1. Relations between T- and B- lymphocyte abnormalities in CVID patients and their age dependency
Ladies a gentlemen, Mr. Chairman
I would like to introduce my presentation to you : Relations between T- and B- lymphocyte abnormalities in CVID patients and their age dependency
Marcela Vlková
Ústav klinické imunologie a alergologie, FN u sv. Anny v Brně

2. Common variable immunodeficiency ( CVID)
Clinically heterogeneous group of primary humoral immunodeficiency diseases, characterized by impaired antibody production  decreased levels of IgG, IgA and sometimes also IgM.
B-cell percentages range from very low to normal
Etiology unknown (except for deficits ICOS, BAFF-R, TACI).
Various T- and B-lymphocyte abnormalities were described: defective T-cell activation and impaired cytokine production, abnormal T-cell phenotypes (for example: reduction number of CD45RA, CD62L, increasing number of CD45RO and HLA-DR). For B-cell: reduced number of memory cell – CD27+IgD-IgM-.
Common variable immunodeficiency - abbreviation C V I D - comprise heterogeneous group of humoral immunodeficiencies of unknown etiology. It is characterised by impaired antibody production. Decreased serum levels of immunoglobulin isotypes – IgG, IgA and sometimes also IgM are observed in these patients.
Determination of B lymphocyte subpopulations showed normal or reduced values
Various T and B lymphocytes abnormalities were described, for example : defective activation, impaired cytokine production of T cells and abnormal T cell phenotypes - reduced expression of CD45RA and CD62L, or increased expression of CD45RO, HLA-DR. Recent results showed that CVID patients have reduced numbers of memory CD27+ IgD-IgM- B-cells, but pathogenetic role of these abnormalities are unclear.
Based on diminution of memory B cells new classification of CVID patients into more homogeneus patient subgroups was created in Freiburg.

3. Goals
A division of CVID patients according to „Freiburg classification“ - based on flow cytometric analysis markers associated with B cell development (CD21, CD27, IgD, IgM).
Analyse CD4+ T lymphocyte subpopulations, which characterize T-cell development (CD45RA, CD45RO, CD62L) and activation (CD25, CD27, CD28, CD29, HLA-DR) for individual subgroup of CVID patients and controls.
Analysis of mutual relations between numbers of T and B cells in various developmental and activation stages.
Based on diminution of memory B cells new classification of CVID patients into more homogeneus patient subgroups was created in Freiburg.
The goal of my study was to classify our CVID patients in accordance with the “Freiburg classification”, because our institute was integrite into multi European study of CVID patients.
Together with the classification I analysed CD4+ T lymphocyte subpopulations for individual subgroup CVID patients and also for controls.
Consequently , I analysed of mutual relations between numbers of T- and B- cells in various developmental and activation stages of CVID patients.

4. Patients and methods
42 CVID patients (10-75 years, mean 43,7±15 years; 28 females, 13 males)
33 healthy controls (21-59 years, mean 37,5 ±10,7 years; 26 females, 7 males)
For B cell analysis - isolation of peripheral blood mononuclear cells (density gradient centrifugation) and stained by standard procedure by flow cytometry
For T cell analysis - whole blood stained by standard procedure by flow cytometry
Antibodies: anti: CD25, 27, 28, 29, 62L FITC,
anti: CD45RA, CD45RO, CD21, HLA-DR, IgD PE
anti: CD3, IgM PC5 and anti: CD4, 8, 19 ECD
Phenotyping by four-color cytometer Coulter EPICS MCL
In our study were examined B- and T-cell subpopulations of 42 CVID patients and 33 healthy controls. Patients fulfilled the ESID diagnostic criteria for CVID and were on regular substitution by intravenous immunoglobulin. Blood samples were collected before the IVIG infusion was given after an informed consent was obtained. Healthy donors were studied in parallel with the patients.
For B-cell analysis peripheral blood mononuclear cells were isolated from heparinized blood by density gradient centrifugation and consequential stained by a standard procedure by flow cytometry. It includes labeling by appropriate antibodies, lysis of erytrocyte, stabilization and fixation
For T cells analysis – whole blood stained by a standard procedure by flow cytometry. The folowing monoclonal antibodies was used. Phenotyping was performed by four-colour cytometer Epics MCL.

5. B-cell differentiation
Bone marrow
Plasma cells
IgM
Plasma cells
IgG,IgA, IgE
CD19+/-, CD27+, CD IgG+, IgA+, IgE+,
Immature
trans.
cells
CD19+, CD IgM+, IgDlow CD21-
CD19+/-, CD27+, CD IgM+
Spleen
Naive
cells
Secondary lymphoid organ
CD19+, CD CD38-, IgG+, IgA+,
IgE+
Freiburg CVID classification it based on flow cytometric analysis of different B-cell subsets in pheripheral blood. This slide shows particular stages of differentiation of B cells, which we can detect in blood. Cells leave the bone marrow as immature transitional B-cells This cells do not express CD21 - marker of the progression of immature via transitional into mature naive B cells. When the naive B cells are stimulated by antigenin the secondary lymphoid organ in the presence of the appropriate costimulation, they develop into plasma cells or memory B cells in a germinal center . CD27 is a general marker for memory B cells in humans.
In most patients with CVID have a decreased number of both class-switched memory B cells and plasma cells.This indicate defect in late B cell differentiation.
* jednotlivá diferenciační stádia B lymfocytů. Které můžeme detegovat v krvi.
CD19+, CD IgM+, IgD+ / CD21+
Memory
IgG,IgA,IgE
cells
CD19+, CD IgM+, IgD CD21+
IgM
Memory
cells

6. „Freiburg“ classification
11 patients
Ia > 20% immature B cells
Group I < 0,4% switched memory B cells of total PBLs
15 patients
Ib normal % immature B cells
CVID patients
Group II >0,4% switched memory B cells of total PBLs
6 patients
Group III <1% B cells of total PBLs
On this slide, we can see CVID patients subdived according to Freiburg classification. CVID patients were divided into 4 groups and subgroups: group I comprises patients with decreased number of switched memory B cells (below 0.4 % of total peripheral blood lymphocytes - PBLs) and group II includes all patients with normal number of switched memory B cells (>0.4 of PBLs). Group III had less than 1 % B-cells of PBLs. Patients of group I were subdivided into group Ia with increased number of immature B cells (higher than 20 % of CD19 number IgM+CD21-) and Ib with normal number of IgM+CD21- immature B-cells. Accumulation of immature B cells in the blood points toward a disturbed maturation or prematuration exodus of immature B cells from the bone marrow.

7. CD4+ T-lymphocyte subpopulation
In this slide we can see the CD4+ subpopulation abnormalities in differred CVID subgroups. Although the increase in CD45RO+ activated/memory CD4+ T cells was documented in all CVID subgroups, abnormalities in other T-lymphocyte activation markers (increase in CD29, HLA-DR, decrease in CD27, CD62L, CD45RA) were observed specifically in group Ia, which has the most severe B cell diferentiation deffect: diminution of class-switched memory B cells and increased number immature B cells. Also the differences from the control group were more significant in Ia subgroup than in the remaining groups..
Surprisingly, when comparing T-lymphocyte abnormalities in various CVID subgroups, we observed most differences between subgroups Ia and Ib, which are both characterised by decreased numbers of memory (CD27+) B cells; . We also showed that subgroup Ib was more similar to normal donors than the other subgroups.

8. Correlations between the expresion of maturation/activation markers on B and T lymphocytes
Immature B cells
Memory B cells
We searched for correlations between examined differentiation markers of B lymphocytes and CD4 T cells activation markers in CVID patients and control group. No significant correlations were observed for the control group.
In CVID patients no significant correlations were observed between T cell activation markers and the percentage of naive, IgM memory and switched memory B cells
The only correlations we obserwed were between subpopulation of immature B-cells and several CD4+T-cell markers (CD27, CD62L, CD45RA, CD29, HLA-DR, CD45RO). These were the same markers, which were most frequently abnormal in group Ia. (decreased number of memory B cells and increased number immature B cells).

9. Age distribution in CVID subgroups
When we compared age of individual subgroups of CVID patients, we found a significant difference between groups Ia and Ib (P=0.008; Student’s t-test, and P=0.009; Mann-Whitney test). We did not find any significant differences in age between the remaining groups.
CVID group

10. Age dependent correlations in the CVID groups and control groups
Naive B cell
IgM memory B cells
CVID patients
Patients CVID
Control
Because we found the differences in the age of patients in groups Ia and Ib, we searched for correlations between T-lymphocyte and B-lymphocyte markers and the age in both patient and control groups We observed that the percentage of naive B-cells (IgD+CD27-) and IgM memory B cells (IgD+CD27+) was age-dependent in the CVID group, such as the percentage of CD27+, CD45RA+ and CD45RO+ in CD4+ cells. The only significant age-dependent correlation in the control group was the percentage of CD45RO+ in CD4+ cells.
Age
Age

11. B-cell based classification of CVID patients may be age-related
Conclusion
T- and B-cell abnormalities in CVID patients are partially related to each other (correlation between immature B cells and CD4+ T activation (CD27, CD29, HLA-DR) and differentiation (CD45RA, CD45RO, CD62L))
Abnormalities in B-cell subpopulation in CVID patients are not definitive but may evolve with the age of patients
B-cell based classification of CVID patients may be age-related
Our study demonstrates that T- and B-lymphocyte abnormalities in CVID are partially related each other – as I have mentioned we have observed correlation between immature B cells and CD4+ T activation (CD27, CD29, HLA-DR) and differentiation ( CD45RA, CD45RO, CD62L) Some of those abnormalities are not fixed but may evolve with the age of patients - These includes in T cells CD27, CD45RA, CD45RO, and naive and IgM memory in B cells. Consequently B-cell based classification of CVID patients may be age-related

12. Thank you for your attention
Ladies a gentlemen, Mr. Chairman
I would like to introduce my presentation to you : Relations between T- and B- lymphocyte abnormalities in CVID patients and their age dependency

13. Age dependent correlations in the CVID groups and control groups
Patients CVID
Controls
% Naive
B-cells
% IgM
memory
B-cells
% CD27+
of CD4+ %
CD45RA+
of CD4+ %
CD45RO+
of CD4+
Age

14. CD8+ T-lymphocyte subpopulation

15. Correlations between the expresion of maturation/activation markers on B and CD8+T lymphocytes

16. Acknowledgments
I am most grateful tu my tutor prof. Jiří Litzman for advice and comments
I thank my collegues in our institute and my family for a patience and you for your attention.
I am most grateful tu my tutor prof. Jiří Litzman for advice and comments. I thank my co-workers: Mária Šárfyová, Luděk Bláha and Vojtěch Thon and my family for a patience.

17. Statistical analysis

18. Various stages of B cell differentiation
Healthy control
CVID patients
Naive B cells: 60,5%
Naive B cells: 93%
IgM memory: 5,6%
IgM memory: 15,3%
IgD
IgD
Memory B cells: 0,1%
This slide show, that we can be subdivide B cells into 3 subpopulation by means of CD27 as a marker for memory B cell and IgD: – naive B cell areCD27-IgD/M+, non-switched IgM memory B cells areCD27+IgD/M+ and switched memory B cells are CD27+IgD-. Here is example of healthy control and here is example of CVID patient.
Memory B cells: 21,3%
CD 27
CD 27

19. Acknowledgments
I am most grateful tu my tutor prof. Jiří Litzman for advice and comments
I thank my co-workers: Mária Šárfyová, Luděk Bláha and Vojtěch Thon
and my family for a patience
I would like to thank to all my co-workers. I am very happy that the article was just recently accepted for publication in Clinical a nd experimental immnunology

20. Immature B cells Healthy control CVID patient IgM IgM CD21 CD21
Here we can see he progression of immature via transitional to mature B cells can be monitored by the expression CD21. The part of this patient have a increased number immature B cell IgM+CD21-.
CD21
CD21