Molecular Analysis of B cell Subsets in Common Variable Immunodeficiency (CVID) A Ridley, S Harris, J Burden, B Ferry, A Janda, Z Davis, D Oscier, AP Williams, JL Smith, E Hodges

CVID 1 in 50,000 Most common primary antibody deficiency Diagnosed between 20 & 40 years Heterogeneous syndrome Decreased Ig levels Recurrent infections Other causes of immunodeficiency excluded The cause of the disorder is unclear, however it is thought that it may arise from a problem in the development of B cells from mature B cells into plasma cells. It may also be dependant on the mutational status of the antibody.

Classification of CVID naive memory CD27 Ig M/D IgM Memory ~4% Class Switched Memory ~1% MB0 Reduced memory B cells LPD Granulomatous Splenomegaly naive memory CD27 Ig M/D IgM Memory ~20% Class Switched Memory ~4% MB1 Reduced class switched memory B cells Splenomegaly Current classifications methods are based on B cell phenotype, looking a class switched and non-class switched memory B cells. These classifications are essentially quantitative, looking at numbers of B cells, but not qualitative, which could be ascertained by looking at the quality of the antibody produced by these B cells. This slide illustrates a widely use classification system defined by Piqueras’s group. The were able to delineate 3 subgroups and correlate there to clinical complications experienced by the patients. In the first group …………………………………………. CD27 Ig M/D naive memory IgM Memory ~20% Class Switched Memory ~20% MB2 Class switched memory B cells Clinical complications less severe

IgD+/CD27+ IgD-/CD27+ Somatic hypermutation (SHM): nucleotide substitution in V region to increase affinity for antigen Class switch recombination (CSR): B-cells substitutes expression of IgM and IgD for IgG, IgA or IgE by deleting DNA between switch regions This slide shows the passage of a normal B cell through the germinal centre of a secondary lymphoid organ & subsequent maturation. Naïve B cells that have never encountered antigen are CD27 negative and IgD positive. In the GC they encounter foreign antigen on follicular DC, become activated and acquire CD27 expression.   It is here that they may undergo the process of SHM and class switching. Somatic hypermutation is the process by which nucleotides are substituted in the gene segments of the variable regions of the BCR to increases the affinity for foreign antigen. In the process of class switching B-cells substitutes expression of IgM and IgD for expression of IgG, IgA or IgE. CSR arises by means of a specialized nonhomologous DNA recombination mechanism. Finally B cells terminally differentiate and leave the germinal center as an antibody producing plasma cell or a memory B cell. All of the memory B cells express CD27 while only those that have not class switched retain the expression of IgD. Class switched B cells loss the expression of IgD. IgD+ and CD27-

Aim Molecular methodology to develop CVID classification to a molecular level Investigation of the pattern and frequency of somatic hypermutation in B cell subgroups as defined by CD27 expression Restriction enzyme-based hot-spot mutation assay (REHMA): screening test to detect the presence of SHM in B cells of CVID patients To allow improved characterisation, prognosis and management of CVID patients The aim of the project is to make use of molecular methodology to develop the classification of CVID to a molecular level. This was done by investigating the pattern and frequency of somatic hypermutation in B cell. These B cell subgroups have previously been classified by phenotypic expression by investigating surface of CD27, a marker of mature memory B cells. It is also found on some T lymphocytes. This involved establishing a REHMA; a non-radioactive, rapid method of looking SHM and investigating the quality of the antibody produced by these B cells. This involved modification of the original methodology by Bonhommes group to make use of fluorescently labelled primer. The rational for carrying out this project is that not only would a molecular classification of this disease would allow better characterisation of these patients but it would also allow greater insight into prognosis and management of the disease.

3 CLL patients 9 healthy controls 10 CVID patients Flow cytometric analysis of B cells using CD19/IgM /IgD/CD27 antibodies Separation of CD27+ peripheral blood B cells Extract RNA & set up RT-PCR reactions to obtain cDNA Amplification with: IgGVH3-23 and C FR1 and FR3 primers Cloning & sequencing of PCR products to identify the pattern of somatic hypermutation RE digestion & fragment length analysis by capillary electrophoresis The method was established using 9 h.c and 3 patients with CLL who had known mutations in the IgVH3-23 gene. 10 CVID patients were then investigated from a well defined cohort of CVID patients, both in terms of phenotypic classification and clinical disease. Peripheral blood was taken and …………………….. IgVH3-23 gene transcripts were studied as they are expressed by between 4 and 10% of circulating B cells. The second round of amplification used primers specific for framework regions 1 and 3 allowing V gene amplification. Cloning and sequencing was carried out to allow confirmation of the REHMA. Correlate levels of somatic hypermutation to previous phenotypic classification

Ig Heavy Chain SHM Analysis Specific product 202 bp IgVH3-23 Cγ FR1 FR3 175 bp 164 bp 158 bp 119 bp Alu I 202 bp 2nd round PCR product Ser31 Ser35 Ala50 Mutated 202 bp Ava II Alu I RE digest Here we can see where the primers bind in the first and second rounds of PCR and the fluorescently labelled FR3 primer.   Following 2 rounds of amplifications specific product of 202 bp is generated, as seen by fragment length analysis. 2 restriction enzymes were then used. 1. The first, Alu I, recognises 3 hot spots that are targeted for SHM. If these hot spots are unmutated because SHM has not occurred, fragments of 119, 164 and 175 base pairs are generated. If all the sites in all the gene transcripts are mutated this restriction enzyme will not digest and the specific product remains, as shown here. 2. The second RE, Ava II, recognises a site that remains unaltered regardless of the process of somatic hypermutation, generating a fragment of 158 base pairs, providing a positive control for the experiment. Unmutated 164 bp 119 bp 175 bp 202 bp Alu I RE digest

RE digestion of CLL patients 158 bp 164 bp Ava II Alu I 119bp 1 2 3 These patients were used as positive controls for establishing the method as they have clearly defined mutations in 1 or more of the 3 hotspots. The 1st example is from a CLL patient with a know mutations at Ser31 and Ser35 but not Ala50. The clonal PCR product from this patient was unmutated at the Ala50 hotspot such that a fragment of 119bp was generated. In the 2nd & 3rd example the CLL patients have a know mutation at the Ala50 hotspot. The clonal PCR product from these patients was mutated at the Ala50 hotspot such that a fragment of 119bp was not generated. In contrast the Ser35 hotspot remained unmutated and a peak of 164 base pairs is seen. Ala50 Ser35 Ser31 Hot spots

RE digestion of a healthy control 202 bp 158 bp 119 bp 164 bp 175 bp Specific product Ava II Alu I This first example shows fragment length analysis of a healthy control. Here much of the specific product remains, suggesting many of the gene transcripts are mutated. Of the peaks generated the peak at 164bp is the predominant peak, suggesting the Ser35 hot spot harbours the least mutations. Ala50 Ser35 Ser31 Hot spots

Alu1 digestion of MB0 CVID patients Control 202 bp 175bp 164 bp 119bp CVID 2 Compared to the control all the patients have less specific product less suggesting there is less SHM occurring. The 1st CVID patient has little mutation at the Ser35 hot spot. The 2nd CVID patient as little mutation at the Ala50 and Ser35 hot spots. The final CVID patient has the least mutations at the Ala50 and Ser31 hot spots. CVID 3 Ala50 Ser35 Ser31 Hot spots

Alu1 digestion of MB1 CVID patients Control 202 bp 119 bp 175 bp 164 bp As compared to the control all except CVID 4 have little specific product, suggesting the IgVH3-23 transcripts from these patients are relatively unmutated compared to the control. CVID 5: predominant fragment at 164bp=Ser35 harbours the least mutations CVID 6: Slightly larger peak at 164bp CVID 7 & 8: predominant peaks at 119 and 164bp indicating Ser31 and Ser35 have fewer mutations than the Ala50 hotspot. Ala50 Ser35 Ser31 Hot spots

Alu1 digestion of MB2 CVID patients Control CVID 9 CVID 10 202 bp 119 bp 175 bp 164 bp 2 CVID patients classified as MB2; similar proportion of memory B cells to healthy individuals. However REHMA analysis reveals 2 distinct patterns within this subgroup. CVID 9: only specific product remaining, indicating all the sequences are mutated. CVID 10: little specific product, suggesting many of the sequences are unmutated & of the hotspots the Ser35 is the least mutated. Ala50 Ser35 Ser31 Hot spots

Mutational status by sequence analysis This slide summarises the cloning and sequencing data. We investigated the percentages of sequences with mutations across the entire IgVH3-23 gene investigated by RHEMA in a healthy control, and a patient classified as MB2 & MB1. We looked at a healthy control and saw that the majority of IgVH3-23 gene transcripts had between 21 &30 mutations. This MB2 patient was the CVID patient 9 seen in the previous slide and it can be seen that all the IgGVH3-23 gene transcripts had between 21 & 30 mutations, confirming the REHMA as an effective assay for rapid analysis of SHM. This MB1 patient was the MB1-a patient looked at on the previous slide, which was seen to have fragments of all 3 sizes generated suggesting little SHM. By cloning and sequencing it can be seen that only one mutation occurred in any of the sequences looked at, again confirming the REHMA as an effective assay for rapid analysis of SHM. Percentages of sequences with 0-1, 2-10, 11-20 and 21-30 mutations from a healthy control, an MB2 patient & an MB1 patient

Summary Rapid, non-radioactive screening method to look at SHM in CVID REHMA confirmed by sequence analysis REHMA patterns heterogeneous in the CVID subgroups defined by phenotype Correlation to clinical disease Investigation of IgM IgVH3-23 transcripts Use of PBMC In summary this project has established a molecular screen for determining antibody affinity. This is non-radioactive, rapid screening test, which could be used in a diagnostic laboratory for improved patient management. The efficiency of the REHMA at detecting the pattern and frequency of SHM was confirmed by sequence analysis. When the method was applied to CVID patients who were well defined phenotypically, highly heterogeneous patterns of SHM were seen even within the defined subgroups. For completeness the pattern of SHM now needs to be correlated to clinical disease to see if this qualitative defect in SHM can explain the severity of clinical complications. Would now like to look at IgM transcripts to assess SHM since CVID patients have more IgM expressing naïve cells and this would give us more information Furthermore we are looking at the results generated using PBMC compared to the results already obtained since the use of PBMC would mean fewer cells are required for the assay, making the assay more viable on a practical basis.

Acknowledgments SGH, Immunology & Molecular Pathology Dr E Hodges Dr J Smith S Harris A Williams Z Shah Churchill Hospital, Oxford J Burden Dr B Ferry Dr A Janda Royal Bournemouth Hospital Dr D Oscier Z Davis