Thymic microenvironment reconstitution after postnatal human thymus transplantation

Abstract

A functional thymus develops after cultured thymus tissue is transplanted into subjects with complete DiGeorge anomaly. To gain insight into how the process occurs, 7 post-transplantation thymus biopsy tissues were evaluated. In 5 of 7 biopsies, the thymus appeared to be predominantly cortex with thymocytes expressing cortical markers. Unexpectedly, the epithelium expressed both cortical [cortical dendritic reticulum antigen 2 (CDR2)] and medullary [cytokeratin (CK) 14] markers. Early medullary development was suggested by epithelial cell adhesion molecule (EpCAM) reactivity in small areas of biopsies. Two other biopsies had distinct mature cortex and medulla with normal restriction of CK14 to the medulla and subcapsular cortex, and of CDR2 to cortex. These data are consistent with a model in which thymic epithelium contains CK14+ "progenitor epithelial cells". After transplantation these cells proliferate as CK14+CDR2+ thymic epithelial cells that are associated with cortical thymocytes. Later these cells differentiate into distinct cortical and medullary epithelia.

Copyright © 2011 Elsevier Inc. All rights reserved.

Figures

Figure 1. T cell generation after thymus transplantation

(A) The number of naïve CD4 and total CD3 T cells are shown in 7 subjects after transplantation. Subject ID numbers are included. The value of the last naïve CD4 data point of DIG410 (*, not shown) was 1836/mm3 at 9 months after transplantation. The value of the first CD3 data point of DIG413 (*, not shown) was 7264/mm3 at 21 days before transplantation. (B) Representative dot plots of CD4 T cells co-expressing CD62L and CD45RA in one of 7 subjects (DIG413) at 12 days before transplantation, day 70 (the day of biopsy), and 385 days after transplantation. The percentages of CD62L+CD45RA+ cells in the upper right quadrant are included in each panel. (C) CDR3 spectratyping profiles of CD4 T cells isolated from subjects DIG410 and DIG412 at 330 days and 388 days post thymus transplantation. (D) CD4 TCRBV repertoires were evaluated after transplantation by flow cytometry in 7 subjects. The time points post transplantation for the assays were 1476 days for DIG012, 749 days for DIG024, 379 days for DIG309, 466 days for DIG409, 783 days for DIG410, 388 days for DIG412, and 385 days for DIG413. The symbols for the thymus transplant recipients are the same as in Panel A. The two solid grey lines indicate the normal adult range (3 standard deviations (SD) above and below the mean).

Figure 2. Real-time PCR analysis of Aire and CK14 mRNA expression in fresh donor thymuses, cultured thymic tissues and allograft biopsies after thymus transplantation

(A) Aire and CK14 mRNA expression was evaluated in fresh harvest day donor thymuses. (B) The levels of Aire and CK14 mRNA expression were evaluated in samples of cultured donor thymic tissues from 9 infants, labeled 1 to 9. The tissue of sample 1 was cultured for 19 days; tissues of sample 2, 3, 4, 16 days; the tissue of sample 5, for 15 days; tissues of sample 6 to 9, for 14 days. The average length of culture was 15.3 days. (Sample 4 was transplanted into DIG409.) (C) Aire and CK14 mRNA expression in allograft biopsies was assessed by real-time PCR. Biopsies were obtained from DIG413 at 70 days after transplantation; from DIG412, at 66 days after transplantation; from DIG410, at 84 days after transplantation; from DIG409, at 84 days after transplantation; and from DIG309, at 75 days after transplantation. The insert has a Y axis of 0 to 0.25 to allow visualization of Aire expression in the DIG413 biopsy. The same freshly harvested thymus RNA was used as a reference control for all PCR assays. The level of expression of that sample was set as 1. Skeletal (SK) muscle RNA was a negative control. Error bars show the mean +/− standard deviation of the RNA samples which were run in duplicate.

Figure 3. Cytokeratin and T cell detection in freshly harvested thymus by IHC

Thymus tissue from the thymus donor of DIG413 is presented as a representative thymus tissue. The harvest day thymus tissue was reacted with antibodies to (A) AE1/AE3, (B) CK14, (C) CDR2, (D) EpCAM, (E) CK5, (F) CD3, (G) CD1a, and (H) Ki-67. Positive reactivity is seen as a brown color in thymus tissue. Hassall’s bodies are indicated by arrows in panel A; the length of the bar is 500 μm. The “M” and “C” in panel B indicate the medullary and cortical areas, respectively. The bar in the low power insert in panel A is 1 mm. All panels have the same magnification.

Figure 4. CK10+ Hassall’s bodies, CK14+ and pan-CK+ epithelium in cultured thymus tissue

Cultured thymus tissue, prior to transplantation into DIG413 is shown. The IHC was performed on frozen sections of tissue after 21 days of culture. (A) pan-CK, (B) CK14 and (C) CK10 antibodies were reacted with the tissue. Multiple Hassall’s bodies are indicated by the arrows. Original magnification ×10. The bar in (C) is 100μm.

Figure 5. Cytokeratin and T cell detection in allograft biopsies by IHC

Thymus tissue in the biopsy of the allograft of subject DIG413 was evaluated. The biopsy tissues were reacted with antibody to (A) AE1/AE3, (B) CK14, (C) CDR2, (D) EpCAM, (E) CK5, (F) CD3, (G) CD1a, and (H) Ki-67. An area showing early medullary development is circled in panels D and H. Positive reactivity is seen as a brown color in thymus tissue. The magnification and bar sizes are the same as in Figure 3.

Figure 6. CK14+ TECs are restricted to medullary areas in allograft biopsies displaying corticomedullary distinction

Thymus allograft biopsies were evaluated in subject DIG024 (top panel) obtained 91 days after transplantation and DIG012 (bottom panel) obtained 218 days after transplantation. The biopsy tissues were reacted with antibody to CK10 (left panel), CK14 (middle panel), and Ki-67 (right panel). Positive reactivity is seen as a brown color in thymus tissue. The bar is 100 μm.

Figure 7. Foxn1 and CK14 protein expression in freshly harvested thymus and the thymus allograft biopsy from DIG410

Biopsy sections are shown in the top panels; freshly harvested thymus sections are in the lower panels. The biopsy of allograft in subject DIG410 was obtained 84 days after transplantation. The tissue was reacted in panels A, B, D, and E with EpCAM (red), and in panels C and F with CK14 (red) and Foxn1 (brown) in two-color double staining IHC. Panels B and E are high power views of panels A and D respectively. Arrows in panels B and E show Foxn1+EpCAM+ TECs. Arrows in panels C and F show Foxn1+CK14+ TECs. The bar is 100 μm in panel D and 50 μm in panel F.

Figure 8. Real-time PCR detection of Foxn1 RNA in thymus tissue specimens of 5 transplant recipients

In each panel the RNAs displayed were obtained from: 1) donor thymus on the day of harvest, 2) donor thymus after culture on the day of transplantation, 3) a biopsy specimen that also contained CK14 and CK5 RNA, 4) biopsy specimens that had no CK14 nor CK5 RNA (thus, this sample was muscle and fat from the surgical site without evidence of allograft), 5) commercial human skeletal muscle RNA, 6) thymocytes, and 7) a fresh thymus control sample. The same fresh thymus control RNA was run in every experiment to allow for comparisons between samples. The control thymus Foxn1 RNA level was set to 1. All RNA levels were normalized to 18S RNA and GAPDH. The “*” in the DIG412 panel indicated that CK-negative allograft biopsy material was not available for testing.

Figure 9. Foxn1 protein detection by IHC

Foxn1 antibody was reacted with formalin-fixed paraffin-embedded tissue from A) freshly harvested thymus on day of harvest from a 9-day infant (later transplanted into DIG410), B) the 20-day cultured thymus which was later implanted into DIG410, and C) the biopsy from DIG410 obtained 82 days after transplantation. These three samples were sections of the tissues used for RNA preparation for Figure 8, Panel DIG410Bx, column 1, 2 and 3, respectively. Bar in main panel, 50 μm, insert bar, 200 μm.

Figure 10. Pax1, Pax9, and Eya1 RNA expression in freshly harvested thymus and biopsies from allograft recipients

For 3 subjects, RNAs from freshly harvested donor thymus, cultured donor thymus, a CK14+CK5+ positive biopsy sample, and a CK14−CK5− biopsy sample were evaluated by real-time PCR. In each panel the freshly harvested and cultured thymus was the thymus used in transplantation for the subject identified to the right of the panel. The same skeletal muscle, thymocyte, and thymus tissue control RNAs were included as in Figure 2 and 8. In each panel, the control thymus RNA expression is set at 1.0. Abbreviation: bx; biopsy.