Titolo della tesi: Memory B cell differentiation in the germinal center
Memory B cells (MBCs) generated by the response to infection or vaccination protect us from diseases cause by previously encountered pathogens. In order to describe novel molecular mechanisms involved in the generation of MBCs in the germinal center (GC) we used, as a model, the Common Variable Immune Deficiency (CVID) disorder. I first studied an Italian family (IT4) with an inherited form of CVID, transmitted as an autosomal dominant trait over five generations. Genetic analysis identified a large duplication (~880 kb) in the chromosome region 3q27.3 of affected IT4 members, containing three genes of immunological interest: the Receptor Transporter Protein 4 (RTP4), B-cell lymphoma 6 protein (BCL6) and ST6 Beta-Galactoside Alpha-2,6-Sialyltransferase 1 (ST6GAL1). We focused our attention on the possible correlation between these genes and the pathological status of the family members analysed.
All affected IT4 members were unable to produce switched MBCs and antibodies of switched isotypes but had normal numbers of IgM MBCs. However, patients B cells responded perfectly to in vitro stimulation, generating plasmablasts and secreting antibodies of every isotype. RTP4 and BCL6 were not overexpressed either in unstimulated patients’ cells or in patients’ lymphoblastoid cell lines. The ST6GAL1 enzyme, that catalyzes the transfer of sialic acid from CMP-sialic acid to galactose-containing substrates, was overexpressed in the lymphocytes and plasma of all IT4 members. B cells from IT4 patients had an increased amounts of sialic acid their surface. CD22 is a negative regulator of BCR signalling that is recruited to the B cell receptor upon stimulation and terminate B-cell signalling. The extracellular domain of CD22 binds to α 2,6-linked sialic acid ligands linked to galactose, synthesized by ST6GAL1. BCR signals are of increased intensity and duration in IT4 and cannot be modulated by CD22. We hypothesize that CD22 is sequestered by the increased sialic acid on B cells of IT4 family members resulting in excessive strength of BCR signals. Consequently, patients’ B cells may undergo apoptosis in the GCs during the affinity maturation process, when, instead of inducing survival, exaggerated signalling strength of the B cell receptor may kill high affinity B cells.
During the time of my thesis the COVID-19 pandemics exploded. The rapid development of SARS-CoV-2 vaccines and the vaccination campaigns of healthy controls and immunodeficient patients has given the unique opportunity to study the results of the GC response in a large number of individuals. We are running a study with the aim to define the short- and long-term mechanisms of impaired or preserved immune responses to COVID-19 mRNA BNT162b2 immunization in a population of adult primary antibody deficiency (PAD) patients including two IT4 members. We assessed the duration of immunity, the generation and persistence of MBC and the effects on individuals with previous asymptomatic or mild SARS-CoV-2 infection. Immunocompetent subjects produced specific antibodies and generated MBCs and activated MBCs with high binding capacity that significantly increased after immunization. A minority of PAD patients showed adaptive, atypical immune responses after SARS-CoV-2 immunization. Immunized CVID patients generated atypical memory B cells (ATM) possibly by extra-follicular or incomplete GC reactions; convalescents CVID patients responded to infection by generating spike-specific MBCs that were improved by the subsequent immunization. Poor spike-specific T cell responses were measured independently from the immunological challenge. In conclusion, our observations underline the need of vaccination in convalescent CVID patients. If these responses to vaccination might result in a partial protection from infection or reinfection is now unknown. It should be demonstrated whether a third vaccine dose may induce a protective response in CVID patients.