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© 2008 American Society for Nutrition J. Nutr. 138:806-812, April 2008

Nutritional Immunology

Diminished Primary CD8 T Cell Response to Viral Infection during Protein Energy Malnutrition in Mice Is Due to Changes in Microenvironment and Low Numbers of Viral-Specific CD8 T Cell Precursors1–3,

Janel Hart Chatraw4,5, E. John Wherry6,7, Rafi Ahmed6 and Zoher F. Kapasi4,5,*

4 Department of Rehabilitation Medicine, Division of Physical Therapy, 5 Graduate Program in Nutrition Health Sciences, and 6 Emory Vaccine Center and Department of Microbiology and Immunology, Emory University School of Medicine, Atlanta, GA 30322

* To whom correspondence should be addressed. E-mail: zkapasi{at}emory.edu.


   ABSTRACT
TOP
 ABSTRACT
Introduction
 Materials and Methods
 Results
 Discussion
 LITERATURE CITED
 
Protein energy malnutrition (PEM) increases the incidence and severity of infection, causing morbidity and mortality in malnourished populations. Viral-specific cells are an important component of protective immunity. We hypothesized that reduction in the expansion of viral-specific cells and the microenvironment of the PEM host leads to increased incidence and severity of infections. We tested this hypothesis using a mouse model of lymphocytic choriomeningitis virus (LCMV) infection and an adoptive transfer system using P14 transgenic mice cells bearing T cell receptors specific for the Db-restricted LCMV glycoprotein 33–41 epitope. We transferred equal numbers of P14 cells from mice fed either an adequate, 18% protein or low, 0.6% protein diet into C57BL/6 mice that had been fed adequate-protein (AP) or low-protein (LP) diets for 2 wk, infected them with LCMV, and followed them 1 wk postinfection. During PEM, the expansion of primary viral-specific CD8 T cells diminished; in LP diet-fed mice, it was only 2–3% of that in the AP diet-fed mice. Furthermore, the diminished primary CD8 T cell response during PEM may in part have been due to low numbers of viral-specific CD8 T cells and an altered microenvironment.


   Introduction
TOP
 ABSTRACT
 Introduction
Materials and Methods
 Results
 Discussion
 LITERATURE CITED
 
Malnutrition and infection are interrelated. It is generally accepted that malnutrition causes increased susceptibility to infection and infection causes increased susceptibility to malnutrition (1). Protein energy malnutrition (PEM)8 is a major form of malnutrition and has been defined as an imbalance between food intake (protein and energy) and the body's requirement for them to ensure the most favorable growth and function (2). Insufficient protein intake can cause PEM (3,4). PEM is the most fatal form of malnutrition in developing countries, accounting for over one-half of the 11.6 million deaths among children <5 y in 1995 (2,5). Although there has been some progress toward halting the decline of malnutrition in many developing countries, malnutrition in sub-Saharan Africa is still rising (6). PEM increases the incidence and severity of infection, causing morbidity and mortality in malnourished populations (2,5,7–10).

Very few studies have examined the nature of immune deficits during PEM during a viral infection. In 1 study, the Sendai virus was studied in PEM mice; they had a lower delayed-type hypersensitivity response than control mice fed an adequate-protein (AP) diet. Additionally, lymphocytes from PEM mice underwent higher proliferation in response to lipopolysaccharide and irradiated allogeneic cells, but had similar or lower proliferation in response to concavalin A than those from control mice. However, mortality was higher in the PEM mice than in the controls (11). Other studies have shown an impaired proliferative response to mitogen and antigen stimulation in vitro (12,13). Additionally, the recirculating lymphocyte pool is reduced in mice who have PEM (14). The lymphoid organs are also greatly reduced in size during PEM (15).

Although these studies show that PEM is associated with reduced immune function in response to infection and may be responsible for the failure of these animals to clear infection, we do not know the exact nature of the deficit in the viral-specific T cell response. For example, the failure of immune cells to clear infection during PEM may be due to low numbers of viral-specific CD8 T cells, intrinsic changes in viral-specific CD8 T cells, or changes in the microenvironment (local areas and milieu in which cells reside and are composed of tissue-specific stromal cells, dendritic cells, other cell types, and matrix components, and factors such as cytokines, chemokines, and other factors) during PEM that affects the functioning of viral-specific CD8 T cells. Clearly, more than 1 of the above-listed factors may be responsible for the decreased ability of viral-specific CD8 T cells to clear the infection.

To address these questions, we used the mouse model of lymphocytic choriomeningitis virus (LCMV) infection. LCMV causes a natural infection in mice and provides a useful model for studying the interaction between a viral infection and host immunity (16). I.v. or intraperitoneal injection of adult immunocompetent mice with the Armstrong strain of LCMV results in acute infection. Following infection, the virus replicates rapidly in many tissues such as the lung, liver, spleen, and lymph nodes. Levels of infectious virus peak ~3 d after the infection (17). By d 5, there is a rapid decline in virus titers and the infection is completely resolved by d 8. Evaluation of the immune response shows that around d 5, LCMV-specific cytotoxic T cells appear. The peak of CD8 and CD4 T cell response occurs at d 8–9 of infection and falls off rapidly over the next 5–10 d. LCMV is a noncytocidal virus and intraperitoneal injection of AP-fed mice with 2 x105 plaque-forming units (pfu) of Armstrong strain of LCMV causes the host immune response to quickly control the spread of the virus and thus there is little tissue damage. In short, because the LCMV model does not cause early death, it allows us to examine the emerging immune response.

Finally, using equal numbers of naïve antigen-specific CD8 T cells bearing T cell receptors (TCR) specific for the Db-restricted LCMV glycoprotein (GP) 33–41 epitope from P14 transgenic mice allowed us to assess the influence of intrinsic and/or micro-environmental changes on CD8 T cell function during PEM following LCMV infection.


   Materials and Methods
TOP
 ABSTRACT
 Introduction
 Materials and Methods
Results
 Discussion
 LITERATURE CITED
 
    Mice, virus. In both Expt. 1 and 2, we obtained naïve female C57BL/6 mice from Jackson Laboratory. For Expt. 2, Thy1.1+ P14 transgenic mice that express Db GP33–41 specific TCR on their CD8 T cells were backcrossed on a C57BL/6 background and generated in our laboratory (18). LCMV Armstrong was generated in our laboratory (19). In Expt. 1, LCMV Armstrong was administered at 2 x 105 pfu to naïve female C57BL/6 mice. In Expt. 2, the virus was given to naïve female C57BL/6 mice 1 d after adoptive transfer of cells from naïve female Thy1.1+ P14 mice. Plaque assays on infected mice organs and serum were performed as described previously (20).

All mice were fed either a low-protein (LP) or AP diet, consumed water ad libitum, and were housed in climate-controlled conditions (25°C, 12-h-light/-dark photoperiod). We measured mouse body weights and food intakes once or twice per week. Coprophagy was permitted. The study was conducted in accordance with ethical procedures and policies approved by the Emory University's Institutional Animal Care and Use Committee.

    Diets. Mice were randomly assigned to different diet groups. The custom diets were prepared by Harlan Teklad (Supplemental Table 1). The diets were isocaloric, but the LP diet contained 0.6% protein and the AP diet contained 18% protein. This LP diet has been shown to cause the features of the human pathology of PEM in mice. In addition to edema as a late-stage outcome, the LP diet produces fatty liver (within 14 d in CBA/J mice and within 21 d in the C57BL/6J strain), low plasma albumin, and moderate anemia. Finally, ~33% mortality begins to appear between wk 5 and 6 of the LP diet (21). Before any adoptive transfers or infections, mice were given either a LP diet or an AP diet for 2 wk, the time needed to induce PEM (22). Mice consumed the same diet throughout the experiment.

    Antibodies and major histocompatibility complex tetramers. All antibodies used were from PharMingen. The construction and purification of the major histocompatibility complex (MHC) class I LCMV tetramers Db nucleoprotein (NP)396–404 and Db GP33–41 has been described (20).

    Cell preparation, staining, and flow cytometry. Mice under isofluorane anesthesia were bled retro-orbitally and blood was collected in a 4% sodium citrate solution. Preparation of spleen cells and staining has been described (20).

    Intracellular cytokine staining. Intracellular cytokine staining was performed as described (20). For intracellular staining, we used fluorescein isothiocyanate-conjugated monoclonal rat anti-mouse interferon (IFN){gamma} antibody (clone XMG 1.2), allophycocyanin-conjugated interleukin (IL)-2 (clone JES6–5H4), and allophycocyanin-conjugated tumor necrosis factor {alpha} (clone MP6-XT22).

    Adoptive transfers and infection. In Expt. 1, weanling C57BL/6 female mice were fed either an isocaloric 18% AP diet or a 0.6% LP diet for 2 wk and then infected with 2 x 105 pfu of LCMV Armstrong. Eight days postinfection mice were killed by overdosing with the anesthetic isoflurane. Spleens were removed and stained with surface stains. Additionally, the splenocytes underwent intracellular cytokine staining. Viral titers were calculated from serum collected at d 8 postinfection. In Expt. 2, adoptive transfers using P14 mice were conducted as follows. Naïve female Thy1.1+ P14 and C57BL/6 mice were fed either an AP or LP diet for 2 wk. Thy1.1+ P14 mice were then killed. An equal number of naïve splenocytes (~100,000 Thy1.1+ P14 cells) bearing TCR specific for the Db-restricted LCMV GP33–41 epitope were adoptively transferred i.v. via the tail vein into naïve C57BL/6 mice fed the AP or LP diet for 2 wk. Following are the 4 groups of mice: 1) LP/AP: We transferred 100,000 Thy1.1+ P14 cells from LP diet-fed P14 mice to AP-diet fed C57BL/6 mice; 2) AP/AP: We transferred 100,000 Thy1.1+ P14 cells from AP diet-fed P14 mice to AP diet-fed C57BL/6 mice; 3) AP/LP: We transferred 100,000 Thy1.1+ P14 cells from AP diet-fed P14 mice to LP diet-fed C57BL/6 mice; and 4) LP/LP: We transferred 100,000 Thy1.1+ P14 cells from LP diet-fed P14 mice to LP diet-fed C57BL/6 mice.

One day post-transfer of Thy1.1+ P14 cells, different groups of mice were infected with 2 x 105 pfu of LCMV Armstrong intraperitoneally. Eight days postinfection, mice were killed and single-cell suspensions of spleens, free of erythrocytes, were prepared in complete RPMI medium and stained as described above (see Fig. 1 for a schematic of Expt. 2). The viral-specific CD8 T cell response in spleen was measured by staining with Thy1.1+ P14 cells (donor cells) and for phenotypic surface markers (CD62L, CD122, CD127, CD27, CD44, CD25, CD69, PD-1, and 1B11) and intracellular markers (Bcl-2 and granzyme B). The function of viral-specific cells was evaluated by measuring production of IFN{gamma}, tumor necrosis factor {alpha}, and IL-2 upon restimulation in vitro. Viral titers were determined from serum, spleen, and kidney collected at d 8 postinfection.


Figure 1
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  		FIGURE 1  Experimental design (Expt. 2) of the transfer of Thy1.1+ P14 naïve cells into C57BL/6 mice and subsequent infection with LCMV Armstrong.

 
    Statistical analysis. Data were analyzed using commercial software (Minitab and SPSS). In Expt. 1, body weight and food intake data were tested using a 2-way (infection x diet) repeated measures ANOVA. In Expt. 2, body weights and food intakes were compared before and after cell transfer and infection by a 2-way repeated measures (LP/AP, AP/AP, AP/LP, and LP/LP) ANOVA. We separately compared the body weights and food intakes of LP or AP diet-fed P14 mice during the 2-wk period using repeated measures ANOVA. Various indicators of immune function were analyzed by 1-way ANOVA. Tukey's honestly significant difference post hoc test was performed when the F-test was significant. All statistical tests were 2-tailed and differences were considered significant at P ≤ 0.05. Values in the text are means ± SD.


   Results
TOP
 ABSTRACT
 Introduction
 Materials and Methods
 Results
Discussion
 LITERATURE CITED
 
    Effects of a LP diet on body weight and food consumption in mice (Expt. 1). As early as 12 d of consuming the LP diet, mice weighed 41% less than AP mice (P = 0.002). By 2 wk, mice fed the LP diet weighed 45% less than controls (P = 0.0003; Fig. 2A). LP diet-fed mice consumed 1.7 ± 0.2 g/d compared with AP diet-fed mice that consumed 2.4 ± 0.02 g/d (P = 0.006). Moreover, when expressed relative to body weight, LP diet-fed mice consumed more food than AP diet-fed mice (P < 0.05; Fig. 2B). Because the LP diet-fed mice consumed more energy relative to body weight than the mice fed the AP diet, we conclude that most of the effects of the LP diet can be attributed to a deficit of protein rather than of energy. For instance, from d 1–4, the LP diet-fed mice consumed ~0.14 ± 0.03 g protein/100 g body weight, whereas the AP diet-fed mice consumed 4.28 ± 0.90 g protein/g body weight (P = 0.0001). Body weights and food intakes did not differ between infected and uninfected AP or LP diet-fed mice (data not shown).


Figure 2
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  		FIGURE 2  Body weight (A) and relative food intake (B) of C57BL/6 mice fed the LP or AP diet for 2 wk and the infected with LCMV Armstrong (Expt. 1). Values are means ± SD, n = 5 (LP) or 3 (AP). *Different from AP at that time, P < 0.05.

 
    Diminished primary CD8 T cell response and viral clearance in mice during PEM (Expt. 1). We next evaluated the effects of PEM on the immune response to acute LCMV infection by studying the development of the primary CD8 T cell response to LCMV in mice fed either the LP or AP diet. At d 8 postinfection, mice fed the LP diet had 89.2% fewer spleen cells (22.8 ± 15.1 x 106) than the mice fed the AP diet (210.7 ± 103.9 x 106; P = 0.006). Although mice fed the LP diet had fewer CD8 T cells (7.4 ± 6.7 x 106) than mice fed the AP diet (76.6 ± 16.4 x 106; P = 0.0001), the percentage of CD8 T cells did not differ between mice fed the LP (29.4 ± 6.2%) and AP (40.1 ± 11.8%) diets.

We assessed CD8 T cell activation and found the percentage of CD8CD44hi T cells to be comparable in uninfected mice fed the LP diet and AP diets (Supplemental Fig. 1 and Fig. 3A). By d 8 post-LCMV infection, the percentage of CD8 T cells expressing the activated (CD44hi) phenotype was lower in LP diet-fed mice compared with AP diet-fed mice (P = 0.02; Fig. 3A) and was reflected in fewer activated splenic CD8 T cells (P = 0.0001; Fig. 3B).


Figure 3
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  		FIGURE 3  Percentage (A) and number (B) of splenic CD44hi T cells 8 d after LP and AP diet fed C57BL/6 mice were infected with LCMV Armstrong (Expt. 1). Values are means ± SD, n = 5 (infected LP), 3 (infected AP), or 2 (uninfected LP and uninfected AP). *Different from infected AP, P < 0.05.

 
At d 8 of the primary response, the frequency of CD8 T cells specific for the different LCMV cytotoxic T lymphocyte epitopes was determined by means of MHC class I tetramer binding and intracellular cytokine staining (functional assay). MHC class I tetramers complexed with defined viral peptide epitopes helped determine the number of CD8 T cells specific for those viral peptide epitopes. Whether these cells are also functional can be determined by measurement of the production of cytokines in response to stimulation with synthetic peptides. Collectively, these methods have allowed the CD8 T cell response to be quantitated with great accuracy and are some of the most sensitive assays available to detect viral-specific T cell response (20). Typically, in normal mice, we do not see a difference in frequency of viral-specific CD8 T cells detected by MHC class I tetramer staining or intracellular cytokine staining, suggesting that cells detected by MHC class I tetramer staining are also functional cells (20).

Similarly, we did not see a difference in frequency of viral-specific CD8 T cells detected by MHC class I tetramer staining or intracellular cytokine staining for mice fed either the LP or AP diet (data not shown), suggesting that cells detected by MHC class I tetramer staining are also functional cells. However, the magnitude of the virus-specific response differed between mice fed LP and AP diets. The frequency of viral-specific CD8 T cells measured by IFN{gamma} production after stimulation with the NP396–404 peptide was lower in mice fed the LP diet compared with those fed the AP diet (P = 0.0003; Fig. 4A,B). Additionally, the frequency of viral-specific CD8 T cells measured by the IFN{gamma} production after stimulation with the GP33–41 peptide was lower in mice fed the LP diet compared with controls (P = 0.003; Fig. 4A,B). Lower frequencies of viral-specific CD8 T cells by intracellular cytokine staining in LP diet-fed mice compared with AP diet-fed mice were also reflected in fewer viral-specific CD8 T cells by intracellular cytokine staining (Fig. 4C). NP396–404 specific CD8 T cells by intracellular cytokine staining in LP diet-fed mice was 1.7% of those fed the AP diet (P = 0.00003; Fig. 4C). GP33–41 specific CD8 T cells by intracellular cytokine staining in LP diet-fed mice was 3.1% of those fed the AP diet (P = 0.0005; Fig. 4C). Clearly, AP diet-fed mice showed a stronger response to the immunodominant epitopes compared with LP diet-fed mice. Spleen cells from LCMV-infected LP and AP diet-fed mice made no IFN{gamma} in the absence of stimulation. In addition, spleen cells from uninfected LP or AP diet-fed C57BL/6 mice did not produce any IFN{gamma} after stimulation with any of the peptides (data not shown).


Figure 4
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  		FIGURE 4  Percentage (A,B) and numbers (C) of IFN{gamma} producing CD8 T cells after stimulation with NP396–404 or GP33–41 peptide of LP or AP diet fed C57BL/6 mice 8 d after infection with LCMV Armstrong and viral titers (D) in serum at d 8 postinfection (Expt. 1). A, A representative flow cytometric analysis of splenocytes stimulated with NP396–404 peptide and GP33–41 peptide indicate the percentage of CD8 T cells that produce IFN{gamma} when stimulated by viral peptide NP396–404 or GP33–41. Dashed line indicates the level below which titer of virus cannot be detected by the assay. Values are means ± SD, n = 5 (infected LP) or n = 3 (infected AP). *Different from infected AP, P < 0.05.

 
Finally, by examining viral clearance in mice fed AP or LP diets, we found AP diet-fed mice cleared the LCMV Armstrong virus from serum by d 8 postinfection (Fig. 4D). The LP diet-fed mice had fewer LCMV-specific cells and although these cells were functional, they did not clear the virus from the serum by d 8 postinfection. Normally, in naive mice infected with LCMV Armstrong, virus is cleared from the serum very quickly and remains in the tissues until ~d 8 postinfection. These data suggest that in LP diet-fed mice, LCMV clearance is severely compromised compared with AP diet-fed mice. Although the serum concentrations of LCMV were high in LP diet-fed mice, we did not see any illness response in LP diet-fed mice.

    Decreased numbers of viral-specific cells and changes in the microenvironment cause diminished primary CD8 T cell response in mice with PEM (Expt. 2). Next, we wanted to see if the difference in starting numbers of viral-specific CD8 T cells was responsible for the difference in the viral-specific CD8 T cell response between LP and AP diet-fed mice following the infection. Furthermore, intrinsic changes in T cells due to the LP diet and/or the differences in the environment (LP or AP) could also be responsible for the differences in the viral-specific CD8 T cell response. To address all these possibilities, we used an adoptive transfer system using P14 transgenic mice that were fed either AP or LP diets for 2 wk.

Prior to transfer into C57BL/6 mice, a sample of the P14 splenocytes from AP and LP diet-fed mice was stained for several activation and functional markers (CD122, CD127, CD25, CD27, CD62L, CD69, and CD44) and no differences in the surface marker expression among Thy1.1+ P14 cells were detected. Moreover, P14 mice fed the LP or AP diet for 2 wk had weight loss, food consumption, and immune variable changes similar to those of C57BL/6 mice (data not shown). This suggests that P14 and C57BL/6 mice respond similarly to the LP diet.

We compared LP/AP and AP/AP mice to assess whether CD8 T cells are permanently affected in an LP environment (intrinsic changes) and whether they recover when transferred into an AP environment. At d 8 following the infection, spleen cell numbers did not differ between the LP/AP (110.2 ± 17.0 x 106) and AP/AP (101.4 ± 55.5 x 106) groups. CD8 T cell numbers and percentages also did not differ between LP/AP (35.4 ± 21.1 x 106, 34.3 ± 4.9%) and AP/AP (35.7 ± 7.3 x 106, 32.3 ± 2.9%) mice.

Surface staining showed that Thy1.1+ P14 CD8 T cell (donor cells) numbers in LP/AP and AP/AP groups were similar (Fig. 5A). Phenotypic surface and intracellular markers showed no marked differences in donor Thy1.1+ P14 CD8 T cells between LP/AP and AP/AP (data not shown) groups. We also measured the ability of donor Thy1.1+ P14 CD8 T cells to produce antiviral cytokine IFN{gamma} upon restimulation in vitro and found no difference in numbers or percentages of Thy1.1+ P14 CD8 T cells between LP/AP (18.7 ± 2.4 x 106, 52.8 ± 4.0%) and AP/AP (17.8 ± 9.6 x 106, 51.3 ± 3.4%) groups. The numbers and percentages of Thy1.1+ P14 cells that made IFN{gamma} and also made IL-2 did not differ between LP/AP and AP/AP (Fig. 5B–D) groups. Furthermore, a subset of LP/AP and AP/AP C57BL/6 mice were longitudinally bled through the contraction and memory phase of the viral-specific CD8 T cell response and the percentage of Thy1.1+ P14 cells in the blood at d 8, 16, 23, 36, 50, and 78 did not differ (data not shown). These data indicate that in an AP environment, viral-specific cells derived from a LP environment function similarly to viral-specific cells derived from an AP environment, suggesting that there may be no intrinsic changes in viral-specific CD8 T cells in a LP environment.


Figure 5
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  		FIGURE 5  Numbers (A) of Thy1.1+ P14 cells and percentages (B, C) and numbers (D) of IL-2 and IFN{gamma} producing cells of LP/AP, AP/AP, AP/LP, or LP/LP C57BL/6 mice 8 d after infection with LCMV Armstrong (Expt. 2). A, Db GP33–41 specific CD8 T cells and Thy1.1 cells in the spleen 8 d postinfection were calculated. B, A representative flow cytometric analysis of cells stimulated with GP33–41 peptide indicates the percentage of IFN{gamma} producing CD8 T cells that also produce IL-2 when stimulated with peptide. C, Percentages and numbers (D) of IFN{gamma} producing CD8 T cells that also produce IL-2. Values are means ± SD, n = 3 except AP/LP, n = 2. *Bracketed means differ, P < 0.05.

 
Because intrinsic changes in viral-specific CD8 T cells may not be responsible for the deficit in the viral-specific CD8 T cell response in LP diet-fed mice, we next addressed the role of the LP environment on the function of viral-specific CD8 T cells. Eight days following the infection, the number of spleen cells, number of CD8 T cells, and CD8 T cell percentages did not differ between AP/LP (35.0 ± 15.6 x 106, 12.8 ± 6.0 x 106, 36.5 ± 1.0%) and AP/AP (101.4 ± 55.5 x 106, 35.4 ± 21.1 x 106, 34.3 ± 4.9%) mice.

Surface stains showed that Thy1.1+ P14 CD8 T cell (donor cell) numbers in AP/LP and AP/AP mice did not differ (Fig. 5A). Of the phenotypic and intracellular markers we stained for, the most noticeable difference between the AP/AP and AP/LP groups of the activated cells that were transferred (Thy1.1+ P14 cells), was in granzyme B (Supplemental Fig. 2). The mean fluorescent intensity of granzyme B in Thy1.1+ P14 T cells was higher in AP/LP mice (74.4 ± 4.0) than in AP/AP mice (30.2 ± 2.0; P = 0.001).

We also measured the ability of donor Thy1.1+ P14 CD8 T cells to produce antiviral cytokine IFN{gamma} upon restimulation with GP33–41 epitope in vitro and found no difference in numbers or percentages of Db GP33–41 specific CD8 T cells between AP/LP mice (6.6 ± 3.4 x 106, 50.4 ± 2.9%) and AP/AP mice (17.8 ± 9.6 x 106, 51.3 ± 3.4%). However, the percentage of Thy1.1+ P14 CD8 T cells that made IFN{gamma} that also made IL-2 was lower in AP/LP mice than in AP/AP mice (P = 0.001; Fig. 5C). Although there was a corresponding 77% decrease in the number of CD8 T cells that made IFN{gamma} and also IL-2 in AP/LP mice compared with AP/AP mice, the difference was not significant (P = 0.06; Fig. 5D).

To further identify differences due to the environment, we compared LP/AP mice to LP/LP mice following infection as described previously. Eight days following the infection, LP/LP mice (23.2 ± 1.1 x 106) had fewer spleen cells than LP/AP mice (110.2 ± 17.0 x 106; P = 0.05). However, CD8 T cell numbers (9.1 ± 0.1 x 106 in LP/LP mice and 35.7 ± 7.3 x 106 in LP/AP mice) and percentages (39.2 ± 1.5% in LP/LP mice and 32.3 ± 2.9% in LP/AP mice) were similar in the 2 groups.

Surface stains showed that Thy1.1+ P14 cell (donor cell) numbers were lower in LP/LP mice than in LP/AP mice (P = 0.04; Fig. 5A). In LP/LP mice, there was a 240-fold expansion of Thy1.1+ P14 T cells, whereas in LP/AP, there was a 660-fold expansion. Of the phenotypic and intracellular markers, the most noticeable difference between LP/LP and LP/AP, among the activated cells that were transferred (Thy1.1+ P14 cells), were in granzyme B (Supplemental Fig. 2). The mean fluorescent intensity of granzyme B in Thy1.1+ P14 T cells was higher in LP/LP mice (63.7 ± 8.5) than in LP/AP mice (34.9 ± 9.2; P = 0.006). The percentage of Thy1.1+ P14 T cells that made granzyme B was higher in LP/LP (38.6 ± 3.4%) than LP/AP (24.7 ± 7.2%; P = 0.02) mice. However, the numbers of these cells that made granzyme B were similar between the groups (0.9 ± 0.06 x 106 in LP/LP mice and 1.6 ± 0.5 x 106 in LP/AP mice).

We also measured the ability of donor Thy1.1+ P14 CD8 T cells to produce antiviral cytokine IFN{gamma} upon restimulation with the GP33–41 epitope in vitro and found lower numbers (P = 0.05) but similar percentages of Db GP33–41 specific CD8 T cells in LP/LP mice (4.2 ± 0.2 x 106, 46.5 ± 2.2%) and LP/AP mice (18.7 ± 2.4 x 106, 52.8 ± 4.1%). The percentage of Thy1.1+ P14 CD8 T cells that made IFN{gamma} that also made IL-2 was lower in LP/LP mice compared with LP/AP mice (P = 0.002; Fig. 5C). There was a corresponding 85.2% decrease in the number of CD8 T cells that made IFN{gamma} and also IL-2 (P = 0.03; Fig. 5D).

The detrimental role of the LP environment on the function of viral-specific CD8 T cells is all the more remarkable, because the viral-specific response of LP/LP did not differ from AP/LP. For instance, surface and intracellular markers between AP/LP and LP/LP did not differ (data not shown). Similarly, there was no difference in the numbers or percentages of Thy1.1+ P14 CD8 T cells that made IFN{gamma} and also made IL-2 between LP/LP and AP/LP mice (Fig. 5C,D).

When we examined the viral titers in the spleen, serum, and kidney, we found that all groups had cleared the virus by d 8 postinfection. This is not surprising considering that very few viral-specific CD8 T cells are required to clear LCMV Armstrong infection.


   Discussion
TOP
 ABSTRACT
 Introduction
 Materials and Methods
 Results
 Discussion
LITERATURE CITED
 
In this study, we show for the first time, to our knowledge, that decreased immunity to viral-specific infection during PEM is due to low numbers of viral-specific CD8 T cells and changes in the microenvironment during PEM. Furthermore, CD8 T cells from an LP environment function normally when transferred into an AP environment, suggesting that feeding AP diets to PEM individuals may restore the immune function of CD8 T cells. Collectively, these findings have important clinical implications.

The specific mechanisms of immune changes caused by malnutrition have not been elucidated. Low numbers of viral-specific CD8 T cells following infection may be due to a decreased number of viral-specific precursors and limited expansion of precursor cells following infection. Decreased numbers of viral-specific T cell precursors is likely given that PEM is associated with profound thymic atrophy with the resultant loss of well-developed thymus-dependent areas of peripheral lymphoid compartments and marked decrease in the production of thymic hormones that are essential for the differentiation and maturation of immunocompetent T lymphocytes (23). Moreover, protein albumin acts as an extracellular antioxidant (24). Hypoalbuminemia is a common feature in PEM (25). There exists, therefore, a possibility of reactive oxygen species production in PEM creating a state of oxidative stress in mice fed LP diets. Reactive oxygen species lead to lymphocyte apoptosis and may reduce the availability of viral-specific naïve T cell precursors for clonal expansion during PEM (26).

Finally, PEM is associated with high concentrations of glucocorticoids (27). Glucocorticoids mediate lymphocyte apoptosis and may also reduce the availability of viral-specific naïve T cell precursors for clonal expansion during PEM (28). However, when equal numbers of viral-specific cells from mice fed either an LP or AP diet were transferred into mice fed an AP diet and then infected, we found similar levels of expansion of viral-specific cells irrespective of whether they were derived from mice fed an LP or AP diet. These data suggest that PEM leads to fewer viral-specific precursors, but the low numbers of precursor cells are capable of expansion in an AP environment.

Limited expansion of viral-specific precursor T cells following infection during PEM may be due to a number of factors in the microenvironment of mice fed an LP diet. For example, in a recent study, mice fed a PEM diet had reduced numbers and functions of dendritic cells (29). Because dendritic cells play a role in viral-specific stimulation of CD8 T cells, it is possible that altered function of dendritic cells may play a role in limited expansion of viral-specific T cells. Additionally, mice fed a LP diet, which induces PEM, have deficiency in protein intake and thus of the essential amino acid glutamine. Being the most abundant free amino acid in the body, glutamine has been shown to affect cell proliferation in several cell types including lymphocytes. Dividing lymphocytes have been shown to have a high uptake of glutamine (30), which is required for synthesis of both purines and pyrimidines, an essential component of replicating DNA, in dividing cells (31). Thus, decreased expansion of viral-specific T cells in LP diet-fed mice may be due to deficiency of glutamine. Additionally, cytokine production is affected by glutamine concentrations (32). IL-2 is a positive factor for generation of T cell responses and our data show a significant decrease in the number of CD8 T cells that made IFN{gamma} and also IL-2 in mice fed a LP diet.

Granzyme B is upregulated in effector CD8 T cells after infection (33). Infected cells can be killed via the release of granzyme B by viral-specific effector T cells. Our data show that mice fed an LP diet, irrespective of where the antigen-specific cells came from, contain higher amounts of granzyme B in the antigen-specific cells compared with mice fed an AP diet. Perhaps upregulation of granzyme B occurs to compensate for the reduction of expansion in antigen-specific cells in LP-fed mice.

In conclusion, this study shows that decreased immunity to viral-specific infection during PEM is due to low numbers of viral-specific CD8 T cells and changes in the microenvironment during PEM.


   ACKNOWLEDGMENTS
 
We thank Bogumila Koneiczny and Yelena Blinder for excellent technical assistance.


   FOOTNOTES
 
1 Supported by NIH Research grant AI30048 (to R.A.). Back

2 Author disclosures: J. H. Chatraw, E. J. Wherry, R. Ahmed, and Z. F. Kapasi, no conflicts of interest. Back

3 Supplemental Table 1 and Supplemental Figures 1 and 2 are available with the online posting of this paper at jn.nutrition.org. Back

7 Current address: Immunology Program, The Wistar Institute, Philadelphia, PA 19104. Back

8 Abbreviations used: AP, adequate protein (18% of diet); AP/AP, 100,000 Thy1.1+ P14 cells from AP diet fed P14 mice were transferred to AP diet fed C57BL/6; AP/LP, 100,000 Thy1.1+ P14 cells from AP diet fed P14 mice were transferred to LP diet-fed C57BL/6; GP, glycoprotein; LCMV, lymphocytic choriomeningitis virus; LP, low protein (0.6% of diet); LP/AP, 100,000 Thy1.1+ P14 cells from LP diet fed P14 mice were transferred to AP diet-fed C57BL/6; LP/LP, 100,000 Thy1.1+ P14 cells from LP diet-fed P14 mice were transferred to LP diet-fed C57BL/6; MHC, major histocompatibility complex; NP, nucleoprotein; PEM, protein energy malnutrition; pfu, plaque-forming unit; TCR, T cell receptor. Back

Manuscript received 5 July 2007. Initial review completed 3 August 2007. Revision accepted 3 January 2008.


   LITERATURE CITED
TOP
 ABSTRACT
 Introduction
 Materials and Methods
 Results
 Discussion
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