Replication history of B lymphocytes reveals homeostatic proliferation and extensive antigen-induced B cell expansion

The contribution of proliferation to B lymphocyte homeostasis and antigen responses is largely unknown. We quantified the replication history of mouse and human B lymphocyte subsets by calculating the ratio between genomic coding joints and signal joints on kappa-deleting recombination excision circles (KREC) of the IGK-deleting rearrangement. This approach was validated with in vitro proliferation studies. We demonstrate that naive mature B lymphocytes, but not transitional B lymphocytes, undergo in vivo homeostatic proliferation in the absence of somatic mutations in the periphery. T cell–dependent B cell proliferation was substantially higher and showed higher frequencies of somatic hypermutation than T cell–independent responses, fitting with the robustness and high affinity of T cell–dependent antibody responses. More extensive proliferation and somatic hypermutation in antigen-experienced B lymphocytes from human adults compared to children indicated consecutive responses upon additional antigen exposures. Our combined observations unravel the contribution of proliferation to both B lymphocyte homeostasis and antigen-induced B cell expansion. We propose an important role for both processes in humoral immunity. These new insights will support the understanding of peripheral B cell regeneration after hematopoietic stem cell transplantation or B cell–directed antibody therapy, and the identification of defects in homeostatic or antigen-induced B cell proliferation in patients with common variable immunodeficiency or another antibody deficiency

Molecular Monitoring of Lymphoma

This chapter provides the background information of the PCR targets for molecular MRD monitoring (i.e., Ig/TCR gene rearrangements and chromosome aberrations), explains how these targets can be identified. [...

Molecular Monitoring of Lymphoma

This chapter provides the background information of the PCR targets for molecular MRD monitoring (i.e., Ig/TCR gene rearrangements and chromosome aberrations), explains how these targets can be identified. [...

Ig heavy chain gene rearrangements in T-cell acute lymphoblastic leukemia exhibit predominant DH6-19 and DH7-27 gene usage, can result in complete V-D-J rearrangements, and are rare in T-cell receptor αβ lineage

Rearranged IGH genes were detected by Southern blotting in 22% of 118 cases of T-cell acute lymphoblastic leukemia (ALL) and involved monoallelic and biallelic rearrangements in 69% (18/26) and 31% (8/26) of these cases, respectively. IGH gene rearrangements were found in 19% (13/69) of CD3- T- ALL and in 50% of TCRγδ+ T-ALL (12/24), whereas only a single TCRαβ+ T- ALL (1/25) displayed a monoallelic IGH gene rearrangement. The association with the T-cell receptor (TCR) phenotype was further supported by the striking relationship between IGH and TCR delta (TCRD) gene rearrangements, ie, 32% of T-ALL (23/72) with monoallelic or biallelic TCRD gene rearrangements had IGH gene rearrangements, whereas only 1 of 26 T-ALL with biallelic TCRD gene deletions contained a monoallelic IGH gene rearrangement. Heteroduplex polymerase chain reaction (PCR) analysis with VH and DH family- specific primers in combination with a JH consensus primer showed a total of 39 clonal products, representing 7 (18%) VH-(DH-)JH joinings and 32 (82%) DH- JH rearrangements. Whereas the usage of VH gene segments was seemingly random, preferential usage of DH6-19 (45%) and DH7-27 (21%) gene segments was observed. Although the JH4 and JH6 gene segments were used most frequently (33% and 21%, respectively), a significant proportion of joinings (28%) used the most upstream JH1 and JH2 gene segments, which are rarely used in precursor-B-ALL and normal B cells (1% to 4%). In conclusion, the high frequency of incomplete DH-JH rearrangements, the frequent usage of the more downstream DH6-19 and DH7-27 gene segments, and the most upstream JH1 and JH2 gene segments suggests a predominance of immature IGH rearrangements in immature (non-TCRαβ+) T-ALL as a result of continuing V(D)J recombinase activity. More mature αβ-lineage T-ALL with biallelic TCRD gene deletions apparently have switched off their recombination machinery and are less prone to cross-lineage IGH gene rearrangements. The combined results indicate that IGH gene rearrangements in T-ALL are postoncogenic processes, which are absent in T-ALL with deleted TCRD genes and completed TCR alpha (TCRA) gene rearrangements.</p

Ig heavy chain gene rearrangements in T-cell acute lymphoblastic leukemia exhibit predominant DH6-19 and DH7-27 gene usage, can result in complete V-D-J rearrangements, and are rare in T-cell receptor αβ lineage

Rearranged IGH genes were detected by Southern blotting in 22% of 118 cases of T-cell acute lymphoblastic leukemia (ALL) and involved monoallelic and biallelic rearrangements in 69% (18/26) and 31% (8/26) of these cases, respectively. IGH gene rearrangements were found in 19% (13/69) of CD3- T- ALL and in 50% of TCRγδ+ T-ALL (12/24), whereas only a single TCRαβ+ T- ALL (1/25) displayed a monoallelic IGH gene rearrangement. The association with the T-cell receptor (TCR) phenotype was further supported by the striking relationship between IGH and TCR delta (TCRD) gene rearrangements, ie, 32% of T-ALL (23/72) with monoallelic or biallelic TCRD gene rearrangements had IGH gene rearrangements, whereas only 1 of 26 T-ALL with biallelic TCRD gene deletions contained a monoallelic IGH gene rearrangement. Heteroduplex polymerase chain reaction (PCR) analysis with VH and DH family- specific primers in combination with a JH consensus primer showed a total of 39 clonal products, representing 7 (18%) VH-(DH-)JH joinings and 32 (82%) DH- JH rearrangements. Whereas the usage of VH gene segments was seemingly random, preferential usage of DH6-19 (45%) and DH7-27 (21%) gene segments was observed. Although the JH4 and JH6 gene segments were used most frequently (33% and 21%, respectively), a significant proportion of joinings (28%) used the most upstream JH1 and JH2 gene segments, which are rarely used in precursor-B-ALL and normal B cells (1% to 4%). In conclusion, the high frequency of incomplete DH-JH rearrangements, the frequent usage of the more downstream DH6-19 and DH7-27 gene segments, and the most upstream JH1 and JH2 gene segments suggests a predominance of immature IGH rearrangements in immature (non-TCRαβ+) T-ALL as a result of continuing V(D)J recombinase activity. More mature αβ-lineage T-ALL with biallelic TCRD gene deletions apparently have switched off their recombination machinery and are less prone to cross-lineage IGH gene rearrangements. The combined results indicate that IGH gene rearrangements in T-ALL are postoncogenic processes, which are absent in T-ALL with deleted TCRD genes and completed TCR alpha (TCRA) gene rearrangements.</p

Involvement of Artemis in nonhomologous end-joining during immunoglobulin class switch recombination

DNA double-strand breaks (DSBs) introduced in the switch (S) regions are intermediates during immunoglobulin class switch recombination (CSR). These breaks are subsequently recognized, processed, and joined, leading to recombination of the two S regions. Nonhomologous end-joining (NHEJ) is believed to be the principle mechanism involved in DSB repair during CSR. One important component in NHEJ, Artemis, has however been considered to be dispensable for efficient CSR. In this study, we have characterized the S recombinational junctions from Artemis-deficient human B cells. Sμ–Sα junctions could be amplified from all patients tested and were characterized by a complete lack of “direct” end-joining and a remarkable shift in the use of an alternative, microhomology-based end-joining pathway. Sμ–Sγ junctions could only be amplified from one patient who carries “hypomorphic” mutations. Although these Sμ–Sγ junctions appear to be normal, a significant increase of an unusual type of sequential switching from immunoglobulin (Ig)M, through one IgG subclass, to a different IgG subclass was observed, and the Sγ–Sγ junctions showed long microhomologies. Thus, when the function of Artemis is impaired, varying modes of CSR junction resolution may be used for different S regions. Our findings strongly link Artemis to the predominant NHEJ pathway during CSR

The nature of circulating CD27+CD43+ B cells

Letter to the Editor.-- et al.M.C. van Zelm is supported by fellowships from the Erasmus University Rotterdam (EUR-Fellowship) and the Erasmus MC, and by Veni grant 916.110.90 from ZonMW/NWO.Peer Reviewe

Autoantibody subclass predominance is not driven by aberrant class switching or impaired B cell development

A subset of autoimmune diseases is characterized by predominant pathogenic IgG4 autoantibodies (IgG4-AID). Why IgG4 predominates in these disorders is unknown. We hypothesized that dysregulated B cell maturation or aberrant class switching causes overrepresentation of IgG4+ B cells and plasma cells. Therefore, we compared the B cell compartment of patients from four different IgG4-AID with two IgG1-3-AID and healthy donors, using flow cytometry. Relative subset abundance at all maturation stages was normal, except for a, possibly treatment-related, reduction in immature and naïve CD5+ cells. IgG4+ B cell and plasma cell numbers were normal in IgG4-AID patients, however they had a (sub)class-independent 8-fold increase in circulating CD20-CD138+ cells. No autoreactivity was found in this subset. These results argue against aberrant B cell development and rather suggest the autoantibody subclass predominance to be antigen-driven. The similarities between IgG4-AID suggest that, despite displaying variable clinical phenotypes, they share a similar underlying immune profile.</p

Autoantibody subclass predominance is not driven by aberrant class switching or impaired B cell development

A subset of autoimmune diseases is characterized by predominant pathogenic IgG4 autoantibodies (IgG4-AID). Why IgG4 predominates in these disorders is unknown. We hypothesized that dysregulated B cell maturation or aberrant class switching causes overrepresentation of IgG4+ B cells and plasma cells. Therefore, we compared the B cell compartment of patients from four different IgG4-AID with two IgG1-3-AID and healthy donors, using flow cytometry. Relative subset abundance at all maturation stages was normal, except for a, possibly treatment-related, reduction in immature and naïve CD5+ cells. IgG4+ B cell and plasma cell numbers were normal in IgG4-AID patients, however they had a (sub)class-independent 8-fold increase in circulating CD20-CD138+ cells. No autoreactivity was found in this subset. These results argue against aberrant B cell development and rather suggest the autoantibody subclass predominance to be antigen-driven. The similarities between IgG4-AID suggest that, despite displaying variable clinical phenotypes, they share a similar underlying immune profile.</p

New insights on human T cell development by quantitative T cell receptor gene rearrangement studies and gene expression profiling

To gain more insight into initiation and regulation of T cell receptor (TCR) gene rearrangement during human T cell development, we analyzed TCR gene rearrangements by quantitative PCR analysis in nine consecutive T cell developmental stages, including CD34+ lin− cord blood cells as a reference. The same stages were used for gene expression profiling using DNA microarrays. We show that TCR loci rearrange in a highly ordered way (TCRD-TCRG-TCRB-TCRA) and that the initiating Dδ2-Dδ3 rearrangement occurs at the most immature CD34+CD38−CD1a− stage. TCRB rearrangement starts at the CD34+CD38+CD1a− stage and complete in-frame TCRB rearrangements were first detected in the immature single positive stage. TCRB rearrangement data together with the PTCRA (pTα) expression pattern show that human TCRβ-selection occurs at the CD34+CD38+CD1a+ stage. By combining the TCR rearrangement data with gene expression data, we identified candidate factors for the initiation/regulation of TCR recombination. Our data demonstrate that a number of key events occur earlier than assumed previously; therefore, human T cell development is much more similar to murine T cell development than reported before