Quantitation of rare memory B cell populations by two independent and complementary approaches

Abstract

Current methodology for quantitation of memory B cells (MBC) in clinical samples is limited and often does not allow for detection of multiple MBC specificities in a single assay. Here we describe two independent approaches to antigen-specific MBC quantitation. First, a sensitive flow cytometry (FC) assay was developed for simultaneous quantitation of two prototypical B cell antigens, tetanus and diphtheria. Second, an ELISA-based MBC limiting dilution assay (LDA) was developed that provides quantitative analysis of up to 8-12 different MBC frequencies from a single blood sample. Cross-validation studies indicated that MBC numbers measured by FC correlated significantly with the frequencies obtained by LDA (R(2)=0.92, p=0.0002). These two functionally distinct approaches will be useful for accurate quantitation of rare MBC populations specific for either simple antigens (e.g. tetanus and diphtheria) or complex antigens (e.g. vaccinia or other viruses), and is amenable for use in a variety of model systems.

Figure 1. Dual FC staining for MBC increases specificity while maintaining sensitivity

Splenocytes from DTaP-immune and DTaP-naïve mice were divided into three fractions and stained with a single antigen-specific fluorescent reagent, or dual fluorescent reagents. (a) Live cell gates were established and class-switched B cells were defined as IgD⁻CD19⁺ for both immune and naïve mice. For cells stained only with TT-FITC or TT-APC, a FITC positive gate or APC positive gate was defined. A TT-specific MBC gate was also defined for dual stained cells (TT-FITC + TT-APC). The numbers in each dot plot diagram refer to the frequency per 1x10⁶ CD19⁺ cells. Frequencies listed below the dot plots are immune frequencies with the naïve frequencies subtracted. Signal-to-noise ratios refer to the immune frequencies divided by the naïve frequencies (b) Mice were analyzed as described above using DT-specific fluorescent reagents. Mice were Tripedia®-immune (3 doses) with splenocytes collected ~45 days after the last immunization.

Figure 2. Enumeration of TT- and DT-specific MBC by FC

Mouse splenocytes or human PBMC were analyzed for the presence of TT- or DT-binding MBC FC. (a) Live cell gates were established and Ig class-switched B cells were defined as IgD⁻CD19⁺ (mice) or IgD⁻CD20⁺ (human subjects). (b) TT binding within class-switched B cells was identified using TT-APC and TT-FITC conjugated reagents. TT-specific MBC frequencies were calculated from the region gates as shown and indicate the number of TT binding B cells per 10⁶ CD19⁺ (mouse) or CD20⁺ (human) B cells. (c) DT-specific MBC were measured as described for TT, except that DT-Alexa Fluor® 700 and DT-Pacific Blue™ conjugated DT reagents were used. (d) Human TT-specific and DT-specific MBC populations were gated and analyzed for staining of fluorochromes specific for the reciprocal MBC populations as an added test of antigen specificity. Immune mice were vaccinated with Tripedia® (3 doses) with splenocytes prepared 1.5 months after the last immunization (representative of 3 mice in 3 experiments). An adult volunteer at ~5 months post-tetanus/diphtheria booster immunization is shown (representative of 13 subjects from 7 experiments), and naïve human cells were collected from UCB (representative of 3 subjects from 3 experiments).

Figure 3. Quantitation of TT-specific MBC in three mammalian species with internal specificity controls

Mouse splenocytes, RM PBMC, and human PBMC were analyzed for the presence of TT-specific MBC. Live cell gates were determined and class-switched B cells were defined as IgD⁻CD19⁺ (mice) or IgD⁻CD20⁺ (RM and humans). Frequencies shown indicate the number of TT-binding B cells per 10⁶ total B cells (CD19⁺ or CD20⁺ lymphocytes) within the designated region gates. To determine the optimal TT-specific MBC gate, cells from the same sample in each species were divided into two fractions, and stained with either TT reagents (TT FITC + TT-APC) or labeled with antigen-mismatched reagents (TT-FITC + HSA-APC). Splenocytes from Tripedia®-immune mice were analyzed at 1.5 months after the last of three immunizations (representative of 3 mice from 3 experiments). RM were Tripedia®-immune with PBMC collected ~3 years after the last of four Tripedia® immunizations (representative of 6 RM tested in 3 experiments). An adult volunteer at ~6 months post-tetanus/diphtheria booster immunization is shown (representative of 13 subjects from 7 experiments).

Figure 4. Optimization and validation of a human multi-antigen MBC LDA

(a) LDA was performed with human PBMC, using different feeder cells with or without an optimal cytokine cocktail. IgG-specific ELISA performed on LDA supernatants identified cultures containing IgG-producing B cells. The percent of negative cultures was plotted versus the number of CD22⁺ B cells per culture. Regression analysis was performed as described (Miller et al., 1977), and MBC frequencies (per CD22⁺ B cell) were calculated from the 37% intercept. (b) Using optimal culture conditions of NIH/3T3 feeder cells and IL-2, IL-6, IL-10 (as well as SAC, PWM, LPS and CpG-2006), purified peripheral B cells were analyzed by LDA. MBC were detected by testing LDA supernatants (15–25 μL/per test) on antigen-specific ELISA plates (vaccinia, measles, mumps, TT, DT) or by an anti-IgG ELISA for quantitating total IgG-secreting cultures. The subject shown was seropositive for each antigen except vaccinia. (c) Vaccinia-specific MBC frequencies were determined by LDA for both vaccinia naïve (n = 6) and vaccinia immune (n = 17) subjects to demonstrate the specificity and sensitivity of the assay. (d) TT-specific MBC frequencies were compared by FC and LDA. PBMC from adult human donors (n = 13) were analyzed for TT-specific MBC as described in Fig. 3. Purified B cells from the same PBMC samples were analyzed for TT-specific MBC by LDA and compared to the frequencies derived from FC. Least-squares linear regression was used to compare the data sets, and calculate the associated p-value. Open symbols represent data points that were at or below detection limits by either FC or LDA and were not included for correlation analysis.