Skip to main content
NCBI home page
NIHPA Author Manuscripts logoLink to NIHPA Author Manuscripts
. Author manuscript; available in PMC: 2014 Mar 1.
Published in final edited form as: Arthritis Rheum. 2013 Mar;65(3):764–769. doi: 10.1002/art.37790

Increased expression of a short splice variant of CTLA-4 exacerbates lupus in MRL/lpr mice

Kunihiro Ichinose *, Zheng Zhang *, Tomohiro Koga *, Yuang-Taung Juang *, Katalin Kis-Tóth *, Arlene H Sharpe †, Vijay Kuchroo ‡, José C Crispín *, George C Tsokos *
PMCID: PMC3582760  NIHMSID: NIHMS419476  PMID: 23203389

Abstract

Background

CTLA-4 is a negative regulator of the immune response expressed by regulatory T cells and activated T cells. Polymorphisms in the CTLA4 gene have been associated with autoimmune diseases including systemic lupus erythematosus. Disease-associated polymorphisms have been shown to affect the production of the different CTLA-4 variants through an effect on alternative splicing.

Methods

We have generated a MRL/lpr mouse strain that transgenically over-expresses a short isoform of CTLA-4 (1/4 CTLA-4) by backcrossing C57BL/6.1/4CTLA4 transgenic mice into the MRL/lpr strain for 9 generations. A new antibody was generated to detect the expression of the 1/4 CTLA-4 isoform. Routine methods were used to evaluate kidney pathology, humoral and cellular immunity.

Results

We show that expression of the 1/4 CTLA-4 isoform accelerates autoimmune disease. Transgenic mice display early onset of mortality, increased renal pathology and higher titers of anti-DNA antibodies, when compared to wild type MRL/lpr mice. Acceleration of autoimmunity and disease pathology by the presence of the short (1/4) isoform of CTLA-4 was linked to increased numbers of activated T cells and B cells and heightened interferon gamma production, but not to altered expression of the full length CTLA-4 molecule or regulatory T cell numbers.

Conclusions

Our results indicate that the presence of the alternatively spliced 1/4 CTLA-4 isoform can further promote autoimmunity and autoimmune pathology in lupus-prone mice and suggest that altered splicing of CTLA4 contributes to the expression of autoimmune disease.

Cytotoxic T Lymphocyte Antigen-4 (CTLA-4) is a costimulatory receptor in the immunoglobulin superfamily closely related to CD28 and ICOS, and binds to CD80 and CD86 (1;2). Expression of CTLA-4 is constitutive on regulatory T cells (3) and induced following activation on effector T cells (1). It exerts an essential inhibitory role and its absence causes an early and lethal autoimmune disease in mice (4).

The CTLA4 gene is highly conserved (76% homology between human and mouse) (5). It is comprised of 4 exons: exon 1 codes for the signal peptide; exon 2 for the ligand-binding domain; exon 3 for the transmembrane region; exon 4 for the intra-cytoplasmic tail (Figure 1A) (6). Human peripheral blood lymphocytes express 3 isoforms of CTLA-4 produced by alternative splicing: full length CTLA-4 (flCTLA-4; all four exons), soluble CTLA-4 (exons 1, 2, and 4), and a short variant that lacks both the ligand-binding domain and the transmembrane domain (1/4 CTLA-4) (6). Allelic variations and single nucleotide polymorphisms in the CTLA4 gene have been associated to several human autoimmune diseases including autoimmune thyroid disease (6), rheumatoid arthritis (7) and systemic lupus erythematosus (SLE) (8;9). Interestingly, the polymorphisms associated with autoimmune disease affect CTLA4 splicing and thus the relative expression of each variant isoform (6). How the differential expression of CTLA-4 isoforms impacts susceptibility to autoimmune disease is not yet clear.

Figure 1. The 1/4 CTLA-4 splice variant codes for a secreted protein.

Figure 1

Open in a new tab

A, Exons 2 and 3 that code for the ligand binding and transmembrane domains, respectively, are spliced out when the short variant 1/4 CTLA-4 is produced. B, A ~10 KDa band was detected by Western blot in sera of MRL/lpr.1/4CTLA4 mice. C, Abundance of 1/4 CTLA-4 was significantly higher in transgenic mice than in MRL/lpr and C57BL/6 mice. Shown is mean + SEM of the densitometry analysis. Albumin was used as loading control. D, 1/4 CTLA-4 transcripts were detected in spleen cells from transgenic mice by Taqman PCR.

The 1/4 CTLA-4 isoform lacks the CD80/86-binding domain and the transmembrane portion and thus its function remains unclear. Forced expression of the 1/4 CTLA-4 isoform in T cells was shown to induce spontaneous autoimmune disease and facilitate the development of experimental allergic encephalomyelitis in C57BL/6 mice through an unknown mechanism (10). Here we demonstrate that in lupus-prone mice increased expression of 1/4 CTLA-4 accelerates autoimmunity, exacerbates disease pathology, and causes early mortality.

MATERIALS AND METHODS

Mice

Female MRL/MpJ-Tnfrsf6lpr (MRL/lpr) mice were purchased from The Jackson Laboratory. The generation of B6.1/4CTLA4Tg mice has been previously described (10). MRL/lpr.1/4CTLA4Tg mice were generated by backcrossing B6.1/4CTLA4Tg mice into MRL/lpr mice (nine generations). Mice were sacrificed at the end of their 12th or 15th week of age. All mice were maintained in a SPF animal facility and all experiments were approved by the Institutional Animal Care Committee of Beth Israel Deaconess Medical Center.

Urine analysis

Proteinuria and pyuria were measured in a semiquantitative manner. Briefly, mice in each group (n=4) were placed together overnight in a Nalgene metabolic cage to collect urine. This procedure was repeated in 3 independent experiments, so that the presented data display the average from a total of 12 mice per group.

Western blotting

Equal aliquots of the diluted serum samples (1:100) were fractionated on NuPAGE 4–12% Bis-Tris Gel (Invitrogen) and transferred to 0.2 µm PVDF membrane (Millipore). The membrane was blocked for 1 h with 3% skimmed milk in TBS-T buffer. The membrane was probed with anti-1/4 CTLA antibody (custom antibody from Yenzym antibodies, LLC CA, USA). The membrane was washed with TBS-T and incubated with a 1:5000 dilution of goat anti-rabbit IgG coupled with horseradish peroxidase (HRP; Jackson Immunoresearch, West Grove, PA, USA). The enhanced chemiluminescence (ECL) system (Amersham, Buckinghamshire, UK) was used for detection.

Measurement of cytokines in cell supernatants

Two million splenocytes were incubated in 1 mL of RPMI 1640 supplemented with 10% FCS and stimulated with anti-CD3 (0.25 µg/ml) and anti-CD28 (0.5 µg/ml) for 24 hr. At the end of the culture period supernatants were collected. IFN-γ concentration was detected with ELISA kits (R&D Systems) as per the manufacturer’s protocols.

Flow cytometry

Thymus, spleens, axillary and inguinal lymph nodes (pLN), mesenteric lymph nodes (mLN) and Peyer’s pathes (PP) were excised from MRL/lpr mice, and single-cell suspensions were obtained by teasing the organs through a nylon mesh. Cells were stained for flow cytometry with antibodies against CD25, CD80, CD86 and CTLA-4 (eBioscience), CD3e, CD4, CD44, CD62L, CD19, F4/80 and CD11c (BioLegend) and Foxp3 (BD Biosciences) for 30 min at 4°C. For intracellular staining of Foxp3, the Foxp3-Staining Buffer Set (fixation/permeabilization and permeabilization buffers; eBioscience) was used according to the manufacturer's protocol. Total cell numbers were determined by counting live cells. Absolute cell numbers were calculated on the basis of the percentage of each population and represented as median. Samples were acquired in a LSRII flow cytometer (BD Biosciences). Analysis was performed with FlowJo v. 7.6.1 (Tree Star).

Real-time PCR

Total mRNA was isolated from spleen cells using the RNeasy Mini Kit (Qiagen). cDNA was produced using random primers from an equal amount of RNA. Taqman quantitative PCR was performed on an Applied Biosystems Step One Plus. Gene expression was assessed by comparative CT method. Primer sequences are available upon request.

Statistical analyses

All values are expressed as mean + SD. Kruskal-Wallis test with post-hoc comparisons using the Scheffe’s test were used for inter-group comparisons of multiple variables. Survival was analyzed by the Kaplan-Meier method and the log rank test. Statistical analyses were performed by StatView software (Abacus Concepts). A level of p<0.05 was considered significant.

RESULTS

The 1/4 CTLA-4 variant isoform codes for a secreted protein

To determine whether increased expression of 1/4 CTLA-4 affects the onset and/or severity of systemic autoimmunity and related pathology, we generated MRL/lpr mice that transgenically expresses the 1/4 CTLA-4 variant isoform in T cells by backcrossing C57BL/6.1/4CTLA4Tg mice into MRL/lpr mice. To ensure the transgene produced a protein product, we generated a rabbit polyclonal antibody directed against the protein domain encoded by exon 4. Consistent with the fact that it lacks the transmembrane domain (exon 3), we could detect a protein corresponding to the size of 1/4 CTLA-4 in the sera of transgenic mice (Figure 1B). Serum levels of this protein were significantly higher in MRL/lpr.1/4CTLA4Tg mice than in non transgenic MRL/lpr and B6 mice (Figure 1C). Consistent with these results, we could detect 1/4 CTLA-4 transcripts using a Taqman assay that only amplifies this species (and not other Ctla4 splice variants) (10) in spleen cells from transgenic mice (Figure 1D).

Expression of the 1/4 CTLA-4 variant exacerbates autoimmunity

The presence of the 1/4CTLA-4-coding transgene was associated with accelerated disease onset and early mortality in MRL/lpr mice (Figure 2A). Lymphoproliferation secondary to Fas deficiency is a prominent feature of the disease in the MRL/lpr mice. To determine if early mortality was associated to increased lymphoproliferation and/or accelerated autoimmunity, we analyzed the secondary lymphoid organs of 12 and 15 week old mice. Spleen weight and cellularity were modestly increased in transgenic mice as compared to non-transgenic littermates. However, no differences in lymph node weight or size were detected (data not shown). In contrast, MRL/lpr.1/4CTLA4Tg mice expressed a more severe glomerulonephritis compared to MRL/lpr. This was manifested by increased levels of proteinuria (P=0.01) and pyuria (P=0.03), as well as significantly more damage of the glomeruli as indicated by the presence of more hyaline thrombi, a sign of extensive immune complex deposition in the capillary loops. Moreover, inflammatory cell infiltration into tubule-interstitial zones was more severe in MRL/lpr.1/4CTLA-4 mice compared to MRL/lpr littermates (Figure 2B). Humoral autoimmunity was likewise more intense in 1/4 CTLA-4 transgenic mice as manifested by statistically significant higher levels of serum IgG and anti-double stranded DNA antibodies (Figure 2C).

Figure 2. Expression of 1/4 CTLA-4 exacerbates autoimmunity in MRL/lpr mice.

Figure 2

Open in a new tab

A, Survival of MRL/lpr mice that over-express 1/4 CTLA-4 is significantly decreased when compared to that of non-transgenic MRL/lpr mice (n≥8 mice per group; empty circles MRL/lpr; filled circles MRL/lpr.1/4CTLA4). B, Autoimmune kidney damage was analyzed in 12 week old mice. Shown are representative images of glomerular and tubule-interstitial areas of PAS stained kidney sections. Black bars indicate 50 µm (glomeruli) and white bars 200 µm (tubule-interstitium). C, Total IgG and anti-double stranded DNA IgG were quantified in serum by ELISA. D, Naïve (CD62L+ CD44−) and activated (CD44+) CD4 T cells were quantified in spleens of 12 week old wild type and transgenic mice by flow cytometry. Interferon-gamma was analyzed by ELISA in supernatants of spleen cells stimulated with anti-CD3 and anti-CD28. CD80 and CD86 expression on B cells (CD19+) and on splenic macrophages (CD86) was quantified by flow cytometry. Values are expressed as mean + SD of ≥6 mice per group. White bars represent MRL/lpr mice and black bars MRL/lpr.1/4CTLA4 mice (C, D).

Increased T and B cell activation in 1/4 CTLA-4-expressing mice

The recorded increased antibody and autoantibody production, as well as increased signs of renal inflammation, suggested that the presence of 1/4 CTLA-4 facilitated further the autoimmune response and kidney pathology. We analyzed the activation state of T and B lymphocytes in 12 week old MRL/lpr and MRL/lpr.1/4CTLA-4 mice. In concordance with the pathology findings, transgenic mice had significantly higher numbers of activated (CD44+) T cells (P<0.01) along with a reciprocal decrease in naïve (CD62L+ CD44v) T cells (P=0.01). After in vitro stimulation, cells from MRL/lpr.1/4CTLA-4 mice secreted significantly higher amounts of the pro-inflammatory cytokine interferon-gamma (Figure 2D). The activation state of B cells was also increased as demonstrated by augmented expression of the co-stimulatory molecules CD80 and CD86 (Figure 2D). Expression of CD86 was also increased in spleen macrophages (Figure 2D).

Regulatory T cells are not affected by 1/4 CTLA-4 overexpression

CTLA-4 is constitutively expressed by regulatory T cells (Treg) (3) and has been shown to be necessary for their suppressive function (11;12). Moreover, 1/4 CTLA-4 is highly expressed by Treg since its transcription closely resembles that of full length CTLA-4 in terms of kinetics and cellular distribution (10). For this reason, we considered that Treg numbers and/or homeostasis could be affected by the presence of the 1/4 CTLA-4-coding transgene. Using flow cytometry, we quantified the abundance of CD4+ FoxP3+ cells in the thymus, spleen, axillary, inguinal and mesenteric lymph nodes and Peyer’s patches of transgenic and wild-type MRL/lpr mice. There were no differences in the number or distribution of Treg between MRL/lpr mice expressing the 1/4 CTLA-4 transgene compared to those who did not (Figure 3A).

Figure 3. Abundance of regulatory T cells and expression of full length CTLA-4 are not affected in transgenic mice.

Figure 3

Open in a new tab

A, FoxP3+ cells were quantified in MRL/lpr and MRL/lpr.1/4CTLA4 mice by flow cytometry in thymus, spleen, axillary and inguinal lymph nodes (pLN), mesenteric lymph nodes (mLN) and Peyer’s patches (PP). Results (mean + SD) are expressed as the percentage of FoxP3+ cells within CD4+ T cells. B, Expression of full length Ctla4 mRNA was quantified by real time PCR in freshly isolated lymph node cells (left panel) or after stimulation with anti-CD3 and anti-CD28 (right panel). Results are expressed as fold change relative to wild type mice (ΔΔCT). C, Membrane expression of CTLA-4 was analyzed by flow cytometry in freshly isolated lymph node T cells. Lymphoid cells were gated according to size (FSC) and complexity (SSC). Then T cells were gated based on CD3 expression. Shown are representative dot plots of cells stained with anti-CTLA-4 (lower panels) or isotype control (upper panels). D, Splenic CD3+ CD4+ cells were divided based on their expression of CD25 in Teff (CD25−) and Treg (CD25+) and CTLA-4 expression was quantified as mean fluorescence intensity (MFI).

1/4 CTLA-4 overexpression does not affect the expression of full length CTLA-4

Certain autoimmune-associated polymorphisms in CTLA4 are located in non-coding regions and have been shown to affect splicing of CTLA4 (6). To determine whether forced expression of 1/4 CTLA-4 altered the transcription of the full length native molecule, we quantified CTLA-4 levels at the mRNA and the protein levels. As shown in Figure 3B, mRNA levels of full length CTLA-4 were not modified in cells freshly isolated from lymph nodes. Likewise, the expression of full length CTLA-4 on the surface membrane of lymph node cells was comparable between MRL/lpr and MRL/lpr.1/4CTLA-4 mice determined as the percentage of CTLA4+CD3+ cells (Figure 3C), or as mean fluorescence intensity (MFI; Figure 3D). To assess if the transgene affected CTLA-4 expression in Treg, we compared by flow cytometry CTLA-4 levels in CD4+CD25+ (Treg) cells and CD4+CD25− (Teff) cells from transgenic and non transgenic mice. As expected, CTLA-4 expression was significantly higher in Treg than in Teff cells (Figure 3D). However, no difference in CTLA-4 Treg expression was detected in the presence of 1/4 CTLA-4. To rule out differential regulation upon T cell stimulation, we activated T cells isolated from peripheral lymph nodes of MRL/lpr and MRL/lpr.1/4CTLA-4 mice with anti-CD3 and anti-CD28 antibodies. No differences were observed in CTLA-4 mRNA expression after 72 hours of cell stimulation (Figure 3B, right panel).

DISCUSSION

In this study we have investigated whether forced expression of an alternatively spliced CTLA-4 variant isoform (1/4 CTLA-4) in T cells can alter systemic autoimmune disease in a murine model of SLE. Our results show that increased levels of 1/4 CTLA-4 exacerbate autoimmunity and cause premature demise of MRL/lpr mice. These data have clinical relevance, because single nucleotide polymorphisms of CTLA4 that have been associated to human autoimmune disease have been shown to regulate alternative splicing of this gene (6).

CTLA-4 is an essential negative regulator of the immune system. Its expression (13) and function (14;15) have been suggested to be abnormal in patients with SLE. However, the relationship between these defects and the polymorphisms associated to SLE in genetic studies is unknown. Moreover, the function(s) of the splice variants of CTLA-4 in health and autoimmune disease are unknown. Our results show that 1/4 CTLA-4 may exist in the serum as a secreted protein. Its lack of the CD80/86 binding domain suggests that it does not block the binding of CTLA-4 but rather exerts its effects through an independent mechanism. Our data indicate that its presence does not affect the expression of full length CTLA-4. Moreover, numbers and distribution of FoxP3+ regulatory T cells are not affected by the transgene. This confirms previous findings in C57BL/6 mice where numbers and in vitro function of regulatory T cells were not altered in mice that express 1/4 CTLA-4 (10).

Our work indicates that the presence of 1/4 CTLA-4 variant facilitates the activation of T and B cells and this probably explains the acceleration in the autoimmune disease observed in the transgenic mice. Further work will investigate more thoroughly the mechanism(s) through which CTLA-4 promotes lymphoid activation. This study highlights the importance of the regulation of the expression of distinct splice variants of complex genes.

Acknowledgments

This work was supported by National Institute of Health Grant R01 A1068787 (G.C.T.)

Footnotes

The authors declare no conflict of interest.

Reference List

  • 1.Walunas TL, Lenschow DJ, Bakker CY, Linsley PS, Freeman GJ, Green JM, et al. CTLA-4 can function as a negative regulator of T cell activation. Immunity. 1994;1(5):405–413. doi: 10.1016/1074-7613(94)90071-x. [DOI] [PubMed] [Google Scholar]
  • 2.Walunas TL, Bakker CY, Bluestone JA. CTLA-4 ligation blocks CD28-dependent T cell activation. J Exp Med. 1996;183(6):2541–2550. doi: 10.1084/jem.183.6.2541. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 3.Takahashi T, Tagami T, Yamazaki S, Uede T, Shimizu J, Sakaguchi N, et al. Immunologic self-tolerance maintained by CD25(+)CD4(+) regulatory T cells constitutively expressing cytotoxic T lymphocyte-associated antigen 4. J Exp Med. 2000;192(2):303–310. doi: 10.1084/jem.192.2.303. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 4.Tivol EA, Borriello F, Schweitzer AN, Lynch WP, Bluestone JA, Sharpe AH. Loss of CTLA-4 leads to massive lymphoproliferation and fatal multiorgan tissue destruction, revealing a critical negative regulatory role of CTLA-4. Immunity. 1995;3(5):541–547. doi: 10.1016/1074-7613(95)90125-6. [DOI] [PubMed] [Google Scholar]
  • 5.Dariavach P, Mattei MG, Golstein P, Lefranc MP. Human Ig superfamily CTLA-4 gene: chromosomal localization and identity of protein sequence between murine and human CTLA-4 cytoplasmic domains. Eur J Immunol. 1988;18(12):1901–1905. doi: 10.1002/eji.1830181206. [DOI] [PubMed] [Google Scholar]
  • 6.Ueda H, Howson JM, Esposito L, Heward J, Snook H, Chamberlain G, et al. Association of the T-cell regulatory gene CTLA4 with susceptibility to autoimmune disease. Nature. 2003;423(6939):506–511. doi: 10.1038/nature01621. [DOI] [PubMed] [Google Scholar]
  • 7.Plenge RM, Padyukov L, Remmers EF, Purcell S, Lee AT, Karlson EW, et al. Replication of putative candidate-gene associations with rheumatoid arthritis in >4,000 samples from North America and Sweden: association of susceptibility with PTPN22, CTLA4, and PADI4. Am J Hum Genet. 2005;77(6):1044–1060. doi: 10.1086/498651. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 8.Hudson LL, Rocca K, Song YW, Pandey JP. CTLA-4 gene polymorphisms in systemic lupus erythematosus: a highly significant association with a determinant in the promoter region. Hum Genet. 2002;111(4–5):452–455. doi: 10.1007/s00439-002-0807-2. [DOI] [PubMed] [Google Scholar]
  • 9.Lee YH, Harley JB, Nath SK. CTLA-4 polymorphisms and systemic lupus erythematosus (SLE): a meta-analysis. Hum Genet. 2005;116(5):361–367. doi: 10.1007/s00439-004-1244-1. [DOI] [PubMed] [Google Scholar]
  • 10.Liu SM, Sutherland APR, Zhang Z, Rainbow DB, Quintana FJ, Paterson AM, et al. Overexpression of the CTLA-4 isoform lacking exons 2 and 3 causes autoimmunity. J Immunol. 2012;188:155–162. doi: 10.4049/jimmunol.1102042. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 11.Tang Q, Boden EK, Henriksen KJ, Bour-Jordan H, Bi M, Bluestone JA. Distinct roles of CTLA-4 and TGF-beta in CD4+CD25+ regulatory T cell function. Eur J Immunol. 2004;34(11):2996–3005. doi: 10.1002/eji.200425143. [DOI] [PubMed] [Google Scholar]
  • 12.Wing K, Onishi Y, Prieto-Martin P, Yamaguchi T, Miyara M, Fehervari Z, et al. CTLA-4 control over Foxp3+ regulatory T cell function. Science. 2008;322(5899):271–275. doi: 10.1126/science.1160062. [DOI] [PubMed] [Google Scholar]
  • 13.Liu MF, Liu HS, Wang CR, Lei HY. Expression of CTLA-4 molecule in peripheral blood T lymphocytes from patients with systemic lupus erythematosus. J Clin Immunol. 1998;18(6):392–398. doi: 10.1023/a:1023226621966. [DOI] [PubMed] [Google Scholar]
  • 14.Hirashima M, Fukazawa T, Abe K, Morita Y, Kusaoi M, Hashimoto H. Expression and activity analyses of CTLA4 in peripheral blood lymphocytes in systemic lupus erythematosus patients. Lupus. 2004;13(1):24–31. doi: 10.1191/0961203304lu488oa. [DOI] [PubMed] [Google Scholar]
  • 15.Jury EC, Flores-Borja F, Kalsi HS, Lazarus M, Isenberg DA, Mauri C, et al. Abnormal CTLA-4 function in T cells from patients with systemic lupus erythematosus. Eur J Immunol. 2010;40(2):569–578. doi: 10.1002/eji.200939781. [DOI] [PubMed] [Google Scholar]

RESOURCES