Louis J. Rezanka

Autoreactive B Cells Escape Clonal Deletion by Expressing Multiple Antigen Receptors

IgH and L chain transgenes encoding a phosphocholine (PC)-specific Ig receptor were introduced into recombinase-activating gene (Rag-2−/−) knockout mice. The PC-specific B cells that developed behaved like known autoreactive lymphocytes. They were 1) developmentally arrested in the bone marrow, 2) unable to secrete Ab, 3) able to escape clonal deletion and develop into B1 B cells in the peritoneal cavity, and 4) rescued by overexpression of bcl-2. A second IgL chain was genetically introduced into Rag-2−/− knockout mice expressing the autoreactive PC-specific Ig receptor. These dual L chain-expressing mice had B cells in peripheral lymphoid organs that coexpressed both anti-PC Ab as well as Ab employing the second available L chain that does not generate an autoreactive PC-specific receptor. Coexpression of the additional Ig molecules rescued the autoreactive anti-PC B cells and relieved the functional anergy of the anti-PC-specific B cells, as demonstrated by detection of circulating autoreactive anti-PC-Abs. We call this novel mechanism by which autoreactive B cells can persist by compromising allelic exclusion receptor dilution. Rescue of autoreactive PC-specific B cells would be beneficial to the host because these Abs are vital for protection against pathogens such as Streptococcus pneumoniae.

Partial dissociation of subgroup C phenotype and in vivo behaviour in feline leukaemia viruses with chimeric envelope genes

Feline leukaemia viruses (FeLVs) are classified into subgroups A, B and C by their use of different host cell receptors on feline cells, a phenotype which is determined by the viral envelope. FeLV-A is the ubiquitous, highly infectious form of FeLV, and FeLV-C isolates are rare variants which are invariably isolated along with FeLV-A. The FeLV-C isolates share the capacity to induce acute non-regenerative anaemia and the prototype, FeLV-C/Sarma, has strongly age-restricted infectivity for cats. The FeLV-C/Sarma env sequence is closely related to that of common, weakly pathogenic FeLV-A isolates. We now show by construction of chimeric viruses that the receptor specificity of FeLV-A/Glasgow-1 virus can be converted to that of FeLV-C by exchange of a single env variable domain, Vr1, which differs by a three codon deletion and nine adjacent substitutions. Attempts to dissect this region further by directed mutagenesis resulted in disabled proviruses. Sequence analysis of independent natural FeLV-C isolates showed that they have unique Vr1 sequences which are distinct from the conserved FeLV-A pattern. The chimeric viruses which acquired the host range and subgroup properties of FeLV-C retained certain FeLV-A-like properties in that they were non-cytopathogenic in 3201B feline T cells and readily induced viraemia in weanling animals. They also induced a profound anaemia in neonates which had a more prolonged course than that induced by FeLV-C/Sarma and which was macrocytic rather than non-regenerative in nature. Although receptor specificity and a major determinant of pathogenicity segregate with Vr1, it appears that sequences elsewhere in the genome influence infectivity and pathogenicity independently of the subgroup phenotype.

p52-independent nuclear translocation of RelB promotes LPS-induced attachment

The NF-kappaB signaling pathways have a critical role in the development and progression of various cancers. In this study, we demonstrated that the small cell lung cancer cell line (SCLC) H69 expressed a unique NF-kappaB profile as compared to other cancer cell lines. The p105/p50, p100/p52, c-Rel, and RelB protein and mRNA transcripts were absent in H69 cells but these cells expressed RelA/p65. The activation of H69 cells by lipopolysaccharide (LPS) resulted in the induction of RelB and p100 expression. The treatment also induced the nuclear translocation of RelB without the processing of p100 to p52. Furthermore, LPS-induced beta1 integrin expression and cellular attachment through an NF-kappaB-dependent mechanism. Blocking RelB expression prevented the increase in the expression of beta1 integrin and the attachment of H69. Taken together, the results suggest that RelB was responsible for the LPS-mediated attachment and may play an important role in the progression of some cancers.

T15-idiotype-negative B cells dominate the phosphocholine binding cells in the preimmune repertoire of T15i knockin mice.

T15i knockin (KI) mice express a H chain that is encoded by a rearranged T15 VDJ transgene which has been inserted into the JH region of chromosome 12. This T15H chain combines with a κ22–33 L chain to produce a T15-Id+ Ab having specificity for phosphocholine (PC). Inasmuch as T15-Id+ Abs dominate the primary immune response to PC in normal mice, it was surprising to find that 80% of the PC-dextran-binding B cells in unimmunized homozygous T15i KI mice were T15-Id−. Analysis of L chains expressed in these T15-Id−, PC-specific B cells revealed that two L chains, κ8–28 and κ19–15, were expressed in this population. The Vκ region of these L chains was recombined to Jκ5, which is typical of L chains present in PC-specific Abs. When T15i KI mice were immunized with PC Ag, T15-Id+ B cells expanded 6-fold and differentiated into Ab-secreting cells. There was no indication that the T15-Id− B cells either proliferated or differentiated into Ab-secreting cells following immunization. Thus, T15-Id− B cells dominate the PC-binding population, but they fail to compete with T15-Id+ B cells during a functional immune response. Structural analysis of T15H:κ8–28L and T15H:κ19–15L Abs revealed L chain differences from the κ22–33 L chain which could account for the lower affinity and/or avidity of these Abs for PC or PC carrier compared with the T15-Id+ T15H:κ22–33L Ab.

The M603 idiotype is lost in the response to phosphocholine in terminal deoxynucleotidyl transferase-deficient mice.

The majority of anti‐phosphocholine (PC) antibodies induced by the PC epitope in Proteus morganii (PM) express the M603 idiotype (id), which is characterized by an invariant Asp to Asn substitution at the VH:DH junction. To elucidate the molecular basis by which M603‐like B cells acquire the mutations resulting in this invariant substitution, we analyzed the immune response to PC‐PM in terminal deoxynucleotidyl transferase (TdT) gene knockout (KO) mice. In the absence of TdT, T15‐id antibodies comprised 80–100% of the primary response to PC‐PM. Less than 10% of the response in wild‐type mice is T15‐id+. In TdT KO mice, the secondary response to PC‐KLH was higher than in wild‐type mice and was dominated by the germ‐line T15‐id. About 10% of this response, in both TdT KO and wild‐type mice, comprised M167‐id+ antibodies. Additionally, none of the functionally rearranged V1/DFL16.1/JH1 cDNA isolated from PC‐PM‐immunizedTdT KO mice showed the Asp/Asn substitution characteristic of PC‐binding, PC‐PM‐induced M603‐like antibodies. These data indicate that production of M603‐id antibody is TdT dependent, while generation of M167‐id antibody is TdT independent, and that in the absence of competition from M603‐like B cells, T15‐id B cells can respond to PC‐PM.

Transforming Growth Factor β1 (TGF-β1) Suppresses Growth of B-cell Lymphoma Cells by p14ARF-dependent Regulation of Mutant p53

Background: TGF-β1 suppresses growth of B-cell lymphoma cells. Results: TGF-β1-induced down-regulation of mutant p53 via p14ARF renders B-cell lymphoma cells sensitive to TGF-β1. Conclusion: Overexpression of p14ARF possibly causes TGF-β1 resistance. Significance: p14ARF is a potential therapeutic target for B-cell lymphoma. Previously we reported that TGF-β1-induced growth suppression was associated with a decrease in mutant p53 levels in B-cell lymphoma cells. The goal of the present study was to understand the mechanism involved in TGF-β1-mediated down-regulation of mutant p53. In RL and CA46, two B-cell lymphoma cell lines, TGF-β1 treatment caused down-regulation of E2F-1 transcription factor resulting in the down-regulation of both p14ARF and mutant p53, leading to growth arrest. Experimental overexpression of E2F-1 increased p14ARF level and blocked TGF-β1-induced down-regulation of p14ARF. Overexpression of p14ARF blocked the down-regulation of mutant p53 and prevented growth arrest. p14ARF also attenuated TGF-β1-induced p21Cip1/WAF1 induction, which was reversible by p53 siRNA, indicating the involvement of mutant p53 in controlling the TGF-β1-induced expression of p21Cip1/WAF1. The interaction observed between phospho-Smad2 and mutant p53 in the nucleus could be the mechanism responsible for blocking the growth-suppressive effects of TGF-β1. In RL cells, p14ARF is present in a trimer consisting of mutant p53-Mdm2-p14ARF and in a dimer consisting of Mdm2-p14ARF. Because it is known that Mdm2 can degrade p53, it is possible that, in its trimeric form, p14ARF is able to stabilize mutant p53 by inhibiting Mdm2. In its dimeric form, p14ARF may be sequestering Mdm2, limiting its ability to degrade p53. Collectively, these data demonstrate a unique mechanism in which the inhibition of TGF-β1-mediated growth suppression by mutant p53 can be reversed by the down-regulation of its stabilizing protein p14ARF. This work suggests that the high levels of p14ARF often found in tumor cells could be a potential therapeutic target.