Characterization data for T cell-specific Blimp-1 transgenic C57BL/6 mice

This article is the first to provide characterization data regarding naive C57BL/6 transgenic mice with overexpression of B lymphocyte-induced maturation protein 1 (Blimp-1) under a T cell-specific pLCK promoter. The data presented are related to phenotype, Blimp-1 overexpression levels, T cell development and T cell proliferation for Blimp-1 transgenic mice. For further Blimp-1 overexpressed T cell findings regarding skin allotransplantation, please refer to the research article “Blimp-1 prolongs allograft survival without regimen via influencing T cell development in favor of regulatory T cells while suppressing Th1” (Wang et al., 2018) [1].


Subject area
Molecular biology and immunology More specific subject area Transplant immunology Type of data Figure and table How data was acquired Flow cytometry (BD FACSCantoII) and real-time PCR (Applied Biosystems StepOnePlus)

Data format
Raw and analyzed data Experimental factors The comparison of phenotype, Blimp-1 expression, lymphocyte populations and T cell proliferation between Tg(−) and Tg( þ ) mice Experimental features The phenotype and characterization for naive Blimp-1 transgenic C57BL/ 6 mice Data source location Taoyuan, Taiwan Data accessibility Data are available in this article

Value of the data
The first characterization data for T cell-specific Blimp-1 transgenic C57BL/6 mice. Lymphocyte proliferation data can be used for a further understanding on the Blimp-1 overexpressed T cell-mediated immunology.
Lymphocyte population data is valuable for researchers interested in Blimp-1-modulated T cell development.

Data
This article provides detailed characterization data of Blimp-1 transgenic mice for macroscopic phenotype and organ comparison of one-year-old Tg(−) and Tg(þ ) mice ( Fig. 1A and B). The schematic diagram depicting the transgene construct with a pLck-proximal driven promoter, PCR genotyping and mRNA overexpression of Blimp-1 are indicated in Fig. 2A, B and C, respectively. Table 1 shows the primer sequence information used in the PCR experiments of Fig. 2. The Blimp-1 protein overexpression levels of T cells under unstimulated and stimulated conditions are shown in Fig. 3A and B. T cell development in thymus, spleen and lymph node was examined between naïve Tg(þ ) and Tg(−) mice (Fig. 4A, B and C). Pathological evaluation of various organs from naïve one-year-old mice in both groups is presented in Fig. 5 and Table 2. CD3-dependent lymphocytic and sorted CD4 þ T cell proliferation in both naïve mice are presented in Fig. 6A and B. Fig. 6C evaluates CD4 þ T cell alloreactivity using mix lymphocyte reaction in both naive mice. Fig. 7A and B show blood lymphocyte and CD4 þ T cell subsets in both skin transplanted mice. Fig. 8 shows lymphocyte and CD4 þ T cell subsets from spleen ( Fig. 8A and B) and LNs ( Fig. 8C and D) in both skin transplanted mice. For further Blimp-1 overexpressed T cell findings regarding skin allotransplantation, please refer to the research article "Blimp-1 prolongs allograft survival without regimen via influencing T cell development in favor of regulatory T cells while suppressing Th1" [1].

Mice
T cell-specific Blimp-1 transgenic C57BL/6 mice were kindly provided by Professors HK Sytwu and SJ Chen of the National Defense Medical Center, Taiwan. Transgenic mice were overexpressed with Blimp-1 under a T cell-specific pLCK promoter. All murine procedures were carried out in full compliance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the Chang Gung Memorial Hospital Animal research guidelines. Animal protocols were approved by the Committee on the Ethics of Animal Experiments of the Chang Gung Memorial Hospital (CGMH) in Taiwan and Institutional Animal Care and Use Committees (IACUC) of CGMH in Taiwan under permit numbers IACUC2014032502 and IACUC2016031109.

Real-time polymerase chain reaction (qPCR)
The expression of mRNA for Blimp-1 in the lymphoid organs was analyzed with TaqMan gene expression assays (Thermo Fisher Scientific, Waltham, MA). The expression was normalized to that of Rn18s [3].

Lymphocyte proliferation
Lymphocytes were harvested from naive transgenic mice and stained with Violet Proliferation Dye 450 (VPD450) (BD Biosciences, San Jose, CA). VPD450 labeled lymphocytes (2 × 10 5 cells per well) were stimulated with different concentrations of bound anti-CD3 antibodies for three days. T cell proliferation was then assessed by flow cytometry.

Mixed lymphocyte reaction
Sorted CD4 þ T cells from naive Tg( þ) and Tg(−) mice were stained with VPD450 proliferation dye and responders (2 × 10 5 cells) were co-cultured with stimulator irradiated donor lymphocytes which were irradiated with 2500 rads in a 96-well round bottom plate for three days. T cell proliferation was assessed by flow cytometry.  The thymus from six to eight-week-old Tg(þ ) and Tg(−) mice were harvested and stained for CD4 þ and CD8 þ . (B, C) The spleen and lymph nodes from Tg(þ ) and Tg(−) mice were harvested and stained for CD4 þ , CD8 þ and CD19 þ . The absolute cell number of each cell type was quantified using flow cytometry. The data was collected from 5 mice in each group and from five independent experiments. The statistical data was represented as a mean 7 SEM. *P o 0.05.  Table 2 One year H&E pathological evaluation in organs of naïve Tg(−) and Tg(þ ) mice.
"-" represents negative pathological findings on microscopic examination of various organs. Fig. 6. Lymphocytic proliferation in naïve Tg(þ ) and Tg(−) mice. (A) CD3 receptor-dependent lymphocytic proliferation. Naive lymphocytes were harvested from splenocytes and LNs of eight to ten-week-old Tg( þ ) and Tg(−) mice. Lymphocytes were stimulated with varying concentrations of anti-CD3 mAb for 3 days. The CD4 þ and CD8 þ T cell proliferation were evaluated by VPD-450 dye dilution using flow cytometry. (B) CD3 receptor-dependent sorted CD4 þ T cell proliferation. Naive sorted CD4 þ T cells were harvested and purified from the LNs of eight to ten-week-old Tg( þ) and Tg(−) mice using autoMACS with a purity of more than 90%. Methods used are similar to (A). (C) The proliferation of sorted CD4 þ T cells in response to alloantigens. Mix lymphocyte reaction assay was used to evaluate the CD4 þ T cell alloreactivity. S represents stimulators whereas R the responders. Sorted CD4 þ T cells (responder) from splenocytes and LNs of eight to ten-week-old Tg( þ) and Tg(−) were labeled with VPD-450. They were then cocultured with irradiated CD90.2-depleted lymphocytes from BALB/c donor mice (stimulator) for 3 days. Analysis and quantification was performed using flow cytometry. Similar results were obtained from three independent experiments and represented as a mean 7 SEM. Fig. 7. Pseudocolor plots of flow cytometry for blood lymphocyte and CD4 þ T cell subsets in both skin transplanted mice.
(A) Blood lymphocyte subsets. Blood was taken from each group at POD 10 for lymphocyte subset analysis. The data was collected from 10 mice in each group. (B) Blood CD4 þ T cell subsets. Blood was taken from each group at POD 10 for CD4 þ T cell subset analysis. Intracellular staining technique, using the following stains, was performed: Th1 cells using CD4 þ IFN-γ þ , Th17 cells using CD4 þ IL-17 þ , Th2 using CD4 þ IL-4 þ , IL-10-producing T cells using CD4 þ IL-10 þ and lastly Treg using CD4 þ CD25 þ FoxP3 þ markers. The data was collected from 6 mice in each group. Percentage of inflammatory and antiinflammatory cell subsets in spleen and lymph nodes of Tg( þ ) and Tg(−) mice respectively. Spleen and lymph nodes were harvested from each group at POD 10 for CD4 þ T cell subset analysis. The cells were stained for CD4 þ IFN-γ þ Th1 cells, CD4 þ IL-17 þ Th17 cells, CD4 þ CD25 þ FoxP3 þ Tregs and IL-10-producing T cells using CD4 þ IL-10 þ . The data was collected from 3-6 mice in each group.