IMMUNOLOGICAL PROFILE OF FANCONI ANEMIA. A MULTICENTRIC

RETROSPECTIVE ANALYSIS OF 61 PATIENTS

Running title: Immunological profile of Fanconi Anemia

Korthof ET1, Svahn J

2, Peffault de Latour R

3, Terranova P

2, Moins-Teisserenc H

4, Socié G

3, Soulier

J5, Kok M

1, Bredius RGM

1, van Tol M

1, Jol-van der Zijde, CM

1, Pistorio A

7, Corsolini F

8, Parodi

A9, Battaglia F

9, Pistoia V

6, Dufour C

2 and Cappelli E

2

Affiliations

1Department of Pediatrics / Willem-Alexander Children’s Hospital, Division of Immunology,

Haematology and Stem Cell Transplantation, Leiden University Medical Center, Leiden, the

Netherlands; 2

Experimental and Clinical Haematology Unit, G. Gaslini Children’s Hospital,

Genova, Italy; 3

HSCT Unit, Hopital St Louis, Paris, France; 4Immunology HLA unit, Hopital St

Louis, Paris France, 5Hematology & Fanconi Anemia Unit, Hopital St Louis, Paris, France;

6

Oncology Laboratory, G.Gaslini Children’s Hospital, Genova, Italy; 7Servizio Epidemiologia

Clinica e Biostatistica, G.Gaslini Children’s Hospital, Genova, Italy; 8

Laboratorio Diagnosi Pre e

Postnatale Malattie Metaboliche, G. Gaslini Children’s Hospital, Genova, Italy; 9Centre of

Excellence for Biomedical Research (CEBR) , University of Genova, Genova, Italy.

Corresponding Author

Enrico Cappelli, Experimental and Clinical Haematology Unit, G. Gaslini Children’s Hospital, L.go

Gaslini, 5 – 16148 Genova, Italy. Tel. 0039 010 5636693 - Fax 0039 010386204

Email: enricocappellispedale-gaslini.ge.it

American Journal of Hematology

This article has been accepted for publication and undergone full peer review but has not beenthrough the copyediting, typesetting, pagination and proofreading process which may lead todifferences between this version and the Version of Record. Please cite this article as an‘Accepted Article’, doi: 10.1002/ajh.23435

2

WORD COUNT

Abstract 189

Text 2392

Tables and figures 5 (2 + 3) + 1 supplementary table

Keywords: Fanconi Anemia, immunophenotype, cytokines, immunoglobulin, immunology,

lymphocytes.

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ABSTRACT

In this study we analysed the immunological status of 61 Fanconi Anemia (FA) patients with

advanced marrow failure before Hematopoietic Stem Cell Transplantation by assessing the

phenotype of peripheral blood lymphocytes, serum immunoglobulin levels and inflammatory

cytokines.

In FA patients total absolute lymphocytes (p<0.0001), B (p<0.0001) and NK (p=0.003) cells were

reduced compared to normal controls. T cells (CD3), i.e., cytotoxic T cells, naïve T cells and

regulatory T cells showed a relative increase as compared to controls.

Serum immunoglobulin G (p< 0.0001) and M (p=0.004) levels were significantly lower, whereas

IgA was higher (p< 0.0001) than in normal controls. TGF-beta (p= 0.007) and interleukin (IL) 6 (p=

0.0007) were increased in serum of patients compared to controls, whereas sCD40L decrease (p<

0.0001). No differences were noted in serum levels of IL-1β, IL-2, IL-4, IL-10, IL-13, IL-17, IL-23

between FA subjects and controls.

This comprehensive immunological study shows that FA patients in advanced marrow failure have

an altered immune status. This is in keeping with some characteristics of FA such as the pro-

inflammatory and pro-apoptotic status. In addition, B lymphocyte failure may make tight and early

immunological monitoring advisable.

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INTRODUCTION

Fanconi Anemia (FA) is an autosomal or X-linked recessive disease characterized by marrow

failure, somatic malformations and cancer proneness, primarily leading to Acute Myeloid

Leukaemia (AML) and head and neck carcinomas (1).

The disease is due to lesions in one of the at least 15 genes currently known to be responsible for

DNA repair mechanisms that render the cells sensitive to interstrand cross linkers leading to a block

in the G2 phase of the cell cycle. There is evidence that FA proteins, apart from their function in

DNA repair, are also implicated in cytokine hypersensitivity, response to oxidative stress and the

immune response.

Scanty information is available on the immunological status in FA patients. It has been suggested

that some FA children have a generally increased infection susceptibility, not completely explained

by neutropenia alone (2). Castello et al. (3) found a grading of immunological defects in FA

patients and their family members. Recently, Myers et al. (4) demonstrated that FA patients have

reduced absolute numbers of NK and B cells and impaired cytotoxic function of NK and T cells. FA

patients have an increased susceptibility to human papilloma virus (HPV)-associated cancer (5) and

Holmgren et al. (6) showed a significant decrease of serological response in FANC-C mice after

HPV vaccination, suggesting that in FA a primary immune dysfunction may occur independently

from bone marrow failure and may play a role in the pathogenesis of this disease.

Also plasma levels of cytokines have been investigated in FA patients . TNF-alpha was thoroughly

studied and data show that FA cell lines produce high amounts of this cytokine (7) and that TNF-

alpha is enhanced in plasma (8) as well as in the marrow of FA patients where it contributes to

marrow failure (9). TNF-alpha was shown in mouse models to contribute to the progression of stem

cells to AML (10). Studies on IL-1β in FA-A patients (11) showed over-expression of this cytokine

in plasma of patients with a constitutively activated PI3K (phosphoinositide 3-kinase)-Akt pathway.

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However, apart from these consistent data on TNF-alpha, findings on other cytokines were

contradictory (12, 7, 13).

In order to address the issue of the immunological status of FA patients in advanced marrow failure

phase ,we conducted a retrospective multi-center study on 61 FA patients before hematopoietic

stem cell transplant (HSCT) assessing the immunophenotype of peripheral blood lymphocytes,

serum levels of immunoglobulins (Ig) and cytokines other than TNF-alpha that are involved in the

immune response.

MATERIAL AND METHODS

Patients and Controls

Frozen lymphocytes and sera of 61 Fanconi Anemia patients referred to Gaslini Children’s

Hospital, Genova, Italy, to the Department of Pediatrics, Leiden University Medical Center, Leiden,

the Netherlands and to the HSCT Unit of Hospital St Louis, Paris, France, were used for the study.

Recruited subjects included patients with FA who were assessed during the work-up process before

HSCT and thus were all in advanced marrow failure. We are able to collect information about

transfusion in 56/61 patients. Forty two (75%) subjects were transfusion dependent for red cells

and/or platelets. Median value of WBC in FA patients was 3075 x109/L (1st quartile 2.100 x109/L -

3rd

quartile 4.157 x109/L). No patients younger than 4 years entered the study. No patients received

specific treatments apart from transfusions at the time of sampling and all were infection free at

least 2 weeks before sampling. Healthy controls were hospital controls i.e. children who were

hospitalized for minor surgery or traumas and whose clinical indicators and laboratory markers

turned out to be negative for infections, autoimmune and inflammatory diseases. Three different

control groups were used: the first, composed of fifty-one controls was for total lymphocyte and

lymphocyte subset comparison, the second of 346 subjects for immunoglobulin and the third of 23

individuals for cytokine serum levels measurement. Informed consent was obtained from patients

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and/or their relatives during the work-up process according to locally approved informed consent

procedures.

.Lymphocyte immunophenotyping

Lymphocyte subset analysis was performed by a six colour immunostaining panel and by a lysis

and wash procedure. Briefly, 100 µL of EDTA anticoagulated whole blood has been incubated

with monoclonal antobodies (mAbs) directed against surface expressed antigens for 20 min at 4°C

and lysed with FACS lysis solution (Becton Dickinson, BD, city, NJ, USA) for 10 min at room

temperature (RT). Data acquisition and analysis have been performed on a FACSCanto flow

cytometer (BD) equipped with two lasers (Argon 488 and HeNe 633) and 6-colour analysis was

performed using FACS Diva™ software (BD). Applying fluorochrome-labelled mAbs (all

produced by BD) the following peripheral blood lymphocyte subsets were investigated: CD3+ T

cells, CD3+ CD4

+ T helper cells (Th), CD3

+ CD8

+ T cytotoxic cells (Tc), CD16

-CD56

+CD3

-

Natural Killer cells (NK), CD19+ B cells, CD4

+ CD25

high T regulatory cells (T regs), CD3

+

CD45RO+ T memory cells, CD3

+ CD45RA

+ T naive cells, HLA-DR

+ T cells (T activated cells).

Leukocyte and lymphocyte absolute numbers were evaluated by a cell counter (Abbott Cell-Dyn

Sapphire; Abbott Diagnostics, Abbott Park, IL, USA).

Immunoglobulin levels

IgG, IgA and IgM were determined during the clinical workup in each center by using standard

methods.

Serum cytokine level measurement

Based on the knowledge of the pro-inflammatory status of FA patients (9, 14), we determined

serum levels of a large panel of 10 pro-inflammatory cytokines involved in the immune response by

adopting a bead-based immunoassay (FlowCytomixTM

Comboplex Bender MedSystems, city,

country) for measurement of IL-1β, IL-2, IL-4, IL-6, IL-10, IL-13 and of sCD40L by flow

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cytometry according to manufacturer's instructions. Briefly, the protocol is based on a sandwich

immunoassay combining two bead populations of different size each one including multiple subset

of beads, differentiated by varying intensities of internal fluorescent dye. Acquisition was

performed with FACSCanto cytometer. FlowCytomix Pro Software was used to calculate cytokine

concentrations in each sample. Serum cytokine concentrations are expressed as pg/mL. IL-17, IL-23

and TGF-β1 were quantified by ELISA Kits (Ready set go – eBioscience, San Diego, CA, USA).

Statistical analysis

Quantitative data describe medians and 1st-3rd quartiles. Comparisons between patients and

controls were done with non-parametric Mann-Whitney U test; the data were not normally distributed

and the homoscedasticity assumption was not fulfilled and for these reasons the non parametric tests

have been used. The normality of the distributions was evaluated by the Shapiro-Wilk test. Bonferroni’s

correction was applied to avoid multiple comparison error in analysis of subgroups. The statistical

package “Statistica” has been used for all the analyses (StatSoft Corp., Tulsa, OK, USA).

RESULTS

Sixty one patients (31 males) entered the study. The median age was 8 years (range, 4 to 43 years).

Complementation groups, available in 49 patients, were: 39 FANCA, 3 FANCC, 3 FANCD2, and 1

each for FANCB, FANCF, FANCG and FANCL, respectively. Median absolute WBC were 3075

x109/L (1st quartile 2100 x109/l - 3

rd quartile 4157.5 x109/l). All patients had platelets < 30 x109/L

and some were already on red cell and/or platelet transfusions.

Absolute counts of total lymphocytes and of lymphocyte subsets

In comparison to controls (absolute total lymphocyte count (ALC) median 2.700 x109/L; 1st quartile

2.020 x109/L - 3rd

quartile 3.400x109/L), FA patients had reduced ALC (median 1.609 x109/L; 1st

quartile 1.236 x109/L - 3rd

quartile 1.978 x109/L) (p<0.000; Fig. 1A) and reduced proportions of B

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(CD19+) (p<0.0001; Fig. 1B) and NK (CD3-CD16/56

+) cells (p=0.003; Fig. 1C). Percentage of T

cells (CD3+) was increased in FA patients compared to controls (p<0.0001; Fig 1D). As for the

different T cell subsets, in comparison to controls, FA patients did not have a significantly different

proportion of Th (CD3+CD4

+) (p=0.90; Fig. 2A), but did have increased percentages of Tc

(CD3+CD8

+ ) (p<0.0001; Fig. 2B) and T naïve (CD3

+CD45RA

+) cells (p< 0.0001; Fig. 2C). No

difference was found in the percentages of the T memory compartment (CD3+CD45RO

+) (p<0.25;

Fig. 2D). The proportion of activated T cells (CD3+HLA-DR+) was decreased (p=0.005; Fig. 2E),

whereas that of T reg cells (CD4+CD25

high) was increased (p< 0.0001; Fig 2F) compared to controls

(Tables I A and I B). Also when we divided the patients in different age subgroup (4-5 years; 6-11

years; 12-18 years and >18 years) the results overlapped those obtained in the whole population

(data not shown). Comparison between A and non A patients did not sort out any significant

difference (not shown).

Immunoglobulin serum levels

In FA patients IgG (p<0.0001; Fig. 3A) and IgM (p=0.004; Fig. 3B) were found significantly

lower, whereas IgA (p<0.0001; Fig. 3C) was significantly higher than in age-matched controls.

Cytokine serum levels

Serum level of TGF-β was increased in FA patients (491 pg/mL) compared to healthy controls

(median 453 pg/mL; 1st - 3rd quartile 465.7-556.0) (p=0.007) (Table II). The same observation was

made for IL-6 (FA median 8.5 pg/mL; 1st -3

rd quartile 0-52.3; controls median 0 pg/mL) (p=0.0007)

(Table II). Soluble CD40 ligand was reduced in FA sera (median 542 pg/mL) compared to normal

controls (5102 pg/mL, p<0.0001) (Table II). No differences were found between FA and control

group with respect to all other tested cytokines (IL-1β, IL-2, IL-4, IL-10, IL-13, IL-17 and IL-23,

data not shown).

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DISCUSSION

The immunological profile of FA patients has been previously evaluated in some descriptive studies

(3, 4, 14) relying on a limited number of patients who were tested for few immunological

parameters. Although our study is also descriptive, it is based on a high number of patients (n=61)

who were assessed in a comprehensive way including an extensive lymphocyte subset testing,

serum Ig level determination and serum level measurements of a large panel of 10 cytokines.

Overall, the immunological profile of FA patients in advanced bone marrow failure is characterized

by absolute lymphopenia with reduced B and NK cells, by a relative increment of T cells, i.e. Tc, T

naive and Treg, by a reduction of IgM and IgG and an increase of IgA serum levels, by increased

serum IL-6 and TGFβ and decreased sCD40L.

In a progressive disorder like FA, these findings can not be considered as the true immunological

profile of FA, but rather representing the immunological status detectable in an advanced marrow

failure phase. However, our findings are consistent with others from literature (3, 4, 14), and may

impact on some clinical decisions concerning the management of FA patients. The reduced B cells

counts and related serum Ig deficiency may prompt tight monitoring of Ig serum levels and, though

FA subjects do not usually have an exceptionally high infection susceptibility, in those subjects

with low IgG levels and recurrent infections, immunoglobulin replacement can be considered. A

more intense protection policy including vaccination of contacts and of patients with killed products

and polysaccharide antigens can also be taken into account. Since T memory cells look less

hampered than other subsets it may be debated if vaccination with attenuated virus like MMRV

could be inserted in this protective plan.

This policy of infection prevention in FA may find some support by in vitro data indicating that

stimulation of TLR4 and 8, mimicking the in vivo effect of infectious agents, induce over-

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production of the myelosuppressive cytokine TNF-alpha (16) that may accelerate bone marrow

failure.

Regarding the causes of the findings emerging from our study, we can only provide speculations.

Absolute lymphopenia has no unequivocal explanation. Obviously it may is likely to reflect, at

least in part, the advanced marrow failure. Reasons why T cells look less affected than other

subsets are not obvious. In fact FA fetal CD 34+ and iPS cells are known to exhibit the classical FA

fragility features (27) which thing would point to an intrinsic deficiency of early FA haematopoetc

cells . However the fact that T cells early migrate out of the pro-apoptotic environment of the bone

marrow might at least in part contribute to the reduced tendency of these cells to undergo apoptosis

as compared to other subsets (B cells, committed hematopoietic cells) that are exposed for longer

times to the harmful pro-apoptotc marrow environment.

Regarding naïve T cells, an additional explanation of their relative increase may derive from the

homeostatic lymphopenia-induced proliferation (LIP) (17) which occurs as compensatory

mechanism when lymphocyte count drops and leads to cell cycle activation and is associated with

the acquisition of a naive phenotype (18).

IgG and IgM serum levels were much lower in FA than in controls. This finding is consistent with

the B lymphopenia and with a recent study (6) indicating that the FA mouse has an impaired

development of the B-cell compartment as illustrated by suboptimal serological responses to

standard vaccinations.

IgA levels were increased as compared to controls. This can be in keeping with prolonged

stimulation of barrier defences and with the increased levels of TGF-β1 and IL-6 we observed in

FA subjects that stimulate the switch for IgA synthesis as part of the pro-inflammatory response

(19).

As for the cytokine profile, since the role of TNF-alpha in marrow failure of FA is well established

(9, 10, 20) and limited material was available, the analysis was restricted to the assessment of

cytokines on which information was scarce.

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The increase of serum TGF-β1 in FA patients seems in keeping with the pro-apoptotic state and

with other findings of our study like the increase of regulatory T cells (21) and the low levels of IgG

and IgM the production of which is known to be inhibited by this cytokine (22). Noteworthy, TGF-

β1 has been shown to selectively inhibit the growth and differentiation of early HSCs ( 23, 24) and

it can not be excluded that this may contribute to marrow failure of FA.

The increased IL-6 levels are in keeping with the known pro-inflammatory status of FA that is also

expressed by the increased production of IgA we observed in our FA group.

Soluble CD40L is released mainly by platelets during their production and thus its low level may

reflect marrow failure related thrombocytopenia. It is of note that CD40L promotes activation and

proliferation of B cells, immunoglobulin heavy chain switching, IgM response in vivo (25, 26) and

the development of NK cells. Therefore, sCD40L reduction is consistent with the low B and NK

cells and IgM levels we found in our patients.

In summary, FA patients in advanced marrow failure show immunological alterations that might

reflect this condition and possibly the pro-apoptotic and pro- inflammatory status of FA. Though

immunological studies initiated in an earlier phase may provide additional clinically relevant

information, awareness of the alterations we found in advanced marrow failure may still be helpful

to reduce the infectious risk of these patients.

Authors contribution

Korthof ET, Peffault de Latour R, contributed essential samples and analysed the data

Svahn J, Socié G, Soulier J, Pistoia V, analysed the data

Pistorio A, performed statistical analysis

Corsolini F, Parodi A, Battaglia F, Moins H, Kok M, Bredius RGM, Tol M van, Jol-van der Zijde,

CM, performed the research

Terranova P, performed the research and designed the research study

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Dufour C, analysed the data and wrote the paper

Cappelli E, designed the research study, analysed the data and wrote the paper

ACKNOWLEDGEMENTS AND DISCLOSURES

ERG Spa, Rimorchiatori Riuniti, Cambiaso & Risso, SAAR Depositi Oleari Portuali, and AIRFA

(Associazione Italiana Anemia di Fanconi) are acknowledged for supporting the activity of the

Clinical and Experimental Haematology Unit, G. Gaslini Children’s Hospital.

Carlo Dufour received small honoraria for chairing a sponsored symposium from Alexion and for

consultation from Pfizer in 2012 and a small Grant Research from Pfizer in 2010.

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FIGURE LEGENDS

Figure 1. Box plots of lymphocyte (A), B cells (B), NK (C) and T cells (D) in FA patients and

controls. Boxes represent median values with first and third quartiles. P values refer to the Mann-

Whitney U test.

Figure 2. Box plots of percentage distribution of T helper (A), T cytotoxic (B), T naive (C), and T

memory (D), T activated (E) and T regulatory (F) in FA patients and controls. Boxes represent

median values with first and third quartiles. P values refer to the Mann-Whitney U test.

Figure 3. Box plots represent serum level of immunoglobulin G, M and A in FA patients and

controls. Boxes represent median values with first and third quartiles. P values refer to the Mann-

Whitney U test.

Table I. Comparisons of absolute value of lymphocyte subsets between FA patients and controls.

Tables represent median values with first and third quartiles in round parentheses. P values refer to

the Mann-Whitney U test.

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Table II. Cytokines serum level in FA patients and controls. Table represent median values with

first and third quartiles in round parentheses. P values refer to the Mann-Whitney U test.

Supplementary table I. Comparisons of percentage value of lymphocyte subsets between FA

patients and controls. Tables represent median values with first and third quartiles in round

parentheses. P values refer to the Mann-Whitney U test.

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Figure 1. Box plots of lymphocyte (A), B cells (B), NK (C) and T cells (D) in FA patients and controls. Boxes represent median values with first and third quartiles. P values refer to the Mann-Whitney U test.

254x190mm (96 x 96 DPI)

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Figure 2. Box plots of percentage distribution of T helper (A), T cytotoxic (B), T naive (C), and T memory (D), T activated (E) and T regulatory (F) in FA patients and controls. Boxes represent median

values with first and third quartiles. P values refer to the Mann-Whitney U test.

254x190mm (96 x 96 DPI)

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Figure 3. Box plots represent serum level of immunoglobulin G, M and A in FA patients and controls. Boxes represent median values with first and third quartiles. P values refer to the Mann-Whitney U test.

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Absolute counts (109/L)

Patients

Median (1st – 3

rd quartile)

Controls

Median (1st – 3

rd quartile)

p-value

Lymphocytes 1601 (1236.0 – 1978.8) 2700.0 (2020.0 – 3400.0) <0.0001

CD3+ CD4+ Th cells 646.2 (471.9 – 822.8) 1018.5 (729.8 – 1261.8) <0.0001

CD3+ CD8

+ Tc cells 608.1 (465.0 – 706.3) 561.0 (421.9 – 727.4) 0.72

CD3+ T cells 1343.1 (1093.7 – 1663.4) 1836.8 (1411.2 – 2273.8) <0.0001

CD19+ B cells 107.9 (25.6 – 211.9) 421.9 (314.9 – 603.2) <0.0001

CD16-56+ CD3- NK cells 103.4 (56.0 – 174.2) 357.2 (144.8 – 499.4) <0.0001

CD3+ HLA-DR+ T activated 55.4 (21.1 – 103.1) 139.1 (93.5 – 241.5) <0.0001

CD4+ CD45RO

+ Th memory 240.2 (176.3 – 265.8) 313.6 (230.6 – 392.4) 0.0006

CD4+ CD45RA+ Th naive 418.1 (257.5 – 576.6) 618.0 (490.8 – 860.9) 0.0007

CD8+ CD45RO

+ Tc memory 80.0 (44.3 – 112.6) 118.5 (84.4 – 170.7) 0.0004

CD8+ CD45RA+ Tc naive 459.0 (293.3 – 639.0) 427.8 (311.1 – 621.0) 0.9

CD3+ CD45RO

+ T memory 293.8 (196.7 – 367.4) 536.5 (402.4 – 676.8) 0.009

CD3+ CD45RA+ T naive 892.0 (658.2 – 1184.8) 1074.0 (831.1 – 1566.4) <0.0001

CD4+ CD25++ Treg 33.2 (14.1 – 54.0) 31.2 (19.2 – 39.8) 0.92

Table I. Comparisons of absolute value of lymphocyte subsets between FA patients and controls. Tables represent median

values with first and third quartiles in round parentheses. P values refer to the Mann-Whitney U test..

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5051525354555657585960

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Supplementary Table I.

Percentages

Patients

Median (1st – 3rd quartile)

Controls

Median (1st – 3rd quartile) p-value

CD3+ CD4+ 37.5 (32 – 45.1) 39.1 (33.1– 42.9) 0.90

CD3+ CD8+ 35.6 (31.2 – 43.4) 21.5 (19.8 – 24.2) <0.0001

CD3+ 84.0 (75.3 – 87.5) 69.4 (66.2 – 73.9) <0.0001

CD19+ 7.7 (3.0 – 13.7) 16.6 (14.3 – 20.1) <0.0001

CD16-56+ CD3- 7.6 (4.4 – 11.7) 10.2 (7.8 – 16.4) 0.003

CD3+ HLA-DR+ 3.3 (1.9 – 6.5) 5.0 (4.0 – 7.0) 0.005

CD4+ CD45RO+ 13.2 (9.1 – 15.3) 11.3 (9.3 – 14.7) 0.31

CD4+ CD45RA+ 24.1 (20.8 – 30.3) 26.4 (20.2 – 29.4) 0.64

CD8+ CD45RO+ 4.3 (2.8 – 7.1) 5.0 (3.4 – 6.6) 0.98

CD8+ CD45RA+ 29.3 (22.2 – 34.1) 17.4 (13.9 – 19.8) <0.0001

CD3+ CD45RO+ Memory 17.9 (12.5 – 24.3) 19.7 (16.1 – 23.9) 0.25

CD3+ CD45RA+ Naive 57.8 (47.0 – 68.4) 44.6 (40.4 – 49.8) <0.0001

CD4+ CD25+++ 2.1 (1.4 – 3.1) 1.2 (1.0 – 1.5) <0.0001

Supplementary Table I. Comparisons of percentage value of lymphocyte subsets between FA patients and controls. Tables represent

median values with first and third quartiles in round parentheses. P values refer to the Mann-Whitney U test.