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. Author manuscript; available in PMC: 2013 May 15.
Published in final edited form as: J Inherit Metab Dis. 2011 May 24;35(1):159–168. doi: 10.1007/s10545-011-9349-x

Aberrant expression of costimulatory molecules in splenocytes of the mevalonate kinase-deficient mouse model of human hyper-IgD syndrome (HIDS)

Elizabeth J Hager 1, Jon D Piganelli 2, Hubert M Tse 3, K Michael Gibson 4,✉
PMCID: PMC3654530  NIHMSID: NIHMS318261  PMID: 21607759

Abstract

Objective

We sought to determine the activation status and proliferative capacities of splenic lymphocyte populations from a mevalonate kinase-deficient mouse model of hyper-IgD syndrome (HIDS). We previously reported that murine mevalonate kinase gene ablation was embryonic lethal for homozygous mutants while heterozygotes (Mvk+/−) demonstrated several phenotypic features of human HIDS including increased serum levels of IgD, IgA, and TNFα, temperature dysregulation, hematological abnormalities, and splenomegaly.

Methods and results

Flow cytometric analysis of cell surface activation markers on T and B lymphocytes, and macrophage populations, demonstrated aberrant expression of B7 glycoproteins in all splenic cell types studied. Differences in expression levels between Mvk+/− and Mvk+/+ littermate controls were observed in both the basal state (unstimulated) and after Concanavalin A (Con-A) stimulation in vitro of whole splenocyte cultures. In Mvk+/− CD4 and CD8 T cells, alterations in expression of CD25, CD80, CD152, and CD28 were observed. Mvk+/− splenic macrophages expressed altered levels of CD80, CD86, CD40, and CD11c while Mvk+/− B lymphocytes had differential expression of CD40, CD80, and CD86. Mvk+/− splenocyte subpopulations also exhibited altered proliferative capacities in response to in vitro stimulation.

Conclusion

We postulate that imbalances in the expression of cell surface proteins necessary for activation, proliferation, and regulation of the intensity and duration of an immune response may result in defective T cell activation, proliferation, and effector functions in our model and potentially in human HIDS.

Hyper-immunoglobulin D (IgD) syndrome (HIDS) is a human inherited autoinflammatory disorder that arises from mutations in the mevalonate kinase gene (MVK), and is associated with recurrent inflammatory episodes (Goldfinger 2009). HIDS has been classified as a primary immunodeficiency disease (Notarangelo et al. 2009). Autoinflammatory disorders usually result from mutations in proteins that are important to functions of the innate arm of the immune system that ultimately affect the efficiency of T and B lymphocyte adaptive immune responses.

T cell activation, proliferation, and T helper (TH) cell subset differentiation (TH1, TH2) are functions of the immune response that are dependent on T cell receptor (TCR)-antigen/major histocompatibility antigen (MHC) recognition and costimulatory signals. Multiple costimulatory signals are critical for the activation and differentiation of naïve T cells and consist of interactions between T cells and antigen presenting cells (APCs), and also T cell-T cell interactions (Salomon and Bluestone 2001; Greenwald et al. 2005). A major costimulatory pathway involves interactions of the B7 family of glycoproteins expressed on lymphocytes, macrophages, dendritic cells, and tissues involved in antigen presentation in disease states. Expression of B7 glycoproteins in this study included CD28, CD152 (CTLA-4), CD80 (B7-1) and CD86 (B7-2). The B7 ligands CD80 and CD86 are expressed constitutively at low levels in resting APCs and T cells and are upregulated during activation. It is believed that basal constitutive expression may limit T cell activation, thereby maintaining self-tolerance, and also maintaining a population of T regulatory cells (Treg) (Lohr et al. 2003).

CD28 is constitutively expressed on naïve T cells and is rapidly upregulated within hours of activation (Freeman et al. 1993). CD152 expression on T cells is very low in naïve T cells but is upregulated within 24–48 hours of T cell activation in vitro (Perkins et al. 1996). Both CD28 and CD152 bind CD80 and CD86 but with differing affinities. CD152 binds CD80 with greater affinity than CD86 (Freeman et al. 1993). CD28 interaction with CD80 or CD86 provides a strong stimulatory signal to mature T cells that have been activated through the T cell receptor (TCR). Moreover, CD28 ligation induces IL-2 production and upregulation of surface CD25, the IL-2 receptor α chain, and proliferation (Jenkins et al. 1991; Butscher et al. 1998). It has been suggested that CD28-CD86 interactions are more important for initial T cell activation while CD28-CD80 interactions are important for sustained T cell activation (Sethna et al. 1994).

CD152 ligation induces an inhibitory signaling pathway that decreases IL-2 synthesis and inhibits T cell responses including proliferation by restricting cell cycle progression from G1 to S phase (Krummel and Allison 1996). CD152 may play a role in peripheral T cell tolerance through maintenance of a Treg population that is dependent on CD152-CD80 interactions (Lohr et al. 2003; Perez et al. 2008). Together, the interactions of B7 glycoproteins on cell surfaces function to regulate the intensity and duration of T cell activation and therefore, the outcome of immune responses involved in transplantation, antitumor immunity, autoimmunity, infectious diseases, and asthma and allergy (Blazar et al. 1999).

In the current study, we postulated that because HIDS patients experience chronic bouts of fever and autoinflammation, it is possible that their peripheral lymphocyte subpopulations may exhibit imbalances in cell types, activation states, cytokine responses, and proliferative capacities while suppressing chronic inflammation to achieve homeostasis. To test this hypothesis, we first compared cell surface activation marker status of lymphocytes and macrophages from wild type and mevalonate kinase-deficient mice.

Materials and methods

Animals

Mvk mice were bred and housed under specific pathogen-free conditions under IACUC-approved protocols. Male mice~30 weeks of age were used in five experiments in these studies since the HIDS-like phenotype is more pronounced in aged animals (Hager et al. 2007). Age-matched female mice were used in one experiment that measured proliferation, B7 expression, and Foxp3 expression.

Splenocyte isolation

For each experiment, spleens were removed from 5 Mvk+/+ and 5 Mvk+/− mice. Spleens were pooled according to genotype in order to have sufficient numbers of cells to simultaneously assess 44 markers at four different time-points in culture. Genotypic pooling of cells also offset variances in individual phenotypes. Splenocytes were obtained from intact spleens and red blood cells removed as described (McAdam et al. 2000). Cells were divided into two portions. One cell aliquot was used for basal cell surface marker immunostaining. The second cell aliquot was cultured in the presence of the T cell mitogen, Con-A, to induce direct stimulation of cell surface marker expression on activated T cells or, indirect induction of markers in other splenocyte populations through T cell cytokines and T cell-cell interactions.

Con-A splenocyte stimulation

Splenocytes were seeded at 5 × 105 cells per well of 96-well tissue culture plates and were stimulated with Con-A (2.5 μg/mL) for 24, 48, and 72 hours.

Flow cytometry

One million cells were stained directly with four fluorochrome-conjugated cell marker-specific antibodies, followed by fixation in 2% paraformaldehyde and analysis by FACSCalibur (BD Biosciences). Expression of a particular cell marker in basal (unstimulated, freshly-isolated) and activated (Con-A stimulated) splenocyte populations was assessed by measurement of the cell surface marker density indicated by mean fluorescent intensity (MFI) and the % positive of splenocytes staining with the respective marker. For analysis of forkhead box P3 (Foxp3), Con-A activated CD4 T cells were treated with reagents provided in the Foxp3 staining buffer set and fluorochrome-labeled anti-Foxp3 antibody (eBioscience). Isotype staining controls were performed to validate the specificity of all antibodies used for immunostaining.

T cell isolation, anti-CD3ε/anti-CD28 costimulation culture, and MTT proliferation assay

T cells were isolated from whole splenocyte preparations by magnetic bead negative selection using the CD4 T cell isolation kit (Miltenyi Biotec). Purified CD4 T cell populations (> 90% purity by flow cytometry) were collected and used for cell proliferation experiments. Anti-CD3 ε and anti-CD28 antibodies (BD Biosciences), at concentrations of 0.1 μg/mL and 1 μg/mL, respectively, were employed with overnight incubation. The following day, purified T cells were added and the plates were incubated at 37°C for up to 96 hours. Plates were prepared with six replicate wells per time-point for each genotype. Colorimetric quantification of cell proliferation was performed with the cell proliferation kit I (MTT) (Roche Applied Science). At each time-point (24, 48, 72, and 96 hours), fold-differences were determined for stimulated cultures compared to unstimulated cultures.

5-, 6-Carboxyfluorescein diacetate, succinimidyl ester (CFSE) labeling of splenocytes

Cells were labeled with 5 μM CFSE (Invitrogen; Kearney et al. 1994) for 15 minutes, followed by plating with Con-A. At harvest, replicate wells were pooled at each time-point (48, 72 hours). One million cells per sample were then stained with fluorochrome-labeled antibodies to specific cell surface lineage markers in order to determine the number of cell divisions of CD4 T cells, CD8 T cells, B cells, and macrophages.

Study design and statistical analysis

Two preliminary screenings of numerous cell surface markers described in Table 1 in male splenocyte populations revealed reproducible directional differences in certain cell marker expression in Mvk+/− mice as compared to Mvk+/+. In three subsequent experiments, results were combined for each cell marker queried (not all markers were queried in every experiment due to insufficient cell numbers at harvest) and the differences between mean values ± SEM for each experimental group were analyzed using a one-tailed Student’s t test for significance (p<0.05) since we had observed directionality in data collected in two preliminary staining experiments. The MFI data is displayed as bar graphs that show mean marker expression between multiple experiments which, in total, consisted of 10–20 mice of each genotype. The data were analyzed using GraphPad Prism 5 software.

Table 1.

Cell markers evaluated in this study

B lymphocytes T lymphocytes Macrophages
IgD CD3 F4/80
IgM CD4 CD16/32
IgG CD8 CD40
CD19 CD25 CD80
CD45R CD28 CD86
CD25 CD152 CD11a
CD11a CD154 CD11b
CD11b CD80 CD11c
CD11c CD86 CD62L
CD23 ICOS MHC I
CD62L CD62L MHC II
CD69 CD69
CD80 CD11a
CD86 CD44
CD40 MHC I
CD17/32
MHC I
MHC II
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Results

Expression levels of cell markers in basal and Con-A stimulated splenic CD4 T lymphocytes

CD4 T lymphocytes expressed all T cell markers listed in Table 1 similarly between Mvk+/− and Mvk+/+ mice with the exception of the surface densities of CD4; the B7 glycoproteins CD80, CD152 and CD28; and CD25. Basal CD4 densities were consistently ~ 2-fold lower in Mvk+/− as compared to Mvk+/+ (Fig. 1) although the percentages of CD4 cells were similar between genotypes (39.6 +/− 3.7% and 32.6 +/− 2.5%, respectively). CD4 surface densities remained similar to basal levels in Con-A stimulated culture although there was an approximate 10% increase in percentages of CD4 T cells in both genotypes at 72 hours.

Fig. 1.

Fig. 1

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Basal CD4 expression in CD3ε+ T cells of Mvk+/+ (wild-type, W) and Mvk+/− (heterozygote, T) splenocyte populations. The differences in the means (± SEM) of MFI in two experiments were not significant (ns), p>0.05. Data were analyzed by one-tailed Student’s t test

Altered expression levels of B7 glycoproteins were observed in CD4 T cells in Mvk+/− mice. Basal levels of CD80 were consistently ~ 2-fold higher in Mvk+/− than Mvk+/+ (Fig. 2a) indicating that these cells may be in a more activated state than basal Mvk+/+ CD4 T cells (Salomon and Bluestone 2001; Taylor et al. 2004). Additionally, the basal density of CD152, the CD80/CD86 inhibitory receptor, was significantly elevated in Mvk+/− CD4 T cells (p=0.0169, Fig. 2b), a further indication of an activated T cell phenotype (Perkins et al. 1996; Allegre et al. 2006). CD152 levels were also ~ 2-fold higher in activated Mvk+/− versus Mvk+/+ CD4 T Con-A stimulated cell cultures, with CD152 levels peaking in both genotypes at 24 hours. In these experiments, increased Mvk+/− CD152 levels coincided with decreased expression of CD25 (the IL-2 receptor α chain) in Con-A stimulated cultures (p = 0.0108, Fig. 2c) and decreased IL-2 accumulation (unpublished observation). These observations are consistent with CD152 ligation inhibiting T cell growth (Krummel and Allison 1996). Unlike CD152, basal levels of CD28, the CD80/CD86 stimulatory B7 receptor, were similar in both genotypes in CD4 T cells, but were consistently elevated by ~ 2-fold at 72 hours in activated Mvk+/− CD4 T cells (Fig. 2d) although IL-2 levels were decreased which is opposite to what would normally be expected. These results suggest that in Mvk-deficient CD4 T cells, CD152 mediated inhibitory regulation of T cell activation may occur to a greater degree than that of Mvk+/+.

Fig. 2.

Fig. 2

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(a) Basal CD80 expression in CD4 T cells of Mvk+/+ (wild-type, W) and Mvk+/− (heterozygote, T) splenocyte populations. The difference in the means (± SEM) of MFI in two experiments was not significant (ns), p>0.05. (b) Basal CD152 expression in CD4 T cells of Mvk+/+ and Mvk+/− splenocyte populations. The difference in the means (± SEM) of MFI in two experiments was significant, p=0.0169. (c) Con-A stimulated CD25 expression in CD4 T cells of Mvk+/+ and Mvk+/− splenocyte populations. The difference in the means (± SEM) of MFI in two experiments was significant, p=0.0108. (d) Con-A stimulated CD28 expression in CD4 T cells of Mvk+/+ and Mvk+/− splenocyte populations. The difference in the means (± SEM) of MFI in two experiments was not significant, p > 0.05. Data were analyzed by one-tailed Student’s t test

Enhanced ligation of CD152 by an agonistic antibody induces IL-10 and TGF-β resulting in expansion of the anti-inflammatory Foxp3 Tregs (Vasu et al. 2004; Li et al. 2007), and blockade of the mevalonate pathway via statin administration results in generation of Foxp3 Treg cells (Kim et al. 2010). Based on these observations, we stained for intracellular levels of Foxp3 in CD4 T cells. In this experiment, we observed a significant increase in Foxp3% positive Mvk+/− CD4 T cells compared to Mvk+/+ (p=0.0246, data not shown) suggesting that Mvk+/− mice have mobilized this Treg subset. Taken together, we postulate that blockade of mevalonate kinase in CD4 T cells results in alteration of B7 glycoprotein surface expression which may perturb signaling in the T-APC immunological synapse, favoring increased inhibition through CD152 and recruitment of Foxp3+Tregs.

Expression levels of cell markers in basal and Con-A stimulated splenic CD8 T lymphocytes

Unlike CD4, the CD8 lineage marker for cytotoxic T cells was expressed in Mvk+/+ and Mvk+/− T cells at similar basal densities (10,337±1,722 MFI and 8,690±893 MFI, respectively) and percentages of the splenocyte population (10.1± 1.2% and 10.1±1.4%, respectively). Similar to Mvk+/− CD4 T cells, Mvk+/− CD8 T cells consistently expressed ~ 2-fold higher basal levels of CD152, but unlike Mvk+/− CD4 T cells, activation-induced CD152 expression was similar between genotypes in CD8 T cells. CD80 basal expression was significantly decreased in Mvk+/− CD8 T cells (p = 0.0046, Fig. 3a) whereas it was consistently increased in Mvk+/− CD4 T cells (Fig. 2a). Additionally, CD80 expression was consistently decreased ~ 2-fold in activated Mvk+/− CD8 T cells, whereas CD80 expression in activated Mvk+/− CD4 T cells was similar to that of Mvk+/+. Mvk+/− CD8 T cells consistently expressed ~ 2-fold lower basal levels of CD28 (Fig. 3b) while activated levels of CD28 were similar between genotypes unlike that of Mvk+/− CD4 T cells. CD25 basal levels were similar between genotypes in CD8 T cells as was observed in CD4 T cells. After 72 hours in Con-A stimulated cultures, Mvk+/− CD25 levels were significantly decreased (p=0.0364, Fig. 3c) similar to Mvk+/− CD4 T cells.

Fig. 3.

Fig. 3

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(a) Basal CD80 expression in CD8 T cells of Mvk+/+ (wild-type, W) and Mvk+/− (heterozygote, T) splenocyte populations. The difference in the means (± SEM) of MFI in three experiments was significant, p=0.0046. (b) Basal CD28 expression in CD8 T cells of Mvk+/+ and Mvk+/− splenocyte populations. The difference in the means (± SEM) of MFI in two experiments was not significant (ns), p>0.05. (c) Con-A stimulated CD25 expression in CD8 T cells of Mvk+/+ and Mvk+/− splenocyte populations. The difference in the means (± SEM) of MFI in three experiments was significant, p=0.0364. Data were analyzed by one-tailed Student’s t test

Expression levels of macrophage markers in basal and Con-A stimulated splenic cultures

Macrophages were identified based on expression of F4/80 antigen, a marker of mature murine macrophages. Of the macrophage markers listed in Table 1, male Mvk+/− manifested altered basal expression of CD80, CD86, CD40, and CD11c. Basal levels of Mvk+/− CD80 and CD86 were significantly decreased (p=0.0387, Fig. 4a) and (p=0.0291, Fig. 4b.), respectively. Basal expression levels of Mvk+/− macrophage CD40 were similar to Mvk+/+; however, CD40-% positive macrophages in the Mvk+/− basal population were significantly decreased as compared to that of Mvk+/+ (p=0.0098, Fig. 4c). In Con-A stimulated splenocyte cultures, the number of Mvk+/− CD40-% positive macrophages reached that of Mvk+/+ suggesting that CD40 upregulation is not intrinsically impaired in Mvk+/− macrophages. Lastly, basal CD11c-% positive Mvk+/− macrophages were significantly increased as compared to Mvk+/+ splenic populations (p=0.00487, Fig. 4d). It is noteworthy that CD11c is an integrin subunit of the complement iC3b and fibrinogen receptor associated with the macrophage M1 phenotype which is proinflammatory (producing TNFα, MCP-1, IL-1β,-6, -8, -10) (Wentworth et al. 2010). Together, these results indicated that Mvk+/− macrophages were deficient in CD80, CD86, and CD40 expression in the basal state but were responsive to signaling induced in stimulated mixed splenocyte culture to increase expression of these markers to normal levels.

Fig. 4.

Fig. 4

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(a) Basal CD80 expression in F4/80+ macrophages of Mvk+/+ (wild-type, W) and Mvk+/− (heterozygote, T) splenocyte populations. The difference in the means (± SEM) of MFI in three experiments was significant, p=0.0387. (b) Basal CD86 expression in F4/80+ macrophages of Mvk+/+ and Mvk+/− splenocyte populations. The difference in the means (± SEM) of MFI in three experiments was significant, p=0.0291. (c) Basal CD40 % positive of F4/80+ macrophages in Mvk+/+ and Mvk+/− splenocyte populations. The difference in the means (± SEM) of % positive in two experiments was significant, p=0.0098. (d) Basal CD11c % positive of F4/80+ macrophages in Mvk+/+ and Mvk+/− splenocyte populations. The difference in the means (± SEM) of % positive in two experiments was significant, p=0.0487. Data were analyzed by one-tailed Student’s t test except for CD11c which was analayzed by two-tailed Student’s t test

Expression levels of B lymphocyte markers in basal and Con-A stimulated splenic culture

B lymphocytes were identified based on the presence of CD19 and surface IgM and IgD. Basal level expression of the B cell markers listed in Table 1 were normal in Mvk+/− as compared to Mvk+/+ B cells except for a consistent ~ 2-fold decrease in CD40-% positive Mvk+/− B cells similar to the observed basal state of Mvk+/− macrophages. Like the other Mvk+/− splenic cell types we studied, Mvk+/− B cells also had altered expression of B7 glycoproteins, particularly in Con-A stimulated splenic cultures. Stimulated Mvk+/− B cells expressed significantly increased levels of CD80 (p=0.0017, Fig. 5a) as compared to Mvk+/+ B cells. Increased CD80 levels were concomitant with significantly decreased expression of CD86 in Mvk+/− B cells (p = 0.0243, Fig. 5b) in Con-A stimulated splenocyte cultures. CD86-% positive B cells were also significantly decreased (p=0.0133, Fig. 5c) in these cultures. These data suggest that CD80-CD152 inhibitory signaling may predominate at the T-B cell immunological synapse in Mvk+/− mice.

Fig. 5.

Fig. 5

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(a) Con-A stimulated CD80 expression in CD19+ B cells of Mvk+/+ and Mvk+/− splenocyte populations. The difference in the means (± SEM) of MFI in two experiments was significant, p=0.0017. (b) Con-A stimulated CD86 expression in CD19+ B cells of Mvk+/+ and Mvk+/− splenocyte populations. The difference in the means (± SEM) of MFI in two experiments was significant, p=0.0243. (c) Con-A stimulated CD86 % positive of F4/80+ macrophages in Mvk+/+ and Mvk+/− splenocyte populations. The difference in the means (± SEM) of % positive in two experiments was significant, p=0.0133. Data were analyzed by one-tailed Student’s t test

Gender differences in CD80 expression and proliferation of splenocyte subpopulations

CD80 is expressed abnormally in both Mvk+/− female and male splenocytes, though oppositely. Basal female Mvk+/− macrophage and B cell CD80 levels were expressed at~2-fold higher levels than Mvk+/+ females. Recall that in Mvk+/− males, basal CD80 levels were significantly decreased in macrophages (Fig. 4a). During Con-A stimulation Mvk+/− male macrophages expressed CD80 at levels similar to Mvk+/+. This was not observed in Mvk+/−female macrophages and B cells. In females, CD80 expression was ~ 3-fold higher in macrophages and ~ 6-fold higher in B cells in splenocyte Con-A stimulated cultures as compared to expression in Mvk+/+. Mvk+/+ females also had increased CD80 expression following Con-A stimulation as compared to Mvk+/+ males with macrophage CD80 levels ~ 4-fold higher and B cell levels ~ 2-fold higher. Curiously, there were no notable differences between genders in any of the markers queried (Table 1) with the exception of CD80.

To determine if splenic subpopulations had altered proliferative capacities, we performed experiments in which cultured male splenocytes were labeled with CFSE in order to assess cellular proliferation. Mvk+/− CD4 and CD8 T cells both increased in number by ~ 2-fold greater than those of Mvk+/+; Mvk+/− B cells had increases ~ 3-fold greater than those of Mvk+/+ B cells in number; and the number of Mvk+/− macrophages had increased ~ 5-fold greater than those of Mvk+/+. We also purified CD4 T cells from male mice and stimulated growth in vitro by anti-CD28/anti-CD3ε costimulation. We observed that similar to CD4 T cells in Con-A stimulated splenocyte cultures, Mvk+/− CD4 T cell growth in two MTT proliferation assays was heightened on average 15-fold as compared to that of Mvk+/+ at 72 hours (data not shown). This may have been due to increased expression of CD28 on these cells.

In addition, we performed MTT proliferation assays in age- and sex-matched whole splenocyte cultures. At all time-points, Mvk+/− female splenocytes proliferated significantly less than those of Mvk+/+ (24 hours, p=0.0046; 48 hours, p=0.0009; 72 hours, p<0.0001; analyzed by two-tailed Student’s t test). The photograph in Fig. 6 illustrates an interesting pattern of proliferation in female Mvk+/+ and Mvk+/− splenocytes stimulated with Con-A at 48 hours. Note that in Mvk+/− there are more foci of proliferation than that of Mvk+/+ but the clones do not expand to as high a density as those of Mvk+/+.

Fig. 6.

Fig. 6

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Photomicroscopic images of female Mvk+/+ (left) and Mvk+/− (right) Con-A stimulated cultures after 48 hours of proliferation. Magnification 400x

Discussion

In these studies, we aimed to establish the activation status of splenic lymphocytes and macrophages in the gene-ablated mevalonate kinase-deficient mouse model. The Mvk+/− mouse model is a nearly faithful phenocopy of HIDS with elevated serum levels of TNFα, IgA and IgD, temperature dysregulation, splenomegaly and/or hepatomegaly, and hematological abnormalities including lymphocytosis, increased macrophage counts in peripheral blood, anemia, and abnormal platelet precursors. Because HIDS patients have periodic autoinflammatory crises, we postulated that Mvk+/−mice may also undergo periodic or chronic inflammatory processes. If this is the case, inflammatory effects may be reflected in the status of basal levels of lymphocyte activation markers and also in the ability of an in vitro stimulus such as Con-A to induce activation of mixed splenocyte populations. The studies presented here describe quantitative alterations in expression of activation markers in Mvk+/− splenocyte subpopulations at basal levels and in Con-A stimulated cultured cells as compared to Mvk+/+ cells. The mechanism(s) by which these alterations occur have yet to be elucidated.

B7 glycoproteins are essential for initial T cell activation and regulation of the intensity of the ensuing immune response to antigenic stimuli. The B7 pathway provides a costimulatory signal to the initial signal transduced through the TCR-antigen/MHC complex. Balance between stimulatory and inhibitory signals is required for effective immune responses to pathogens and for maintaining self-tolerance. Without the B7 costimulatory signal, antigen-stimulated T cells become anergic (Chen and Nabavi 1994; Hara et al. 2006). Several disease states have been described where B7 glycoproteins CD80 and CD86 are abnormally expressed on the cell surface of APCs and also as soluble molecules that are able to bind T cell-associated B7 receptors and mediate the outcome of the disease state (Wong et al. 2005; Ip et al. 2006).

CD152 regulates T cell tolerance and autoimmunity (Fife and Bluestone 2008). Polymorphisms in the B7 loci have been linked to autoimmune susceptibility in humans and mice (Keir and Sharpe 2005). In two mouse models of autoimmunity, experimental autoimmune encephalomyelitis (EAE) and non-obese diabetic (NOD) autoimmune diabetes, B7 molecules have been shown to regulate peripheral T cell tolerance. Certain CD152 splice variants correlate with increased susceptibility to autoimmune diabetes in NOD mice and in Grave’s disease, autoimmune hypothyroidism, and type I diabetes in humans. Aberrant expression of B7 family members are associated with other tissue-specific autoimmunity that develop in certain mouse strains as they age ranging between 5–14 months (Fife and Bluestone 2008). In our experiments, we used mice that were aged approximately 30 weeks.

B7 family protein structure, function, and expression depend on at least three products of the mevalonate pathway: 1) dolichol which mediates protein glycosylation; 2) cholesterol which is the main component of membrane lipid rafts; and 3) geranylgeranyl-diphosphate (GGDP) which is involved in protein prenylation. CD80 is one of the most glycosylated proteins on leukocyte surfaces (Davis et al. 2001). It has been suggested that changes in glycosylation of CD80 or other activation markers may greatly impact the outcome of an immune response (Greenfield et al. 1997). For example, altered glycoslyation patterns can lead to systemic autoimmune disease (Chui et al. 2001). Human IgA nephropathy has been linked to defective O-glycosylation of IgA1 (Baharaki et al. 1996; Hiki et al. 1999).

B7 glycoproteins, like many other membrane proteins, are prenylated by farnesyl-diphosphate or GGDP. Due to pleiotropic effects, and the complexity of regulation of the mevalonate pathway, it is not surprising that many experimental findings involving this pathway are paradoxical. This is reminiscent of our experimental system, in which the differences in expression level, either up or down, of B7 and other activation molecules were often approximately two-fold, sometimes having statistical significance, sometimes not. Nevertheless, it was striking that two-fold changes were so predominant. This phenomenon implies that a dose-dependent effect may be operative in our experiments. In this case, it may be a mevalonate kinase gene dosage effect in Mvk heterozygotes, directly or indirectly, impacting expression levels of several leukocyte activation markers.

It is possible that the aberrant expression of B7 glycoproteins we observed in Mvk+/− mice may be due to an aging effect on development of autoimmunity although aged-matched wildtype littermates were not affected in the same way. Perhaps Mvk mice carry an additional mutation such as one in the B7 or MHC loci that may predispose them for an “autoimmune” phenotype, however, one would expect wildtype littermates to exhibit similar trends unless a mevalonate kinase deficiency exacerbates the phenotype. We believe the likely explanation for B7 expression aberration is due to defective post-translational modification, i.e., deficient prenylation and/or glycosylation which affects surface expression and may or may not play a role in establishment of autoimmunity in the HIDS mouse model. The results of our studies suggest the hypothesis that Mvk+/− mice undergo chronic immune stimulation from the environment, which in this disease model would deplete mevalonate products that B7 glycoproteins are dependent on for normal cell surface expression and function.

We also examined splenic Mvk+/− lymphocyte proliferation since the mevalonate pathway is critical to cell growth and its regulation (Cuthbert and Lipsky 1990; Mantha et al. 2005). We found that Mvk+/− male T cells and splenocytes had increased kinetics and levels of proliferation for up to 72 hours in culture. On the other hand, cultured Mvk+/− female splenocytes had significantly lower levels of proliferation than Mvk+/+ females at all time-points. Supporting our observations are reports of gender effects during the use of statins in humans (Allen and Canadian Academic Detailing Corporation 2006; Ferrario 2008), although there have been no reports of gender effects in mevalonate kinase-deficient patients such as the association between gender and creatine kinase activity observed with statin consumption (Chan et al. 2006). We propose that the differences in proliferation between Mvk+/+ and Mvk+/− females can be explained by the overexpression of CD152 on T cells and CD80 on APCs in Mvk+/− splenocytes. Increased expression of both CD152 and CD80 likely downregulated Mvk +/− female T cell proliferation through ligation of CD152 and CD80 evoking the B7 inhibitory pathway, especially in light of a significantly increased presence of CD4+Foxp3+ Tregs in Mvk+/− female spleens (unpublished observation). Consistent with this observation, human T cell proliferation is inhibited through CD80-CD152 interaction and is dependent on Tregs (Manzotti et al. 2002).

In conclusion, we report here that there are significant alterations in expression of B7 costimulatory molecules and other markers of activation in Mvk+/− splenocyte subpopulations. The aberrant expression of CD80 and CD152 support the classification of HIDS as an immunodeficiency disorder since the prevailing B7 interaction may have an inhibitory effect on the initiation of the T cell response to specific antigen. The data presented here provide the foundation for mechanistic studies to explain some of the immunological defects in HIDS patients and also provide possible cellular biomarker candidates of human HIDS that can be readily monitored for treatment efficacy in future clinical trials.

Acknowledgments

The authors would like to thank Sherri Wiseman, Tara Rutledge, and Stephanie Guimond for their excellent technical assistance.

Supported in part by a grant from the Research Advisory Committee (RAC) Children’s Hospital of Pittsburgh, NIH HD57864 (KMG), and the Sterol and Isoprenoid Diseases (STAIR) consortium. STAIR is a part of NIH Rare Diseases Clinical Research Network (RDCRN). Funding and/or programmatic support for this project has been provided by a grant (1U54HD061939) from NICHD and the NIH Office of Rare Diseases Research (ORDR). The views expressed in written materials or publications do not necessarily reflect the official policies of the Department of Health and Human Services nor does mention by trade names commercial practices, or organizations imply endorsement by the U.S. Government.

Abbreviations

APC

Antigen presenting cell

CFSE

5-6-carboxyfluorescein diacetate, succinimidyl ester

Con-A

Concanavalin-A

GGDP

Geranylgeranyl-diphosphate

EAE

Experimental autoimmune encephalomyelitis

Foxp3

Forkhead box P3 transcription factor

HIDS

Human hyper-IgD syndrome

IL-

Interleukin-

MCP-1

Macrophage chemoattractant protein 1

MFI

Mean fluorescence intensity

MHC

Major histocompatibility antigen

MVK

Mevalonate kinase

NOD

Non-obese diabetic

TCR

T cell antigen receptor

TH

T helper cell

TNF

Tumor necrosis factor

Treg

Regulatory T cell

Footnotes

Competing interest: None declared.

Contributor Information

Elizabeth J. Hager, Department of Biological Sciences, DOW ESE Room 742, Michigan Technological University, 1400 Townsend Drive, Houghton, MI 49931, USA

Jon D. Piganelli, Division of Immunogenetics, Department of Pediatrics, Children’s Hospital of Pittsburgh, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA

Hubert M. Tse, Department of Microbiology, Comprehensive Diabetes Center, Shelby 1202, University of Alabama at Birmingham, Birmingham, AL, USA

K. Michael Gibson, Email: kmgibson@mtu.edu, Department of Biological Sciences, DOW ESE Room 742, Michigan Technological University, 1400 Townsend Drive, Houghton, MI 49931, USA.

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