severe reduced level of memory b cells – is it diagnostic tool for cvid?
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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
Belarus – the country of lakes
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