CD56 (the majority of NK cells are CD56dim),have lower natural cytotoxicity compared with CD56dim cells
but have similar levels of cytotoxicity after activation with IL-2
or IL-12, proliferate more vigorously than CD56dim NK cells,
and are the primary source of NK-derived immunoregulatory
cytokines (IFN␥, tumor necrosis factor-␤, IL-10, IL-13, granulocyte macrophage colony-stimulating factor) (31). The natural ligand of NK-T cells is thought to be hydrophobic (probably lipids and glycolipids), and is presented by major
histocompatibility complex class I–like molecules (CD1d proteins) on antigen-presenting cells (32). NK-T cells produce
high levels of immunoregulatory cytokines, including IFN-␥
and IL-4 (32). They also exhibit potent cytolytic activity (31)and have been shown to increase during infancy (16) and withchronologic aging (33). The majority of CD57⫹ NK-T cells
express CD8 (␣␤ heterodimer) but lack the CD28 costimulatory molecule. These cells increase during human cytomegalovirus, or human immunodeficiency virus infections,and after bone marrow and kidney transplantation (34,35).
CD56⫹ NK-T cells are thought to be liver-derived (36),whereas CD57⫹ NK-T cells are bone marrow– derived (37).
Evidently, nucleotides influence some lymphocytes of the innate immune system, which may lead to an increased ability toeliminate intracellular pathogens and/or tumor cells.
With regard to clinical outcomes, there were no significant
differences between HMF and formula-fed infants in physician
visits due to infectious [otitis media, respiratory tract infections899(independent of otitis media), other infections] or noninfectious causes (12). Nucleotide effects might be clinically apparent in infant populations different from those currently studied.
In summary, dietary nucleotides affected T lymphocyte subsets (memory/effector T cells and naive T cells), and NK
lymphocyte subsets (CD56⫹CD16⫺ NK cells, and NK-T cells)
in infants. Collectively, these results suggest an increased
maturation of the cellular immune system in the nucleotidegroup compared with the control group. Furthermore, thenucleotide-associated changes were consistent with thosefound in HMF infants. These changes represent shifts in lymphocyte populations capable of responding to a plethora ofantigens. Consistent with previous data (16), the addition of
nucleotides did not result in any shifts in the overall distribution of the major classes of immune cells. The five major WBC
categories studied included neutrophils, eosinophils, basophils,
monocytes, and lymphocytes; there were no differences between formula groups, or between either formula and HMF
groups (except at 6 mo, where lymphocyte numbers werehigher in F versus HMF). Furthermore, there were no differences in the major lymphocyte types (total T, B, or NK cells)
between the formula groups. However, also consistent withprevious reports, infants fed either formula had significantly
increased numbers and percentages of T cells resulting indecreased percentages of B cells and NK cells compared with
HMF infants (10,11).
The present data provided additional observations, namely,
granulocyte phagocytic ability and granulocyte extravasation(migration) potential were not affected by supplemental nucleotides. Similarly, supplemental nucleotides did not affect T-B
lymphocyte cooperation or lymphocyte activation as mediated,
respectively, by CD40 ligand or CD69 expression on T cells(data not shown).
CONCLUSIONS
Nucleotide-associated increases in memory/effector T-cell
populations and changes in NK cell subtypes provide evidence
that infant formula supplemented with levels of nucleotides
similar to levels in human milk (72 mg/L) may facilitate
maturation and immunoregulatory shifts in some lymphocyte
populations consistent with HMF infants. These shifts might
support increased antibody responses and immune cell protection. The nucleotide-associated changes in immunoregulatory
NK cell subsets may also enhance innate immune responses
against tumors and/or intracellular pathogens. Finally, thisstudy established the important role that certain key nutrients
may play when included in infant formula in helping to provide
some of the benefits associated with breast-feeding.
Acknowledgments. The authors thank Amy Bates, Joe Laco,
Pete Yakimovich, Karen Goehring, Melissa Nameth, Pam
Newton, R.N., Tim Murphy, M.S., John Lasekan, Ph.D., andPeter Hu, Ph.D. for technical assistance, and Marc Masor,
Ph.D. for editorial review. We also thank the following physicians who participated in this study: Wilson Andrews, Mark
Blatter, Iqbal Choudhry, David Ferrera, William Forti, Martin
Greenberg, Terry Jefferson, Susan Kerns, Jacob Lohr, Edgardo

900

BUCK ET AL.

Malacaman, Michael Pichichero, Bruce Pistorius, Robert Reis,
Allan Robinson, Laurie Schnell, and Michael Smith.
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