Severe reduced level of memory B cells – is it diagnostic tool for CVID?

Belarusian Research Center for Pediatric

Oncology and Hematology

ESID – Prague

May 2009Svetlana Sharapova Immunology Department

Sasha Migas Molecular Biology Department

Galina Kachan Clinical Department

Mihael Belevtsev Immunology Department

Belarusian Center for Primary Immunodeficiencies

Common variable immunodeficiency (CVID) is a heterogeneous disorder that is associated with low serum immunoglobulin concentrations, defective specific-antibody production and an increased susceptibility to bacterial infections of the respiratory and gastrointestinal tracts. (Ochs H. et alPrimary Immunodeficincy Disease: Oxford University Press 2007)

CVID has two picks of manifestation: from 6 to 10 and from 26 to 40 years.

Gastrointestinal complications are fairly common in CVID —up to 50% of patients with CVID have chronic diarrhoea with malabsorption. Other gastrointestinal diagnoses in patients with CVID include Crohn’s disease, intestinal granulomatous disease, intestinal parasitic bacterial or viral infections, coeliac sprue, and intestinal lymphangiectasia. Autoimmune thrombocytopenic purpura and autoimmune haemolytic anaemia are the most common autoimmune consequences, occurring in 5–8% of all patients with CVID. (Lancet, 2008)

Patients with CVID often have defects in post-antigenic B-cell differentiation:

-- a reduction of CD27+ memory Bcells; -- impaired class switching; -- expansion of CD21low immature Bcells; -- poor diferentiation to plasma cells. (Warnatz et al.)

Common Variable Immunodeficiency

Male was born to healthy unrelated parents. Delivery at term. Body weight: 3 530g. Early neonatal period was without features. At his first year he grown up and developed according to his age, get over some respiratory infections (not severe disease course).

(4 years 9 months) Pt had an infectious mononucleosis (diagnose was made on clinical data: fever, intoxication, lymphadenopathy, hepatosplenomegaly, in blood there were found atypical mononuclear cells – 28%).On the background of which appears:pneumonia in left S5, thrombocytopenia 12,000 per liter, agranulocytosis 100* 10(9)/L.

That was the reason for admittance to our hospital.

Clinical case HistoryDate

1999

December2003

Clinical case

In our hospital pneumonia progressed (despite antibacterial therapy);

It was supposed to be tuberculosis;

The patient was sent for clinical examination and treatment at the Institute of Tuberculosis, where specific treatment was not effective. The effect was reached at single dosing IV Ig (0,8g/kg) + Zienam (Imipenem), prednisolone 2mg/kg.

Tuberculosis was excluded;

Consequently appeared hemolysis on the basis of antituberculous therapy.

Thr-penia: 12,000/L Leu-penia: 3,3*10(9)/L Agran-cytosis: 100* 10(9)/L, Anemia: Hbg 92 g/L

NORMAL: Urine analysis, electrolyte levels, lymphocyte subsets, Ig concetration, blood coagulation, Liver enzymes.

Lab Evaluation

Date

February 2004

March

2004

Immune thrombocytopenia (ITP) is mediated by platelet autoantibodies that accelerate platelet destruction and inhibit their production.

Was stopped by prednisolone, IV Ig

Autoimmune haemolytic anaemia(AIHA) is an immune disorder is an immune disorder caused by antibodies directed against unmodified autologous red cells.Most AIHA are caused by warm antibodies, whereas cold antibodies are less commonly detected.

Wien Klin Wochenschr. 2008;120(5-6):136-51.

Repeated hemolytic crises (at the attempt to reduce the dose of corticosteroids)

Patient’s data

Autoimmune Manifestation

Date

Lab Evaluation

BM cytology: a lot of megakaryocyte, absent functioning it’s

Reticulocytosis: 140 ‰, norma 5-10‰; Haptoglobin: <13g/L norma > 30 Anemia:

Hbg: 40 g/L;

Coombs test: direct positive 1:8

From December 2003

to April 2004

From April 2004

to December 2004

Severe hemolytic crisis was the reason for admittance to hospital (intensive care unit)

Our patient with severe autoimmune hemolysis with both cold and warm antibodies was previously refractory to conventional treatments was treated with weekly infusions of Rituximab.

The only treatment with steroids, was allowed during the period of rituximab administration.

Rituximab reacts specifically with the CD20 antigen and induces B-cell depletion. This could interfere with the production of autoantibodies in some immune diseases.

Intractable hemolysis Treatment

December 2004

IV Ig 0,2 g/kg

Prednisolone 2mg/kg

Rituximab 375mg/m2, for 2 weeks

Date

Patient Data Treatment Result

Complete hematologic response (+90)Hemolysis was stopped, Hb became

normal (+10).The hematologic improvement was prompt, appearing by the second infusion of rituximab.

(8-12 times ) watery stool with negative bacterial seeding. Skin: hyperemic spotswith desquamation (atopy) possible reaction to medicines. Despite intensive antibiotic therapy, IVIg the patient is constantly ill (sinusitis, ethmoiditis)Secondary malabsorbtion syndrome with secondary lactase deficiency was revealed;Clinically important electrolytic disturbances; Physical development delay, Osteoporosis (as manifestation of secondary hyperparathyroidism)

Date

April 2005

Hbg: 112 g/L Coombs test: negative

June 2005

Hypo-electrolytemia

(K, Ca, Ph, Mg,)

Patient’s Lab Data

Recovery humoral immunity

Evolution of Ig concentration

00,20,40,60,8

11,2

Period of time before Rituximab and after

Ig g

/L IgM

IgA

2004 2005 2007 2009

IV Ig replacement 0,2g/kg

Patient’s Lab Data

Recovery humoral immunity

Evolution of B cells % in PB

05

10152025

- 6 m

onth

- 4 m

onth

- 3 m

onth

- 2 m

onth

-1 m

onth

+ 1

mont

h

+ 5

mont

h

+ 9

mont

h

+ 12

month

+ 24

month

+ 36

month

+ 48

month

Period of time before Rituximab and after

% f

rom

e C

D45

+C

D14

-

CD19+

2004 2005 2007 2009

CVID Diagnosis

After absence of recovery of humoral immunity was suggested CVID as diagnosis

The well-accepted definition of CVID includes three key features: 1) the presence of hypogammaglobulinaemia

Pt has decreasing of of two or more immunoglobulin isotypes

three isotypes(low IgG, IgA, or IgM),

2)recurrent sinopulmonary infections,

Pt has constant sinusitis,

ethmoiditis

3) impaired functional antibody responses Pt (I-0):

α-antibodies - very low

β-antibodies - absent

In addition to these date, there can be other clinical findings including autoimmunity, granulomatous disease, and neoplasia.

Pt – Evans syndrome

Common variable immunodefi ciency: a new look at an old disease, Lancet, 2008

Humoral immunity has not recovered during 4 years and 4 months after Rituximab treatment!!

Diagnostics procedures

Patient’s Bone Marrow Investigation

Data.019

100 101 102 103 104

CD10 FITC

Data.002

100 101 102 103 104

CD10 FITC

Data.002

100 101 102 103 104

CD19 PerCP-Cy5.5

Data.019

100 101 102 103 104

CD19 PerCP

Data.012

100 101 102 103 104

CD3 FITC

Healthy Donor

Patient

HANDBOOK OF DIAGNOSTIC HEMATOPATHOLOGY TESTS, 2001

Pro-B cells

0.09%

Pro-B cells

4,7%

Pre-B cells

55,9%

Pre-B cells

0,05%

Gated on CD45+CD14-

B-cell precursors

4,9%

B-cell precursors

0%

April 2008

+52months after treatme

nt

Date

0%

CD19 CD34 CD34

Diagnostics procedures

Patient’s Bone Marrow Investigation

Data.011

100 101 102 103 104

CD19 PerCP

R1

Data.011

100 101 102 103 104

CD10 FITC

Data.002

100 101 102 103 104

CD10 FITC

Data.003

100 101 102 103 104

CD3 FITC

Healthy Donor

Patient

February

2009

+62months after treatme

nt

Pro-B cells

7,5%

Pre-B1 cell

75,4%

Pro-B cells

Pre-B1 cell

17,1%

3%

Date

CD19 CD34

28,2%

69,1%

2,3%

Data.002

100 101 102 103 104

CD19 FITC

Data.005

100 101 102 103 104

CD27 FITC

Patient data Extended Immunological Investigations

Memory B cells (CD19+CD27+IgD-) detectionData.002

100 101 102 103 104

CD27 FITC

Data.002

100 101 102 103 104

CD19 FITCIgD CD21

Healthy

Donor

Patient

18,6%

11,1%

57,3%

97,3%

1,9%

0%

51,4%

48,6%

4,02%

95,9%

Patient data Mutational analysis

Resequencing of 1-5 exons of TNFRSF13B gene has not revealed any mutations;

The only nucleotide substitution was synonymous homogenic SNP in exon 2 : rs8072293 C/T;

Data.003

100 101 102 103 104

CD4 FITC

Patient data Extended Immunological Investigations

CD4+CD45RA+ CD4+CD45RO+Data.004

100 101 102 103 104

CD4 FITC

The immune dysregulation in our patient was extended to a reduction of naive CD4+CD45RA+ T

cells

CD45RA CD45RO

14,5% 35,9%

Patient

Phenotyping of naïve and memory CD4 T cells (gated on CD3+)

At present moment our Pt’s intestinal problems (severe malabsorbtion) occupy the first place. Since 2005 Pt has had 8-12 times stool in a day. Most days of his life he spends in our Hospital.The Pt moves in hospital room with great difficulties (severe osteoporosis).He has stopped to grow since 2005 and has poor quality of life.

-- According to literature data our patient may have CVID (severe phenotype) but usually such patient’s respond to IV Ig replacement therapy, treatment with steroids etc.-- Severe clinical data is a full manifestation of CVID in first age-specific period or complication of rituximab administration?-- We observed B-cells populations disturbance in BM of patien’t with CVID before but have never seen absolute absence of CD19+ in BM ?-- We do not know if it is possible to regarded that synonymous homogenic SNP in exon 2 : rs8072293 C/T formed such severe phenotype?-- It is possible to consider BMT as curable therapy?

Summary

Thank you for your attention

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