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Blood First Edition Paper, prepublished online May 4, 2009; DOI 10.1182/blood-2009-03-211029
EARLY DEFECTS IN HUMAN T-CELL DEVELOPMENT SEVERELY AFFECT
DISTRIBUTION AND MATURATION OF THYMIC STROMAL CELLS: POSSIBLE
IMPLICATIONS FOR THE PATHOPHYSIOLOGY OF OMENN SYNDROME
Pietro Luigi Poliani, M.D. Ph.D.1, Fabio Facchetti, M.D. Ph.D.1, Maria Ravanini, Ph.D.1, Andrew
Richard Gennery, M.D.2, Anna Villa, M.D.3, 4, Chaim M. Roifman, M.D.5,8, Luigi D. Notarangelo,
M.D.6,7,8
1
Department of Pathology, University of Brescia, Italy; 2Department of Paediatric Immunology,
Newcastle upon Tyne Hospitals Foundation Trust, Newcastle upon Tyne, UK; 3TIGET- H. San
Raffaele, Milano, Italy 4Consiglio Nazionale delle Ricerche-ITB Segrate (MI); 5Hospital for Sick
Children, Toronto, Canada; 6Division of Immunology and 7The Manton Center for Orphan Diseases
Research, Children’s Hospital, Boston, USA.
8
These authors equally contributed
Running Title: Thymus pathology in human T-cell immunodeficiencies
Scientific category: Immunobiology
Correspondence to:
Luigi D. Notarangelo
Division of Immunology, Children’s Hospital Boston
Karp Family Building, 9th floor, Room 9210
1 Blackfan Circle
Boston, MA 02115 – U.S.A.
Tel: (617)-919-2276
FAX: (617)-730-0709
e-mail: [email protected]
Copyright © 2009 American Society of Hematology
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Abstract
Thymocytes and thymic epithelial cells (TECs) cross-talk is crucial to preserve thymic architecture
and function, including maturation of TECs and dendritic cells (DCs), and induction of mechanisms
of central tolerance. We have analyzed thymic maturation and organization in nine infants with
various genetic defects leading to complete or partial block in T-cell development. Profound
abnormalities of TECs differentiation (with lack of AIRE expression) and severe reduction of
thymic DCs were identified in patients with T-negative SCID, reticular dysgenesis and Omenn
syndrome (OS). The latter also showed virtual absence of thymic Foxp3+ T cells. In contrast, an
IL2RG-R222C hypomorphic mutation permissive for T-cell development allowed for TEC
maturation, AIRE expression and Foxp3+ T-cells.
Our data provide evidence that severe defects of thymopoiesis impinge on TEC homeostasis and
may affect deletional and non-deletional mechanisms of central tolerance, thus favoring immune
dysreactive manifestations, as in OS.
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Introduction
Severe combined immunodeficiency (SCID) comprises a heterogeneous group of genetic disorders
that affect early stages in T-cell development resulting in the virtual lack of circulating Tlymphocytes and, depending on the nature of the genetic defect, in B and/or NK lymphocyte
depletion1. In contrast, hypomorphic mutations in these genes may result in a more complex
phenotype, with residual development of T lymphocytes. Oligoclonal expansion of T lymphocytes
that infiltrate peripheral tissues is typically observed in Omenn syndrome (OS) and is associated
with significant tissue damage2. Although OS is often due to hypomorphic mutations in RAG1 and
RAG2 genes,3 mutations in other genes that control early stages in T-cell development, have also
been identified, suggesting a common pathophysiological mechanism4. Our previous studies in
RAG-mutated patients5 and mice6 indicate that impairment of deletional and non deletional
mechanisms of central tolerance may play a key role in the pathophysiology of OS.
Studies in mice indicate that signals delivered by thymocytes are crucial to induce maturation of
cortical and medullary thymic epithelial cells (cTECs, mTECs) from a common precursor and to
support maintenance of thymic architecture7-11. A subset of mature mTECs and thymic dendritic
cells (DCs) express Aire, a transcription factor driving the expression of tissue-restricted antigens
(TRAs)12 that are presented to autoreactive T cells, hereby inducing deletional central tolerance13.
In addition, interaction between epithelial cells of Hassall’s corpuscles and thymic DCs has been
implicated to mediate differentiation of T-cell precursors into Foxp3+ cells that include natural
regulatory T cells (nTregs).14
We have hypothesized that genetic defects that abrogate or severely affect early stages of T-cell
development in humans may result in profound abnormalities in the distribution and function of
TECs and thymic DCs, and may affect induction of the mechanisms of central tolerance, thus
providing a basis for the genetic heterogeneity of OS. To test this hypothesis, we have performed a
detailed analysis of thymic architecture and maturation in nine patients representative of six
different genetic defects that variably affect early stages of T-cell development.
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Material and Methods
Patients and thymic biopsies
Clinical, immunological and molecular features of the patients studied are listed in Table 1. Thymic
biopsies from patients P3, P4, P6, P7 and P9 were obtained prior to hematopoietic cell
transplantation (HCT) in 5 patients, in agreement with protocols approved by the Institutional
review Board (IRB), The Hospital for Sick Children, Toronto (Canada) with informed consent
obtained in accordance to the Declaration of Helsinki. Thymic samples from patients P1, P2, P5 and
P8 were retrieved during post-mortem examination. Anonymous normal thymi were obtained from
infants with no immunological abnormalities who underwent elective surgery for cardiovascular
defects correction, according to the protocol approved by the IRB, Spedali Civili, Brescia (Italy)
and Department of Paediatric Immunology, Newcastle upon Tyne Hospitals Foundation Trust, UK.
Histological and Immunohistochemical procedures
Formalin-fixed paraffin embedded thymic sections have been used for routine hematoxylin and
eosin (H&E) and single/double immunohistochemical staining using the following reagents:
claudin-4 (Cld4), Ulex Europaeus Agglutinin-1 (UEA-1) ligand and Aire as markers of TEC
maturation; S-100, CD208/DCLAMP, CD11c and CD303/BDCA2 as markers of DCs; CD163 to
identify CD11c+ macrophages; Foxp3 as a marker of nTreg. Double immunofluorescence staining
for cytokeratin 5 (CK5) and 8 (CK8) have been used to evaluate TEC maturation and distribution.
Detailed information is available in Supplementary Materials online.
Images have been acquired by Olympus DP70 camera using CellF imaging software (Soft Imaging
System GmbH). Aire+ cells, when present, have been counted on ten different high power fields for
each section and values expressed as number of cells/ mm2 ± standard deviation (s.d.).
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Results and Discussion
As shown in Figure 1 and Figure 1S, thymic sections from patients with null mutations in the AK2
gene (reticular dysgenesis, P1), IL2RG (P2 and P3), CD3D (P6) or RAG2 (P7) genes, or with
complete lack of adenosine deaminase (ADA) activity (P5), showed severe atrophy, loss of corticomedullary demarcation (CMD) and lobular architecture with thin septa. Similar findings, with loss
of CMD and lack of Hassall’s corpuscles, were also observed in patients P8 and P9, with OS due to
hypomorphic mutations in RAG2 and RMRP, respectively. Both of these patients had a markedly
reduced number of CD3+ T cells, as previously reported.2,15 In contrast, biopsies from patient P4
(whose hypomorphic R222C mutation in the IL2RG gene was permissive for normal
thymopoiesis16) showed preserved thymic architecture.
Development of TECs is marked by differential expression of CK5 and CK8, with TEC progenitors
being mostly CK5+CK8+ and mature cTECs and mTECs CK8+CK5- and CK8-CK5+, respectively17
(Figure 1). Thymic biopsies from all patients, with the exception of P4, showed a diffuse epithelial
network mostly composed of CK5+CK8+ cells, with predominance of CK8 expression. In contrast,
normal compartmentalization of CK8+CK5- cTECs and CK8-CK5+ mTECs was detected in patient
P4 (Figure 1 and Figure 1S).
Maturation of mTECs is marked by expression of Cld4 and UEA-1 binding18, with a subset of
mature Cld4+UEA-1+ mTECs expressing Aire18,19 (Figure 1). With the exception of patient P4,
thymic sections from all other patients in our series showed virtual absence of Cld4+UEA-1 +
mTECs and lack of Aire expression, regardless of the genetic defect (Figure 1 and Figure 1S). In
contrast, preserved UEA-1 binding (data not shown) and normal Cld4 and Aire expression (Figure
1S) were observed in the thymus from patient P4, with no significant difference in the number of
Aire+ mTECs as compared to control thymus (392.2±94.5 vs 414±106.2 cells/mm2).
Staining for S-100, CD11c (Figures 1 and 1S) and CD303/BDCA2 (data not shown), covering the
large majority of DCs populations, showed virtual absence of thymic DCs in all patients with the
exception of P4. Although a significant number of CD11c+ cells was detected in all patients, these
cells largely co-expressed CD163, and hence represented macrophages (Figures 1 and 1S). The
presence of CD163+ and CD11c+CD163+ macrophages in patient P1 (Figure 1) is in keeping with
the notion that the myeloid differentiation arrest in reticular dysgenesis affects the granulocytic but
not the monocytic lineage. Overall, these data extend previous observations that the number of
thymic DCs is markedly decreased in patients with X-linked SCID. 20
Thymic DCs have been implied to mediate a critical role in the conversion of autoreactive T-cells
into nTregs14. Staining of control thymi confirmed that Foxp3+ cells were distributed around
mature/activated (CD208/DCLAMP+) DCs (Figure 1). No (P8) or very rare (P9) Foxp3+ cells were
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observed in thymic biopsies from patients with OS (Figures 1 and 1S), in spite of the residual
ability to generate T cells. In contrast, a normal number of Foxp3+ cells were present in patient P4,
carrying the hypomorphic IL2RG R222C mutation that was permissive to T-cell development
(Figure 1). To our knowledge, this is the first study in which thymic architecture and maturation of
thymic stromal cells have been analysed in detail in patients with a variety of defects that affect
early stages in T-cell development. The lack of Aire expression and the severe depletion of thymic
Foxp3+ cells may provide a unifying mechanism for the pathophysiology of OS in patients whose
genetic defects severely reduce, but do not completely abrogate, T-cell development.
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Acknowledgements
This manuscript was partially supported by NIH grant P01AI076210 and by The Manton
Foundation. We thank Network Operativo per la Biomedicina di Eccellenza in Lombardia (NOBEL
to AV, LDN, FF), Fondazione CARIPLO to AV and PLP, and Italian Telethon Foundation (AV)
Authorship Contributions and Disclosure of Conflicts of Interest
P.L.P. performed the study and participated in the critical interpretation of the findings and in the
writing of the manuscript; F.F. participated in the critical interpretation of the findings and in the
research design; M.R. performed some of the experiments; A.R.G. provided biological specimens
and participated in the writing of the manuscript; A.V. participated in the writing and in the critical
interpretation of the findings; C.M.R. provided most of the thymic samples and participated in the
critical interpretation of the findings; L.D.N. designed the study and participated in the critical
interpretation of the findings and in the writing.
The authors declare no conflict of interest.
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the T-cell repertoire in Omenn syndrome. Blood. 1999;94:3468-3478.
Villa A, Santagata S, Bozzi F, et al. Partial V(D)J recombination activity leads to Omenn
syndrome. Cell. 1998;93:885-896.
Villa A, Notarangelo LD, Roifman CM. Omenn syndrome: inflammation in leaky severe
combined immunodeficiency. J Allergy Clin Immunol. 2008;122:1082-1086.
Cavadini P, Vermi W, Facchetti F, et al. AIRE deficiency in thymus of 2 patients with
Omenn syndrome. J Clin Invest. 2005;115:728-732.
Marrella V, Poliani PL, Casati A, et al. A hypomorphic R229Q Rag2 mouse mutant
recapitulates human Omenn syndrome. J Clin Invest. 2007;117:1260-1269.
Hollander GA, Wang B, Nichogiannopoulou A, et al. Developmental control point in
induction of thymic cortex regulated by a subpopulation of prothymocytes. Nature.
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van Ewijk W, Hollander G, Terhorst C, Wang B. Stepwise development of thymic
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Akiyama T, Shimo Y, Yanai H, et al. The tumor necrosis factor family receptors RANK and
CD40 cooperatively establish the thymic medullary microenvironment and self-tolerance.
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Irla M, Hugues S, Gill J, et al. Autoantigen-specific interactions with CD4+ thymocytes
control mature medullary thymic epithelial cell cellularity. Immunity. 2008;29:451-463.
Hikosaka Y, Nitta T, Ohigashi I, et al. The cytokine RANKL produced by positively
selected thymocytes fosters medullary thymic epithelial cells that express autoimmune
regulator. Immunity. 2008;29:438-450.
Anderson MS, Venanzi ES, Klein L, et al. Projection of an immunological self shadow
within the thymus by the aire protein. Science. 2002;298:1395-1401.
Zuklys S, Balciunaite G, Agarwal A, Fasler-Kan E, Palmer E, Hollander GA. Normal
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Watanabe N, Wang YH, Lee HK, et al. Hassall's corpuscles instruct dendritic cells to induce
CD4+CD25+ regulatory T cells in human thymus. Nature. 2005;436:1181-1185.
Roifman CM, Gu Y, Cohen A. Mutations in the RNA component of RNase mitochondrial
RNA processing might cause Omenn syndrome. J Allergy Clin Immunol 2006;117:897-903.
Sharfe N, Shahar M, Roifman CM. An interleukin-2 receptor gamma chain mutation with
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Figure Legends
Figure 1.
Thymic compartmentalization and maturation of mTEC, dendritic and natural regulatory T
cells is abrogated in patients with severe defects in T-cell development, but is preserved in a
patient whose genetic defects is largely permissive for T-cell development.
Detailed analysis of the thymic biopsy from a representative normal thymus (Panel A) shows
defined CMD (H&E staining) with normal compartmentalization of CK8+CK5- cTECs (CK8, red
staining) and CK8-CK5+ mTECs (CK5, green staining). Mature mTECs express Cld4 and Aire
(brown staining, upper and lower images, respectively).
In contrast, mutations that abrogate T cell development (reticular dysgenesis, Panel B; γc-null
SCIDX1, Panel C; RAG2-null T-B-SCID, Panel D) or that are only partially permissive for T cell
development (Omenn Syndrome associated with RMRP mutations, Panel E), are associated with
profound atrophy and loss of CMD (H&E staining), highlighted by the presence of a diffuse
epithelial network mostly composed of CK5 and CK8 double positive immature TECs (yellow
staining). No expression of claudin-4 (Cld4) and Aire was detected in these samples. In contrast, as
shown in Panel F, the biopsy from the patient carrying a hypomorphic R222C mutation in the
IL2RG gene, permissive for T-cell development shows normal thymic architecture with CMD
(H&E staining), normal distribution of CK8+CK5- cTECs (CK8, red) and CK8-CK5+ mTECs (CK5,
green) and expression of Cld4 and Aire (brown staining, upper and lower images, respectively).
In the normal thymus (Panel A) CD11c+ (upper image, red staining) and S-100+ (lower image,
brown staining) dendritic cells are distributed in the medullary areas.
Combined CD11c and CD163 staining differentially marks CD11c+ DCs in the medullary region
and CD163+ macrophages, that are primarily distributed into the cortex, with only rare CD11c and
CD163 double positive cells. Conversely, severe depletion of DCs is present in the thymic biopsies
of all patients whose genetic defects severely compromize T cell development. Panels B, C, D and
E show absence of S-100+ cells in all patients. In addition, CD11c+cells, although present in good
number, largely co-express CD163, indicating a macrophage phenotype. Rare CD11c+CD163- DCs
have been observed in the patient with OS (Panel E). In the control thymus, Foxp3+ nTreg clusters
around mature activated CD208+ (DC-LAMP) DCs, as highlighted by double staining procedures
(Panel A). In contrast, thymic biopsies from patients with genetic defects that are non permissive
for T cell development, show absence of mature activated CD208+ (DC-LAMP) DCs and Foxp3+
nTregs (Panel B, C and D). The thymic biopsy from the OS patient (Panel E) shows absence of
CD208+ (DC-LAMP) DCs but focal expression of rare Foxp3+ cells (Panel E, arrow). In contrast,
the thymus from the patient with hypomorphic R222C mutation in the IL2RG gene demonstrates
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normal distribution of both S-100+ and CD11c+ dendritic cells (Panel F, CD11c+ upper image, red
staining; S-100+ lower image, brown staining) along with the evidence of Foxp3+ nTreg interacting
with mature activated CD208+ (DC-LAMP) DCs (Panel F).
H&E staining, 10x original magnification; Immunofluorescence stainings, 20x original
magnification: CK5 (green), CK8 (red), nuclei (blue), merge (yellow). Single immunohistochemical
stainings, 40x original magnification: Cld4, Aire, S-100 (brown) and CD11c (red); Double
immunohistochemical stainings, 40x original magnification: CD11c (blue) and CD163 (brown);
Foxp3 (blue) and DCLAMP (CD208) (brown).
Table 1. Clinical, immunological and genetic features of the patients.
Age at
biopsy (mo)
CD3+
ALC
(cells/μL) (cells/μL)
CD4+
(cells/μL)
CD8+
(cells/μL)
CD19+
(cells/μL)
CD16+/56+
Clinical features
(cells/μL)
Mutation
P1
10 a,b
400
<1%
<1%
<1%
<1%
<1%
Pancytopenia, IUGR
AK2: c.400-401delCT/c. 614-615delGG RD
729
114
0
40
721
16
PCP
IL2RG: IVS3+1G>C
SCIDX1
a
Diagnosis
P2
7.5
P3
2
860
35
13
21
750
9
pneumonia, FTT
IL2RG: R258Q
SCIDX1
P4
10
2500
1600
1000
475
475
225
PCP
IL2RG: R222C
SCIDX1
P5
2a
323
45
6
39
26
13
disseminated
candidiasis
unknown
ADASCIDc.
P6
2
1090
3
0
0
676
391
positive family history CD3δ: R68X/R68X
CD3δSCID
P7
3
900
0
9
1
2
280
positive family history RAG2: g.4953delT/4953delT
T-B- SCID
P8
3a
8600
3698
946
1462
NA
3956
ED, CD, FTT,
myocarditis, IP
RAG2: R229Q/R229Q
OS
924
ED, FTT, CD,
bronchiolitis,
lymphadenopathy,
hepatosplenomegaly
RMRP:
Allele 1: ins ACTCTGTGAATACTCT
GTGAAGCTGA at -10
Allele 2: C4T
OS
P9
6.5
4591
2670
1669
125
1437
a
post-mortem examination; bdied of disseminated adenovirus after T-cell depleted haploidentical hematopoietic cell transplantation; cDiagnosis of
ADA deficiency was established based on lack of ADA activity in red blood cells.
NA, not available; IUGR: intrauterine growth retardation; PCP, Pneumocystis pneumonia; FTT: failure to thrive; ED, erythrodermia; CD, chronic
diarrhea; IP: interstistial pneumonia; RD, reticular dysgenesis; SCID, severe immunodeficiency; ADA, Adenosine deaminase; OS: Omenn
syndrome; RMRP, ribonuclease mitochondrial RNA-processing mutation.
11
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Patient #
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Prepublished online May 4, 2009;
doi:10.1182/blood-2009-03-211029
Early defects in human T-cell development severely affect distribution and
maturation of thymic stromal cells: possible implications for the
pathophysiology of Omenn syndrome
Pietro Luigi Poliani, Fabio Facchetti, Maria Ravanini, Andrew Richard Gennery, Anna Villa, Chaim M.
Roifman and Luigi D. Notarangelo
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