Terminal deoxynucleotidyl transferase is required for an optimal response to the polysaccharide α-1,3 dextran

Abstract

An understanding of Ab responses to polysaccharides associated with pathogenic microorganisms is of importance for improving vaccine design, especially in neonates that respond poorly to these types of Ags. In this study, we have investigated the role of the lymphoid-specific enzyme TdT in generating B cell clones responsive to alpha-1,3 dextran (DEX). TdT is a DNA polymerase that plays a major role in generating diversity of lymphocyte AgRs during V(D)J recombination. In this study, we show that the DEX-specific Ab response is lower, and the dominant DEX-specific J558 idiotype (Id) is not detected in TdT(-/-) mice when compared with wild-type (WT) BALB/c mice. Nucleotide sequencing of H chain CDR3s of DEX-specific plasmablasts, sorted postimmunization, showed that TdT(-/-) mice generate a lower frequency of the predominant adult molecularly determined clone J558. Complementation of TdT expression in TdT(-/-) mice by early forced expression of the short splice variant of TdT-restored WT proportions of J558 Id+ clones and also abrogated the development of the minor M104E Id+ clones. J558 Id V(D)J rearrangements are detected as early as 7 d after birth in IgM-negative B cell precursors in the liver and spleen of WT and TdT-transgenic mice but not in TdT(-/-) mice. These data show that TdT is essential for the generation of the predominant higher-affinity DEX-responsive J558 clone.

Figure 1. Ontogeny of DEX-responding B cell clones

Figure adapted from (46). (A) Percent of DEX-responsive spleen precursors in BALB/c mice from donors of the indicated ages was determined using the splenic focus assays. (B) The idiotype expression profile in each assay was determined by ELISA using anti-Id antibodies to detect J558 Id, M104E and IdX. IdX, the cross-reactive idiotype, is expressed by most DEX responding precursors. (C) Amino acid sequence alignment of the heavy chain of the two dominant anti-DEX clones M104E and J558 shows that the two clones are identical except for the CDR3 region.

Figure 2. TdT−/− mice elicit a lower antibody response to DEX and fail to generate the J558 Id+ clone

Adult WT and TdT−/− mice were immunized i.v. with DEX-expressing Enterobacter cloacae and serum was collected at the indicated time points. ELISA was used to determine (A) DEX-specific IgM antibodies and (B) J558-specific antibodies (using EB3-7 anti-J558 Id).

Figure 3. TdTS x TdT−/− restore J558 Id+ antibody levels to DEX

Adult WT, TdT−/− and TdTS x TdT−/− mice were immunized i.v. with DEX-expressing Enterobacter cloacae and serum collected day 7 post-immunization. ELISA was used to determine (A) DEX-specific IgM antibodies and (B) J558-specific antibodies. *** p < 0.0001, * p < 0.05 by One Way ANOVA post-hoc test, ns; not significant by Kruskal Wallis post-hoc analysis.

Figure 4. Adult WT and TdT−/− pre-immune mice have similar numbers of DEX-binding B cells

(A) A representative FACS density plot showing the percentage of CD19+ B cells in the spleen that are DEX+ JC5-1+ (λ+) in adult WT BALB/c and TdT−/− mice. C57BL/6 mice were used as negative control. (B) A graphical representation of the absolute cell numbers of DEX+ λ+ B cells in the spleens and peritoneal cavity cells (PEC) of WT and TdT−/− mice. The numbers were back-calculated from the FACS plots represented in (A). LM: littermate control mice.

Figure 5. WT mice response to DEX shows higher J558 Id expression and greater diversity of anti-DEX antibodies than TdT−/− and TdTS x TdT−/−

DEX+ λ+ plasmablasts were sorted 7 days post-immunization with Enterobacter cloacae and V(D)J rearrangements were amplified from genomic DNA from WT, TdT−/− and TdTS x TdT−/− mice. (A) Frequency of DEX+ λ+ plasma cells utilizing J558 (RY), M104E (YD) or other sequences. ND; not detected. The frequency of plasmablasts expressing J558 or M104E was back calculated from the frequency of total DEX+ λ+ plasmablasts (as determined by FACS analysis, 7 days post-immunization) and the percentage of J558+ or M104E+ sequences obtained out of the total V(D)J sequences amplified from bulk DEX-specific plasmablasts. (B) Representative nucleotide and deduced amino acid sequences of heavy chain CDR3 regions amplified from WT, TdT−/− and TdTS x TdT−/−. In the case of sequence overlap nucleotides were arbitrarily assigned to the D_H gene segment except when P-nucleotides to J_H were identified, in which case they were assigned to J_H gene segment. P-nucleotides are underlined. AA: the two amino acids that differentiate each DEX-specific heavy chain sequence from the others. Data is representative of four independent FACS sorts for WT, five for TdT−/− and three for TdTS x TdT−/−.

Figure 6. TdTS expression correlates with J558 expression when J558 Id starts to emerge

B220+ IgM- B cell progenitors were sorted from the spleens, liver and bone marrow of WT, TdT−/− and TdTS x TdT−/− mice at 7 days of age. (A) RT-PCR showing the expression of TdTS and Actin in the liver, spleen and bone marrow. (B) Percent of J558 sequence expression from J558.3-J_H1 V(D)J rearrangements amplified from sorted neonatal B cell progenitors in the liver and spleen. The figure is representative of two independent FACS sorts (one sort for TdTS x TdT−/−) from an entire litter of 7-day-old pups. ND: not detected. N= 17 for WT liver, 11 for WT spleen, 25 for TdT−/− liver, 18 for TdT−/− spleen, 9 for TdTS x TdT−/− liver and 18 for TdTS x TdT−/− spleen. The J558 sequence was not detected in bone marrow B cell progenitors at this time point.