February Core Spotlight: Transgenic Mouse Core

FEBRUARY, 2018

In this DF/HCC News Spotlight Edition, we present the Transgenic Mouse Core at Harvard Medical School. The Transgenic Mouse Core was established in 1992 operating at the BWH, and is now located at Harvard Medical School and the newest DF/HCC core. The Transgenic Mouse Core provides services for the generation of transgenic and knockout mice using state-of-the-art facilities and equipment.

Recent Updates 

The Core has been added to DF/HCC. 

The core's newest services include CRISPR injections, sperm and embryo cryopreservation, and reanimation with fresh or frozen sperm via IVF or the thawing and implanting embryos. 

Key Services

Project Highlights

An obligate cell-intrinsic function for CD28 in Tregs.

PI:  L. TurkaBIDMC, MGH (Cancer Immunology Program)

Tregs expressing the transcription factor FOXP3 are critical for immune homeostasis. The costimulatory molecule CD28 is required for optimal activation and function of naive T cells; however, its role in Treg function has been difficult to dissect, as CD28 is required for thymic Treg development, and blockade of CD28-ligand interactions has confounding effects in trans on nonregulatory cells. To address this question, we created Treg-specific Cd28 conditional knockout mice. Despite the presence of normal numbers of FOXP3+ cells, these animals accumulated large numbers of activated T cells, developed severe autoimmunity that primarily affected the skin and lungs, and failed to appropriately resolve induced experimental allergic encephalomyelitis. This in vivo functional impairment was accompanied by dampened expression of CTLA-4, PD-1, and CCR6. Disease occurrence was not due to subversion of Cd28-deficient Tregs into pathogenic cells, as complementation with normal Tregs prevented disease occurrence. Interestingly, in these "competitive" environments, Cd28-deficient Tregs exhibited a pronounced proliferative/survival disadvantage. These data demonstrate clear postmaturational roles for CD28 in FOXP3+ Tregs and provide mechanisms which we believe to be novel to explain how interruption of CD28-ligand interactions may enhance immune responses independent of effects on thymic development or on other cell types. 

Treg homeostasis. (Left Image)
(A and B) BrdU incorporation assay. 4- to 7-week-old WT or Cd28-ΔTreg mice were pulsed with BrdU every 12 hours for 3 days and sacrificed, and relevant tissues were analyzed by flow cytometry. Representative BrdU staining of gated CD4+FOXP3+ cells is shown in A, and results of each experiment is plotted in B (Het, male Cd28fl/+ × Foxp3YFP-Cre mice; cKO, male Cd28fl/fl × Foxp3YFP-Cre mice). Symbols represent individual mice; horizontal bars indicate the mean. (C) BrdU incorporation in thymectomized mice, as per the protocol in A. CD4+FOXP3+ cells from blood were analyzed at the indicated times after the last BrdU pulse. (D and E) Sorted WT Tregs or Cd28-ΔTregs were labeled with CellTrace Violet and were stimulated by soluble CD3 plus mitomycin-treated T cell–depleted splenocytes (D) with or without rIL-2 or (E) with B7-1/B7-2 double-knockout T cell–depleted splenocytes. 

Cd28-ΔTreg mice develop autoimmunity with skin inflammation.(Right Image)
(A) Gross appearance and lymph nodes and spleens from representative WT littermate control (labeled 1 and 3) and Cd28-ΔTreg (labeled 2 and 4) mice. (B) Cell numbers in lymph nodes and spleens from 2- to 5-month-old mice. 6 littermate pairs were analyzed. Data represent mean ± SEM. (C) H&E staining of skin and lung tissue in a 3-month-old Cd28-ΔTreg mouse. Original magnification, ×400. (D) CD44+CD62Lhi cells in lymph nodes of a representative 4-month-old Cd28-ΔTreg mice. (E) Proportions of CD44+ cells from lymph nodes of 2-month-old mice. 5 littermate pairs were analyzed. Data represent mean ± SEM. (F) Percentage of IFN-γ+ cells in stimulated splenocytes from 2- to 5-month-old littermate pairs. Symbols represent individual mice; horizontal bars indicate the mean. 

Contribution of the Core: The Core carried out the embryo stem cell work on the C57BL/6 background to generate the Cd28-ΔTreg mice. 

Publication:  Zhang R, Huynh A, Whitcher G, Chang J, Maltzman JS, Turka LA.  An obligate cell-intrinsic function for CD28 in Tregs.  J Clin Invest. 2013 Feb;123(2):580-93. PMC3561819.

Deletion of CTLA-4 on regulatory T cells during adulthood leads to resistance to autoimmunity.

PIs: Z.  A. SharpeHMS (Cancer Genetics Program, Cancer Immunology Program); V. KuchrooBWH (Cancer Immunology Program); and G. FreemanDFCI (Cancer Immunology Program, Melanoma Program)

Cytotoxic T lymphocyte antigen-4 (CTLA-4) is an essential negative regulator of T cell responses. Germline Ctla4 deficiency is lethal, making investigation of the function of CTLA-4 on mature T cells challenging. To elucidate the function of CTLA-4 on mature T cells, we have conditionally ablated Ctla4 in adult mice. We show that, in contrast to germline knockout mice, deletion of Ctla4 during adulthood does not precipitate systemic autoimmunity, but surprisingly confers protection from experimental autoimmune encephalomyelitis (EAE) and does not lead to increased resistance to MC38 tumors. Deletion of Ctla4 during adulthood was accompanied by activation and expansion of both conventional CD4+Foxp3 (T conv) and regulatory Foxp3+ (T reg cells) T cell subsets; however, deletion of CTLA-4 on T reg cells was necessary and sufficient for protection from EAE. CTLA-4 deleted T reg cells remained functionally suppressive. Deletion of Ctla4 on T reg cells alone or on all adult T cells led to major changes in the Ctla4 sufficient T conv cell compartment, including up-regulation of immunoinhibitory molecules IL-10, LAG-3 and PD-1, thereby providing a compensatory immunosuppressive mechanism. Collectively, our findings point to a profound role for CTLA-4 on T reg cells in limiting their peripheral expansion and activation, thereby regulating the phenotype and function of T conv cells.

Deletion of Ctla4 leads to EAE resistance and expanded, apparently activated, T cell populations. Ctla4fl/fl mice that were either UBCCre/ERT2- (Cre) or UBCCre/ERT2+ (Cre+) were treated with tamoxifen and immunized with MOG35-55 to induce EAE. Clinical scores are shown in A and representative CNS pathology is shown in C. CD4+ cells from cervical LNs of mice immunized with MOG35-55 to induce EAE were analyzed at the peak of disease for (B) the number of CD4+Foxp3 and CD4+Foxp3+ cells, (D) the percentage of Foxp3+ cells, (E) Ki-67 expression by intracellular staining, and (F) CD62L and CD44 expression by surface staining within the cervical LN. (E, right) Representative dot plots gated on CD4+, for which mean data are plotted on the left. (G) Brains and spinal cords from MOG35-55 immunized mice were analyzed at the onset of disease for percentage of Foxp3+ cells and CTLA-4 expression. Data are gated on CD4+ T cells. Data in each panel are representative of at least two independent experiments (mean and SEM for at least 7 mice per group). Bars: 200 µm (100× magnification); 50 µm (400× magnification). *, P < 0.05; ***, P < 0.001.

Contribution of the Core: The core carried out the embryo stem cell work to generate the CTLA-4 conditional knockout mouse Es cells on the C57BL/6 background and performed the blastocyst injection to generate the mice. 

Publication: Paterson AM, Lovitch SB, Sage PT, Juneja VR, Lee Y, Trombley JD, Arancibia-Cárcamo CV, Sobel RA, Rudensky AY, Kuchroo VK, Freeman GJ, Sharpe AH. Deletion of CTLA-4 on regulatory T cells during adulthood leads to resistance to autoimmunity. J Exp Med. 2015 Sep 21;212(10):1603-21.PMC4577848.

Defect in regulatory B-cell function and development of systemic autoimmunity in T-cell Ig mucin 1 (Tim-1) mucin domain-mutant mice.  

PI:  V. KuchrooBWH (Cancer Immunology Program); J. BonventreBWH (Kidney Cancer Program); and S. XiaoBWH (Cancer Genetics)

Tim-1, a type I transmembrane glycoprotein, consists of an IgV domain and a mucin domain. The IgV domain is essential for binding Tim-1 to its ligands, but little is known about the role of the mucin domain, even though genetic association of TIM-1 with atopy/asthma has been linked to the length of mucin domain. We generated a Tim-1–mutant mouse (Tim-1Δmucin) in which the mucin domain was deleted genetically. The mutant mice showed a profound defect in IL-10 production from regulatory B cells (Bregs). Associated with the loss of IL-10 production in B cells, older Tim-1Δmucin mice developed spontaneous autoimmunity associated with hyperactive T cells, with increased production of IFN-γ and elevated serum levels of Ig and autoantibodies. However, Tim-1Δmucin mice did not develop frank systemic autoimmune disease unless they were crossed onto the Fas-mutant lpr mice on a C57BL/6 background. Tim-1Δmucinlpr mice developed accelerated and fulminant systemic autoimmunity with accumulation of abnormal double-negative T cells and autoantibodies to a number of lupus-associated autoantigens. Thus, Tim-1 plays a critical role in maintaining suppressive Breg function, and our data also demonstrate an unexpected role of the Tim-1 mucin domain in regulating Breg function and maintaining self-tolerance.

Generation and characterization of Tim-1Δmucin mice. (A) The gene structures of the WT Tim-1 allele, the Tim-1Δmucin targeting construct, and the targeted Tim-1 allele. Colored boxes represent coding sequences; Roman numerals represent exons. E, EcoR I sites. (B) (Left) CD19+ B cells isolated from WT and Tim-1Δmucin mice were used to determine Tim-1 mRNA expression by RT-PCR. (Right) 293T cells were overexpressed with WT Tim-1 and Tim-1Δmucin and then were used to determine Tim-1 protein expression by Western blot. (C and D) RT-PCR products were sequenced, and parts of cDNA and predicted encoding amino acid sequences of WT Tim-1 (C) and Tim-1Δmucin (D) are shown. (E) Isolated WT and Tim-1Δmucin B cells were examined for Tim-1 expression by flow cytometry. (F) Schematic representation of Tim-1 and Tim-1Δmucin protein structures.

Contribution of the Core: The Core carried out the embryo stem cell work to generate the Tim-1Δmucin mice on the C57BL/6 background. 

Publication: Xiao S, Brooks CR, Zhu C, Wu C, Sweere JM, Petecka S, Yeste A, Quintana FJ, Ichimura T, Sobel RA, Bonventre JV, Kuchroo VK. Defect in regulatory B-cell function and development of systemic autoimmunity in T-cell Ig mucin 1 (Tim-1) mucin domain-mutant mice. Proc Natl Acad Sci U S A. 2012 Jul 24;109(30):12105-10. PMC3409739.

More Information

For more information for the Transgenic Mouse Core, visit the core website here