Retinoic acid (RA), a bioactive retinoid, and polyriboinosinic:polyribocytidylic acid (PIC) are known to promote immunity in vitamin A-deficient animals. In this study, we hypothesized that RA, PIC, and the combination can provide significant immunoadjuvant activity even in the vitamin A-adequate state. Six-week-old C57BL/6 mice were immunized with tetanus toxoid (TT) and treated with RA and/or PIC at priming in three independent studies of short and long duration. RA and PIC differentially regulated both primary and secondary anti-TT IgG isotypes, whereas the combination of RA + PIC stimulated the highest level of anti-TT IgG production and, concomitantly, a ratio of IgG1 to IgG2a similar to that of the control group. The regulation of Ab response was strongly associated with type 1/type 2 cytokine gene expression. Whereas RA reduced type 1 cytokines (IFN-γ and IL-12), PIC enhanced both type 1 and type 2 cytokines (IL-4 and IL-12) and cytokine-related transcription factors. Despite the presence of PIC, the IL-4:IFN-γ ratio was significantly elevated by RA. In addition, RA and/or PIC modulated NK/NKT cell populations and the level of expression of the costimulatory molecules CD80/CD86, evident 3 days after priming. Notably, the NKT:NK and CD80:CD86 ratios were correlated with the IL-4:IFN-γ ratio, indicative of multiple converging modes of regulation. Overall, RA, PIC, and RA + PIC rapidly and differentially shaped the anti-tetanus Ig response. The robust, durable, and proportionate increase in all anti-TT IgG isotypes induced by RA + PIC suggests that this combination is promising as a means to enhance the Ab response to TT and similar vaccines.

Soon after its discovery, vitamin A was characterized as the anti-infective vitamin due to the observation that vitamin A-deficient animals succumbed to infections that vitamin A-adequate animals survived (1). In humans, vitamin A deficiency is now recognized as a highly significant risk factor associated with increased mortality in children and pregnant women (2, 3). Intervention studies in at-risk populations have clearly demonstrated that providing vitamin A to children, ranging from newborn to 5 years of age, decreases child mortality rates by an average of 23%, with a 50% reduction observed in some studies (4, 5). Furthermore, vitamin A supplementation has reduced measles-related mortality and the severity of several infectious diseases, including measles, diarrhea, malaria, and HIV infection (6, 7, 8, 9). These encouraging results have prompted the distribution of vitamin A to children in at-risk populations, sometimes in concert with immunization programs, including vaccinations against measles and tetanus (10, 11, 12).

The anti-infective effect of vitamin A is thought to be attributable to immune stimulation and/or regulation. Vitamin A and its transcriptionally active metabolite, retinoic acid (RA),3 have been shown to modulate several indices of innate and adaptive immunity, such as dendritic cell (DC) maturation, cytokine production, T and B cell activation and Ab responses, as well as mucosal immunity (1, 13, 14). Whereas most immunological research has addressed the effects of vitamin A and RA in remediating vitamin A deficiency, it is also important to evaluate vitamin A and/or RA in the normal state, not only because these nutrients may potentially be useful in strategies to improve vaccine efficiency, but because many of the recipients of current vitamin A distribution-immunization programs are not themselves vitamin A deficient (10). Therefore, understanding the consequences of vitamin A and its metabolite RA on the development of Ag-specific Ab responses can aid in their appropriate use in both clinical and public health settings.

Polyriboinosinic:polyribocytidylic acid (PIC), a synthetic double-stranded polyribonucleotide and a mimetic of dsRNA viruses, is known for its ability to induce type I/type II IFNs and other cytokines (15), increase antiviral and antitumor reactions in several models (16, 17, 18), and activate both innate and adaptive immunity (16, 19, 20, 21). Multiple mechanisms appear likely in that PIC has promoted DC maturation, stimulated NK cell cytotoxicity (20, 21, 22), and also increased Ag-specific total IgG and IgG isotypes (21). Besides immune effects of PIC alone, it is of interest that combinations of IFNs and retinoids have shown promise in cancer therapy, which may in part be due to their regulatory effects on the immune system (23).

Tetanus toxoid (TT) is a classical T cell-dependent Ag and a clinically important vaccine especially in young children and women of child-bearing age (24). Previously, we demonstrated that the combination of RA and PIC synergistically increased anti-TT Ab production in vitamin A-deficient rats and mice (25), and elevated primary anti-TT IgG response in vitamin A-adequate rats (26). However, little is yet known of the independent and combined effects of these agents on the production of IgG subtypes, memory responses to TT, and cellular and molecular markers of type 1/type 2 immunity. The aim of the present study was to evaluate RA and PIC, alone and in combination, as modulators of molecular and cellular aspects of immunity in nonimmunocompromised vitamin A-sufficient mice.

Animal protocols were approved by the Institutional Animal Use and Care Committee of Pennsylvania State University. Six-week-old C57BL/6 female mice (Charles River Laboratories) were divided into four groups: control, RA, PIC, and RA + PIC, and fed a nutritionally complete diet (LabDiet 5001, which contains 22 IU of vitamin A/g diet; Purina Mills) throughout the experimental period. Three independent experiments were conducted. For the first study (11-day study), mice were immunized with 10 μg of TT (Aventis Pasteur) by i.p. injection, and cotreated with 50 μg of RA (Sigma-Aldrich) orally in 25 μl of canola oil and/or PIC (stabilized with poly(l-lysine) and carboxymethycellulose, provided by the late H. Levy, National Institutes of Health, Bethesda, MD), at a dose of 2 μg i.p. on the first day of the experiment (day 0). From days 1–10 after immunization, the mice were fed the same dose of RA or oil daily. Blood was collected 7 and 10 days after priming for Ab quantification. On day 11, the mice were reimmunized with TT and cotreated with same doses of RA and/or PIC as on day 0. After 24 h, spleens were collected for later analysis. For the second study (3-day study), mice were immunized with TT and cotreated with RA and/or PIC as described for the first study, except that blood and spleen were collected 3 days after priming. For the third study (secondary anti-TT Ab response), mice were immunized with TT ± RA (37.5 μg, orally) and/or PIC (2 μg, i.p.) on day 0, followed by an additional 6-day treatment with RA or oil. Five weeks after priming, without further treatment with either RA or PIC, mice were reimmunized with TT, and blood and spleen were collected 7 days later. Throughout the studies, the treatment groups did not differ in body weight, and liver and spleen weights did not differ at the end of study.

Serum anti-TT IgM, total anti-TT IgG, and anti-TT IgG isotypes were quantified by ELISA, as previously described (27). Serum was serially diluted to assure measurements were in a linear dose-response range. A standard of serially diluted pooled immune serum was included on every plate, and titers of Ab were calculated based on this standard curve, in which 1 U was defined as the dilution fold that produced 50% of maximal OD for the standard sample.

Spleen cells were isolated, as reported previously (26). Briefly, spleen tissues were gently pressed through a sterile wire screen mesh, and the resulting cell suspensions were individually layered over Ficoll-Hypaque (1.083 g/ml; Sigma-Aldrich), followed by centrifugation at 2500 rpm for 20 min at 20°C. The mononuclear cells were carefully collected, washed twice, and suspended in RPMI 1640 medium (Invitrogen Life Technologies) with 10% FBS.

TT-specific ASCs were assessed by ELISPOT assay, as previously described (28). Spleen cell suspensions (106 cells/ml) from individual mice were serially diluted and incubated in TT-precoated filtration plates (Multiscreen HA; Millipore) at 37°C for 4–5 h. The plates were then washed and incubated with alkaline phosphatase-conjugated goat anti-mouse Abs (The Binding Site) at 4°C, overnight, followed by reaction with alkaline phosphatase-substrate solution (Bio-Rad). The developed spots were counted under a dissection microscope and calculated as number of spots per 106 cells.

Splenic mononuclear cells were suspended in RPMI 1640 medium (Invitrogen Life Technologies) containing 1% FBS and incubated with combinations of appropriately diluted fluorochrome-conjugated mAbs (BD Pharmingen) at room temperature for 40 min in the dark. For T cell population analysis, the cells were double stained with PE CD4 (H129.19) and FITC CD8 (53-6.7). B cells were stained with PE B220 (RA3.6B2) or FITC CD19 (1D3). For NK/NKT cell populations, the cells were double stained with FITC CD3 (17A2) and PE NK1.1 (PK136). For APCs, the cells were double stained with PE CD11b (M1/70) and FITC CD80 (16-10A1) or FITC CD86 (GL1). The cells were then washed, fixed with 1% paraformaldehyde in PBS, and analyzed on a Corixa XL-MLC flow cytometer (26).

A total of 1 μg of total RNA was subjected to reverse-transcriptase reaction (Promega), followed by 33P-labeled PCR, as described previously (29). Sense and antisense primers were as reported previously (26, 30) or were as follows: IL-4 (NM 021283), 5′-AGAGCTATTGATGGGTCTCA, 3′-GGCTTTCAAGGAAGTCTTTC, 401 bp; T-bet (AF241242), 5′-GATCGTCCTGCAGTCTCTCC, 3′-AACTGTGTTCCCGAGGTGTC, 413 bp; GATA-3 (NM008091), 5′-CTTATCAAGCCCAAGCGAAG, 3′-CAGGGATGACATGTGTCTGG, 311 bp; IL-12Rβ2 (NM008354), 5′-TGACAGCTGCTGGTGAAAGTCC, 3′-ATGATCAGGGGCTCAGGCTCTTCA, 269 bp; and 18S rRNA (M35283), 5′-AATGGTGCTACCGGTCATTC, 3′-ACCTCTCTTACCCGCTCTCC, 193 bp. The PCR products were separated on a nondenaturing polyacrylamide gel and then exposed to x-ray film. For quantification, individual bands were excised and counted in liquid scintillation fluid (ScintiVerse; Fisher Scientific), and normalized with 18S or GAPDH mRNA that was similarly quantified.

Data are reported as mean ± SE. The main effects of RA and PIC, and the interaction of RA and PIC were evaluated by two-way ANOVA. Differences among groups were determined using Fisher’s protected least significant difference test (SuperAnova; Abacus Concepts). When group variances were unequal, data were subjected to log10 or square-root transformation before statistical analysis. Simple linear regression was determined using the same software. A value of p < 0.05 was considered statistically significant.

Primary anti-TT IgM and IgG production was determined 7 and 10 days after priming, respectively. IgM was elevated only by PIC alone and RA + PIC, each about 4-fold (Fig. 1,a). Whereas RA and PIC alone significantly increased IgG, the combination of RA + PIC increased anti-TT IgG levels >80-fold compared with the response of normal control mice (Fig. 1,b). Surprisingly, RA and PIC differentially regulated anti-TT IgG isotypes (Fig. 1, c–e). RA alone selectively increased IgG1 and IgG2b, and elevated the IgG1/IgG2a ratio, an indicator of type 1/type 2 balance. PIC alone strongly increased all anti-TT IgG isotypes (IgG1, IgG2a, and IgG2b), while the IgG1/IgG2a ratio was not different from control. Compared with PIC alone, RA + PIC more potently increased ant-TT IgG1, but attenuated the induction of IgG2a, thereby maintaining the IgG1/IgG2a ratio similar to control group (Fig. 1,f). Two-way ANOVA confirmed that RA was a positive regulator for IgG1 and IgG2b, but a negative regulator for IgG2a, whereas PIC was a positive regulator for all of the IgG isotypes (Fig. 1, c–e). Hence, RA and PIC cooperatively promoted a robust primary anti-TT IgG response, but differentially regulated IgG isotypes in vitamin A-sufficient mice.

FIGURE 1.

RA and PIC synergistically enhance primary anti-TT IgG, but differentially regulate IgG isotypes. Six-week-old mice were immunized with TT ± RA and/or PIC, followed by an additional 10-day treatment with RA or oil (control). Plasma anti-TT IgM (a) and IgG (b), including IgG isotypes (c–f), were measured by ELISA on day 7 (IgM) and day 10 (IgGs) after priming. The Ab titers were calculated based on a standard curve, in which 1 U was defined as the dilution fold that gave 50% of ODmax. Bars show mean ± SE, n = 12–16 mice/group. Different letters above bars within panels indicate significant differences (p < 0.05, a < b < c < d). Results of two-way ANOVA for each factor (RA, PIC, and interaction) are also shown in each panel.

FIGURE 1.

RA and PIC synergistically enhance primary anti-TT IgG, but differentially regulate IgG isotypes. Six-week-old mice were immunized with TT ± RA and/or PIC, followed by an additional 10-day treatment with RA or oil (control). Plasma anti-TT IgM (a) and IgG (b), including IgG isotypes (c–f), were measured by ELISA on day 7 (IgM) and day 10 (IgGs) after priming. The Ab titers were calculated based on a standard curve, in which 1 U was defined as the dilution fold that gave 50% of ODmax. Bars show mean ± SE, n = 12–16 mice/group. Different letters above bars within panels indicate significant differences (p < 0.05, a < b < c < d). Results of two-way ANOVA for each factor (RA, PIC, and interaction) are also shown in each panel.

Close modal

Having observed that RA and PIC differentially regulated anti-TT IgG isotypes, we next asked whether type 1/type 2 cytokines, the key regulators of Ig isotype switching, were also regulated. The effects of RA and PIC on type 1/type 2 cytokine mRNA levels were evaluated in two independent studies: an 11-day study with reimmunization of TT with and without RA, PIC, or RA + PIC 24 h before spleens were collected, and a 3-day study to examine effects in the early stage of priming. On day 11, RA alone selectively reduced the mRNA expression of type 1 cytokine genes (IFN-γ and IL-12; Fig. 2, a and b), and elevated the ratio of IL-4 to IFN-γ (IL-4/IFN-γ), a commonly used index of the balance of type1/type 2 responses (Fig. 2, a, b, and e). PIC alone significantly enhanced both type 1 cytokines (IFN-γ and IL-12) and type 2 cytokines (IL-4 and IL-10; Fig. 2, c and d) without altering the ratio of IL-4 to IFN-γ compared with the ratio in the control group. The combination of RA + PIC abrogated the induction of IFN-γ and IL-12 by PIC (RA + PIC < PIC), thereby elevating the ratio of IL-4 to IFN-γ (Fig. 2,e). Moreover, RA and PIC also regulated several transcriptional factors and receptors involved in type 1/type 2 (Th1/Th2) responses. T-bet and GATA-3 are two major transcriptional factors essential for Th1 and Th2 differentiation, respectively (31). IFN regulatory factor-1 is such a factor involved in Th1 differentiation. IL-12Rβ2, a subunit of IL-12R, is required for IL-12-induced Th1 differentiation (31, 32). Overall, RA slightly suppressed Th1-related gene expression, while PIC alone enhanced both Th1- and Th2-related gene expression, especially IFN regulatory factor-1 (IRF-1), which was significantly induced by PIC. The combination of RA + PIC tended to attenuate the induction of Th1-related genes by PIC (Table I). Despite weaker regulation of Th1/Th2-related transcription factors than cytokines, RA was still a negative regulator for each of these Th1-related genes, as shown by two-way ANOVA, whereas PIC was a positive regulator for both Th1- and Th2-related genes, patterns consistent with the regulation of type 1/type 2 cytokines.

FIGURE 2.

RA and PIC differentially regulate mRNA levels of type 1/type 2 cytokines. Six-week-old mice were immunized with TT ± RA and/or PIC, followed by additional 10-day treatment with RA or oil. On day 11 after priming, the mice were rechallenged with TT ± RA and/or PIC. Spleens were collected 24 h later, and RNA was extracted. The mRNA levels of IL-4 (a), IL-10 (b), IL-12 (c), and IFN-γ (d) were quantified by 33P-labeled PCR, normalized, and then shown as fold induction relative to control. The ratio of IL-4 mRNA to IFN-γ mRNA was calculated and shown in e. Bars shown are means ± SE, n = 12–16/group. Bands from two representative mice per group are shown for illustration below the group means. Different letters above bars within panels indicate significant differences (p < 0.05, a < b < c). Results of two-way ANOVA for each factor (RA, PIC, and interaction) are also shown in each panel.

FIGURE 2.

RA and PIC differentially regulate mRNA levels of type 1/type 2 cytokines. Six-week-old mice were immunized with TT ± RA and/or PIC, followed by additional 10-day treatment with RA or oil. On day 11 after priming, the mice were rechallenged with TT ± RA and/or PIC. Spleens were collected 24 h later, and RNA was extracted. The mRNA levels of IL-4 (a), IL-10 (b), IL-12 (c), and IFN-γ (d) were quantified by 33P-labeled PCR, normalized, and then shown as fold induction relative to control. The ratio of IL-4 mRNA to IFN-γ mRNA was calculated and shown in e. Bars shown are means ± SE, n = 12–16/group. Bands from two representative mice per group are shown for illustration below the group means. Different letters above bars within panels indicate significant differences (p < 0.05, a < b < c). Results of two-way ANOVA for each factor (RA, PIC, and interaction) are also shown in each panel.

Close modal
Table I.

Regulation of Th1/Th2-related genes by RA, PIC, and RA/PIC in combinationa

Th1-Related Genes (mRNA Fold Induction)Th2-Related Gene (mRNA Fold Induction)
T-betIRF-1IL-12Rβ-2GATA-3
Control 1.00 ± 0.30ab 1.00 ± 0.33a 1.00 ± 0.38ab 1.00 ± 0.26ab 
RA 0.67 ± 0.17a 0.66 ± 0.20a 0.45 ± 0.17a 0.87 ± 0.25a 
PIC 1.20 ± 0.34b 2.14 ± 1.24b 1.43 ± 0.65b 1.20 ± 0.25ab 
RA/PIC 0.97 ± 0.46ab 1.34 ± 0.84a 1.06 ± 0.69b 1.24 ± 0.43b 
Two-way ANOVAb     
RA p < 0.05 (−) p < 0.01 (−) p < 0.05 (−) NS 
PIC p = 0.06 (+) p < 0.05 (+) p = 0.01 (+) p < 0.05 (+) 
Th1-Related Genes (mRNA Fold Induction)Th2-Related Gene (mRNA Fold Induction)
T-betIRF-1IL-12Rβ-2GATA-3
Control 1.00 ± 0.30ab 1.00 ± 0.33a 1.00 ± 0.38ab 1.00 ± 0.26ab 
RA 0.67 ± 0.17a 0.66 ± 0.20a 0.45 ± 0.17a 0.87 ± 0.25a 
PIC 1.20 ± 0.34b 2.14 ± 1.24b 1.43 ± 0.65b 1.20 ± 0.25ab 
RA/PIC 0.97 ± 0.46ab 1.34 ± 0.84a 1.06 ± 0.69b 1.24 ± 0.43b 
Two-way ANOVAb     
RA p < 0.05 (−) p < 0.01 (−) p < 0.05 (−) NS 
PIC p = 0.06 (+) p < 0.05 (+) p = 0.01 (+) p < 0.05 (+) 
a

Mice were treated as described in Fig. 2. The mRNA levels of genes in spleen tissues were quantified by 33P-labeled PCR. Data were normalized with 18S rRNA and shown as fold induction relative to the control group. Values are mean ± SE, n = 8/group. Different superscript letters in a column indicate significant differences (p < 0.05, a < b). IRF-1, IFN regulatory factor 1.

b

The effect of each factor (RA, PIC, and interaction) was evaluated by two-way ANOVA. (+) and (−), Indicate positive and negative regulation, respectively. There were no significant interactions between RA and PIC.

Regression analysis indicated that anti-TT IgG1 titers were positively correlated with IL-4 (r = 0.47, p < 0.01) and IL-10 (r = 0.39, p < 0.05), while anti-TT IgG2a titers were highly correlated with the level of IL-12 (r = 0.60, p < 0.01). Interestingly, IgG2b titer was correlated with both IL-4 (r = 0.43, p < 0.05) and IL-12 (r = 0.58, p < 0.01). These data imply that RA and/or PIC in vivo differentially modulated type 1/type 2 responses, which in turn contributed to the regulation of anti-TT Ab response and IgG isotypes.

To further determine whether RA and PIC could regulate type 1/type 2 cytokines in the early stage of response to immunization (before primary Ab response and before or during Ig isotype switching), we also measured IL-4 and IFN-γ mRNA levels on day 3 after priming (Fig. 3). PIC alone selectively induced IFN-γ mRNA (Fig. 3,a), whereas RA alone only slightly enhanced IL-4 mRNA (Fig. 3,b). The combination of RA + PIC significantly increased IL-4, while it abrogated the induction of IFN-γ. In consequence, the ratio of IL-4:IFN-γ was significantly increased in mice that received RA + PIC compared with PIC alone (Fig. 3 c). Two-way ANOVA confirmed that PIC was a positive regulator for IFN-γ, and RA was a positive regulator for IL-4, but a negative regulator for IFN-γ. Thus, RA and PIC were able to manipulate the expression of type 1/type 2 cytokine genes in the early stage of the response to immunization.

FIGURE 3.

RA and PIC significantly regulate mRNA levels of IL-4 and IFN-γ 3 days after priming. Six-week-old mice were immunized with TT ± RA and/or PIC, followed by additional 2-day treatment of RA or oil. Spleens were collected on day 3 after priming, and RNA was extracted. The mRNA levels of IL-4 (a) and IFN-γ (b) were quantified by 33P-labeled PCR, normalized, and then shown as fold induction relative to control. The ratio of IL-4 mRNA to IFN-γ mRNA was calculated and shown in c. Bars shown are means ± SEM, n = 12–16/group. Bands from two representative mice per group are shown for illustration. Different letters above bars within panels indicate significant differences (p < 0.05, a < b < c). Results of two-way ANOVA for each factor (RA, PIC, and interaction) are also shown in each panel.

FIGURE 3.

RA and PIC significantly regulate mRNA levels of IL-4 and IFN-γ 3 days after priming. Six-week-old mice were immunized with TT ± RA and/or PIC, followed by additional 2-day treatment of RA or oil. Spleens were collected on day 3 after priming, and RNA was extracted. The mRNA levels of IL-4 (a) and IFN-γ (b) were quantified by 33P-labeled PCR, normalized, and then shown as fold induction relative to control. The ratio of IL-4 mRNA to IFN-γ mRNA was calculated and shown in c. Bars shown are means ± SEM, n = 12–16/group. Bands from two representative mice per group are shown for illustration. Different letters above bars within panels indicate significant differences (p < 0.05, a < b < c). Results of two-way ANOVA for each factor (RA, PIC, and interaction) are also shown in each panel.

Close modal

T cells, B cells, APCs, and NK/NKT cells are directly or indirectly involved in the regulation of thymus-dependent Ab production. Therefore, we wanted to determine whether RA and PIC modulate these cell types. The 3-day study showed that T and B cell populations were not affected by the treatments, except for CD8+ T cells, which were significantly reduced by PIC (Table II). RA and PIC differentially regulated NK and NKT cell populations. The proportion of NK cells (CD3NK1.1+) was increased by PIC alone, whereas the proportion of NKT cells (CD3+NK1.1+) was increased by RA. Two-way ANOVA confirmed that RA was a positive regulator for the NKT cell population, and PIC was a positive regulator for the NK cell population (Table II).

Table II.

Regulation of T cell and NK/NKT cell populations by RA, PIC, and RA/PIC in combinationa

ControlRAPICRA + PICTwo-Way ANOVAb
RA (p)PIC (p)
T cell population       
 CD4+ (%) 14.21 ± 1.40 14.82 ± 1.68 11.14 ± 1.30 13.03 ± 1.06 NS NS 
 CD8+ (%) 8.28 ± 1.27b 7.58 ± 1.24ab 5.02 ± 0.81a 5.30 ± 0.75a NS 0.01 (−) 
 CD4+ to CD8+ ratio 1.85 ± 0.14a 2.11 ± 0.20ab 2.40 ± 0.21ab 2.72 ± 0.34b NS < 0.05 (+) 
B cell population       
 B220+ (%) 74.20 ± 1.98 71.92 ± 2.97 77.61 ± 2.38 75.58 ± 1.92 NS NS 
 CD19+ (%) 72.31 ± 2.11 71.90 ± 2.08 73.14 ± 2.13 76.13 ± 2.24 NS NS 
NK/NKT cell populations       
 NK1.1+ (%) 5.27 ± 0.62a 6.37 ± 0.59ab 6.54 ± 0.75ab 7.40 ± 0.59b NS NS 
 CD3NK+ (%) 3.52 ± 0.45a 3.98 ± 0.46ab 5.06 ± 0.60b 4.75 ± 0.43ab NS < 0.05 (+) 
 CD3+NK+ (%) 1.20 ± 0.17ab 1.71 ± 0.12c 0.99 ± 0.15a 1.54 ± 0.11b < 0.01 (+) NS 
 NKT to NK cell ratio 0.34 ± 0.03bc 0.43 ± 0.03c 0.20 ± 0.03a 0.34 ± 0.03b < 0.01 (+) < 0.01 (−) 
ControlRAPICRA + PICTwo-Way ANOVAb
RA (p)PIC (p)
T cell population       
 CD4+ (%) 14.21 ± 1.40 14.82 ± 1.68 11.14 ± 1.30 13.03 ± 1.06 NS NS 
 CD8+ (%) 8.28 ± 1.27b 7.58 ± 1.24ab 5.02 ± 0.81a 5.30 ± 0.75a NS 0.01 (−) 
 CD4+ to CD8+ ratio 1.85 ± 0.14a 2.11 ± 0.20ab 2.40 ± 0.21ab 2.72 ± 0.34b NS < 0.05 (+) 
B cell population       
 B220+ (%) 74.20 ± 1.98 71.92 ± 2.97 77.61 ± 2.38 75.58 ± 1.92 NS NS 
 CD19+ (%) 72.31 ± 2.11 71.90 ± 2.08 73.14 ± 2.13 76.13 ± 2.24 NS NS 
NK/NKT cell populations       
 NK1.1+ (%) 5.27 ± 0.62a 6.37 ± 0.59ab 6.54 ± 0.75ab 7.40 ± 0.59b NS NS 
 CD3NK+ (%) 3.52 ± 0.45a 3.98 ± 0.46ab 5.06 ± 0.60b 4.75 ± 0.43ab NS < 0.05 (+) 
 CD3+NK+ (%) 1.20 ± 0.17ab 1.71 ± 0.12c 0.99 ± 0.15a 1.54 ± 0.11b < 0.01 (+) NS 
 NKT to NK cell ratio 0.34 ± 0.03bc 0.43 ± 0.03c 0.20 ± 0.03a 0.34 ± 0.03b < 0.01 (+) < 0.01 (−) 
a

Six-week-old mice were immunized with TT and cotreated with RA and/or PIC. On day 3 after priming, spleen cells were isolated and stained with fluorochrome-conjugated Abs, as described in Materials and Methods. Values are mean ± SE, n = 8/group. Different superscript letters within a row indicate significant differences (p < 0.05, a < b < c).

b

The effect of each factor (RA, PIC, and interaction) was evaluated by two-way ANOVA. (+) and (−), Indicate positive and negative regulation, respectively. There were no significant interactions between RA and PIC.

Moreover, RA and PIC regulated populations and differentiation of APCs. We evaluated markers associated with three types of professional APC: DC (CD11c+), macrophages (CD11b+), and B cells (B220+). Treatment with RA and/or PIC for 3 days did not significantly affect B cell and DC populations (Table II and data not shown); however, RA and RA + PIC up-regulated CD11b, a major marker of macrophages (Table III). Regarding costimulatory molecules, the treatments did not affect MHC II and CD40 (data not shown). However, RA and RA + PIC induced the expression of the costimulatory molecule CD80 (B7-1), whereas PIC alone induced the expression of CD86 (B7-2). Two-way ANOVA indicated that RA was a positive regulator for CD80, but a negative regulator for CD86, while PIC was a positive regulator for CD86. Interestingly, RA and RA + PIC together up-regulated CD80 expression on CD11b+ cells, whereas PIC induced CD86 on CD11b cells (Table III). These data indicated that RA and PIC affected different cell types and regulated the expression of CD80/CD86 differentially.

Table III.

Expression and distribution of CD80/CD86 molecules on splenic lymphocytesa

ControlRAPICRA + PICTwo-Way ANOVAb
RA (p)PIC (p)
CD11b+ (FI)c 639.93 ± 58.39a 927.53 ± 80.56b 636.90 ± 64.65a 915.62 ± 91.02b 0.01 (+) NS 
CD80+ (%) 10.49 ± 0.84ab 12.75 ± 0.91c 9.93 ± 0.42a 12.91 ± 0.71c < 0.01 (+) NS 
 CD80+ CD11b (%) 2.79 ± 0.27 3.19 ± 0.13 3.09 ± 0.17 3.01 ± 0.16 NS NS 
 CD80+ CD11b+ (%) 3.65 ± 0.39a 4.53 ± 0.45ab 3.75 ± 0.20ab 4.80 ± 0.41b < 0.05 (+) NS 
CD86+ (%) 6.25 ± 0.32a 5.62 ± 0.24a 8.30 ± 0.59b 6.86 ± 0.53a < 0.05 (−) 0.001 (+) 
 CD86+ CD11b (%) 2.05 ± 0.17ab 1.19 ± 0.14a 4.59 ± 0.77c 2.62 ± 0.44b < 0.01 (−) 0.0001 (+) 
 CD86+ CD11b+ (%) 1.54 ± 0.23 1.52 ± 0.10 1.61 ± 0.13 1.54 ± 0.15 NS NS 
CD80 to CD86 ratio 1.67 ± 0.08b 2.26 ± 0.11c 1.25 ± 0.10a 1.89 ± 0.14b 0.0001 (+) 0.001 (−) 
ControlRAPICRA + PICTwo-Way ANOVAb
RA (p)PIC (p)
CD11b+ (FI)c 639.93 ± 58.39a 927.53 ± 80.56b 636.90 ± 64.65a 915.62 ± 91.02b 0.01 (+) NS 
CD80+ (%) 10.49 ± 0.84ab 12.75 ± 0.91c 9.93 ± 0.42a 12.91 ± 0.71c < 0.01 (+) NS 
 CD80+ CD11b (%) 2.79 ± 0.27 3.19 ± 0.13 3.09 ± 0.17 3.01 ± 0.16 NS NS 
 CD80+ CD11b+ (%) 3.65 ± 0.39a 4.53 ± 0.45ab 3.75 ± 0.20ab 4.80 ± 0.41b < 0.05 (+) NS 
CD86+ (%) 6.25 ± 0.32a 5.62 ± 0.24a 8.30 ± 0.59b 6.86 ± 0.53a < 0.05 (−) 0.001 (+) 
 CD86+ CD11b (%) 2.05 ± 0.17ab 1.19 ± 0.14a 4.59 ± 0.77c 2.62 ± 0.44b < 0.01 (−) 0.0001 (+) 
 CD86+ CD11b+ (%) 1.54 ± 0.23 1.52 ± 0.10 1.61 ± 0.13 1.54 ± 0.15 NS NS 
CD80 to CD86 ratio 1.67 ± 0.08b 2.26 ± 0.11c 1.25 ± 0.10a 1.89 ± 0.14b 0.0001 (+) 0.001 (−) 
a

Mice were treated as described in Table II. Values are mean ± SE, n = 8/group. Different superscript letters in a row indicate significant differences (p < 0.05, a < b < c).

b

The effect of each factor (RA, PIC, and interaction) was evaluated by two-way ANOVA. (+) and (−), Indicate positive and negative regulation, respectively. There were no significant interactions between RA and PIC.

c

FI (total fluorescence intensity) = % of positive cells × median fluorescence intensity.

Simple regression showed that the ratio of IL-4 to IFN-γ, described above, was significantly correlated with both the ratio of NKT to NK cells (Fig. 4,a) and the ratio of CD80 to CD86 (Fig. 4 b). These results imply a possible functional association between the balance of NK/NKT cells, CD80/CD86 molecules, and type 1/type 2 cytokine responses.

FIGURE 4.

The ratio of IL4/IFN-γ is significantly associated with the ratio of NK/NKT cells, and the ratio of CD80/CD86 costimulatory molecules. Mice were treated as described in Table II and Fig. 3. The relationship between ratio of IL4/IFN-γ and ratio of NK/NKT cells (a) and ratio of CD80/CD86 (b) was assessed by simple regression analysis.

FIGURE 4.

The ratio of IL4/IFN-γ is significantly associated with the ratio of NK/NKT cells, and the ratio of CD80/CD86 costimulatory molecules. Mice were treated as described in Table II and Fig. 3. The relationship between ratio of IL4/IFN-γ and ratio of NK/NKT cells (a) and ratio of CD80/CD86 (b) was assessed by simple regression analysis.

Close modal

Next, we wanted to determine whether the strong effects of RA and PIC on anti-TT Ab response observed in the primary response were durable, as would be crucial for a vaccine adjuvant. As for the primary response, RA and/or PIC given only with priming significantly enhanced secondary anti-TT IgG production (Fig. 5, a–d). Regarding anti-TT IgG isotypes, RA selectively enhanced IgG1 and IgG2b. Although PIC robustly increased all anti-TT IgG isotypes, it significantly reduced the IgG1 to IgG2a ratio as compared with the control group. The combination of RA + PIC stimulated the highest levels of IgG and IgG1, but suppressed induction of IgG2a by PIC, which in turn partially restored the ratio of IgG1 to IgG2a toward the control level (Fig. 5 e).

FIGURE 5.

RA and/or PIC treatments given with the primary immunization enhance secondary anti-TT Ab IgG responses. Six-week-old mice were immunized with TT ± RA and/or PIC, followed by additional 6-day treatment of RA or oil. Five weeks later, the mice were reimmunized with TT only. Secondary anti-TT IgG (a) and IgG isotypes (b–e) in plasma were measured by ELISA 7 days after reimmunization. Different letters above bars within panels indicate significant differences (p < 0.05, a < b < c). Results of two-way ANOVA for each factor (RA, PIC, and interaction) are also shown in each panel.

FIGURE 5.

RA and/or PIC treatments given with the primary immunization enhance secondary anti-TT Ab IgG responses. Six-week-old mice were immunized with TT ± RA and/or PIC, followed by additional 6-day treatment of RA or oil. Five weeks later, the mice were reimmunized with TT only. Secondary anti-TT IgG (a) and IgG isotypes (b–e) in plasma were measured by ELISA 7 days after reimmunization. Different letters above bars within panels indicate significant differences (p < 0.05, a < b < c). Results of two-way ANOVA for each factor (RA, PIC, and interaction) are also shown in each panel.

Close modal

Finally, the effects of RA and PIC on the secondary Ab response were further determined by measuring anti-TT ASCs. Consistent with the plasma Ab response, RA and PIC also differentially regulated splenic ASCs (Table IV). The number of ASCs was highly correlated with the corresponding plasma anti-TT IgG isotypes (IgG1, r = 0.727, p < 0.0001; IgG2a, r = 0.804, p < 0.0001; IgG2b, r = 0.73, p < 0.0001).

Table IV.

ELISPOT assay of splenic anti-TT ASCa

IgG1 (ASC/106 cells)IgG2a (ASC/106 cells)IgG2b (ASC/106 cells)
Control 51.8 ± 15.2a 0.4 ± 0.4a 2.8 ± 1.9a 
RA 79.1 ± 9.1ab 1.3 ± 0.6a 6.3 ± 1.8a 
PIC 247.4 ± 84.4bc 30.6 ± 9.9c 32.4 ± 7.2b 
RA/PIC 396.8 ± 116.7c 12.5 ± 4.4b 26.1 ± 7.1b 
IgG1 (ASC/106 cells)IgG2a (ASC/106 cells)IgG2b (ASC/106 cells)
Control 51.8 ± 15.2a 0.4 ± 0.4a 2.8 ± 1.9a 
RA 79.1 ± 9.1ab 1.3 ± 0.6a 6.3 ± 1.8a 
PIC 247.4 ± 84.4bc 30.6 ± 9.9c 32.4 ± 7.2b 
RA/PIC 396.8 ± 116.7c 12.5 ± 4.4b 26.1 ± 7.1b 
a

Mice were treated as described in Fig. 5. On day 7 after reimmunization, spleen cells were isolated and anti-TT ASCs were detected by ELISPOT assay. Values are mean ± SE, n = 8/group. Different superscript letters in a column indicate significant differences (p < 0.05, a < b < c).

The combination of RA and IFNs has been demonstrated to be an effective strategy for cancer chemoprevention and chemotherapy (33). However, the immunoregulatory effect of RA and IFNs combined has not yet been well characterized. DeCicco et al. (25, 30) reported that RA and PIC synergistically enhanced both primary and secondary anti-TT Ab responses in vitamin A-deficient rats, suggesting that RA and IFNs can interactively promote immune functions. However, whether RA and IFNs, especially in combination, can effectively modulate primary immune responses and promote long-term immunity in the healthy state had not been studied. Therefore, we evaluated the immunoregulatory effects of RA, PIC, and their combination in a model of normal immunocompetent mice. Several new findings resulted from these studies.

First, RA and/or PIC given at priming modulated important immune regulators within a few days of immunization. On day 3 after priming, RA and PIC differentially regulated mRNA levels of IFN-γ and IL-4, the signature cytokines of type 1 and type 2 immune responses, respectively. Previous studies showed that PIC selectively induces type 1 cytokines, such as IFN-γ and IL-12, while suppressing type 2 cytokines, such as IL-4 and IL-5 (21, 34). In contrast, RA suppressed Th1 cytokines, but enhanced Th2 cytokines in both vitamin A-deficient animals and in vitro culture (35, 36). In the present study, PIC significantly induced IFN-γ mRNA 3 days after priming. RA, in contrast, was a positive, albeit modest, regulator for IL-4, but a negative regulator for IFN-γ. In consequence, RA was a strong positive regulator of the ratio of IL-4 to IFN-γ gene expression. Therefore, RA, PIC, and their combination had already shaped the developing type 1/type 2 response within 3 days of immunization and treatment.

Retinoic acid and PIC also differentially regulated NK/NKT cell populations by 3 days after treatment and priming. NK cells are considered as an early source of IFN-γ (37), while NKT cells have been shown to secrete IL-4 and IL-10 and promote a Th2 (type 2) response (38, 39). In the present study, RA was a positive regulator for NKT cells, whereas PIC was a positive regulator for NK cells. The ratio of NKT cells to NK cells was positively correlated with the ratio of IL-4 to IFN-γ mRNAs, implying that RA and PIC treatments might regulate type 1/type 2 cytokines and anti-TT Ab response through modulating NK/NKT cell populations.

Furthermore, RA and PIC significantly affected APC characteristics within 3 days of immunization. PIC has been shown to induce DC maturation and expression of CD80 and CD86 (20). Several studies suggested that RA could regulate immune response by targeting APCs (40, 41). In the present study, RA and RA + PIC significantly induced expression of CD11b, consistent with our previous results in the human monocytic cell line THP-1 (29). Moreover, RA and PIC differentially regulated the expression of CD80 and CD86, the major costimulatory molecules for T cell activation. The regulation of CD80 and CD86 molecules was associated mostly with CD11b+ cells and CD11b cells, respectively, suggesting the interesting and unexpected finding that these costimulatory molecules can be modulated individually. Although APC function was not assessed in the present study, the differences observed in cell markers imply that RA and PIC can modulate APC function, thereby affecting T cell activation as well as downstream Ab responses. Notably, the ratio of CD80/CD86 was positively correlated with the ratio of IL-4/IFN-γ. The differential regulation of CD80 and CD86 by RA and PIC is not readily explained at this time because the possibly distinct functions of CD80 and CD86 in type 1/type 2 responses have not yet been clarified. Kuchroo et al. (42) suggested that CD80 and CD86 are involved in the generation of Th1 and Th2 responses, respectively. Lang et al. (43) observed that CD86 was essential for both Th1 and Th2 responses, while CD80 provided a negative signal for Th1 response. Despite the present uncertainty about the individual roles of CD80 and CD86, the significant positive correlation observed between the ratio of IL-4 to IFN-γ and the ratio of CD80 to CD86 suggests that the early regulation of CD80 and CD86 molecules by RA/PIC might make a significant contribution to the ability of these treatments to rapidly modulate type 1/type2 cytokines.

Later, by days 10–12 after priming, the effects of RA and PIC on immune function became more dramatic as it was evident that RA and/or PIC robustly promoted the primary anti-TT IgG response. Compared with the control level of anti-TT IgG produced by normal mice, treated mice produced levels that were up to 80-fold higher. Moreover, RA and PIC differentially regulated anti-TT IgG isotypes. RA alone shifted anti-TT IgG production toward IgG1 and therefore elevated the ratio of IgG1/IgG2a. In contrast, PIC strongly boosted all of the IgG isotypes, without changing the ratio of IgG1 to IgG2a titers. Surprisingly, whereas RA + PIC synergistically enhanced IgG1, this combination attenuated IgG2a production as compared with PIC alone. Therefore, RA + PIC not only potently stimulated total anti-TT IgG production, but also kept the balance of IgG1/IgG2a Abs similar to that in control mice.

At the same time, the regulation of type 1/type 2 cytokines by RA and PIC was also very significant. PIC, which was expected to induce type 1 cytokines, significantly induced both type 1 and type 2 cytokines as well as Th1/Th2-related genes. The enhancement of type 2 cytokines by PIC was probably due to its ability to induce IFN-β (19), which has been shown to reduce type 1 cytokines (e.g., IL-12), but increase type 2/regulatory cytokines (e.g., IL-10) as observed in treatment of multiple sclerosis (44, 45). Thus, PIC appears to be a relatively indiscriminant, but potent inducer of immune responses, with very broad inducing effects on both type 1 and type 2 immunity. The enhancement of type 1/type 2 cytokines by PIC was well correlated with the increased production of all anti-TT IgG isotypes. Oppositely, RA inhibited type 1 cytokines and Th1-related genes, confirming previous reports (35, 36). Interestingly, this inhibition occurred despite presence of PIC and was strongly correlated with the attenuation of IgG2a, suggesting that RA could abolish part of PIC-induced IgG2a production by suppressing type 1 cytokine expression. Although RA did not significantly induce type 2 cytokines, it consistently suppressed type 1 cytokines and therefore skewed the balance in the type 2 direction, which most likely enhanced the production of anti-TT IgG1. Nevertheless, RA combined with PIC manipulated type 1/type 2 cytokine expression, which in turn contributed to the enhancement of anti-TT Ab response and directed Ig isotype switching toward a nearly normal balance.

Because a strong memory response is a hallmark of successful vaccination, it was important to determine whether the immunoregulatory effects of RA and PIC were durable. Indeed, providing RA and PIC (only at the time of priming) greatly enhanced the secondary anti-TT IgG response. Consistent with increased plasma Ig isotypes, RA and PIC also up-regulated the number of splenic anti-TT ASCs. These data provided insight that RA and PIC enhanced secondary anti-TT IgG responses by regulating the clonal expansion of memory B cells and the differentiation of B cells into effector ASCs.

Formulation of vaccines with potent adjuvants is an important approach for improving vaccine efficiency. When incorporated into vaccines, adjuvants can accelerate, prolong, or enhance the quality of specific immune response to vaccine Ags. In the past decades, many adjuvants have been developed and tested; however, few of them are used for human vaccines because of potential toxicity and adverse effects (46). Therefore, developing effective adjuvants for human vaccines remains a challenge for the vaccine industry. The present study has demonstrated that RA and/or PIC treatments can robustly and durably enhance anti-TT Ab response in vitamin A-sufficient mice, suggesting that a simple nutritional intervention, RA, coupled with PIC can effectively improve vaccine performance. This outcome appears to involve multiple mechanisms, including early regulation of NK/NKT cell and APC populations and shaping of type 1/type 2 cytokine gene expression. Compared with RA or PIC alone, RA + PIC not only was more potent in increasing the TT-specific IgG response, but also maintained the balance of IgG1/IgG2a. Thus, RA + PIC may serve as a promising strategy for increasing vaccine efficiency in not only vitamin A-deficient, but also in healthy populations.

We thank the staff of the Center for Quantitative Cell Analysis, Pennsylvania State University, for their assistance with flow cytometry.

The authors have no financial conflict of interest.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1

Financial support was provided by National Institutes of Health Grant DK-41479 and funds from the Dorothy Foehr Huck chair.

3

Abbreviations used in this paper: RA, retinoic acid; ASC, Ab-secreting cell; DC, dendritic cell; PIC, polyriboinosinic:polyribocytidylic acid; TT, tetanus toxoid.

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