Thymus transplantation for complete DiGeorge syndrome: European experience

E Graham Davies, Melissa Cheung, Kimberly Gilmour, Jesmeen Maimaris, Joe Curry, Anna Furmanski, Neil Sebire, Neil Halliday, Konstantinos Mengrelis, Stuart Adams, Jolanta Bernatoniene, Ronald Bremner, Michael Browning, Blythe Devlin, Hans Christian Erichsen, H Bobby Gaspar, Lizzie Hutchison, Winnie Ip, Marianne Ifversen, T Ronan Leahy, Elizabeth McCarthy, Despina Moshous, Kim Neuling, Malgorzata Pac, Alina Papadopol, Kathryn L Parsley, Luigi Poliani, Ida Ricciardelli, David M Sansom, Tiia Voor, Austen Worth, Tessa Crompton, M Louise Markert, Adrian J Thrasher, E Graham Davies, Melissa Cheung, Kimberly Gilmour, Jesmeen Maimaris, Joe Curry, Anna Furmanski, Neil Sebire, Neil Halliday, Konstantinos Mengrelis, Stuart Adams, Jolanta Bernatoniene, Ronald Bremner, Michael Browning, Blythe Devlin, Hans Christian Erichsen, H Bobby Gaspar, Lizzie Hutchison, Winnie Ip, Marianne Ifversen, T Ronan Leahy, Elizabeth McCarthy, Despina Moshous, Kim Neuling, Malgorzata Pac, Alina Papadopol, Kathryn L Parsley, Luigi Poliani, Ida Ricciardelli, David M Sansom, Tiia Voor, Austen Worth, Tessa Crompton, M Louise Markert, Adrian J Thrasher

Abstract

Background: Thymus transplantation is a promising strategy for the treatment of athymic complete DiGeorge syndrome (cDGS).

Methods: Twelve patients with cDGS underwent transplantation with allogeneic cultured thymus.

Objective: We sought to confirm and extend the results previously obtained in a single center.

Results: Two patients died of pre-existing viral infections without having thymopoiesis, and 1 late death occurred from autoimmune thrombocytopenia. One infant had septic shock shortly after transplantation, resulting in graft loss and the need for a second transplant. Evidence of thymopoiesis developed from 5 to 6 months after transplantation in 10 patients. Median circulating naive CD4 counts were 44 × 106/L (range, 11-440 × 106/L) and 200 × 106/L (range, 5-310 × 106/L) at 12 and 24 months after transplantation and T-cell receptor excision circles were 2,238/106 T cells (range, 320-8,807/106 T cells) and 4,184/106 T cells (range, 1,582-24,596/106 T cells). Counts did not usually reach normal levels for age, but patients were able to clear pre-existing infections and those acquired later. At a median of 49 months (range, 22-80 months), 8 have ceased prophylactic antimicrobials, and 5 have ceased immunoglobulin replacement. Histologic confirmation of thymopoiesis was seen in 7 of 11 patients undergoing biopsy of transplanted tissue, including 5 showing full maturation through to the terminal stage of Hassall body formation. Autoimmune regulator expression was also demonstrated. Autoimmune complications were seen in 7 of 12 patients. In 2 patients early transient autoimmune hemolysis settled after treatment and did not recur. The other 5 experienced ongoing autoimmune problems, including thyroiditis (3), hemolysis (1), thrombocytopenia (4), and neutropenia (1).

Conclusions: This study confirms the previous reports that thymus transplantation can reconstitute T cells in patients with cDGS but with frequent autoimmune complications in survivors.

Keywords: DiGeorge syndrome; athymia; thymus transplantation.

Figures

**Fig 1**
Analysis of cellular composition of thymic slices by using flow cytometry at different time points during culture. A, Dot plots show representative anti-CD45 versus EpCam1 staining. Percentages of EpCam1+CD45− cells are given in the regions shown. Histograms show anti–HLA-DR staining gated on the EpCam1+CD45− population shown in the dot plots. B, Number of live cells recovered showing the overall number of thymocytes and number of CD4/CD8 double-positive *(DP)* thymocytes retrieved per milligram of tissue. C, Percentage of cells that were CD45−EpCam1+HLA-DR+ (as a frequency of the live gate). D, Proportion of cells in each thymocyte subset based on CD4 and CD8 surface expression. E, When stimulated for 5 days, thymocytes from day 15 slices proliferate. *CFSE*, Carboxyfluorescein succinimidyl ester. F, When stimulated for 72 hours, CD4 single-positive thymocytes from day 22 slices upregulate the activation marker CD25.

**Fig 2**
T-cell reconstitution after transplantation. *Dotted lines* indicate the 10th percentile of published lymphocyte subset counts in healthy children aged 1 to 2 years and 2 to 5 years.

**Fig 3**
A, TREC levels determined on CD3 cells with the 10th percentile for in-house normal ranges for children less than 2 years and 2 to 5 years of age. B, PHA responses: maximum counts per minute after stimulation of isolated mononuclear cells stimulated with PHA. The *dotted line* indicates the 10th percentile for in-house normal adult control subjects. C, Frequency of IFN-γ–producing cells in the patient's PBMCs measured by using ELISpot (mean ± SEM) in response to autologous and third-party EBV-transformed LCLs in P3 after primary EBV infection. The 2-tailed Student t test for unpaired samples was applied.

**Fig 4**
A and B, Cells with the Treg phenotype expressed as a percentage of CD4 cells and in absolute numbers in patients (n = 5) and an age-range matched control group (n = 11). C and D, Transendocytosis assay shows CD4+FoxP3+ cells in patients (n = 6) and control subjects (n = 5) incubated with anti-CD3 plus untransfected Chinese Hamster ovary *(CHO)* cells or with anti-CD3 plus CHO transfected with CD80 with or without anti-CTLA4. In Fig 4, C, upregulation of CTLA4 expression (shown as mean fluorescence intensity of Treg cells normalized to mean fluorescence intensity of CTLA4 in that patient's own naive conventional T cells (as an internal negative control) is shown. In Fig 4, D, relative total fluorescence intensity of CD4+FoxP3+ cells that have acquired green fluorescent protein *(GFP)* tagged onto CD80 as a result of transendocytosis of CD80 is shown. This is derived from the mean fluorescence intensity of GFP multiplied by the number of GFP+ cells to get total fluorescence intensity divided by the number of Treg cells acquired. In both panels the patients and control subjects had equivalent results. **P = .0031.

**Fig 5**
Histologic appearances of positive thymic biopsy specimens. A and B, Hematoxylin and eosin staining showing medullary differentiation and Hassall body formation. Original magnification = ×10 and ×40, respectively. C, Expression of FoxP3 within the thymic medulla (brown). Original magnification = ×20. D, Double staining with terminal deoxynucleotidyl transferase (brown, nuclear signal) showing immature thymocytes within the cortical area and CD3 (blue, membrane signal), highlighting maturing T lymphocytes within the medulla. Original magnification = ×40. E, AIRE-expressing cells within the medullary region, Original magnification = ×20. F, Double staining for AIRE (brown) and involucrin (blue) that shows colocalization of AIRE-expressing cells, with fully mature, involucrin expressing mTECs. Original magnification = ×40.

**Fig E1**
Histologic appearance of thymic slices before and after culture. A, Hematoxylin and eosin staining before culture. Original magnification = ×4. B, Hematoxylin and eosin staining after 16 days of culture shows lymphoid depletion but with some remaining lymphoid clusters. Original magnification = ×4. C, CK5 (staining mTECs) at day 16. Original magnification = ×10. D, CK8 (staining predominantly cTECs) at day 16. Original magnification = ×10.

**Fig E2**
Correlation of naive T-cell counts between different staining methods: **A-C,** CD4 cells; **D-F,** CD8 cells. All were stained with CD45RA plus an additional second antibody (CD27, CD31, or CD62L), and the results between different second antibodies were compared. Each *symbol* represents a different patient tested at around 24 months (range, 17-27 months) after transplantation.

**Fig E3**
Correlation of naive cell counts measured by using different methods with TREC levels: **A-C,** CD4; **D-F,** CD8. All were stained with CD45RA plus an additional second antibody (CD27, CD31, or CD62L). Each *symbol* represents a different patient tested at around 24 months (range, 17-27 months) after transplantation.

**Fig E4**
TCRVβ spectratyping performed on CD3+ T cells in patient 8 with atypical cDGS before **(A)** and after **(B)** transplantation, respectively.

**Fig E5**
Flow cytometric strategy for enumerating Treg cells. *FSC*, Forward scatter; *SSC*, side scatter.

**Fig E6**
Negatively selected CD4+ cells (from PBMCs using a kit from STEMCELL Technologies, Vancouver, British Columbia, Canada) cultured 2.5:1 with Chinese hamster ovary *(CHO)* cells and soluble OKT3 for 21 hours. *Unstimulated* is defined as CHO-blank (no transfection) or CHO cells transfected with CD80. Patient and control subject show equivalent upregulation of CD25 and CTLA4 expression upon activation.

**Fig E7**
CTLA4 mediated transendocytosis of green fluorescent protein *(GFP)*–tagged CD80 in a patient and a control subject. A, Gating strategy for CD4+FoxP3+ cells. B, After incubation with Chinese hamster ovary *(CHO)* cells. There was no uptake of GFP with CHO cells alone *(left boxes)*, and cells acquire GFP from CHO-CD80 *(middle boxes)*; this uptake is blocked by anti-CTLA4 at 20 μg/mL *(right boxes)*.

**Fig E8**
TCRVβ spectratyping performed on isolated Treg cells (CD4+CD25hiCD127−) from patient P9 **(A)**, conventional (non-Treg) CD4 cells from P9 **(B)**, isolated Treg cells from an adult control subject **(C)**, and conventional CD4 cells from a control subject **(D)**. *Boxes* in Fig E8, B, C, and D, have the same designations as shown in Fig E8, A.

**Fig E9**
A, B-cell (CD19+) counts over time. Reference lines indicate median values for age-related control subjects aged 1 to 2 and 2 to 6 years.B, Class-switched memory cells over time. Note: There are no data on P2. Reference lines indicate the 25th percentile for age-related control subjects aged 0 to 1, 2 to 3, and 4 to 5 years.

**Fig E10**
Further immunohistochemical staining of thymic biopsy specimens. A, CD3 staining showing T cells throughout the thymic tissue. Original magnification = ×4. B, CD1a staining showing strong staining in cortical areas consistent with thymopoiesis. Original magnification = ×2. C, Ki67 staining showing proliferation of cortical thymocytes. Original magnification = ×4. D, CK staining in TECs (CK14). Original magnification = ×10. E, CK5 staining showing mTEC. Original magnification = ×20. F, Claudin 4 (blue) and AIRE (brown) staining in mature mTECs. Original magnification = ×20.

**Fig E11**
Histologic appearance of biopsy specimens of patients who died of viral infections. All original magnifications are ×20, except image Fig E11, B, which is ×10. **A-D,** Hematoxylin and eosin, CK14, CD3, and CD1a staining, respectively, in patient 7 shows a nest of thymic epithelium present in the muscle but with very few CD3+ or CD1a+ lymphoid cells, suggesting little or no thymopoiesis. E and F, Hematoxylin and eosin and CD31 staining, respectively, of a strand of thymic tissue in the muscles in patient 12 shows extensive neovascularization at 2 weeks after transplantation.

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Source: PubMed

Thymus transplantation for complete DiGeorge syndrome: European experience

Abstract

Figures

References

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