Effects of thymus size and involution on the contribution of recent thymic emigrants to the peripheral T cell pool

The contribution of recent thymic emigrants (RTEs) to the peripheral naïve T cell population is necessary to maintain diversity of the T cell receptor (TCR) repertoire and produce immune responses against newly encountered antigens. The thymus involutes with age, after irradiation or chemotherapy, and due to severe viral infections. Thymus involution results in decreased thymopoiesis and RTE output leading to a reduced diversity of peripheral T cells. This increases susceptibility to disease and impairs immune responsiveness to vaccines. Therefore, studies aimed at maintaining or regenerating thymic function are integral for maintaining and restoring peripheral TCR diversity. Mice that express a K5.CyclinD1 transgene expression have a severely hyperplastic thymus that fails to undergo involution. Both thymocyte and TEC development appear normal in these mice. We have used the K5.CyclinD1 transgenic model to test the hypothesis that preventing thymus involution will sustain RTE output and incorporation into the peripheral T cell pool to prevent naïve T cell depletion with age. The K5.CyclinD1 transgene was crossed to the RAG2p-GFP transgenic model so that RTEs could be tracked by the intensity of the GFP signal. The frequency and number of RTEs in naïve CD4 splenic T cells was analyzed at monthly intervals to 5 months of age. Using this double transgenic approach, we determined that preventing thymus involution does maintain or enhance the number of RTEs in the peripheral T cell pool before and after thymus involution

PREVENTING THYMUS INVOLUTION IN K5.CYCLIN D1 TRANSGENIC MICE SUSTAINS THE NAÏVE T CELL COMPARTMENT WITH AGE

The thymus maintains T cell receptor (TCR) repertoire diversity through perpetual release of self-MHC restricted naive T cells. However, thymus involution during the aging process reduces naïve T cell output, leading to defective immune responsiveness to newly encountered antigens. We have found that early thymus involution precipitates the age-associated shift favoring memory T cell dominancy in young control mice. Furthermore, we have shown that age-related thymus involution is prevented in mice expressing a keratin 5 promoter-driven Cyclin D1 (K5.D1) transgene in thymic epithelial cells (TECs). Thymopoiesis occurs normally in K5.D1 transgenic thymi and sustains T cell output to prevent the age-associated decline of naïve T cells in the periphery. We find that K5.D1 recent thymic emigrants (RTEs) undergo typical phenotypic maturation. In addition, functional studies show that K5.D1 peripheral T cells are responsive to anti-CD3 and anti-CD28 stimulation in vitro. Competitive adoptive transfer studies with K5.D1 and nontransgenic RTEs indicate that K5.D1 RTEs are incorporated into the peripheral T cell pool comparably to nontransgenic controls. However, K5.D1 mature naïve (MN) T cells out-persist control counterparts, which likely occurs, in part, to increased expression of the cytokine receptor IL-7Rα. Collectively, these data show that preventing thymus involution not only sustains T cell output to maintain naïve T cell numbers, but also provides functionally competent MN T cells during aging

Association between Obesity, Race or Ethnicity, and Luminal Subtypes of Breast Cancer

Luminal breast cancers are the most common genomic subtype of breast cancers where Luminal A cancers have a better prognosis than Luminal B. Exposure to sex steroids and inflammatory status due to obesity are key contributors of Luminal tumor development. In this study, 1928 patients with Luminal A breast cancer and 1610 patients with Luminal B breast cancer were compared based on body mass index (BMI), age, race, menopausal status, and expressed receptors (i.e., estrogen (ER), progesterone (PR), and human epidermal growth factor receptor 2 (HER2)). Patients with Luminal B tumors had a significantly higher mean BMI (Δ = 0.69 kgm−2 [0.17, 1.21], p = 0.010) versus Luminal A. Interestingly, the risks of Luminal B tumors were higher among Black/African American patients versus White and Hispanic patients (p < 0.001 and p = 0.001, respectively). When controlled for each other, Black/African American race (p < 0.001) and increased BMI (p = 0.008) were associated with increased risks of Luminal B carcinoma, while postmenopausal status was associated with a decreased risk (p = 0.028). Increased BMI partially mediated the strong association between Black/African American race and the risk of Luminal B carcinoma. Thus, Black/African American race along with obesity seem to be associated with an increased risk of more aggressive Luminal B breast carcinomas

Stat3 Signaling Promotes Survival And Maintenance Of Medullary Thymic Epithelial Cells

<div><p>Medullary thymic epithelial cells (mTECs) are essential for establishing central tolerance by expressing a diverse array of self-peptides that delete autoreactive thymocytes and/or divert thymocytes into the regulatory T cell lineage. Activation of the NFκB signaling pathway in mTEC precursors is indispensable for mTEC maturation and proliferation resulting in proper medullary region formation. Here we show that the Stat3-mediated signaling pathway also plays a key role in mTEC development and homeostasis. Expression of a constitutively active Stat3 transgene targeted to the mTEC compartment increases mTEC cellularity and bypasses the requirement for signals from positively selected thymocytes to drive medullary region formation. Conversely, conditional deletion of Stat3 disrupts medullary region architecture and reduces the number of mTECs. Stat3 signaling does not affect mTEC proliferation, but rather promotes survival of immature MHCII^loCD80^lo mTEC precursors. In contrast to striking alterations in the mTEC compartment, neither enforced expression nor deletion of Stat3 affects cTEC cellularity or organization. These results demonstrate that in addition to the NFkB pathway, Stat3-mediated signals play an essential role in regulating mTEC cellularity and medullary region homeostasis.</p></div

Expression of K5.Stat3C enhances MHCII^loCD80^lo mTEC survival, but not proliferation.

<p><b>(A)</b> Representative FACS histograms showing frequency of BrdU positive cells in MHCII^hi and MHCIl^lo mTEC subsets from K5.Stat3C (thick black line) and control (shaded) thymi. <b>(B)</b> Scatter graphs showing percentage of BrdU positive cells. <b>(C)</b> Representative FACS histograms showing frequency of cleaved caspase 3 (CC3) positive cells in MHCII^hi and MHCIl^lo mTEC subsets from K5.Stat3C (thick black line) and control (shaded) thymi. <b>(D)</b> Scatter graphs showing percentage of CC3 positive cells. <b>(E)</b> Quantitative RT-PCR analysis of <i>Bcl-2</i> expression in MHCII^hi and MHCIl^lo mTECs. Relative expression of <i>bcl-2</i> mRNA was normalized using α-tubulin mRNA and the NTg MHCIl^hi mTEC control was set at 1. Bar graph shows mean ± SEM of four independent experiments with duplicate or triplicate samples in each experiment. *P<0.05 (Student’s unpaired <i>t</i>-test). <b>(F)</b> Representative FACS histograms showing Bcl-2 expression in MHCII^hi and MHCIl^lo mTEC subsets from K5.Stat3C (thick black line) and control (shaded) thymi.</p

K5.Stat3C expression generates medullary regions in RAG-2^-/- thymi.

<p>(a-d) Serial frozen sections of RAG-2^-/- and RAG-2^-/-;K5.Stat3C thymi. <b>(A)</b> H&E stain shows medullary region expansion in RAG-2^-/-;K5.Stat3C thymi. The dotted line separates cortex (C) and medulla (M). (b-d) IHC staining showing expression of <b>(B)</b> K5 and K8; <b>(C)</b> K14, UEA-1 and DAPI; <b>(D)</b> Flag epitope. Data are representative of at least 5 mice of each genotype. <b>(E</b>) FACS analysis of CD4-CD8- thymocyte subsets identified, by CD25 and CD44 expression, from wildtype, RAG-2^-/- and RAG-2^-/-;K5.Stat3C mice. The percentage of cells in each subset is shown on the FACS plots. FACS data are representative of 3 mice from each genotype. Scale bar: 200μm.</p

Reduced thymus cellularity and increased medulla to cortex ratio in K5.Stat3C transgenic mice.

<p><b>(A)</b> IHC staining showing expression of the Flag tag in medullary regions of K5.Stat3C transgenic thymi. <b>(B)</b> Western blots show the Flag tag is expressed in CD45- stromal cells, but not in CD45+ hematopoietic cells. Elevated levels of phosphorylated Stat3 are also restricted to stromal cells. <b>(C)</b> Total thymus cellularity is reduced in K5.Stat3C transgenic (n = 9 for all) compared to control NTg littermates. **<i>P</i><0.005 (Student’s paired <i>t</i>-test) <b>(D)</b> Medulla/cortex ratio in K5.Stat3C (n = 4) and control (n = 5) thymi determined by morphometric analysis of Aperio scanned H&E stained sections. *P<0.05 (Student’s paired <i>t</i>-test) (<b>E-H</b>) H&E and immunofluorescence (IF) stains of serial frozen thymus sections. <b>(E)</b> Low power image of H&E stained thymus section; the area demarcated by the dotted white is magnified in f-g. <b>(F)</b> H&E stain; <b>(G)</b> K5 and K8; <b>(H)</b> K14 and UEA-1. Scale bar: 200μm. Images are representative of sections from at least 5 NTg control and 5 K5.Stat3C transgenic thymi.</p

MHCII^loCD80^lo mTECs are selectively expanded in K5.Stat3C transgenic thymi.

<p><b>(A)</b> Representative FACS plots show percentage of MHCII^loCD80^lo and MHCII^hiCD80^hi mTECs. <b>(B)</b> Bar graph shows number (mean ± SD) of MHCII^lo and MHCII^hi mTECs in K5.Stat3C and control thymi (n = 5 each). <b>(C)</b> FACS plots show percentage of MHCII^hiAire⁺ mTECs. <b>(D)</b> Bar graph shows number (mean ± SD) of MHCII^hiAire⁺ mTECs (n = 5). <b>(E)</b> IHC stains show abundant K14+ and Aire+ mTECs in K5.Stat3C thymi. Scale bar: 200μm. <b>(F)</b> The frequency of involucrin+ mTECs is not increased in K5.Stat3C thymi. Scale bar: 100μm. <b>(G)</b> IHC stains show K8 expressing cTECs and CD11c expressing DCs. Scale bar: 200μm. *P<0.05 (Student’s paired <i>t</i>-test).</p

mTECs are selectively depleted in Stat3 CKO thymi.

<p><b>(A)</b> Representative FACS plots show the frequency of CD45-EpCAM+ TECs in Stat3 CKO and control thymi. <b>(B)</b> Bar graph shows (mean ± SD) cellularity of total TECs, cTECs and mTECs (n = 5 for each) <b>(C)</b> Representative FACS plots show the frequency of MHCII^loCD80^lo and MHCII^hiCD80^hi mTECs in Stat3 CKO and control thymi <b>(D)</b> Bar graph shows number (mean ± SD) of MHCII^lo and MHCII^hi mTECs (n = 5 for each). <b>(E)</b> Representative FACS plots show frequency of MHCII^hiAire⁺ mTECs. <b>(F)</b> Bar graph shows number (mean ± SD) of MHCII^hiAire⁺ mTECs (n = 5). *P<0.05 and **P<0.005 (Student’s paired <i>t</i>-test)</p

Depletion of Stat3 in the TEC compartment disrupts medullary architecture.

<p><b>(A)</b> Thymus cellularity is comparable in Stat3 CKO (n = 9 for both) and controls. (p = 0.9960, Unpaired Student’s T test) <b>(B)</b> FACS plots show comparable frequency of major thymocyte subsets defined by CD4 and CD8 expression. The percentage of cells in each quadrant is shown. Results are representative of 5 mice of each genotype. <b>(C)</b> Low power image of H&E stained frozen thymic sections. <b>(D-H)</b> IHC stains on frozen sections of CKO and control thymi <b>(D)</b> H&E <b>(E)</b> K5 and K8 <b>(F)</b> K14, UEA-1 and DAPI <b>(G)</b> CD11c and K8 <b>(H)</b> K14 and Aire. All images are serial sections with the exception of the control section in H. Results are representative of at least 5 thymi of each genotype. Scale bar: 200μm.</p