RB inactivation in keratin 18 positive thymic epithelial cells promotes non-cell autonomous T cell hyperproliferation in genetically engineered mice

<div><p>Thymic epithelial cells (TEC), as part of thymic stroma, provide essential growth factors/cytokines and self-antigens to support T cell development and selection. Deletion of Rb family proteins in adult thymic stroma leads to T cell hyperplasia <i>in vivo</i>. To determine whether deletion of Rb specifically in keratin (K) 18 positive TEC was sufficient for thymocyte hyperplasia, we conditionally inactivated Rb and its family members p107 and p130 in K18+ TEC in genetically engineered mice (<i>TgK18GT</i>_<i>121</i><i>; K18</i> mice). We found that thymocyte hyperproliferation was induced in mice with Rb inactivation in K18+ TEC, while normal T cell development was maintained; suggesting that inactivation of Rb specifically in K18+ TEC was sufficient and responsible for the phenotype. Transplantation of wild type bone marrow cells into mice with Rb inactivation in K18+ TEC resulted in donor T lymphocyte hyperplasia confirming the non-cell autonomous requirement for Rb proteins in K18+ TEC in regulating T cell proliferation. Our data suggests that thymic epithelial cells play an important role in regulating lymphoid proliferation and thymus size.</p></div

Transgene expression by immunostaining in <i>TgK18GT</i>_<i>121</i> (<i>K18</i>) or induced-<i>K18</i> mice.

<p>(A) Transgene cassette consisting of floxed eGFP stop cassette upstream of truncated SV40 large T antigen (first 121 amino acid; T₁₂₁) was inserted into the 1^st exon of K18 gene on a bacterial artificial chromosome (BAC). Transgene eGFP was driven by K18 regulation. Once K18 mice were crossed to a transgenic mice expressing Cre recombinase, T₁₂₁ was expressed directly under K18 regulation. (B) Representative images of K18 IHC staining in cortex (C) and medulla (M) of <i>WT</i> thymus. Inserts are higher magnification of the images. (C) Representative immunofluorescence images of T₁₂₁ (green), K18 (yellow), K5 (red), and DAPI (blue) in cortex (C) and medulla (M) delineated with a white dotted line, in induced-<i>K18;Cre</i> thymus. Middle and right images are higher magnification of areas in white and red boxes of left image, respectively. Right image: * Cell is positive for T₁₂₁, K18, and K5, and # positive for K5 only. (D) Representative images of K18 (red) and eGFP (green) immunostaining in thymic cortex and medulla (data not shown) of uninduced-<i>K18</i> mice (Cre negative). (E) Representative images of K18 (red) and T₁₂₁ (green) immunostaining in thymic cortex and medulla (data not shown) of induced-<i>K18</i> mice (<i>K18;β-actin Cre</i>).</p

Rb-TS inactivation in K18+ TEC led to decreased survival and thymic hyperplasia.

<p>(A) Kaplan-Meier survival curve of <i>K18;β-actin Cre</i> (n = 74), <i>K18;R26CreER</i> (n = 27), and <i>K18;PbCre4</i> (n = 45) mice with median survival of 94, 41, and 231 days, respectively. Uninduced -<i>K18</i> mice (n = 8) did not develop any gross abnormalities. (B) Gross phenotype of thymuses in <i>WT</i> and <i>K18;β-actin Cre</i> mice. (C) Representative images of H.E. stained thymus sections in <i>WT</i>, <i>K18;R26CreER</i> mice. C: cortex; M: medulla. (D) Representative low magnification images of H.E. stained thymuses in <i>WT</i> and <i>K18;β-actin Cre</i> mice. C: cortex; M: medulla.</p

CBC profile in whole blood of Wildtype and K18;Cre mice.

<p>CBC profile in whole blood of Wildtype and K18;Cre mice.</p