Lin28 regulates thymic growth and involution and correlates with MHCII expression in thymic epithelial cells

Thymic epithelial cells (TECs) are essential for T cell development in the thymus, yet the mechanisms governing their differentiation are not well understood. Lin28, known for its roles in embryonic development, stem cell pluripotency, and regulating cell proliferation and differentiation, is expressed in endodermal epithelial cells during embryogenesis and persists in adult epithelia, implying postnatal functions. However, the detailed expression and function of Lin28 in TECs remain unknown. In this study, we examined the expression patterns of Lin28 and its target Let-7g in fetal and postnatal TECs and discovered opposing expression patterns during postnatal thymic growth, which correlated with FOXN1 and MHCII expression. Specifically, Lin28b showed high expression in MHCIIhi TECs, whereas Let-7g was expressed in MHCIIlo TECs. Deletion of Lin28a and Lin28b specifically in TECs resulted in reduced MHCII expression and overall TEC numbers. Conversely, overexpression of Lin28a increased total TEC and thymocyte numbers by promoting the proliferation of MHCIIlo TECs. Additionally, our data strongly suggest that Lin28 and Let-7g expression is reliant on FOXN1 to some extent. These findings suggest a critical role for Lin28 in regulating the development and differentiation of TECs by modulating MHCII expression and TEC proliferation throughout thymic ontogeny and involution. Our study provides insights into the mechanisms underlying TEC differentiation and highlights the significance of Lin28 in orchestrating these processes.


Introduction
Lin28 is a small mRNA-binding protein that blocks processing of the Let-7 miRNA.Both Lin28 and Let-7 were originally identified by their important roles in developmental timing control in C. elegans (1-3) and are evolutionarily conserved in sequence and function from worms to humans, which highlights the important role of these two ancient small molecules (4,5).Lin28 and Let-7 are frequently expressed in opposing patterns during embryonic development and differentiation, in the early postnatal period, and in adults.In general, Lin28 is expressed in stem and immature cells and is generally considered to promote pluripotency, while Let-7 is expressed in mature cells and promotes differentiation.Lin28 specifically suppresses the expression and function of Let-7, and in turn, Let-7 feeds back on Lin28 (6, 7).Importantly, both Lin28 and Let-7 also have the broad capability of regulating the expression of multiple genes.Thus, the Lin28/Let-7 system forms a regulatory axis that plays critical roles in a wide range of physiological development and pathogenesis in many organisms, including humans (8,9).These processes include stem cell self-renewal and differentiation (10,11), glucose metabolism (8), tissue repair (12), hematopoietic progenitor reprogramming (13)(14)(15), and the attraction of inflammatory endothelium into cancer cells (16).Additionally, Lin28 may act as an oncogene, while Let-7 acts as a cancer suppressor in a variety of human cancers; an imbalance in the expression of both genes has been linked to the formation, progression, diagnosis, and therapy of multiple cancer types (16)(17)(18)(19).
Lin28/Let-7 has different expression patterns with regard to level, timing, and function during the fetal to postnatal period (20-23), with studies focused mostly on tissues of germline, ectodermal, and mesodermal origin (24).Less progress has been made on endodermal cell types; however, the general pattern of Lin28 expression in progenitors and Let-7 expression in more differentiated cells is also seen in the epithelia of the small intestine and the seminiferous epithelium of the testis (25,26).Lin28 has two orthologs in mammals, Lin28a and Lin28b (22,23), which have divergent temporal and cellular expression patterns in the seminiferous epithelium, with LIN28a expressed in undifferentiated A cells and LIN28b in spermatid cells at a later stage (25,26).Let-7s also show opposing profiles to that of Lin28b in this adult tissue (26).There are also temporal dynamics for Lin28 expression; Lin28a mRNA levels peak in neonates and Lin28b in young adults during postnatal maturation (25,26).Lin28b expression in the seminiferous epithelium in mice is also consistent with genome-wide association studies in which the Lin28b locus was associated with the timing of puberty at menarche and height in humans (27).
Although Lin28b (but not Lin28a) and Let-7g have been reported to be expressed in fetal and early postnatal thymocytes (13) and thymic pro B cells (28), their expression and function in thymic epithelial cells (TECs) is poorly characterized.Let-7 has 12 homologs in mice (5,29), of which only Let-7g and Let-7f2 have relatively high expression levels in TECs (30).Let-7g, but not Let-7f2, also has predicted sites in the 3' and 5'UTRs (untranslated regions) of the Lin28a and Lin28b genes (31,32), suggesting that these genes form a negative regulatory loop.The thymic primordium initiates from the endoderm of the third pharyngeal pouches in mid-gestation (33).Endoderm-derived TECs are critical for most aspects of T cell development from thymocytes.FOXN1 is a key transcription factor required for TEC proliferation, differentiation, and homeostasis that is expressed in TECs from embryonic day 11.25 (E11.25)(33)(34)(35)(36).The thymus also undergoes age-associated degeneration, called thymic involution, which is associated with reduced thymocyte number and T cell production.Current evidence suggests that this decline is initiated primarily due to the decline in Foxn1 expression in TECs (37).However, the mechanisms by which FOXN1 regulates these various aspects of TEC biology have not been fully investigated.
In this study, we investigated the expression and function of Lin28 and Let-7 in TECs.Lin28a was expressed at very low levels or undetected in fetal and postnatal TECs.Lin28b and Let-7g were detected in TEC progenitors at E13.5; their expression increased from fetal to adult stages but then declined by 6 months of age, associated with the process of age-related thymic involution.However, their expression showed opposite patterns in relation to major histocompatibility complex (MHC) class II (MHCII) hi and MHCII lo TECs.Lin28b genes were highly expressed in MHCII hi TECs and expressed at very low or undetectable levels in MHCII lo TECs, while the Let-7g gene was highly expressed in MHCII lo and expressed at very low levels in MHCII hi TECs.Immunofluorescence staining showed that both Lin28a and Lin28b were differentially correlated with FOXN1 and MHCII expression.TEC-specific Lin28a gain and Lin28a and Lin28b loss of function showed that Lin28a promoted the proliferation of MHCII lo TECs and that both Lin28a and Lin28b were required for maintaining MHCII expression in TECs.We also showed that Foxn1 expression was correlated with both Lin28b and Let-7g expression during ontogeny and that reducing Foxn1 expression in Foxn1 Z/Z mice reduced Lin28 and Let-7g expression.Lin28b, but not Let-7g, expression increased during thymic rebound induced by sex steroid ablation.In summary, our findings indicate that Lin28 and Let-7 may both modulate MHCII expression and regulate thymic growth and involution by controlling TEC proliferation in the postnatal thymus.

Mice
C57BL6/J mice were purchased from Jackson Laboratory.Foxn1 lacZ/lacZ (Z/Z) and Foxn1 Cre mice were generated by Dr. Manley's laboratory as described previously and are maintained on a C57Bl6/J background (37,38).Lin28a fl/fl b fl/fl mice (Lin28b tm2.1GqdaLin28b tm2.1Gqda /J, stock No. 02395) were purchased from the Jackson Laboratory (39).R26iLin28a inducible transgenic mice were originally provided by Eric Moss (Department of Molecular Biology, Rowan University, Stratford, NJ 08084 USA) and transferred from Dr. Jianfu Chen (Department of Genetics, University of Georgia.Current address: Herman Ostrow School of Dentistry of USC) (40).The Lin28a fl/fl b fl/fl ;Foxn1 Cre TECspecific deletion of Lin28a and b and R26iLin28a;Foxn1Cre TECspecific overexpression of Lin28a mice were generated by crossing the Lin28a fl/fl b fl/fl strain or the R26iLin28a strain with the Foxn1 Cre mouse strain, respectively.All analyses were performed using littermate animals whenever possible.Data from both male and female mice were combined because no difference was detected based on sex.All mice were maintained in a pathogen-free facility at the University of Georgia.The experiments were approved by the University of Georgia Institutional Animal Care and Use Committee.
For TEC isolation, one thymic lobe (fetal thymi were mixed) was cut into approximately 1 mm 3 sections and gently washed in 2% FBS RPMI 1640 medium to remove thymocytes.The thymic pieces were digested in 5 ml of collagenase/dispose (1 mg/ml, Roche) plus DNase I (20 ng/mL, Sigma) in 2% FBS RPMI 1640 medium, placed in a 37°C water bath for 60 minutes, and agitated by passing through an 18G needle 8 times and a 25G needle twice.Cells were then filtered by passing through a 70 mm cell strainer.
For TEC sorting, the digested cells were incubated with anti-CD45 APC, EpCAM PE, and MHCII FITC.The CD45 - Epcan + MHCII + cells were sorted as TECs and separated into MHCII hi and MHCII lo subpopulations by MoFlo-DXP cytometry (Dako Cytomation).All antibodies were purchased from Biolegend if not noted (San Diego, CA).Phenotypical analysis was performed with a Cyan ADP Flow Cytometer (Beckman Coulter, Miami, FL).The data were analyzed by Flowjo ™ Software (Tree Star, Ashland, OR).

BrdU incorporation and Annexin V staining
Each mouse was given a single intraperitoneal injection.with 1 mg of BrdU (Sigma−Aldrich) and hydrated with BrdU-containing water (0.8 mg/ml) for 5 days.One thymic lobe was digested for TEC, and one was ground for thymocyte analysis.For BrdU staining, freshly isolated thymocytes were incubated with anti-CD4-PE-Cy7, CD8-APC-Cy7, CD25-PerCp, and CD44-APC antibodies.The digested cells were incubated with anti-CD45 PE-Cy7, EpCAM APC, MHCII PE, and UEA-1-Biotin following Avidin-APC-Cy7.The surface-stained cells were then fixed and permeabilized in PBS containing 1% paraformaldehyde plus 0.01% Tween 20 for 48 h at 4°C and then incubated with 2 mg/ml DNase I for 15 min in a 37°C water bath.FITC-anti-BrdU Ab (clone 3D4; BD Pharmingen) was used for BrdU staining according to the manufacturer's instructions.For Annexin V staining, freshly isolated thymocytes (5 X 10 5 ) or total digested thymic cells (1 X 10 6 ) were incubated with surface antibodies and then incubated with Annexin V FITC and PI in Annexin V binding buffer.The samples were analyzed within 1 hour following the protocol of the Annexin V kit (Biolegend, San Diego, CA).

RT−PCR and Q-PCR
The fetal thymi and sorted total TECs or MHCII lo and MHCII hi TEC subsets were extracted for mRNA by the RNeasy micro kit (QIAGEN).Reverse transcription PCR was performed with the superscript III system (Invitrogen).Gene expression levels of Lin28a (Mm00524077_m1), Lin28b (Mm01190673_m1), Let-7g (Mm04231484_s1), and H2-IAb (00439216_m1) in total TECs or MHCII lo and MHCII hi subsets of TECs were measured by Q-PCR, with a GAPDH FAM (Mm99999915_g1) primer/probe used as an endogenous control.All primers/probes were ordered from Applied Biosystems.Q-PCR was performed following the manufacturer's instructions in a 10 ml volume using the AB 7500 Sequence Detector.

Sex steroid ablation
Mice were anesthetized using isoflurane anesthesia and then placed in dorsal recumbency.A 0.5-1 cm ventral midline incision was made in the scrotum, and the skin retracted to expose the tunica.The tunica was pierced, and the testes were pushed out sequentially.The testes were raised to expose the underlying blood vessels and tubules.Mouse testes were removed using forceps; cauterization was not needed.All deferential vessels and ducts were replaced back into the tunica.Skin incisions were closed with stainless steel wound clips.All of the excised paired tissues from each animal were retained to verify completeness of removal.Sham castration was performed as described above except for the removal of the testes.

Statistical analysis
All data were collected in a Microsoft Excel file and analyzed using Prism software (GraphPad Software, Boston, MA 02110) by one-way analysis of variance (ANOVA)-Bonferroni test or Student's t test, P value in two-tailed.For statistical analysis, the significance was set at P ≤ 0.05.

Lin28a and Lin28b are differentially expressed in fetal TECs
To assess LIN28 in the fetal thymus, we first performed immunofluorescence (IF) on E13.5 and E18.5 thymi from C57BL6/J WT mice using LIN28a and LIN28bspecific antibodies.No LIN28a signal was detected in the E13.5 and E18.5 thymi (Supplementary Figures 1A, B).At E13.5, LIN28b was detected in the cytoplasm of both FOXN1 -negative cells (FOXN1 -) (Figure 1A, white arrows) and in a minor subset of FOXN1-positive (FOXN1 + ) TECs (Figure 1A, pink arrows).A small number of MHCII + K14 + early medullary TECs (mTECs) were also detected in the center of the thymus but were found to be LIN28b negative (Figure 1B, Supplementary Figures 1C, D).At E18.5, LIN28b was found to be expressed in a subset of TECs in both the cortex and medulla, colocalizing with FOXN1 and MHCII (Figures 1C, D; pink arrows).Quantitative analysis showed the frequency of LIN28b + cells increased in total FOXN1 + cells (Figure 1E).Some LIN28b lo FOXN1 -cells were also detected in the outer area but were absent from the central area of the thymi, which might represent early developing thymocytes (Figures 1C, D; white arrows).
To further investigate expression in fetal TECs, we analyzed the gene expression of Lin28a and b and Let-7g in fetal TEC subsets sorted based on EpCAM and MHCII expression at E13.5 and E18.5.At E13.5, all TECs are MHCII negative or low (Supplementary Figures 2A-D).Lin28a was not detected or was present at very low levels in all fetal TEC subsets.Lin28b was correlated with the expression of MHCII, increasing significantly in MHCII lo compared to MHCII -TEC at E13.5, increasing further at E18.5 in MHCII lo TEC, and increasing two-fold higher in MHCII hi than in MHCII lo at E18.5 (Figure 1F).Let-7g expression also correlated with MHCII expression and was higher in MHCII lo TECs than MHCII - TECs at E13.5 and higher in MHCII hi than MHCII lo TECs at E18.5.However, at E18.5, expression declined in MHCII lo TECs relative to that at E13.5 (Figure 1F), suggesting more complex regulation.These results indicated that Lin28b and Let7g but not Lin28a may be involved in TEC differentiation during fetal thymic development.

Lin28b and Let-7g are anticorrelated in TEC subsets in the postnatal thymus
To understand Lin28 and Let-7 expression in the postnatal thymus, we first assessed LIN28a and LIN28b at the protein level in postnatal TECs.Sections from C57BL6/J WT thymi obtained at 2 months of age were stained along with FOXN1 and MHCII.Both LIN28a and LIN28b in the medulla colocalized with both FOXN1 (Figures 2A, B) and MHCII (Figures 2C, D), indicating that they are each expressed in a subset of mTECs at this stage.Quantitative analysis showed the frequency of LIN28b + cells in total FOXN1 + cells was higher than LIN28a + cells (Figure 2E).We further investigated the gene expression of Lin28a, Lin28b, and Let-7g in sorted MHCII lo and MHCII hi TEC subsets from 2-month-old WT mice (Figure 2F).Both Lin28a and Lin28b were preferentially expressed in MHCII hi TECs with little or no expression in MHCII lo TECs.Lin28b expression was 2.8-fold higher than that of Lin28a.Conversely, Let-7g was almost exclusively expressed in MHCII lo TECs (Figure 2F).This opposing expression profile is consistent with the known reciprocal regulation pattern for Lin28 and Let-7 in other tissues.These patterns for Lin28a and Lin28b are at least broadly consistent with Let-7g expression.However, the Let-7g pattern is the opposite of that observed at E18.5, where Let-7g was in both MHCII subsets but higher in MHCII hi (Figure 1F), which suggests complex regulation of this miRNA gene.

Temporal dynamics of Lin28a, Lin28b, and Let-7g expression with aging
We next traced the temporal dynamics of Lin28a, Lin28b, and Let-7g expression in postnatal MHCII lo and MHCII hi TEC subsets.The expression of both Lin28a and Lin28b gradually increased from 1 week, peaked at 1 month, and then gradually declined at 2 and 6 months in MHCII hi TECs; a parallel pattern was found at a much lower level for Lin28b in MHCII lo TECs (Figures 2G, H).Let-7g showed a similar temporal profile but in MHCII lo TECs, with very low levels in MHCII hi TECs (Figure 2I).
Consistent with these gene expression kinetics, IF staining showed that LIN28b was present at a low level in a subset of FOXN1 + mTECs at day 20 (Figure 2J), increasing in cell number and intensity per cell at 2 months (Figure 2K), then dramatically declined or was undetectable at 6 months (Figure 2L).These data indicate that the expression of Lin28a, Lin28b, and Let-7g is correlated with both thymic growth and the early stages of ageassociated involution.Specific deletion of Lin28a and Lin28b in TECs caused a reduction in TECs and generated fewer thymocytes in the postnatal thymus Lin28a and Lin28b single and double null mutants have a variety of defects that could confound analysis of their function in TECs (20, 21); therefore, we generated TEC-specific deletion of Lin28a and Lin28b using Foxn1 +/Cre (38).Since similar genetic and phenotypic analysis results were shown at multiple time points, we show here representative results from 2-month-old mice.Q-PCR analysis showed that at 2 months, both Lin28a and Lin28b transcripts were undetectable in Lin28a fl/fl b fl/fl ;Foxn1 Cre/+ double mutant TECs and that only Lin28a was detected in the MHCII hi TECs from Lin28a +/+ b fl/fl ;Foxn1 Cre/+ single mutants (Figures 3A, B).Specificity of LIN28a and LIN28b antibody staining on the thymic sections from Lin28a fl/fl b fl/fl ;Foxn1 +/+ control and Lin28a fl/fl b fl/fl ; Foxn1 Cre/+ double knockout adult mice confirmed the specific deletion of Lin28a and b in TECs (Supplementary Figures 3A-D).Deletion of Lin28b alone or of both Lin28a and Lin28b was associated with increased Let-7g expression in MHCII hi TECs and even showed a 1.3-fold increase in MHCII lo TECs (Figure 3C), despite overall low expression of both Lin28a and Lin28b in these cells.These data support a negative regulatory relationship between Lin28 and Let-7g expression in MHCII hi TECs.
We next assessed the role of Lin28 and Let-7g in TEC differentiation and MHCII expression in 2-month-old double mutant mice.TEC-specific deletion of both Lin28a and Lin28b caused a significant reduction in both the percentage of TECs within total CD45 -stromal cells and in total TEC number (Figures 3D-F).Reductions were similar in both MHCII lo and MHCII hi TEC numbers, with a slight decline in the frequency of MHCII hi TECs (Figures 3G-K).Analysis of cortical (cTEC) and mTEC populations showed that this reduction was entirely in mTECs in the Lin28a fl/ fl b fl/ fl Foxn1 Cre/+ double mutants (Figures 3L-Q).These TEC defects caused a significant reduction in thymus size (Figure 3R) and in total thymocyte number (Figure 3S) starting at 6 weeks of age.The overall decline in thymocyte numbers was reflected in small but significant declines in DP and CD4SP numbers (Figures 3T-V).These results suggest that Lin28a and Lin28b impact thymocyte differentiation indirectly, primarily by regulating the differentiation and/or proliferation of MHCII hi mTECs in the postnatal thymus.Lin28a, Lin28b and Let-7g are expressed in postnatal murine thymi.Immunofluorescence staining of thymic sections from BL6 WT mice at 2 months of age (A-D).(A, B). 4% PFA-fixed thymic sections were stained for LIN28a (A, green) or LIN28b (B, green) with FOXN1 (red).(C, D).The frozen thymic sections were stained for anti-LIN28a (C, green), anti-LIN28b (D, green), and anti-MHCII (red).(E) Quantitative analysis showed the frequency of LIN28a + and LIN28b + cells in total FOXN1 + TECs.(F) Gene expression levels of Lin28a, Lin28b and Let-7g were measured by qPCR in MHCII lo and MHCII hi TEC subsets sorted from 2-month-old murine thymi.The mRNA level of Lin28a in MHCII hi subsets was used as a reference with a value of 1, and the mRNA levels of Lin28b and Let-7g were normalized accordingly and expressed as fold changes.(G-I).Gene expression levels of Lin28a, Lin28b, and Let-7g were measured by qPCR in MHCII lo and MHCII hi TEC subsets sorted from 1-, 2-, 3-, 4-, 8-and 24-week-old thymi.The mRNA levels of Lin28a and Lin28b in MHCII hi TECs and Let-7g in MHCII lo TECs at week 1 were used as references with a value of 1, and the mRNA levels of Lin28a, Lin28b, and Let-7g at other time points were normalized accordingly and expressed as fold changes.Each time point represents at least three to five individuals.(J-L).Immunofluorescence staining, 4% PFA-fixed thymic sections from BL6 white mice at 20 days (J), 2 months (K), and 6 months (L) of age were stained for LIN28b (green) and FOXN1 (red) antibodies.The double-positive staining is shown in yellow (purple arrows).

Lin28a overexpression in TECs increased the total TEC number and produced more thymocytes
To further test LIN28 activity in TECs, we overexpressed (OE) Lin28a by activating the iLin28a transgene (iLin28a-Tg) (40) specifically in TECs with Foxn1Cre (38).Activation of the transgene resulted in a 34-fold increase in Lin28a expression in MHCII lo TECs and a 14-fold increase in MHCII hi TECs in 6-weekold iLin28a +/Tg ;Foxn1 Cre/+ (iLin28a OE) transgenic mice (Figure 4A).Flow cytometry analysis was consistent with these data, with a low level of LIN28a signal detected in MHCII + TECs in iLin28a +/+ controls and a strong LIN28a signal in the iLin28a OE mice in both MHCII lo and MHCII hi TECs, although LIN28a overexpression was detected only in a subset of TECs (Figures 4B, C).Consistent with the mRNA levels and flow cytometry, LIN28a was difficult to detect by IF in the iLin28a +/+ control thymus at 3 weeks of age (Figures 4D, F), but LIN28a dramatically increased in iLin28a OE mice ( Figures 4E, G).LIN28a protein in transgenics was found primarily in the cytoplasm but was also present in the nucleus of TECs (Figure 4E, inset).Interestingly, Lin28b expression in MHCII hi subsets was reduced in iLin28a OE mice compared to controls (Figure 4H), which suggests crossregulation of these two related genes.Expression of the transgene was associated with a 60% reduction in Let-7g in MHCII lo TECs but no significant change in Let-7g in MHCII hi TECs (Figure 4I), possibly because of the reduction in Lin28b expression in these cells.
Overexpression of iLin28a in TECs caused an increase in thymus weight and the ratio of thymus weight to body weight in iLin28a OE mice (Supplementary Figures 4A-D), although it did not change the frequencies of cTECs and mTECs (Figures 5A-D).Increased thymus size was associated with significant increases in the number of both cTECs and mTECs (Figures 5E-G).To further analyze TEC phenotypes, we assessed MHCII expression.The frequency of MHCII lo cTECs was increased, and the frequency of MHCII hi mTECs was reduced in iLin28a OE mice (Figures 5H-L).Consistent with the overall increased size, the numbers of all MHCII subsets were increased, except for MHC hi cTECs (Supplementary Figures 4E, F).Overall, the ratio of MHCII lo to MHCII hi cells increased in iLin28a OE mice (Supplementary Figure 4G).
Consistent with increased thymus size and TEC number, total thymocyte number was significantly increased in iLin28a OE mice (Figure 5M) with increases in the number of all subsets except for CD4SP cells, which declined in percentage (Figures 5N, O; Supplementary Figure 4H).The decline in CD4SP cell frequency is consistent with the decline in the frequency of MHCII hi mTECs in iLin28a OE mice (Figure 5L).

Overexpression of Lin28a promotes the proliferation of MHCII lo TECs
The above results showed that TEC-specific deletion of both Lin28a and Lin28b caused a selective reduction in MHCII hi mTECs, while overexpression of Lin28a expanded all TEC subsets but preferentially expanded MHCII lo TECs.Because Lin28a and Lin28b are typically expressed in stem/progenitor cells associated with cell proliferation, we hypothesized that Lin28a and Lin28b promote TEC proliferation in the postnatal thymus, especially in MHCII lo TECs.To test this hypothesis, we measured TEC proliferation in iLin28a OE mice at 6 weeks of age.Compared to control mice, in iLin28a OE mice, the total number of BrdU + TECs was significantly increased in total TECs (Figures 6A, B) but was selectively higher in MHCII lo TECs (Figures 6C-E).LIN28a + TECs incorporated a significantly higher level of BrdU than LIN28a -TECs, indicating that this is a cell-autonomous effect (Figures 6F, G).In addition, the frequency of BrdU + cells did not change in thymocyte subsets (Supplementary Figure 5A), and thymocytes and TECs from both mutant and control mice did not show a significant change in apoptosis (Supplementary Figures 5B-D).These data suggest that overexpressing Lin28a primarily affects TEC subsets by directly or indirectly increasing the proliferation of MHCII lo TECs.

Downregulation of Foxn1 expression causes a reduction of Lin28b in MHCII hi
and Let-7g in MHCII lo TECs TEC differentiation and proliferation are also controlled by the transcription factor FOXN1, and Foxn1 downregulation is considered an initiating event for age-associated thymic involution (37).The dynamic patterns of Lin28a, Lin28b, and Let-7g across age, the loss of MHCII hi mTECs in Lin28a and Lin28b loss-of-function mutants and the selective effect on proliferation of MHCII lo TECs with Lin28a OE all mirror aspects of Foxn1 expression and function (37).To test the relationship between FOXN1 and Lin28b and Let-7g in TECs, we generated an allelic series using two alleles of Foxn1: the hypomorphic allele Foxn1 Lacz/ LacZ (Z/Z) (37) and the null allele Foxn1 nu (42).In Z/Z mutants, Foxn1 expression is downregulated in TECs beginning at P7, resulting in a relative reduction in MHCII hi and an increase in MHCII lo TEC subsets.Combining the Z allele with the nu allele results in a progressive decline in thymus size and TEC phenotypes (37).We first analyzed Lin28b and Let-7g gene expression in total TECs from +/+, +/Z, Z/Z and Z/N (Z/nude) mice at 3 weeks of age.We found that with the progressive reduction in Foxn1 expression, both Lin28b and Let-7g in total TECs were downregulated in a Foxn1 dose-specific manner (Figures 7A, B).Consistent with their wild-type expression patterns, Lin28b was reduced in MHCII hi TECs, and Let-7g was reduced in MHCII lo TECs in the Z/Z mutants compared to +/Z controls (Figures 7C, D).These data indicated that the differential expression of both genes is controlled directly or indirectly by FOXN1.

Increase in Lin28b expression in sex steroid ablation-induced thymic rebound
To further investigate the regulation of Lin28 and Let-7 relative to thymic involution, we performed sex steroid ablation (SSA)induced thymic rebound in 2.5-and 6-month-old Wt mice and analyzed gene expression early at day 3.5 after castration.SSA significantly increased Lin28b but not Lin28a expression specifically in MHCII hi TECs at both 2.5 and 6 months, while expression in MHCII lo TECs remained undetected (Figures 8A, B).SSA also caused a reduction in Let-7g expression in both MHCII lo and MHCII hi TECs at 2.5 months but no change in either subset at 6 months (Figure 8C).Based on microRNA search results, we found that the mouse class II transactivator-encoding gene Ciita (a critical regulator of MHCII gene expression) and several H2 genes had the predicted target sites for Let-7g and some other Let-7s (Table 1).To test the effect of Let-7g, we further measured the changes of these two genes after SSA, and consistent with down-regulation of Let-7g, both Ciita and H2-IAb mRNA were increased in MHCII lo and MHCII hi TECs at 2.5 months, but still increased in MHCII hi TECs at 6 months even without Let-7g change (Figures 8D, E).These changes in gene expression were associated with an increase in the total number of TECs (Figure 8F), including MHCII lo and MHCII hi TECs (Figure 8G).Hiweverthe increase was much greater in MHCII hi TECs (Figure 8H), causing an increase in the ratio of MHCII hi : MHCII lo (Figure 8I).These data suggest that SSAinduced thymic rebound is associated with an increase in Lin28b in MHCII hi TECs, with higher frequency of MHCII hi TECs, and increased MHCII gene expression in these cells.The increase in MHCII gene expression in these cells may be caused by reducing the inhibitory effect of Let-7g to Ciita and H2 genes.

Discussion
In this study, we linked the expression profiles of Lin28a, Lin28b, and Let-7g in MHCII hi and MHCII lo TEC subsets to the development and differentiation of TECs during thymic ontogeny and involution.We found that, as in other systems, at murine postnatal stages, Lin28a, Lin28b, and Let-7g had inversely correlated expression patterns in TECs.Both Lin28a and Lin28b were more highly expressed in MHCII hi TECs but were expressed at low or undetectable levels in MHCII lo TECs (Lin28b was more highly expressed overall than Lin28a), while Let-7g was expressed at the highest level in MHCII lo TECs but was absent or undetectable in MHCII hi TECs.
Several lines of evidence suggest that Lin28 and Let-7 expression and function in TECs are downstream of FOXN1 and are consistent with mediating some of the effects of Foxn1 downregulation that is thought to precipitate age-related involution.Lin28a, Lin28b, and Let-7g expression all peak at 1 month of age in their respective cell types and then decline with aging starting at 3 months, similar to Foxn1 (37).Loss of Lin28b specifically in TECs (which causes an increase in Let-7g) resulted in a smaller thymus with selective loss of MHCII hi mTECs, while overexpression of Lin28a (which causes a loss of Let-7g) causes a larger thymus due to selectively increased proliferation in MHCII lo TECs.Both phenotypes are consistent with known roles for Foxn1 in promoting TEC differentiation and selectively impacting the proliferation of MHCII lo TECs (37).Furthermore, we demonstrated a dose-dependent expression relationship between Foxn1 expression and both Lin28b and Let-7g expression, consistent with Foxn1 acting upstream of both genes.Taken together, these data suggest that at least some functions of Foxn1 are mediated by its regulation of Lin28 and Let-7, both in the steady-state thymus and during involution.
Lin28a and Lin28b have redundant roles in other cell types at both fetal and adult stages (21,25,26).Our data support both unique and redundant functions for Lin28a and Lin28b in TECs.While only Lin28b is expressed at fetal stages, both genes are expressed in postnatal TECs, with similar patterns of expression at least in bulk analysis: higher in MHCII hi than MHCII lo TECs, peaking at 1 month and then declining with age.Comparison of the Lin28b single knockout (KO) and Lin28a and Lin28b double KO phenotypes indicates a redundant function in suppressing Let-7g expression, even in MHCII lo TECs where neither gene has much expression.However, their expression patterns do not directly overlap, and immunofluorescence analysis shows that LIN28a and b proteins are expressed in distinct subsets of TECs, with LIN28a protein tending to correlate with lower FOXN1 levels and LIN28b correlating with higher levels of FOXN1.These data suggest that both genes mediate their effects by acting in different subsets of TECs, and while both may act similarly to suppress Let-7g, the effects could differ in detail.Their impacts could also differ if Let-7g regulates different sets of target genes in different TEC subsets.Regarding the inhibitory effect of LIN28 on Let-7g, while the gene expression of Lin28 and Let-7g showed opposite patterns in MHCII hi and MHCII lo TECs during postnatal thymic ontogeny, there were instances where Let-7g expression did not follow the changes in Lin28 expression in TECs.For example, Lin28b showed a positive correlation with Let-7g expression in E13.5 and E18.5 TECs (Figure 1F); specific deletion of Lin28a and b caused a 1.3-fold increase in MHCII lo TECs (Figure 3C), despite overall low expression of both Lin28a and Lin28b in these cells; overexpression of Lin28a did not lead to Let-7g reduction in MHCII hi TECs (Figures 4H, I); and the rebound of Lin28b reduced Let-7g expression in MHCII lo TECs in 2.5-month-old but not in 6month-old SSA mice (Figure 6G).These findings suggest that Let-7g has additional regulatory inputs beyond LIN28 in TECs (28, 43), which would be a potentially fruitful avenue for future study.The lack of a change in Let-7g expression at 6 months combined with the increased expression of Lin28b suggests that increases in H2-IAb expression correlated specifically with increased Lin28b and may be independent of Let-7g, Another important point is that the preferential expression of Let-7g in MHCII lo TECs, which are typically considered to represent less mature TECs, is the opposite of what would be predicted from its expression in the most differentiated cells in other cell types.The high expression of Let-7g in MHCII lo TECs thus raises the possibility that this expression is due to a subset of MHCII lo postnatal TECs called post-Aire TECs, which represent a senescent, post-proliferative TEC subset that is derived from mature AIRE + mTEC (44,45).In this case, expression could potentially drive cells into the post-AIRE state.Alternatively, Let-7g could have a different pattern and function in TECs compared to other cell types.It may suppress the expression of a critical differentiation marker MHCII in less mature TECs, with upregulation of Lin28 during TEC differentiation acting to suppress Let-7g and thus allow upregulation of MHCII.Supporting this possibility, several H2 genes and the class II transactivator encoding gene Ciita, a critical regulator of MHCII gene expression, have Let-7g binding sites in their 3'UTRs (Table 1).Notably, the expression of Ciita and H2-IAb were consistent with Let-7g down-regulation in 2.5-month SSA mice (Figures 8D, E), suggesting that Lin28 promotes MHCII expression by reducing the inhibitory influence of Let-7g on Ciita and H2 genes.
The data from Foxn1 Lacz mutants showed a Foxn1 dosedependent downregulation of both Let-7g and Lin28b expression in TECs, suggesting that expression of both genes requires FOXN1 at some level.This regulation could be direct or indirect, and other regulators are clearly needed to restrict expression to specific TEC subsets.Indeed, mTECs have been implicated in the regulation of Let-7 levels in developing natural killer T cells in the thymus (46), supporting that TECs can indirectly regulate Let-7.Our own data show that the same signals (RA, vitamin D3) regulate Let-7g levels in thymic progenitor B cells and depend on Foxn1 levels (28).Collectively, our data are consistent with a model in which during postnatal TEC development and differentiation, Let-7g is broadly upregulated in TECs by increasing FOXN1 levels, while Lin28b is upregulated in a subset of TECs due to both FOXN1 and an unidentified regulator(s).In those cells, LIN28b blocks Let-7g, allowing for increased MHCII expression and driving the maturation of TECs to MHCII hi cells.With the downregulation of Foxn1 during aging, both Let-7g and Lin28b expression decline, but the reduction in Lin28b offsets this Let-7g downregulation and results in increased inhibition of MCHII expression, which results in long-term declines in MHCII expression during aging and involution.

Data availability statement
The original contributions presented in the study are included in the article/Supplementary Material.Further inquiries can be directed to the corresponding author.Lin28b increased in sex steroid ablation-induced thymic rebound.2.5 and 6 months BL6 WT mice underwent castration surgery.Three days later, TECs were isolated, and MHCII lo and MHCII hi TEC subsets were sorted for gene expression analysis and phenotypic analysis.

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FIGURE 6 Specific overexpression of Lin28a in TECs increased the proliferation capability of MHCII lo TECs.(A) Representative profiles of MHCII and BrdU staining on total CD45 -cells in +/+ and OE mice and histograms show overlapping profiles of BrdU staining on gated MHCII + cells.(B) Summary of the percentage of BrdU + cells in total TECs.(C) Representative profiles of MHCII and EpCAM staining on gated CD45 -cells and the gates of MHCII lo and MHCII hi TEC subsets in TECs, and histograms show overlapping profiles of BrdU staining on gated MHCII hi and MHCII lo TECs.(D) Percentage of BrdU + cells in MHCII hi TECs.(E) Percentage of BrdU + cells in MHCII lo TECs.(F) Representative profiles of MHCII and Lin28a staining on gated CD45 -cells and histograms show overlapping profiles of BrdU staining on gated Lin28a -(red) and Lin28a + (blue) TECs.(G) Percentage of BrdU + cells in gated Lin28a - and Lin28a + TECs from OE mice only.Data are representative of two individual experiments (+/+: n= 5; OE: n=5).Data are representative of three individual experiments.Mice aged 6-7 weeks were used.Student's t test results between +/+ and OE mice: *P <0.05, **P <0.01.Bars indicate means ± SEMs.ns: not significant.

TABLE 1
Predicted target sites for miRNA Let-7 in Ciita and H2 genes.