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. Author manuscript; available in PMC: 2013 Jan 1.
Published in final edited form as: J Immunol. 2011 Nov 23;188(1):77–84. doi: 10.4049/jimmunol.1004027

1Protein Energy Malnutrition Impairs Homeostatic Proliferation of Memory CD8 T cells

Smita S Iyer 1,*, Janel Hart Chatraw 2,3,*, Wendy G Tan 1, E John Wherry 4, Todd C Becker 5, Rafi Ahmed 3, Zoher F Kapasi 2,3
PMCID: PMC3244573  NIHMSID: NIHMS335089  PMID: 22116826

Abstract

Nutrition is a critical but poorly understood determinant of immunity. There is abundant epidemiological evidence linking protein malnutrition to impaired vaccine efficacy and increased susceptibility to infections; yet, the role of dietary protein in immune memory homeostasis remains poorly understood. Here we show that protein energy malnutrition (PEM) induced in mice by low-protein (LP) feeding has a detrimental impact on CD8 memory. Relative to adequate-protein (AP) fed controls, LP feeding in lymphocytic choriomeningitis virus (LCMV) immune mice resulted in a 2-fold decrease in LCMV-specific CD8 memory T cells. Adoptive transfer of memory cells, labeled with a division tracking dye, from AP mice into naive LP or AP mice demonstrated that PEM caused profound defects in homeostatic proliferation. Remarkably, this defect occurred despite the lymphopenic environment in LP hosts. While antigen-specific memory cells in LP and AP hosts were phenotypically similar, memory cells in LP hosts were markedly less-responsive to poly(I:C)-induced acute proliferative signals. Furthermore, upon recall, memory cells in LP hosts displayed reduced proliferation and protection from challenge with LCMV-clone 13 resulting in impaired viral clearance in the liver. The findings show a metabolic requirement of dietary protein in sustaining functional CD8 memory and suggest that interventions to optimize dietary protein intake may improve vaccine efficacy in malnourished individuals.

Keywords: immunity, recall, amino acids, nutrition

INTRODUCTION

Generation of long-lived immune memory is the basis of vaccination and infection-generated immunity (1). Memory T cells arise after antigen clearance from a small subset of effectors derived from the clonal expansion and differentiation of naive T cells responding to antigen (2, 3). Upon antigen re-encounter, antigen-specific memory T cells respond more rapidly and efficiently than naive T cells, conferring the host with long-term protection (4). This dynamic memory pool is maintained at a relatively constant size for prolonged periods, even up to a lifetime, by homeostatic proliferation (5).

Homeostatic proliferation is a process where influx of newly generated memory cells, by slow turnover, is balanced by death of pre-existing memory cells (6). The signals needed for memory homeostasis are well studied; it is independent of antigen and is dependent on the concerted action of the gamma chain cytokines, interleukin (IL)-7, for survival, and IL-15, for turnover (7, 8). Cell survival and turnover also depend on nutrient availability; very little, however, is known about how nutrition impacts memory T cell homeostasis. Because proliferation of memory cells imposes a metabolic demand on amino acid supply to support protein synthesis, dietary protein may be critical in sustaining T cell memory.

The present study was designed to determine whether homeostatic proliferation of memory CD8 T cells is impaired in protein-energy malnourishment (PEM). PEM is a major form of malnutrition and is defined as an imbalance between intake of protein and energy and the optimal requirement to ensure the most favorable body growth and function (9). Malnutrition is a major health concern among millions in the developing world and accumulating evidence suggests that malnutrition also afflicts HIV-infected patients, individuals with chronic disease, and aged populations in industrialized nations (10, 11). There is abundant epidemiological evidence suggesting that malnutrition impairs vaccine efficacy and increases susceptibility to infections; yet, the underlying mechanisms remain poorly understood (9, 12). A better understanding of the role of nutrition in memory homeostasis may open avenues for nutritional interventions to improve vaccine efficacy in malnourished individuals

We assessed quantitative and qualitative aspects of CD8 T cell memory in lymphocytic choriomeningitis virus (LCMV) immune mice (>40 days post-infection) fed either a low protein (LP) diet to develop PEM, or an adequate protein (AP) control diet. After 4-weeks of dietary intervention, LP mice demonstrated a two-fold reduction in antigen-specific memory CD8 cells compared to AP-fed controls, suggesting impaired proliferation due to PEM. Using adoptive transfer of carboxyfluorescein succinimidyl ester (CFSE)-labeled memory cells from AP mice into naive LP or AP mice we confirmed that PEM caused profound defects in homeostatic proliferation. Interestingly, memory cells in AP or LP hosts were phenotypically similar with respect to the gamma chain cytokine receptors, but were markedly less-responsive to poly(I:C)-induced acute homeostatic proliferation. Additionally, on challenge with LCMV-clone 13 memory cells in LP hosts displayed markedly reduced proliferation resulting in impaired viral clearance in the liver. Together, the data show that dietary protein is critical for sustaining a functional memory CD8 pool.

MATERIALS AND METHODS

Mice, virus, and infections

Four-to 6-week-old female C57BL/6 mice were obtained from the Jackson Laboratory (The Jackson Laboratory, Bar Harbor, ME). Thy1.1+ P14 transgenic mice with CD8 T cells expressing the T cell receptor specific for the Db/GP33–41 epitope of LCMV were obtained from the Jackson Laboratory and backcrossed to B6 mice in our colony. Mice were housed in cages and maintained on a 12-h light-12-h dark cycle at the Division of Animal Resources at Emory University. All experiments were initiated during the light cycle. Mice were infected with 2 × 105 plaque-forming units (PFU) of LCMV Armstrong i.p. For secondary challenge experiments, 2 × 106 PFU of LCMV clone 13 was given i.v. Recombinant vaccinia virus (VV)-GP33 expressing the GP33–41 epitope of LCMV was propagated and used as previously described (13). Mice were infected with 2 × 106 PFU of VV-GP33. All animal protocols were reviewed and approved by the Institutional Animal Care and Use Committee at Emory University.

Diets

Semi-purified diets were custom-prepared (Harlan-Teklad, Madison, WI, USA) in order to test the specific effects of protein energy malnutrition on memory CD8 T cell homeostasis (14). Protein levels were controlled at the desired experimental level by decreasing amounts of essential and non-essential amino acids. The low protein diet contained 0.6% (5 g/kg) of protein and the adequate protein diet contained 18% protein (210 g/kg) by weight. Low protein diets contained more carbohydrate in the form of sucrose and cellulose, compared to adequate protein diets but were otherwise isocaloric and both diets contained equal and identical quantities of energy, fat, and micronutrients. Animals had free access to water and food at all experimental time points.

Flow Cytometry

Single-cell suspensions were prepared from spleen and 106 cells were stained in phosphate-buffered saline containing 2% fetal bovine serum and 0.02% sodium azide (FACS buffer) for 30 minutes at 4°C followed by three washes in FACS buffer. Cells were stained with anti–CD8a-PerCP (53-6.7), Thy1.1-PE (OX-7), CD122-PE (TM-b1), BCL-2–FITC (3F11), IFN-g–FITC (XMG1.2), TNF-a–APC (MP6-XT22), IL-2–PE (JES6-5H4), Ki-67-FITC (B56) (all from BD Biosciences), CD127-PE-Cy7 (A7R34) (eBioscience, San Diego, CA), IL-15Ra-FITC (BAF551) (R&D Systems, Minneapolis, MN), CD44-Pacific blue (IM7) (Biolegend, San Diego, CA). Intracellular staining for BCL-2 and Ki-67 was performed directly ex vivo. Cytokine staining for IFN-g, TNF-a, or IL-2 was done after a 5-h in vitro stimulation with 0.1mg/ml gp33 peptide using the Cytofix/Cytoperm kit, according to the manufacturer’s instructions (BD Biosciences). MHC class I peptide tetramers were generated and used as described (15). Samples were acquired using either FACSCalibur or FACS Canto II instruments (Becton Dickinson, San Jose, CA). Data were analyzed using FlowJo software (Tree Star, Inc).

CFSE labeling and Adoptive Transfer

CFSE labeling was performed as described (16). Splenocytes were obtained from immune C57BL/6 mice or immune P14 chimeric mice. Fifty million CFSE-labeled splenocytes were adoptively transferred intravenously via tail vein injection into naive recipients that had been on the LP or AP diet for 2 weeks. Longitudinal monitoring of T cell proliferation was performed by saphenous vein bleeding into 4% sodium citrate under isofluorane anesthesia.

Poly(I:C) treatment

Mice were treated with 150 ug of poly(I:C) (Sigma-Aldrich, St. Louis, MO) dissolved in 0.5 ml of PBS or PBS alone by i.p. injection, as previously described (17). Splenocytes and bone marrow cells were analyzed 3 days later for cycling memory CD8 T cells by Ki-67 staining.

Statistical Analysis

Data are expressed as the mean + SEM. Statistical analysis was performed by the two-tailed Student t test using Prism software (GraphPad, La Jolla, CA). A probability value of ≤0.05 was used to determine significance.

RESULTS

Protein-energy malnutrition decreases CD8 T cell memory

Increased incidence of infections in protein-malnourished individuals suggests that dietary protein may be a critical determinant of immune memory. But, the role of nutrition in memory homeostasis is yet to be precisely defined. The objective of the present study was to determine whether PEM impairs CD8 memory homeostasis. Because PEM compromises CD8 effector response to LCMV (14), which, in turn, could contribute to decreased CD8 memory, we designed the study to specifically examine the effect of PEM on established CD8 memory.

Mice were infected with the Armstrong strain of LCMV, which initiates a vigorous immune response resulting in clearance of virus and the subsequent generation of a robust memory pool (3). LCMV immune mice (> 40 days after infection) were randomly assigned to receive either an adequate protein diet (18% dietary protein; AP) or a low protein diet (0.6 % dietary protein; LP) (Figure 1A). The diets were formulated to be isocaloric, therefore the LP diet provided excess energy from carbohydrates (14). Because optimal amino acid supply is critical for cell proliferation and function, the effect of LP diet on CD8 T cells may be directly attributed to limited amino acid supply in the LP diet.

Figure 1.

Figure 1

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Effect of protein-energy malnutrition on LCMV-specific memory CD8 T cells. A) Experimental outline; 6–8 week-old B6 mice were infected intraperitoneally with LCMV Armstrong and after 40 days post-infection, mice were assigned to receive either an adequate protein (18% dietary protein) or a low protein diet (0.6% dietary protein). Analysis of LCMV-specific memory CD8 T cells in the spleen was performed after 4 weeks of dietary treatment. B) Db/GP276 and Db/GP33 tetramer staining of CD8 T cells from the spleen of adequate protein (top panel) and low protein-fed (bottom panel) mice. Data are percent of tetramer positive CD8 T cells. C) Absolute numbers of antigen-specific CD8 T cells in the spleen in adequate protein (black bars) and low protein-fed (red bars) mice. One representative experiment of three is shown. Data are mean of four mice per group + SEM; * P < 0.05

As previously described, after 4 weeks on a low-protein diet mice become protein-energy malnourished (14). This experimental set-up allows us to quantitatively assess the effects of PEM on CD8 memory homeostasis without the confounding effects of malnutrition on memory precursors and / or viral control.

As demonstrated previously, weight loss occurs within a week of initiating the low-protein diet and continues throughout the dietary period (14). After 4 weeks, mice on the LP diet showed a significant weight loss of 20% of body weight, while body weight in AP mice remained relatively stable (data not shown). We first examined whether PEM quantitatively impaired CD8 T cell memory. To this end, lymphocytes were isolated from the spleen of AP and LP mice and examined for memory cells specific for the LCMV epitopes Db/GP276–286 and Db/GP33–41. While no differences in percentage of CD44 hi (33% in both groups) or total antigen (Ag)-specific cells were observed between both groups (Figure 1B), estimation of total numbers of Ag-specific T cells in the spleen revealed a 2-fold decrease in LP mice compared to AP mice (P< 0.05; Figure 1B). The size of the total CD44-high memory pool was decreased by 2.8-fold in malnourished mice.

We next determined whether the functional capacity of Db/GP276 memory cells was also impaired in LP mice. For this purpose, splenocytes were stimulated for 5 hours with GP276–286 peptide and cytokine production was determined. As measured by the mean fluorescence intensity (MFI), interferon (IFN)-γ, tumor necrosis factor (TNF)-α, and interleukin (IL)-2 production was not different between the two dietary groups, suggesting that cytokine production on a per-cell basis ex vivo was not impaired by PEM (Figure 2A, B). However, the total number of cytokine producing cells was significantly reduced in LP mice (Figure 2C).

Figure 2.

Figure 2

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Effect of protein-energy malnutrition on cytokine production by LCMV-specific memory CD8 T cells. A) Intracellular cytokine staining for IFN-γ, TNF-a, and IL-2 after 5 h of GP276 peptide stimulation. Data are percent of CD8 T cells that produced cytokine. B) Median fluorescence intensity (MFI) of cytokine expression after GP276 stimulation. C) Total cytokine producing cells in the spleen after GP276 in adequate protein (black bars) and low protein-fed (red bars) mice. One representative experiment of three is shown. Data are mean of four mice per group+ SEM; * P < 0.05

Protein-energy malnutrition decreases acute homeostatic proliferation of CD8 memory T cells

Decreased CD8 memory in LP hosts could be due to defects in homeostatic proliferation; because the receptors for the γ chain cytokines are key signals for homeostatic proliferation (8, 18), we first determined whether expression of IL-7 and IL-15 receptor was decreased on memory cells in LP mice. We found no difference either in % receptor positive cells or MFI of Ag-specific CD8s between both dietary groups (Supplemental Figure 1; solid histogram denotes expression in GP276 tetramer+ CD8 T cells and black lines denote expression levels in CD44 low naive CD8 T cells). This data suggested that memory cells in the LP environment were poised to receive proliferative signals from IL-15. To directly assess responsiveness of memory CD8 T cells to acute proliferative signals, we treated AP and LP LCMV immune mice with poly (I:C). Poly(I:C), an RNA analog which induces IFN-γ which in turn upregulates IL-15 production leading to proliferation of memory CD8 T cells in an IL-15-dependent manner (17, 19). Three days after poly(I:C) treatment, lymphocytes were isolated from the spleen and the bone marrow, a major site for homeostatic proliferation (17), and division of memory cells was assessed by staining lymphocytes with the cell proliferation marker Ki-67 (Figure 3A). As shown in Figure 3B, frequency of Ki-67 positive cells (of total CD44 high CD8 T cells) was lower in the spleen and in the bone marrow in LP mice suggesting that PEM caused defects in acute homeostatic proliferation. Frequency of LCMV-specific cells (gated on GP276 tetramer+ cells) responding to poly (1:C) treatment was also markedly decreased (5% in LP mice compared to 21% in AP controls; S2)

Figure 3.

Figure 3

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Acute homeostatic proliferation of memory CD8 T cells in protein-energy malnutrition. A) Adequate protein and low protein-fed Armstrong immune mice were injected intraperitoneally with poly (I:C) and division of CD44hi CD8 T cells was determined 3 days later. (B) Percentage of CD44hi CD8s that responded to poly (I:C) in the spleen and bone marrow was assessed by intracellular staining with Ki-67. Histograms show median fluorescence intensity of Ki-67 in CD44hi CD8s T cells; grey dashed lines show Ki-67 staining in CD44lo CD8 T cells. One representative mouse of four from two experimental replicates is shown.

Protein-energy malnutrition decreases basal homeostatic proliferation of CD8 memory T cells

In addition to defects in acute homeostatic proliferation, lower numbers of LCMV-specific memory CD8 T cells in LP mice suggested that, PEM may also impair basal homeostatic proliferation. To determine if this was the case, memory cells generated in an AP environment were labeled with the division tracking dye, carboxyfluorescein succinimidyl ester (CFSE), and adoptively transferred into naive recipients that had been maintained either on AP or LP diets for 2 weeks (Figure 4A). Dietary regimes were continued for 4 weeks after cell transfer. At this time, lymphocytes were isolated from the spleen to compare division of CFSE labeled cells between AP and LP mice. Inspection of CFSE profiles revealed that the vast majority of memory cells in LP recipients had failed to divide compared to efficient homeostatic proliferation in AP recipients (Figure 4B). Overall, the number of CD44hi, CFSE-labeled CD8 T cells was 8-fold lower in LP mice compared to AP mice (0.3±0.2 × 105 in LP mice vs. 2.5±0.6 × 105 in AP mice, P<0.001).

Figure 4.

Figure 4

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Basal homeostatic proliferation of memory CD8 T cells in protein-energy malnutrition. A) Splenocytes were prepared from LCMV-immune mice, labeled with CFSE, and adoptively transferred into naive B6 recipients receiving either an adequate protein or a low protein diet. Diets were continued for 4 weeks after cell transfer. (B) Homeostatic proliferation of donor cells was assessed in the spleen by examining CFSE dilution. Histogram shows % of transferred cells that divided. Bar graph shows total numbers of CD44hi T cells per spleen. In (C) donor cells were obtained Armstrong immune P14 chimeric mice. Histogram shows % of divided donor cells. Kinetic analysis of divided donor cells in blood is shown in the graph. One representative experiment of three is shown. Data are mean of three mice per group+ SEM; * P < 0.05

In a separate experiment, CFSE-labeled memory cells from immune mice containing Thy 1.1 P14 transgenic memory cells specific for the Db/ GP33–41 LCMV epitope were transferred to naive recipients that had been on an AP or LP diet for 2 weeks. Dietary regimes were continued 4 weeks after cell transfer. Homeostatic proliferation of transferred cells was tracked by longitudinal bleeding each week for a total of 4 weeks. The data showed that transferred cells in AP mice began to divide while transferred cells in LP mice divided at a much slower rate such that the percentage of divided Thy1.1+ P14 memory cells was significantly higher in the AP mice at days 22 and 29 (Figure 4C). A representative flow plot of donor cells in the blood at day 22 is shown. Thus, the data clearly demonstrate that PEM impaired homeostatic proliferation of CD8 memory cells.

Protein-energy malnutrition impairs recall responses of memory CD8 T cells

Because rapid recall response to secondary infection is a characteristic feature of memory cells (3), we next investigated whether PEM also compromised the ability of memory CD8 T cells to respond to a secondary infection. For this purpose, memory cells from Thy 1.1+ P14 chimeric immune mice were adoptively transferred into naive recipients that had been maintained either on AP or LP diets for 2 weeks. Mice were infected with the clone-13 strain of LCMV at 1 day post-transfer and the secondary memory response was assessed at day 7 post-transfer (Figure 5A).

Figure 5.

Figure 5

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Protective immunity of memory CD8 T cells in protein energy malnutrition. A) To assess protective immunity of memory CD8 T cells in protein-energy malnutrition, memory cells from AP-fed P14 chimeric mouse were labeled with CFSE and transferred to naive adequate protein or low-protein fed recipients. Recipients were challenged with LCMV clone-13 and recall responses were assessed 7 days after transfer. B) shows a representative flow plot of percentage of Thy 1.1 + Db/GP33+ tetramer-specific CD8 T cells in adequate protein and low protein-fed hosts. Bar graph shows total Db/GP33 tetramer-specific CD8 T cells per spleen. In C) intracellular cytokine staining for IFN-γ and IL-2 after 5 h of GP33 peptide stimulation is shown. Data are percent of CD8 T cells that produced both IFN-γ and IL-2. In D) viral titers in liver and lung are shown. In a separate experiment (Figure 6A) malnourished memory cells from LP-fed mice were transferred to naive adequate protein or low protein- fed recipients subsequently challenged with VV-GP33. Recall responses were assessed 7 days after transfer. B) shows a representative flow plot of percentage of Thy 1.1+ Db/GP33+ tetramer-specific CD8 T cells in adequate protein and low-protein hosts. Total number of tetramer positive cells per spleen in shown in C. One representative experiment of three is shown. Data are mean of three mice per group+ SEM; * P < 0.05

At 1-day post-transfer, similar numbers of Thy1.1+ P14 cells were present in adoptive AP and LP hosts (data not shown). However, examination of secondary effector proliferation at day 7 revealed a 10-fold decrease in secondary effectors in LP hosts compared to AP hosts (Figure 5B; AP mice, 30.3 × 105 ± 8.2; LP mice, 3.1 × 105 ± 0.8; P< 0.05). A representative flow cytometric analysis of cells is shown. We stained for several surface activation markers such as CD27, CD127, CD69, CD62L, PD-1, CD122, and CD127 and found no difference in expression of these molecules between AP and LP hosts (data not shown). Furthermore, Thy1.1+ P14 cells in AP and LP hosts were functionally competent and could produce IFN-γ, TNF-α, and IL-2 upon GP33 peptide stimulation ex vivo (Figure 5C). The observation that LP hosts were unable to support robust expansion of secondary effectors would suggest that LP hosts would be less effective at controlling virus compared to AP hosts. Indeed, assay of viral titres in the liver revealed significantly higher titers in LP mice compared to AP mice (LP mice, 5.0±0.07 log10/mg of tissue; AP mice, 4.2±0.4 log10/mg of tissue; P< 0.05; Figure 5D). No difference in viral titres was observed in the lung. In a separate experiment we assessed recall proliferation of malnourished memory cells in AP versus LP hosts (Figure 6A). Representative flow plots of LP donor cells in AP and LP hosts are shown in Figure 6B. The data show that recall proliferation of LP memory cells in AP hosts was superior to that in LP hosts (Figure 6C) suggesting that malnutrition-induced defects in proliferation may be rescued by adequate protein supplementation.

Figure 6.

Figure 6

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Protective immunity of memory CD8 T cells in protein energy malnutrition. A) To assess protective immunity of memory CD8 T cells in protein-energy malnutrition, memory cells from AP-fed P14 chimeric mouse were labeled with CFSE and transferred to naive adequate protein or low-protein fed recipients. Recipients were challenged with LCMV clone-13 and recall responses were assessed 7 days after transfer. B) shows a representative flow plot of percentage of Thy 1.1 + Db/GP33+ tetramer-specific CD8 T cells in adequate protein and low protein-fed hosts. Bar graph shows total Db/GP33 tetramer-specific CD8 T cells per spleen. In C) intracellular cytokine staining for IFN-γ and IL-2 after 5 h of GP33 peptide stimulation is shown. Data are percent of CD8 T cells that produced both IFN-γ and IL-2. In D) viral titers in liver and lung are shown. In a separate experiment (Figure 6A) malnourished memory cells from LP-fed mice were transferred to naive adequate protein or low protein- fed recipients subsequently challenged with VV-GP33. Recall responses were assessed 7 days after transfer. B) shows a representative flow plot of percentage of Thy 1.1+ Db/GP33+ tetramer-specific CD8 T cells in adequate protein and low-protein hosts. Total number of tetramer positive cells per spleen in shown in C. One representative experiment of three is shown. Data are mean of three mice per group+ SEM; * P < 0.05

Impaired memory maintenance in malnourishment is rescued by dietary protein supplementation

Finally, we wanted to assess whether malnourishment-induced impairment in memory maintenance could be rescued by dietary intervention with an adequate protein diet. For this purpose, Armstrong immune P14 chimeric mice were assigned to one of three dietary regimens as outlined in Figure 7A 1) Adequate protein for 8 weeks (AP); 2) Adequate protein for 4 weeks followed by switch to Low protein for 4 weeks (LP) and 3) Low protein for 4 weeks followed by switch to Adequate protein for 4 weeks (LP-Rescue). After 8 weeks of each respective dietary treatment, lymphocytes were isolated from the spleen of AP, LP, and LP-rescue mice and examined for Thy 1.1+ memory cells specific for the LCMV epitope Db/GP33–41 (Figure 7B). Estimation of total numbers of Ag-specific T cells in the spleen revealed that adequate protein supplementation of low protein hosts resulted in a complete quantitative rescue of memory CD8 T (Figure 7C). Together, these data underscore the importance of dietary amino acid availability in optimal CD8 memory homeostasis.

Figure 7.

Figure 7

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Protein supplementation rescues malnutrition-induced defect in CD8 T cell memory maintenance. A) Experimental outline; 6–8 week-old P14 Chimeric mice were infected intraperitoneally with LCMV Armstrong and 40 days post-infection, mice were assigned to receive either an adequate protein (18% dietary protein) or a low protein diet (0.6% dietary protein). A third group of mice received LP diet for 4 weeks followed by switch to AP diet. Analysis of LCMV-specific memory CD8 T cells in the spleen was performed after 8 weeks of dietary treatment. B) Db/GP33 tetramer staining of donor Thy1.1 + CD8 T cells from the spleen of low protein (top panel), adequate protein (middle panel) and low-protein rescue mice (bottom panel). Data are percent of tetramer positive donor CD8 T cells. C) Percentage, absolute numbers of GP33-specific donor CD8 T cells, and total CD44-high memory CD8 T cells in the spleen in adequate protein (black bars), low protein (red bars), and low protein rescue (blue bars) mice. One representative experiment of two is shown. Data are mean of four mice per group + SEM; * P < 0.05

DISCUSSION

While nutrition is recognized as being a critical determinant of immunity, an understanding of the mechanisms by which poor nutrition impairs immune function remains poorly characterized (20–22). The current study bridges this gap by presenting two main findings: first, that maintenance of memory CD8 T cells is impaired in malnourishment due to defects in homeostatic proliferation and; second, that memory cells in malnourished hosts have defects in recall responses leading to impaired viral clearance upon re-infection. These findings have important implications for understanding the basis of increased infections in malnourished individuals in the world’s poorest countries. The data also suggest that malnourishment that frequently accompanies aging and chronic diseases may contribute to impaired immunity observed in these states. In all, the data show that a robust memory response generated under nutritionally optimal conditions is impaired by poor nutrition.

The complex and reciprocal relationship between nutrition and infections has long been recognized. Studies in humans and experimental animals show that infections place a significant metabolic demand on the host; for instance, increased mobilization of amino acids from peripheral tissues, largely, skeletal muscle, supports gluconeogenesis in the liver to meet the heightened glucose needs of rapidly dividing immune cells (12, 23, 24). Cell culture studies show that activated T cells increase amino acid uptake by approximately 6-fold compared to resting cells (25). Collectively, these data indicate that activated T cells have increased amino acid requirements to support at least two fundamental processes- increased bioenergetic demands and increased protein synthesis.

It therefore comes as no surprise that cell-mediated immune responses are compromised in protein-malnourished individuals. Salimonu et al found significantly lower T lymphocyte count in malnourished compared to well-fed children at baseline and at 3,10, and 21 days post-immunization with measles virus (26). Several studies have also shown decreased responses to tuberculin test in infants and children with PEM (21, 27). More recently, we demonstrated that PEM compromises the effector response to LCMV infection in mice (14). Decrease in effector responses led to a 3 log-fold increase in serum viral titers in PEM mice at day 8, while virus was completely cleared in control mice. Thus, there is abundant evidence indicating that malnutrition impairs effector CD8 responses to infection. However, relatively little is known about whether malnutrition impairs memory maintenance.

In the present study, we demonstrate that PEM induced a two-fold decrease in the memory CD8 compartment. Total lymphocyte numbers were also decreased suggesting that mice on LP diet were lymphopenic. Studies have shown that lymphopenia, resulting from severe infection, chemotherapy, irradiation, or malnourishment, induces proliferation of naive cells to memory cells, that are phenotypically indistinguishable from true antigen-experienced memory cells (28). Lymphopenia-induced proliferation (LIP) of naive cells to memory-phenotype cells would lead to an over-estimation of the memory compartment in LP hosts. Thus, the true deficit induced by PEM maybe underrepresented. However, it is also possible that thymic output was decreased in LP mice and/or circulating levels of IL-7 and IL-15 in LP mice were not sufficient to induce proliferation of naive cells to memory-phenotype cells (29).

Impaired memory homeostasis in PEM could result from decreased proliferation, increased cell death, or a combination of both. Adoptive transfer experiments using CFSE-labeled cells showed that at 4 weeks post-transfer 77% of donor cells had an undiluted CFSE profile in LP hosts compared to 25% in AP hosts. This suggests that defects in cell proliferation contributed to decreased memory pool in PEM. Further, comparable levels of the pro-survival protein B-cell lymphoma (Bcl)-2 and Bcl-XL, and the pro-apoptotic protein Bim in memory cells from LP and AP mice argue against a pro-apoptotic memory phenotype in LP hosts (Figure 3).

In addition to defects in basal homeostatic proliferation, memory cells in LP hosts showed impaired responses to poly (I:C), which induces acute proliferation of memory cells in an IFN-γ, IL-15-dependent manner (19). This defect, which was also seen in the bone marrow, one of the major site of homeostatic proliferation, could be due to at-least three reasons. First, cytokine levels-induced by poly(I:C) in LP hosts may not have been sufficient to drive increased proliferation. Indeed, amino acids such as glutamine are critical for production of IFN-γ in T cells (25). Second, impaired activation of antigen presenting cells such as dendritic cells in LP hosts could also be a contributing factor. Third, limited amino acid availability in LP hosts could interfere with efficient cell cycle progression and turnover. In vitro studies by Angelini et al showing that antigen presenting cells control T cell proliferation by controlling availability of cysteine in the extracellular fluid underscore the importance of nutrient availability in T cell turnover (30).

Interestingly, despite demonstrating marked deficiencies in basal and acute proliferation, memory cells in LP hosts did not appear to be phenotypically different from fully functional memory cells. This observation raises the possibility that the effects of malnutrition may not be T cell intrinsic but rather may be environmental and suggest that impaired memory maintenance in malnourished individuals may be amenable to rescue by protein supplementation. Indeed, replenishing protein in malnourished hosts rescued the proliferative defects due malnourishment (Figure 7). Furthermore, recall proliferation was superior during adoptive transfer of LP memory cells into AP hosts compared to LP hosts (Figure 6). While this result points the critical role of environmental/host factors in regulating memory homeostasis, it does not exclude the possibly that any T cell intrinsic defect, resulting from malnutrition, was also corrected as a consequence of protein supplementation. It is likely, therefore, that a combination of factors both environmental and T cell intrinsic contribute to impaired memory homeostasis in malnourishment. Experimental targeting of pathways involved in amino acid transport and metabolism in T cells and subsequent response in well-nourished and malnourished mice would be required to thoroughly dissect the role of T-cell intrinsic versus host/ environmental factors in malnutrition.

It is noteworthy that in malnourished children, an improvement in immune function, as measured by antibody titers, after protein supplementation has been reported (31). In contrast, however, numerous studies have failed to find significant benefit of protein supplementation in the severely malnourished (32, 33). It is possible that there may be a narrow window for successful intervention and the ability to rescue immune function may depend on a number of factors including severity of malnourishment, type of infection, and age of the host. Further studies are needed to determine whether memory homeostasis can be rescued by amino acid supplementation in malnourished individuals. Indeed, rigorous studies are also need to determine the impact of malnutrition on immune responses to vaccines with marginal efficacy as this is of direct clinical and public health relevance.

In summary, the data show that dietary protein intake is critical for the maintenance of a functional CD8 memory pool. The findings suggest that interventions to optimize dietary protein intake may improve vaccine efficacy in malnourished individuals and decrease susceptibility to infections in chronically infected malnourished patients

Supplementary Material

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Figure 8.

Figure 8

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Protein-energy malnutrition impairs CD8 memory homeostasis. Malnourished memory CD8 T cells demonstrate markedly reduced homeostatic proliferation despite no measurable decrease in cell surface expression of the gamma chain cytokine receptors. Malnourished memory CD8 T also conferred reduced protective immunity due to impaired recall proliferation. The data show that dietary protein is a critical for sustaining a long-lived and functional memory CD8 T cell pool and that this defect may be rescued by protein supplementation.

ACKNOWLEDGMENTS

We thank Bogumila Koneiczny and Yelena Blinder for excellent technical assistance

Footnotes

1

This work was supported by the National Institutes of Health Research Grant AI30048 (to R.A.).

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NIHMS335089-supplement-1.docx (58.1KB, docx)
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