La intrincada interface entre la autoinmunidad y la …...SLE (n=300) 3 1 Controls (n=301) 1 2 p 0.624 0.616 No patient or control with Common Variable Immunodeficiency, Hyper-IgM

La intrincada interface entre la autoinmunidad y la

inmunodeficiencia

Associate Professor

Rheumatology Division – UNIFESP

[email protected]

WHO/IUIS/AF

Autoantibody Standardizing

Committee

Medical consultant in Immunology

Fleury Medicine & Health Laboratories

[email protected]

Luís Eduardo Coelho Andrade

72º CONGRESO ARGENTINO DE BIOQUÍMICA

Buenos Aires, 22-25 de agosto de 2017

Dendritic cell trying to reach a micro-organism

Kristine Krafts, 2007

Dendritic cell capturing micro-organisms



Dendritic cell phagocyting micro-organisms

Natural killer cell (top) killing infected cell (bottom)

Natural Killer lysing an infected cell


CD8+ T cells surrounding tumor cell

CD8+ T lymphocytes attacking a cancer cell


White blood cells Cells of the immune system are dispersed in the peripheral blood and tissues

Neutrophil

Basophil

Eosinophil

Lymphocyte

Monocyte

Young

neutrophil


The immune system must also exercise DIPLOMACY!

Fight infection Fight cancer

Tolerance

AUTOIMMUNE LYMPHOPROLIFERATIVE SYNDROME (ALPS)

Mutations in genes related to FAS-induced apoptosis extensive

survival & accumulation of lymphocytes (CD3+TCR+ double negative)

ALPS

• Autoimmune manifestations: 50-70%

• Autoimmune hemolytic anemia

• Autoimmune thrombocytopenia

• Autoimmune neutropenia

• Glomerulonephritis, optic neuritis, Guillain-Barré

• Arthritis, vasculitis, primary biliary cirrhosis

• Autoimmune hepatitis, blistering dermatitis

• SLE-associated autoantibodies

} Evans syndrome

Autoimmune Polyendocrinopathy-Candidiasis-

Ectodermal Distrophy (APECED)

Defect in AIRE gene (autoimmune regulator) deficient

presentation of autoantigens at medullary thymus epithelium

APECED

• Chronic Muco-Cutaneous Candidiasis

• Ectodermal Displasia

• Hypoparathyroidism + Addison`s Disease

• Hypothyroidism, Type 1 DM, Autoimmune Hepatitis

• Pernicious Anemia, Vitiligo, Alopecia

• Primary Biliary Cirrhosis

Immunodysregulation, polyendocrinopathy,

enteropathy, X-linked (IPEX syndrome)

Mutation in FoxP3 gene defect in development of CD4

CD25 T regulatory cells

IPEX YNDROME

• Very early onset and high mortality rate (< 2 years)

• Extensive autoimmune enteritis

• Severe eczema

• Recurrent infections

• Type 1 Diabetes Mellitus

• Hpothyroidism

• Autoimmune Hemolytic Anemia

• Membranous nephropathy

Hyper-IgM syndrome (HIGM)

• Absent IgG and IgA, with normal or high IgM

• Recurrent infections opportunistic germs

• Autoimmune cytopenias, Inflammatory Bowel Disease,

Glomerulonephritis, Rheumatoid-like Arthritis,

Autoimmune Hepatitis, Thyroiditis, SLE-like

• Heterogeneous genetic basis (CD40L, CD40, NEMO,

AID, UNG)

– CD40L X-linked and the most frequent

– NEMO and AID most frequently associated with autoimmunity

Wiskott-Aldrich syndrome (WAS)

• X-linked defect in WAS gene

• Eczema and thrombocytopenia (small platelets)

• Autoimmune and inflammatory manifestations 40-70%

• Autoimmune cytopenia (AIHA, neutropenia)

• Inflammatory Bowel Disease, Glomerulonephritis,

Rheumatoid-like Arthritis, Uveitis

Selective IgA deficiency

• IgA is the most abundant immunoglobulin

• IgG1 predominates in serum IgA predominates in

secretions

• SIgAD 1:200 to 1:1,200 (lower in Asians)

• May be associated with IgG2 or IgG4 deficiency

• Most individuals with SIgAD are asymptomatic

• Otherwise…

– Infections (respiratory and intestinal)

– Allergy

– Autoimmune diseases (celiac disease, type 1 DM)

• SIgAD may progress to CVID

DSIgA

Idiopathic

juvenile

arthritis Rheumatoid

arthritis

Myastenia

gravis

SLE

Autoimmune

thyroiditis

Celiac

disease

Chron`s

disease

Type 1

DM

Cataldo F et al. Gut 1998; 42: 362. Cassidy JT, Kitson RK, Selby CL. Lupus 2007; 16: 647. Jesus AA ET al. Lupus 2011; 20 (12): 1275

5.2% (juvenile)

2.6% (adult)

10x greater

RR

Selective IgA deficiency

Common Variable Immunodeficiency - CIVD

• Most frequent symptomatic PID in adults

• Recurrent infections systemic granulomatosis

• Autoimmune manifestations (25%) women with

granuloma (50%)

• Autoimmune cytopenia, Hashimoto`s thyroiditis, vitiligo

• pernicious anemia, rheumatoid-like arthritis, hepatitis

• Cytopenia may precede infectious manifestations

• Heterogeneous genetic and immunologic basis

CVID

Autoimmune

hemolytic anemia

Immunologic

Thrombo-

cytopenic Purpura

Autoimmune

endocrinopathy

Rheumatoid

Arthritis

SLE Dermatomyositis

Fernández-Castro M et al. Semin Arthritis Rheum 2007; 36: 238-45.

18 CVID with SLE (Fernández-Castro et al)

50% had CVID diagnosed up to 5 years after first SLE manifestations

67% had SLE disease activity decreased after development of CVID

Common Variable Immunodeficiency

Complement deficiency and autoimmunity

• C1 deficiency is rare, but strongly associated with SLE

(pediatric onset) C1q (90%), C1r (50%) and C1s (50%)

• C2 homozygous deficiency is the most common 10-30%

SLE, SLE-like syndromes, ANCA vasculitis

• Heterozygous C2 deficiency (2% population) no apparent

association with autoimmunity

• Complete homozygous C4 deficiency is rare 75% SLE

• Low C4A gene copy number predisposes to SLE

The oxidative burst is

defective in CGD

Mandell, Bennett, & Dolin: Principles and Practice of Infectious Diseases, 6th ed.

Multifocal pneumonia by

Aspergilus fumigatus

Chronic Granulomatous Disease

Chronic Granulomatous Disease & SLE

Several case reports of occurrence of

SLE in CGD patients and carriers

• 368 consecutive CGD patients

10 (2.7%) with Discoid Lupus

2 (0.5%) with SLE

• 290 1st degree CGD relatives

20 with Discoid Lupus

2 with LES

Chronic Granulomatous Disease &SLE

Winkelstein JA et al. Medicine (Baltimore) 2000; 79: 155

• C3 deficiency

• Hereditary C1 inhibitor deficiency

• MAC (membrane attack complex) components deficiency

• Combined Variable Immunodeficiency (CVID)

• Hyper-IgM syndrome (AID or UNG deficiency)

• Autoimmune Lymphoproliferative syndrome (ALPS)

• Prolidase deficiency

• Hyper IgE Syndrome

PID OCCASIONALLY ASSOCIATED WITH SLE

AND SLE-LIKE PRESENTATIONS

Carneiro-Sampaio M et al. J Clin Immunol 2008; 28 (Suppl 1): S34-S41.c

• Autoimmune PolyEndocrinopathy Candidiasis Ectodermal Dystrophy

(APECED)

• Immune deregulation Polyendocrinopathy X-linked (IPEX)

• Wiskott-Aldrich syndrome

• Omenn syndrome

• X-linked agammaglobulinemia

• X-linked Hyper-IgM syndrome

• Hyper IgM syndrome due to CD40/CD40L deficiency

• Defects in IL-12/IL23/INF- axis

• DiGeorge syndrome

• Ataxia-Telangiectasia syndrome

• Chédiak-Higashi syndrome

PID NOT ASSOCIATED WITH SLE


• C1q deficiency

• C4 deficiency

• C1r/s deficiency

• C2 deficiency

• Carriers of X-linked Chronic Granulomatous Disease (CGD)

gene

• X-linked and autosomal recessive CGD

• IgA deficiency

SOME FORM OF PID ARE CONSISTENTLY ASSOCIATED WITH SLE


Author Year PID addressed Nature of communication

Ravin et al 2008 CGD Case report

Winkelstein et al 2000 CGD Series of cases

Cale et al 2007 CGD Series of cases

Cassidy et al 2007 Selective IgA deficiency Series of cases

Rankin et al 1997 Selective IgA deficiency Series of cases

Fernández-Castro et al 2007 Common Variable Immunodeficiency Series of cases

Tsuge I et al 2010 Hyper-IgM syndrome & C1q

deficiency

Case report

Melegari A et al 2007 Hyper-IgM syndrome Case report

Aggarwal et al 2010 C1q & C2 deficiency Series of cases (familial)

Mehta P et al 2010 C1q deficiency Case control

Jönsson G et al 2007 C2 deficiency Series of cases

Kallel-Sellami et al 2007 C2 deficiency Case report

Tsukamoto et al 2005 C3 deficiency Case report

Wu et al 2009 C4 deficiency Series of cases (familial)

SLE-PID association is largely based on case reports and small

series addressing individual primary immunodeficiencies

What is the share of

PID participation in

SLE?

With PID

No PID 3%? 10%?

25%?

50%?

75%?

97%?

Survey of overall PID in SLE

• 300 sequentially retrieved adult SLE patients

• 301 sequentially retrieved healthy individuals

• Structured questionnaire & informed consent

• Blood draw for immune system evaluation

• Exclusion criteria

– Infectious episode within the last 30 days

– Use of immuno-biological therapy within the last 6 months

– Severe nephrotic syndrome

– Co-existence of HIV or malignant disease

Perazzio SF, Salomao R, Carneiro-Sampaio MS, Andrade LEC: Rheumatology (Oxford) 2016; 55:1647

• IgA, IgG, and IgM

– Turbidimetry, Olympus

• IgE

– ELISA, Mabtech AB

• IgG subclasses (IgG1, IgG2, IgG3, IgG4)

– ELISA, Invitrogen

• Isohemmaglutinins

– In vitro agglutination, Diaclon

• Anti-pneumococcal antibodies

– ELISA, home made assay

Investigation of humoral immunodeficiencies

CVID investigation


• CH50 and C2 hemolytic activity

– Radial immunohemolysis, home made assay

• C3 & C4

– Turbidimetry, Olympus

• C1q

– Radial immunodiffusion, The Binding Site

• Mannose binding lectin (MBL)

– ELISA (Bioporto Diagnostics)

• C4 gene copy number determination

– Quantitative Real Time PCR

Investigation of Complement immunodeficiencies


C4 gene copy number variation

qRT-PCR with SYBR Green probes (Applied Biosystems)

Quantification by the -CT method (reference sample gently donated by

Prof. Szilagyi , Hungary)

Rotor-Gene 3000 Real-Time PCR System (Corbett Life Research/Qiagen)

CGD patient CGD gene carrier

Normal individual

Quantification of neutrophil oxidative burst

Oxidation of 2,7-

dichlorofluorescein-

diacetate (DCFH-DA)

measured by flow

cytometry


Blood draw

Abnormal result

Active SLE

Still active SLE

Disease remission

Controls and SLE with no

activity

Repeat exams

(60 days)

Normal results

Annotated in the study Follow-up

Flow chart to confirm abnormal

results and rule out immunological

abnormalities associated with SLE

activity


Primary Immunodeficiencies defined according

to IUIS criteria

SLE Controls p

300 301 ----

Gender (F:M) 284:16 286:15 0.999

Age (years) 39.58 12.54 35.1 11.10 0.07

Disease duration 10.74 8.15 ---- ----

Other autoimmune rheumatic

diseaes

32 ---- ----

APS 16 ---- ----

Sjögren`s syndrome 7 ---- ----

Systemic sclerosis 4 ---- ----

Rheumatoid arthritis 4 ---- ----

Non-rheumatic autoimmune

diseases

20 0 ----

Thyroiditis 15 ---- ----

Psoríasis 3 ---- ----

Vitiligo 3 ---- ----

Primary biliary cirrhosis 1 ---- ----

IgA nepropathy 1 ---- ----

Miscelaneous 140 43 <0.001

SLICC (mean ± DP/median) 0.97 1.10/1 ---- ----

SLEDAI (mean ± DP/median) 1.57 2.77/0 ---- ----

Clinical characteristics of SLE and controls

SLE Controls p

300 301 ----

Gender (F:M) 284:16 286:15 0.999

Age (years) 39.58 12.54 35.1 11.10 0.07



diseaes

32 ---- ----

APS 16 ---- ----





diseases

20 0 ----










SLE Controls p

300 301 ----

Gender (F:M) 284:16 286:15 0.999

Age (years) 39.58 12.54 35.1 11.10 0.07



diseaes

32 ---- ----

APS 16 ---- ----





diseases

20 0 ----










What is the share of

PID participation in

SLE?

With PID

No PID

Normal controls SLE patients

With PID

No PID

* p < 0.001

28% was the share of PID

participation in this SLE cohort

28%

1.6%


1 PID 2 PID 3 PID

SLE (n=300) 80 4 0

Controls (n=301) 8 0 0

p < 0.001 0.05 1.00

Frequency of simultaneous PID

1 PID 2 PID 3 PID

SLE (n=300) 80 4 0

Controls (n=301) 8 0 0

p < 0.001 0.05 1.00

Frequency of simultaneous PID

Frequency of Selective IgA deficiency (<7mg/dL)

and IgG deficiency (<450mg/dL)

Selective IgA

deficiency

Selective IgG

deficiency

SLE (n=300) 3 1

Controls (n=301) 1 2

p 0.624 0.616

No patient or control with Common Variable

Immunodeficiency, Hyper-IgM syndrome and Hyper-IgE

syndrome.


IgG and IgA serum levels were higher in SLE patients

mg/d

L

mg/d

L

Controls Controls SLE

0

100

200

300

400

500

600

700

800

900 IgA

0

500

1000

1500

2000

2500

3000

3500 IgG

SLE

Black transversal

bar: mean

Red dotted line: cut-

off threshold for PID

Blue area: normal

reference range * *

* p < 0.001

Frequency of IgG subclass deficiency

IgG1

(<300mg/dL)

IgG2

(<75mg/dL)

IgG3

(<16mg/dL)

IgG4

(<1mg/dL)

SLE (n=300) 5 40 24 11

Controls (n=301) 1 1 0 0

p 0.122 <0.001 <0.001 0.003


IgG1 IgG2 IgG3 IgG4

Black transversal bar: mean

Red dotted line: cut-off threshold for PID

* * *

* p< 0.001

SLE SLE SLE SLE Controls Controls Controls Controls

IgG subclass serum levels

Frequency of Selective IgM deficiency (<30mg/dL)

and IgE deficiency (<10mg/dL)

Selective IgM

deficiency

Selective IgE

deficiency

SLE (n=300) 24 49

Controls (n=301) 8 37

p <0.001 0.205


ng/m

L

Controls SLE 0

50

100

150

200

250

300

350

400

450

500

550

600

650

700

750

800

850

IgE 4800

mg/d

L

Controls SLE 0

100

200

300

400

500

600

700 IgM

* *

* p < 0.001

IgM and IgE serum levels were reduced in SLE patients

Black transversal

bar: mean

Red dotted line: cut-

off threshold for PID

Blue area: normal

reference range

Chronic Granulomatous Disease

• No SLE patient nor controls

• 1 SLE patient is CGD gene carrier (0.33%!!!)

= - 24.18%

= - 15.76%

S. aureus

P. aeruginosa

No stimulus

MFI= 14.47

MFI = 10.97

MFI = 12.19


Complement System Immunodeficiency

Abnormal results

1st screening

Confirmed abnormal

results (2nd test)

Definite Complement

deficiency

SLE Controls p LES Controles p LES Controles p

C2 12 3 0.033 9 1 0.020 ? ? -----

C3 5 0 0.061 1 0 0.999 ? 0 -----

CH50 14 3 0.012 2 0 0.499 ----- ----- -----



Abnormal results

1st screening

Confirmed abnormal

results (2nd test)

Definite Complement

deficiency


C2 12 3 0.033 9 1 0.020 ? ? -----

C3 5 0 0.061 1 0 0.999 ? 0 -----

CH50 14 3 0.012 2 0 0.499 ----- ----- -----



Abnormal results

1st screening

Confirmed abnormal

results (2nd test)

Definite Complement

deficiency


C2 12 3 0.033 9 1 0.020 2 0 -----

C3 5 0 0.061 1 0 0.999 * 0 -----

CH50 14 3 0.012 2 0 0.499 ** 0 -----

* “Non-pathogenic” C3 mutation

** Isolated C6 low levels


C4A copy number

≤1 2 >2

Controls 20 210 71

SLE 40 200 60

p <0.01 0.431 0.323

C4A gene copy number

Fre

qu

ency

C4A copy number

Controls

SLE


C4B copy number

<2 2 >2

Controles 61 197 43

LES 65 189 46

p 0,689 0,552 0,731

C4B gene copy number

Fre

qu

ency

Controls

SLE

C4B copy number


Therapeutics in SLE patients with & without PID

With PID

n = 84 (%)

No PID

n = 216 (%) p

Current immunosuppressant 61 (72.6) 143 (66.2) 0.335

Previous immunosuppressant 59 (70.2) 139 (64.3) 0.416

Corticosteroids

Total 45 (53.5) 114 (52.7) 0.999

Low dose 22 (26.19) 45 (20.8) 0.355

High dose 23 (27.3) 68 (31.4) 0.576

Hydroxichloroquine 54 (64.2) 152 (70.3) 0.333

Azatioprine 20 (23.8) 46 (21.2) 0.644

Mycofenolate 10 (11.9) 16 (7.4) 0.253

Methotrexate 14 (16.6) 38 (17.5) 0.999

Cyclofosfamide 9 (10.7) 14 (6.4) 0.231

Leflunomide 4 (4.7) 12 (5.5) 0.999

Dapsone 2 (2.3) 1 (0.4) 0.190

Talidomide 0 (0) 2 (0.8) 0.999

Tacrolimus 2 (2.3) 2 (0.8) 0.313

Cyclosporine 2 (2.3) 6 (2.4) 0,999

PID versus disease activity

p = 0.285

PID

Yes No

SL

ED

AI


Current age (years) Disease duration (years)

p = 0.222 p = 0.638

Age at disease onset

(years)

PID versus time variables

p = 0.350

PID Yes No

PID Yes No PID Yes No

PID versus disease severity

p = 0.250

PID Yes No

SL

ICC

-DI


SLE clinical manifestations in the presence of PID

PID (n = 84)

(%)

No IDP (n = 216)

(%) p

Gender (M:F) 5:79 (1:15.8) 11:205 (1:18.6) 0.778

Cli

nic

al fe

atu

res

Cutaneous 77 (91.7) 199 (92.1) 0.999

Mucosa 13 (15.5) 51 (23.6) 0.157

Articular 72 (85.7) 185 (85.6) 0.999

Renal 54 (64.2) 113 (52.3) 0.070

Hematologic 58 (69.0) 144 (66.6) 0.784

Serosa 26 (30.9) 56 (25.9) 0.390

Neurologic 20 (23.8) 44 (20.3) 0.532

Severe infection 4 (4.7) 22 (10.1) 0.172

IgG3 deficiency

IgG4 Def

n = 11

No IgG4 Def

n = 289

No PID

n = 216

Other IDP

n = 73

Glomerulo-

nephritis

% 100 53.9 52.3 58.9

p ---- p < 0.001 p < 0.001 p < 0.01

IgG3 Def

n = 24

No IgG3 Def

n = 276

No PID

n = 216

Other PID

n = 60

Glomerulo-

nephritis

% 75 53.9 52.3 60

p ---- p < 0.05 p < 0.05 NS

IgG4 deficiency

IgG3 & IgG4 deficient SLE patients had more nephritis


Clinical characteristics of IgM deficient SLE patients

IgM D

n = 24

No IgM D

n = 276

No PID

n = 216

Other PIDs

n = 60

Oral ulcers % 4.1 22.8 23.6 20

p ---- < 0.05 < 0.05 NS

Current age Mean ± SD 48.54±11.63 38.80±12.11 39.01±11.92 38.06±12.86

p ---- < 0.01 < 0.01 < 0.01

Disease

duration

Mean ± SD 14.58±9.70 10.40±7.93 10.60±8.05 9.71±7.50

p ---- < 0.05 < 0.05 < 0.05

Age at disease

onset

Mean ± SD 33.58±11.60 28.37±10.75 28.39±10.32 28.30±12.26

p ---- < 0.05 < 0.05 < 0.05


Concluding remarks

• Tolerance is a major function of the immune system

Immunodeficiency contributes to autoimmunity

• An underlying immunodeficiency state was detected in over one fourth of

a large cohort of sequentially retrieved SLE patients

• The main forms of immunodeficiency state observed in SLE were

immunoglobulin deficiencies, especially IgG subclasses and IgM

• There was a significant association between low C4A gene copy number

and SLE

• IgG subclass deficiency, especially IgG3 and IgG4, was strongly

associated with lupus nephritis

Acknowledgments

• Sandro F. Perazzio

• Magda S. Carneiro-Sampaio

• Neusa P. Silva

• Reinaldo Salomão

• National Council for Research Development (CNPq)

• São Paulo State Research Foundation (FAPESP)

Documents

La intrincada interface entre la autoinmunidad y la …...SLE (n=300) 3 1 Controls (n=301) 1 2 p 0.624 0.616 No patient or control with Common Variable Immunodeficiency, Hyper-IgM