Increased number of T cells and exacerbated inflammatory pathophysiology in a human IgG4 knock-in MRL/lpr mouse model

Abstract

Immunoglobulin (Ig) G4 is an IgG subclass that can exhibit inhibitory functions under certain conditions because of its capacity to carry out Fab-arm exchange, inability to form immune complexes, and lack of antibody-dependent and complement-dependent cytotoxicity. Although several diseases have been associated with IgG4, its role in the disease pathogeneses remains unclear. Since mice do not express an IgG subclass that is identical to the human IgG4 (hIgG4), we generated hIGHG4 knock-in (KI) mice and analyzed their phenotypes. To preserve the rearrangement of the variable, diversity, and joining regions in the IGH gene, we transfected a constant region of the hIGHG4 gene into C57BL/6NCrSlc mice by using a gene targeting method. Although the mRNA expression of hIGHG4 was detected in the murine spleen, the serum level of the hIgG4 protein was low in C57BL/6-IgG4KI mice. To enhance the production of IgG4, we established an MRL/lpr-IgG4KI mice model by backcrossing. These mice showed a high IgG4 concentration in the sera and increased populations of IgG4-positive plasma cells and CD3+B220+CD138+ T cells in the spleen. Moreover, these mice showed aggravated inflammation in organs, such as the salivary glands and stomach. The MRL/lpr-IgG4KI mouse model established in the present study might be useful for studying IgG4-related disease, IgG4-type antibody-related diseases, and allergic diseases.

Fig 1. Introduction of the human IGHG4 gene into mice.

A. Genetic design of the human IGHG4 knock-in mouse. B. Quantitative real-time PCR of hIGHG4 and mIghg1 in splenocytes. Data were calculated by the 2-Δ·ΔCt method using GAPDH gene as the control and hIGHG4 and mIghg1 genes as the target genes. C. Serum concentration of mIgG1 and hIgG4 in 5 to 8-week-old mice as assessed by enzyme-linked immunosorbent assay (ELISA). *: P < 0.05, **: P < 0.01, ***: P < 0.001, ****: P < 0.0001 by Bonferroni’s multiple t-test (all pairs). hIGHG4, human IGHG4 gene; mIghg1, mouse Ighg1 gene. Hetero, heterozygous; Homo, homozygous; WT, wild-type.

Fig 2. Protein expression of hIgG4 in MRL/lpr-hIgG4KI mice.

A. Serum hIgG4 concentration. B. IgG4-positive cells in erythrosplenic medulla of MRL/lpr-hIgG4KI mouse spleen (16-week-old) as assessed by labelled streptavidin biotinylated antibody (LSAB) staining. C. Immunostained IgG4⁺ cells in 8 to 16-week-old mice. Circle indicates glomerulus.

Fig 3. Hematoxylin and eosin (H&E)-stained tissue sections in MRL/lpr-hIgG4KI homozygous mice.

A. Marked inflammatory cell infiltration around blood vessels and, partially, inflammatory cell infiltration around islets of the pancreas. B. Diffuse inflammatory cell infiltration in the salivary gland. C. Diffuse inflammatory cell infiltration around follicles of the thyroid gland. D. Prominent inflammatory cell infiltration in submucosa of the stomach. E. Marked perivascular inflammatory cell infiltration in the kidney. F. Marked perivascular inflammatory cell infiltration in the lung. G. Marked inflammatory cell infiltration in the prostate gland.

Fig 4. Analysis of splenocytes in MRL/lpr-IgG4KI mice.

A. Percentages of lymphocytes in splenocytes; FSC, Forward Scatter; SSC, Side Scatter. B. Percentage of activated CD69⁺ cells in lymphocytes. C. Percentage of CD4⁺ SP T cells in CD3⁺B220⁻ cells. D. Percentage of CD8⁺ SP T cells in CD3⁺B220⁻ cells. E. Percentage of CD4SP effector (CD44⁺CD62L⁻) T cells in CD3⁺CD4⁺B220⁻ cells. F. Percentage of CD4SP central memory (CD44⁺CD62L⁺) T cells in CD3⁺CD4⁺B220⁻ cells. G. Percentage of CD138⁺ cells in lymphocytes. H. Percentage of CD38⁺ memory B cells in CD3⁻B220⁺ cells. I. Percentage of CD138⁺ plasma cells in CD3⁻B220⁺ cells in the spleens of 18 to 25-week-old MRL/lpr (wild-type) and MRL/lpr-hIgG4KI homozygous mice. P < 0.05 was considered statistically significant (Welch’s test). Wild-type MRL/lpr: male: n = 2, female: n = 6; homozygous MRL/lpr-IgG4KI, male: n = 3, female: n = 5. The gating strategies are shown in S7 Fig in S1 File.