ALBERT

Description: Induction of antibody-mediated immunity against hematologic malignancies requires CD4+ T-cell help, but weak tumor antigens generally fail to induce adequate T-cell responses, or to overcome tolerance. Conjugate vaccines can harness alternative help to activate responses, but memory B cells may then be exposed to leaking tumor-derived antigen without CD4+ T-cell support. We showed previously using lymphoma-derived idiotypic antigen that exposure to “helpless” antigen silences the majority of memory IgG+ B cells. Transfer experiments now indicate that silencing is permanent. In marked contrast to IgG, most coexisting IgM+ memory B cells exposed to “helpless” antigen survive. Confirmation in a hapten (NP) model allowed measurement of affinity, revealing this, rather than isotype, as the determinant of survival. IgM+ B cells had Ig variable region gene usage similar to IgG but with fewer somatic mutations. Survival of memory B cells appears variably controlled by affinity for antigen, allowing a minority of low affinity IgG+, but most IgM+, memory B cells to escape deletion in the absence of T-cell help. The latter remain, but the majority fail to undergo isotype switch. These findings could apply to other tumor antigens and are relevant for vaccination strategies aimed to induce long-term antibody.

Description: We analyzed lymphocyte morphology, histology, immunophenotype, immunoglobulin heavy chain (IgVH) gene mutations, and clinical course in 80 unselected patients presenting with circulating t(11;14) lymphocytes. Of the 80 patients, 43 had peripheral lymphadenopathy (nodal group), and histology confirmed mantle cell lymphoma (MCL) in all. There were 37 patients with no lymphadenopathy (nonnodal group); 13 of 37 had histology, all showing MCL. IgVH genes were unmutated in 28 (90%) of 31 nodal and 15 (44%) of 34 nonnodal cases (P = .0001); CD38 was positive in 32 (94%) of 34 nodal and 16 (48%) of 33 nonnodal cases (P 〈 .001); 41 (95%) of 43 nodal patients required immediate treatment compared with 18 (49%) of 37 nonnodal patients who had indolent disease (P 〈 .0001). Median survival (95% confidence interval) was 30 months (10-50) in the nodal group and 79 months (22-136) in the nonnodal group (P = .005). Mutation status did not statistically affect survival, but of 6 long-term survivors (〉 90 months) all were nonnodal and 5 of 5 had mutated IgVH genes. Lymphocyte morphology was heterogeneous in both groups: typical MCL in 56 cases (34 nodal, 22 nonnodal), blastoid MCL in 8 cases (3 nodal, 5 nonnodal), and small-cell MCL in 16 cases (6 nodal, 10 nonnodal, P = .12). Matutes immunophenotyping score was 1 in 65 cases and 2 in 15 (8 nodal, 7 nonnodal). We find no evidence against a diagnosis of MCL in the nonnodal group and suggest that mutated IgVH genes may help identify patients with indolent disease.

Description: Abstract 1567 The B-cell receptor (BCR) is critical to survival of normal B-cells, and regulates key aspects of cellular behavior. Of these, response to antigen determines pathways of normal B-cell maturation, including isotype switch events that occur by deletional class switch recombination (CSR), an irrevocable event, to yield IgG/A memory B-cells. Less frequently, CSR via a cryptic site generates IgD+ B-cells whereas IgM+IgD+ antigen experienced B-cells synthesize each isotype by an alternative transcript splicing mechanism. The role of the BCR in survival of malignant B-cells however is less well defined, in particular in response to antigen. Intriguingly, in Hairy cell leukemia (HCL), BCR assembly occurs with multiple surface immunoglobulin (sIg) isotypes (mult-HCL), many co-expressed on individual hairy cells (HCs) in an otherwise monoclonal tumor. Multiple isotypes appear to exclude deletional CSR events, and suggests a RNA processing mechanism of molecular assembly. This phenotype is rare even amongst malignant B-cells, and raises the question of the functional relevance of individual variant isotypes. It also potentially presents a model to dissect roles of multiple isotypes on single B-cells. To examine this, we investigated the BCR in CD19+CD11c+CD103+ mult-HCL cases (n=10), in which 2–4 differing sIg isotypes were present on most HCs, with single or, in 3 cases, dual sIgL expression. In all cases, IGHV genes were mutated, and confirmed monoclonality. Phenotype revealed 2 distinct subsets by sIg isotype co-expression, IgD+ve and IgD-ve. Using Ca2+ flux and ERK phosphorylation assays after cross-linking with specific anti-sIg antibodies, we observed a functional BCR in all mult-HCL examined, in both subsets (10/10 cases Ca2+, 6/6 cases ERK). However, striking differences emerged between the two subsets. In sIgD+ve mult-HCL, IgD mediated persistent Ca2+ flux, with flux also evident via 〉1 sIgH isotype. In marked contrast, in sIgD-ve mult-HCL Ca2+ flux was restricted to a single sIgH isotype, but not via IgM. Flux signals in this subset were transient. In most cases only a single sIgL transduced flux. We next evaluated BCR endocytosis after cross-linking individual isotypes and IgL. In 2 sIgD+ve cases, anti-IgD and anti-Igλ stimulation led to endocytosis of both sIgD and sIgλ, and in 1 case, where examined, anti-IgM stimulation endocytosed both sIgM and sIgλ. In 3 sIgD-ve cases, functional sIgH and sIgL induced endocytosis of the stimulated isotype, but again sIgM was dysfunctional, remaining immobilized on the cell surface. Ca2+ flux through endocytosed isotypes was correspondingly either significantly reduced or ablated in both subsets. In HCs, BCR endocytosis is clearly dependent on functional isotypes and IgL, and parallels events in normal B-cells. Lastly, we examined downstream effects of BCR signalling on cell viability, using soluble (sAb) and bound (bAb) anti-sIg antibodies. In a single IgD+ve mult-HCL case, both sAb and bAb anti-IgM yielded a significant level of apoptosis compared to control antibodies, whereas anti-IgD sAb resulted in no appreciable difference to level of spontaneous apoptosis, suggesting a disengagement of signals from this pathway. This disengagement was also observed in a separate HCL case expressing only IgD, and not in the mult-HCL cohort initially selected, where anti-IgD signals again did not increase levels of apoptosis. In IgD-ve mult-HCL (n=4), sAb and bAb specific cross-linking of IgG/A triggered significant apoptosis. These data demonstrate, for the first time, that mult-HCL retains a functional responsiveness via the BCR, suggesting an absence of anergic effects that may follow chronic antigen exposure in-vivo to self-antigen. Signals via sIgM/G/A isotypes, where functional, induce apoptosis in mult-HCL, whereas sIgD opposes such effects. Despite an apparently unique molecular mechanism of IgD expression in mult-HCL, this isotype appears to be hardwired in B-cells to mediate responses that differ from IgM. The persistent flux observed here indicates a more sustained and robust IgD signaling cascade, as also observed in B-cell models. These data reveal distinctive and opposing effects of individual isotypes on BCR mediated behavior in mult-HCL. While apoptotic responses appear to negate a role for antigen in tumor drive in-vivo, potential antigen engagement via IgD, if dominant leaves this question open. Disclosures: No relevant conflicts of interest to declare.

Description: Hairy cell leukemia (HCL) commonly expresses multiple immunoglobulin isotypes, a feature rare in other B-cell malignancies or in normal B cells. In HCL, there is no phenotypic evidence for subpopulations, and single cells from one previous case contained transcripts for several isotypes. This raises the questions of the differentiation status of the cell of origin and of posttransformation events. We have investigated 9 cases, all expressing multiple immunoglobulin isotypes. Multiple tumor-derived variable-(diversity)-joining-constant μ δ, γ, α (V(D)J-Cμ, δ, γ, α) transcripts were confirmed in single cells of a further case. All cases were negative for germinal center (GC)-associated markers CD27 and CD38. Seven of 9 cases had mutated VH genes, with low levels of intraclonal heterogeneity, but 2 of 9 were unmutated, indicative of pre-GC origin. Eight of 9 cases expressed activation-induced cytidine deaminase (AID), a molecule essential for somatic mutation and isotype switch. All cases expressed germ line heavy-chain I exon (IH)-CH transcripts which paralleled surface immunoglobulin (sIg) isotype. Significantly, no circle transcripts indicative of deletional recombination of switched isotypes were detectable in 9 of 9 cases. These data indicate heterogeneity in the cell of origin in terms of mutational status, but reveal common features of AID expression and isotype-switching events occurring prior to deletional recombination. Both mutational and switching events may be influenced by environmental factors at extrafollicular sites. (Blood. 2004;104:3312-3317)

Description: Isotype switch commonly follows onset of somatic hypermutation in the germinal center (GC), with activation-induced cytidine deaminase (AID) as a prerequisite. Mantle cell lymphoma (MCL) with t(11;14) includes a subset with unmutated (UM) and a minor subset with mutated (MUT) VH genes. Here, we investigated whether switch events and AID expression occur in MCL. In 4 of 6 UM and 4 of 7 MUT MCLs, alternative tumor-derived Cγ,α,ϵ transcripts were identified. AID transcripts, including a splice variant, were common to both subsets. AID expression correlated with switch in 8 of 8 cases, but in 3 of 5 cases it occurred with switch absent. Circle transcripts (Iγ-Cμ/Iα-Cμ) were identified in 5 of 7 evaluated cases. In 1 of 12 cases, 12% of tumor cells expressed immunoglobulin L-restricted surface IgA. Ongoing switch recombination events appear to be a feature of MCL, likely restricted to a minor tumor subpopulation, with occasional variant sIg expression. UM MCLs implicate origins from pre-GC B cells and reveal switch events at ectopic sites. (Blood. 2004;103:2795-2798)

Description: In Waldenstrom’s macroglobulinemia (WM), which locates primarily in the bone marrow (BM), VH gene analysis had previously suggested origins from a post-follicular B-cell arresting prior to isotype switch. Using more sensitive assays, facilitated by amplified cDNA from BM cells, nested PCR unexpectedly revealed tumor-derived isotype-switch transcripts in 7/7 cases. In 5/7 cases, both Cγ and Cα variant transcripts were identified, and Cγ or Cα only in 2/7. Detection of activation induced cytidine deaminase (AID) and germline and circle transcripts confirmed switching activity. Selected gene expression profiles established the memory B-cell marker CD27 as highly expressed in all cases. These findings were evaluated further in additional WM cases where availability of tumor material allowed detailed analysis. In 2/2 cases, phenotype suggested a variable CD27 expression within the tumor clone. In these, tumor IgM transcripts were readily detected in both the CD19+CD27+ and CD19+CD27− fractions, and in 1 of the 2 cases, post-switched tumor-derived Cα transcripts were also identified in each fraction. In this WM case, the frequency of tumor-derived transcripts was then assessed at the single cell level. Switch transcripts were identified in 3/96 cells with no co-expression of the IgM isotype. Similarly, AID transcripts were observed in some cells, not always correlating with switch events or with ongoing somatic mutation, which was apparent in VDJ-Cμ sequences. These findings reveal a dynamic intratumoral diversification, with AID activated and ongoing mutational and switch activity occurring post-transformation in a proportion of the tumor clone. Heterogeneity in CD27 expression is also evident within tumor cells, revealing phenotypic change. Interestingly, these data indicate that although WM tumor cells have arrested at the IgM stage and do not express isotype switched Ig, they retain the capacity to initiate events critical for isotype switch.