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Nutritional Immunology

Dietary Polyunsaturated Fatty Acids Modulate In Vivo, Antigen-Driven CD4⁺ T-Cell Proliferation in Mice¹

Departments of Animal Sciences, Nutritional Sciences and Molecular Microbiology and Immunology, University of Missouri, Columbia, MO 65211

²To whom correspondence should be addressed. E-mail: FritscheK{at}missouri.edu.

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Our objective was to determine whether dietary lipids affect in vivo, antigen-driven, proliferation of naïve CD4⁺ T lymphocytes. To accomplish this, we adoptively transferred lymphocytes from T-cell receptor (TCR) transgenic DO11.10 (i.e., donor) mice into syngeneic, nontransgenic BALB/c (i.e., recipient) mice. Before adoptive transfer, recipient mice were fed for 4 wk AIN93G-type diets that differed only in fat source: lard, low in PUFA, fish oil, rich in (n-3) PUFA, and soybean oil, rich in (n-6) PUFA. One week after transfer, recipient mice were immunized with antigen (i.e., ovalbumin), and expansion of CD4⁺ T^DO11.10 cells in the spleen and draining lymph nodes (LN) was measured by flow cytometry. Five days postimmunization (p.i.), at the peak of expansion, CD4⁺ T^DO11.10 cells in the draining LN and spleen were 5- to 10-fold higher than in unimmunized mice, then quickly declined during the contraction phase (i.e., 7 and 10 d p.i.). Recipients fed the (n-6) PUFA rich diet had 25% greater in vivo expansion of CD4⁺ T^DO11.10 cells than lard- and fish oil–fed recipient mice at 5 d p.i. (P < 0.05). However, at 7 and 10 d p.i., CD4⁺ T^DO11.10 cells in the draining lymph nodes did not differ between groups, nor in the spleen at 5, 7, and 10 d p.i. In summary, we are the first to demonstrate that dietary PUFAs affect antigen-driven expansion of naïve CD4⁺ T cells in vivo. Surprisingly, (n-3) PUFA consumption did not reduce CD4⁺ T-cell expansion.

KEY WORDS: • CD4⁺ T cells • TCR transgenic mice • diet • fatty acids • proliferation

Consumption of fish oil rich in (n-3) PUFA reduces in vitro lymphocyte proliferation in numerous species, including mice and humans (1–4). Typically, these studies involve the isolation of immune cells from either the peripheral blood or secondary lymphoid tissues and the subsequent culturing of these cells in vitro in the presence of a polyclonal activator such as a plant lectin (e.g., concanavalin A, pokeweed mitogen), cross-linking antibodies (e.g., anti-CD23/CD28), or chemicals such as phorbol myristate acetate and Ca²⁺ ionophore, which completely bypass signaling through the cell membrane. In some reports, however, (n-3) PUFA did not reduce in vitro T-cell proliferation (5,6) and under some circumstances, actually increased it (7,8). The many factors that may contribute to such paradoxical findings were discussed elsewhere (9).

Despite the widespread acceptance that (n-3) PUFA are capable of reducing polyclonal lymphocyte proliferation, our knowledge of how (n-3) PUFA affect in vivo T-cell responses remains quite limited. Evidence that (n-3) PUFA can affect in vivo lymphocyte function is based primarily on delayed-type hypersensitivity (DTH)³ studies. Researchers reported that (n-3) PUFA from fish oil can either reduce DTH in humans (10) and mice (11) or have no effect (12). DTH responses involve a complex array of inflammatory mediators, chemokines, and cells, including monocytes/macrophages and antigen-specific T cells (13). Therefore, at best, changes in DTH response provide only indirect evidence that in vivo T lymphocyte function is altered. More importantly, the DTH test is a recall response that is intrinsically different from the initial response to antigen by naïve T cells (14,15).

Studying antigen-specific naïve T-cell responses had not been technically feasible until recently because under normal conditions, the frequency of T cells that are responsive to a given antigen is too small to detect during a primary response (i.e., <1 in 10⁵ T cells) (16). The generation of mice that express transgenic TCR specific for a known antigen overcame this technical problem. During the past decade T-cell receptor (TCR) transgenic mouse models have provided immunologists with a powerful research tool that has greatly expanded our understanding of T-cell biology, antigen-driven signaling, and the processes involved in T-cell proliferation and differentiation (17). Unfortunately, TCR transgenic mice cannot be used directly to study in vivo responses because the frequency of the antigen-specific T cells is so high that antigen stimulation generates a pathologic response (18). To overcome this problem, Jenkins and co-workers (19) developed an experimental approach that relied on the adoptive transfer of a small, but detectable number of TCR transgenic T cells into normal syngeneic recipient mice. We considered this adoptive transfer technique to be an ideal experimental approach for studying the effect of dietary constituents, such as (n-3) PUFA, on antigen-driven proliferation of naïve CD4⁺ T lymphocytes in vivo. Here, we demonstrate for the first time that the dietary fat source can influence in vivo proliferation of naïve CD4⁺ T cells during a primary immune response to a specific antigen.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
    Mice. DO11.10 mice on BALB/c background (a generous gift from Marc Jenkins, University of Minnesota, Minneapolis, MN) were bred and maintained at the Office of Animal Resources, University of Missouri (Columbia, MO). Shortly after, weanling mice were transferred to the Animal Sciences Research Center. In this facility, the mice were housed in autoclaved microisolator cages and provided with free access to food (Autoclavable Laboratory Rodent Diet 5010, Purina Mills) and autoclaved, distilled water. Specific pathogen-free female BALB/cAnNHsd mice were used as recipients in all experiments (Harlan). These mice were also housed at the Animal Sciences Research Center in polycarbonate cages containing aspen wood shavings, with free access to food and water as described above. The room was maintained on a 12-h light:dark cycle at 23°C and 40–50% relative humidity.

    Experimental diets. At the onset of each experiment, recipient mice were switched from the commercial diet to 1 of 3 experimental diets. The experimental diets were nutritionally complete and were based on the semipurified AIN-93G diet (20). Our diets were modified to contain 200 g fat/kg diet, while maintaining the same nutrient-to-energy ratio of the original lower fat diet. Unless noted, diet ingredients were purchased from ICN Biomedicals. The 3 major dietary fat sources used were lard, soybean oil, and refined menhaden fish oil (the latter was a generous gift from Omega Protein). A small amount of corn oil was added to the fish oil to approximate the essential fatty acid (i.e., linoleic acid) content of lard. The fatty acid composition of the experimental diets was reported previously (21). Oils and diets were protected against autooxidation as describe previously (21); this included the addition of a synthetic antioxidant (0.2 g tert-butylhydroquinone/kg fat). Mice were fed the experimental diets for 4 wk before adoptive transfer and an additional week before immunization. Care and treatment of mice were in accordance with federal guidelines and overseen by the Animal Care and Use Committee of the University of Missouri-Columbia.

    Immune cell isolation and adoptive transfer. After administration of anesthesia (an intramuscular injection of ketamine, 200 mg/kg, and xylazine, 16 mg/kg), mice were killed humanely by cervical dislocation. Lymph nodes and spleens were aseptically removed from naïve DO11.10 mice and placed in sterile EHAA media (Gibco BRL Products, Invitrogen). Lymph nodes (LN) and spleens were dispersed into a single cell suspension using sterile tissue sieves. Lymphocytes from all LN and spleen were combined within the same dietary treatment group (i.e., when donors were fed the experimental diets), than enumerated electronically with a Coulter Counter (model ZM; Beckman Coulter). Small aliquots of each immune cell preparation were labeled with anti-CD4 and the transgenic TCR-specific antibodies as described below for determination of the frequency of CD4⁺ T^DO11.10 cells, which was typically 25%. The crude immune cell preparation was resuspended in sterile PBS at a concentration that would result in each recipient mouse receiving 2.5 x 10⁶ CD4⁺ T^DO11.10 cells in a final volume of 0.2 mL via tail vein injection.

    Immunization. Equal volumes of chicken ovalbumin (OVA; 100 µg in 50 µL, Sigma Chemical) and Complete Freund’s Adjuvant (Sigma) were mixed vigorously. This emulsion was then injected into 3 subcutaneous sites on the back 7 d after the transfer of CD4⁺ T^DO11.10 cells.

    Immune cell collection. Mice were anesthetized as described previously, then killed by exsanguination or cervical dislocation. Draining (axillary, brachial, and inguinal) LN and spleen were collected from individual mice, dispersed into single-cell suspensions, and electronically counted as described above for subsequent analysis by flow cytometry. Blood was collected in a heparinized tube, gently inverted for 30 s, and stored at room temperature. RBCs were removed from 100-µL aliquots of heparinized blood by treatment with 0.19 mol/L NH₄Cl for 10 min at room temperature. The peripheral blood leukocytes were rinsed with cold PBS before staining.

    Flow cytometry. The number of DO11.10 cells present in different cell preparations was determined by flow cytometric analysis. Briefly, aliquots of cells were incubated with anti-CD4 (a rat anti-mouse CD4-APC conjugate; Caltag Laboratories) and KJ1–26 monoclonal antibody (a gift of Marc Jenkins, University of Minnesota), specific for the DO11.10 transgenic TCR. Cells were analyzed on a FACSvantage (BD Bioscience). We collected data on a minimum of 50,000 live cells using forward and side scatter gating to avoid dead cells and debris.

    Fatty acid analysis. The effect of dietary fat source on lymphocyte fatty acid composition was determined as described in detail elsewhere (22). Briefly, total cellular lipids were extracted from lymphocytes with chloroform and methanol (2:1). FAME were prepared by base-catalyzed methylation, isolated by TLC, then analyzed by GC. The gas chromatograph (model 5890; Hewlett-Packard) was equipped with a 30 m x 0.25 mm i.d. fused silica capillary column (Supelco). Fatty acids were identified by comparing relative retention times with commercial standards.

    Statistical analysis. Statistical analyses were carried out using ANOVA and the Fisher PLSD test on a Macintosh or PC with GraphPad Prism and InStat (GraphPad Software version 3.0). When variances were heterogenous, data were transformed before ANOVA. Data are expressed as means ± SEM of the untransformed values. Significance of differences was accepted when P < 0.05.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
    Body and organ weights, immune cell yields. Dietary fat source did not significantly affect growth, final body weight or liver and spleen weight of either BALB/c or DO11.10 mice (data not shown). We obtained 6.5 ± 0.4 (x10^–7) total cells from each mouse spleen and 2.2 ± 0.3 (x10^–7) from the LN (axillary, brachial and inguinal), and dietary fat source did not affect these cell yields (P = 0.61 and 0.99, respectively).

    Lipid profiles of immune cells. Lymphocytes isolated from LN of DO11.10 mice readily incorporated dietary (n-3) PUFA into their cellular membranes within 7 d of consuming a diet rich in (n-3) PUFA from fish oil (Table 1). For example, eicosapentaenoic acid increased from undetectable levels to nearly 1.5 mol/100 mol and docosahexaenoic acid increased >10-fold. As expected, immune cells from lard-fed DO11.10 mice were poor in (n-3) PUFA and rich in monounsaturated fatty acids, whereas those from soybean oil–fed mice were rich in linoleic acid.

View larger version (61K): [in this window] [in a new window]   FIGURE 1 Flow cytometric analysis of TCR transgenic mouse CD4+ T cells. The histogram shown is representative data from the draining LN cells of a single mouse. The cells were obtained from BALB/c mice that had received 2.5 x 106 naïve CD4+ TDO11.10 cells via tail vein injection and were then immunized with 100 µg OVA in complete Freund’s adjuvant. Cells preparations were stained for 2-color flow cytometric analysis with a fluorochrome-conjugated anti-CD4 antibodies and KJ1–26 (a mouse antibody specific for the transgenic TCR), followed by a fluorochrome-conjugated goat anti-mouse F(ab2)'. Data were collected on 100,000 events, gating on live cells only. The CD4+ TDO11.10 cells appear in the upper right corner. The number shown represents the frequency of these cells within the total cell population. (Inset) This histogram is representative of the background staining of BALB/c lymph node cells after immunization, but in the absence of adoptive transfer of CD4+ TDO11.10 cells.

View larger version (13K): [in this window] [in a new window]   FIGURE 2 Dietary (n-6) PUFA increased, whereas (n-3) PUFA had no effect on in vivo proliferation of naïve TCR transgenic CD4+ T cells in BALB/c mice fed diets containing lard, soybean oil, or fish oil. After 4 wk of consuming semipurified diets that differed only in fat source, naïve CD4+ TDO11.10 cells were adoptively transferred via tail vein into healthy 8-wk-old, female BALB/c mice. Seven days after adoptive transfer, mice were immunized as described in Figure 1. Five days after immunization, draining LN were isolated, dispersed, stained, and analyzed by flow cytometry as described in Figure 1. Values are means ± SEM, n = 24 mice with data combined from 4 independent experiments. *Different from lard- and fish oil–fed mice, P < 0.05.

View larger version (18K): [in this window] [in a new window]   FIGURE 3 Dietary fat source had no effect on the clonal contraction of a primary immune response 7 (a) or 10 d (b) postimmunization in BALB/c mice fed diets containing lard, soybean oil, or fish oil. BALB/c mice received naïve CD4+ TDO11.10 as described for the previous figure, and were immunized 7 d post-transfer. Draining LN were collected 7 (a) or 10 d (b) later. Immune cells were isolated, dispersed, stained, then analyzed by flow cytometry as described in Figure 1. Values are means ± SEM, n = 8 with data combined from 2 independent experiments.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Over the past decade, adoptive transfer of T cells from various TCR transgenic mice into syngeneic recipient mice has become a widely used approach for investigating antigen-driven activation, expansion, and differentiation of naïve CD4⁺ and CD8⁺ T cells in vivo (24,25). Using this approach, we completed a series of experiments to ascertain the effect of dietary (n-3) PUFA from fish oil and (n-6) PUFA from soybean oil on CD4⁺ T-cell proliferation in vivo. In our experiments, recipient mice (i.e., normal BALB/c) were fed experimental diets, whereas the donors (i.e., DO11.10 mice) were fed an autoclavable, commercial rodent diet before CD4⁺ T^DO11.10 cell isolation and transfer. We waited 1 wk after transfer before immunizing the recipient. The week long delay was designed to provide time for transferred CD4⁺ T^DO11.10 cells to incorporate fatty acids from recipients who had been fed experimental diets for 4 wk and continued to receive the same dietary treatment throughout the primary immune response (i.e., 5 to 10 d). Previous researchers showed that transferred CD4⁺ T^DO11.10 cells persist in recipient mice without stimulation, although their numbers tend to decline over time (19). We were surprised not to find an effect of (n-3) PUFA on in vivo proliferation of naïve CD4⁺ T cells. In contrast, feeding recipient mice a diet rich in (n-6) PUFA resulted in significantly greater CD4⁺ T-cell expansion in vivo compared with recipients fed the low-PUFA control diet (i.e., lard) or the high (n-3)–PUFA diet (i.e., fish oil). Thus, adoptive transfer of TCR transgenic T cells into diet-manipulated recipient mice is an approach that is capable of detecting diet-induced changes in CD4⁺ T-cell proliferation in vivo.

That (n-3) PUFA did not diminish in vivo T-cell proliferation was not the result of an inherent inability of the transgenic T cells to incorporate (n-3) PUFA. We showed that immune cells from the TCR transgenic mice become significantly enriched with (n-3) PUFA after only 1 wk of feeding those mice the (n-3) PUFA–enriched diet (Table 1). We recently reported that after feeding DO11.10 mice a diet rich in (n-3) PUFA (i.e., the same diet used in the current studies), in vitro, antigen-driven proliferation of CD4⁺ T^DO11.10 cells was significantly diminished (26). In that paper, we also reported, however, that culture conditions dramatically influenced our in vitro results and conclusions. Such findings are consistent with data from others (27–29). Thus, the new data described in this article suggest that in vitro assessment of lymphocyte proliferation may not be a reliable means of predicting how diet fatty acids affect these cells in vivo.

Measuring proliferative response of CD4⁺ T cells, rather than total T cells, is also fairly novel. Evidence is mounting that CD4⁺ and CD8⁺ T cells differ in their proliferative responses (30,31). Similarly, it follows that the effect of various dietary components may also differ between these T-cell populations. We are aware of only 1 report in which the effects of (n-3) PUFA on CD4⁺ T cells was demonstrated (7). Those authors found that after 2 wk of dietary treatment, CD4⁺ T cells enriched with (n-3) PUFA demonstrated modestly higher (i.e., 15–30%) in vitro proliferation after stimulation with anti-CD3 in combination with the protein kinase C activator, phorbol myristate acetate. No such effects were noted when cells were stimulated with anti-CD3 alone or with the phorbol and ionomycin, a Ca²⁺ ionophore. In contrast, they reported that CD8⁺ T cells isolated from fish oil–fed mice showed significantly diminished in vitro proliferation (i.e., 50% lower) when stimulated with anti-CD3 alone. The other stimuli combinations resulted in a 5- to 7-fold greater proliferative response, but no diet effects were noted under those conditions. Although it is not entirely clear why (n-3) PUFA affect different T-cell subsets in a stimulus-specific manner, these intriguing data warrant further investigation. In the future, we plan to conduct feeding trials with mice and TCR transgenic CD8⁺ T cells in a manner similar to the studies described in this article.

After expansion, the majority of the responding T cells undergo apoptosis, a phase referred to as clonal contraction (32). This process is an important component of the immune response and is vital for maintaining T-cell homeostasis. The most common method for measuring in vitro lymphocyte proliferation (i.e., ³H-thymidine uptake) cannot distinguish between reduced proliferation and enhanced apoptosis. Researchers reported that (n-3) PUFA enrichment leads to enhanced apoptosis of murine T lymphocytes (33). Therefore, we hypothesized that (n-3) PUFA would affect the clonal contraction phase of a primary immune response through increased apoptosis. In our study, we did not directly measure apoptosis of CD4⁺ T cells; however, our final 2 feeding trials were designed specifically to investigate the effect of our dietary treatments on the clonal contraction phase of a primary, antigen-driven immune response. As expected, we found evidence that shortly after CD4⁺ T^DO11.10 cell numbers peaked, there was a rapid and substantial decline in antigen-specific CD4⁺ T cells in the draining LN and spleen. Dietary (n-3) PUFA did not appear to affect this process. Surprisingly, the relative number of CD4⁺ T^DO11.10 cells in the draining LN 7 and 10 d postchallenge were similar between mice fed all 3 diets. This suggests that despite the greater number of CD4⁺ T^DO11.10 cells at the peak of the proliferative phase, reduction in the TCR transgenic CD4⁺ T cells was greater in (n-6) PUFA–fed mice than in those fed the control or high (n-3) PUFA diets. Whether this greater cell loss occurred through enhanced apoptosis or efflux cannot be determined with the experimental approach used in our studies. Additional studies will be required to further define the effect of dietary PUFA on the dynamics of CD4⁺ T-cell loss and migration after a primary immune response.

Naïve lymphocytes express high levels of CD62L, which facilitates their homing to secondary lymphoid tissues (34). (n-3) PUFA lower surface expression of CD62L on rat lymphocytes (35). Thus, if (n-3) PUFA decreased expression of CD62L on murine lymphocytes, T-cell trafficking could be affected such that after adoptive transfer, fewer naïve CD4⁺ T^DO11.10 cells might end up in the draining LN and spleen. After adoptive transfer, the majority of the CD4⁺ T^DO11.10 cells localized to LN and spleen as expected and as previously reported (15,19). In our studies, we found a higher frequency of transferred cells in the LN of mice fed either (n-3) or (n-6) PUFA-rich diets compared with lard-fed mice. Thus, it appears that PUFA may have a beneficial effect on naïve CD4⁺ T-cell trafficking and/or short-term survival in the absence of immune stimulation. Additional studies are warranted if we are to tease out the individual contribution of each of these processes on PUFA-mediated changes to lymphocyte behavior in vivo.

After antigen stimulation and expansion, among the many changes occurring in the CD4⁺ T^DO11.10 cells is a drop in CD62L expression, a characteristic of effector T cells (36). Effector cells leave the secondary lymphoid tissues and accumulate at sites of antigen deposition (37). In our last study, we did not assess the individual contribution of apoptosis and efflux of effector cells as being responsible for the rapid decline in CD4⁺ T^DO11.10 cells in the draining LN. However, others reported that alterations in fatty acid composition of lymphocyte membranes may alter in vivo localization (38). In light of this fact, the effect of PUFA on immune cell trafficking deserves further study. Once again, it is important to underscore the power of this adoptive transfer/TCR transgenic mouse model. It is possible to immunize mice and at later time points do in situ staining of sections of certain tissues, or even sections of an entire mouse, to examine the distribution of antigen-specific effector/memory cells (37).

Our finding that dietary (n-6) PUFA enhanced CD4⁺ T-cell proliferation in vivo is novel and somewhat unexpected. Results from several in vitro studies suggest that (n-6) PUFA enrichment is associated with reduced proliferation and interleukin-2 production by human and rodent lymphocytes (39–41). Similarly, a number of studies showed that feeding mice or rats diets rich in linoleic acid, a (n-6) PUFA, from corn, soybean, sunflower, or safflower oil, led to reduced ex vivo lymphocyte proliferation after stimulation with polyclonal mitogens (9,42). In contrast to immunosuppression, 2 reports exist that associate high dietary (n-6) intake with enhanced ex vivo lymphocyte proliferation (29,43). In the latter study, an unusual source of (n-6) PUFA, borage oil rich in -linolenic acid [18:3(n-6)], was fed to the mice. Furthermore, this study was an in vitro, recall response with a spleen cell preparation that contained both CD4⁺ and CD8⁺ effector cells, whereas in our study, we followed the primary, OVA peptide-specific responses of naïve CD4⁺ T cells in vivo. Rather than discuss the merits of these possible sources of experimental variance, we and others (23,44) think that it is important to acknowledge the inherent limitations in relying on in vitro immunoassays when understanding the behavior and function of immune cells in vivo.

In summary, this is the first demonstration that dietary lipids can affect antigen-driven expansion of naïve CD4⁺ T cells in vivo. We found that consumption of a diet rich in (n-6) PUFA enhanced proliferation, whereas (n-3) PUFA were without effect compared with mice fed a control lard-containing diet low in (n-6) and devoid of (n-3) PUFA. We believe that the adoptive transfer of TCR transgenic T cells as described in this manuscript is a powerful experimental approach. It is suitable not only for investigating antigen-driven lymphocyte proliferation, but one can also study nutrient modulation of in vivo differentiation of naïve CD4⁺ T cells into T-helper 1 (Th1) vs. T-helper 2 (Th2) phenotypes.

	ACKNOWLEDGMENTS

 
The authors thank Marc Jenkins (University of Minnesota) for providing us with a breeding pair of DO11.10 mice, the KJ1–26 hybridoma, and technical advice. We also extend our thanks to Louise Barnett, Meijuan Zhang, Robert Irons, Tommi White, Lisa Pompos, and David Harah for their technical assistance with these studies.

	FOOTNOTES

 
¹ Supported by U.S. Department of Agriculture-CSREES National Research Institute grant #00–35200–9115, the University of Missouri’s College of Agriculture, Food and Natural Resources Food-for-the 21st Century Nutrition Program and the Agricultural Experiment Station.

³ Abbreviations used: DTH, delayed-type hypersensitivity; LN, lymph nodes; OVA, ovalbumin; TCR, T-cell receptor.

Manuscript received 29 March 2004. Initial review completed 7 May 2004. Revision accepted 26 May 2004.

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