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Limits for Antibody Affinity Maturation and Repertoire Diversification in Hypervaccinated Humans This information is current as of June 14, 2017. Tine Rugh Poulsen, Allan Jensen, John S. Haurum and Peter S. Andersen J Immunol published online 19 September 2011 http://www.jimmunol.org/content/early/2011/09/16/jimmun ol.1000928 http://www.jimmunol.org/content/suppl/2011/09/16/jimmunol.100092 8.DC1 Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Email Alerts Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2011 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Downloaded from http://www.jimmunol.org/ by guest on June 14, 2017 Supplementary Material Published September 19, 2011, doi:10.4049/jimmunol.1000928 The Journal of Immunology Limits for Antibody Affinity Maturation and Repertoire Diversification in Hypervaccinated Humans Tine Rugh Poulsen,1 Allan Jensen,2 John S. Haurum,3 and Peter S. Andersen W hen challenged with a foreign substance, the immune system mounts a genetically diverse polyclonal response of specific Abs, which initially are of predominantly low to intermediate binding affinity (1–3). Binding strength of subsequent generations of Abs is improved through further diversification by introducing point mutations in the V regions, and the resulting variants are selected on the basis of improved binding to Ag (2–4). Recent advances in cloning of Ab genes from single human B cells have provided important new insights into the molecular composition of Ab responses from single individuals (5–11). What remain to be determined are the actual limits of the repertoire diversification processes and their functional outcome in terms of affinity maturation of Abs. Such knowledge can be gained by studying the progression of Ab repertoires in individuals repeatedly exposed to an invariant B cell Ag. Determination of such limits is scientifically important as it reveals some basic features of the adaptive immune system in the protection against infectious diseases and hence is clinically important in the design and implementation of vaccines and development of Ab-based drugs. Ab V gene repertoires in response to antigenic challenges have been reported to display extensive genetic diversity, utilize most of Symphogen A/S, 2800 Lyngby, Denmark 1 Current address: Novo Nordisk A/S, Måløv, Denmark. 2 Current address: Pfizer, Aberdeen, U.K. 3 Current address: ImClone, New York, NY. Received for publication March 25, 2010. Accepted for publication August 1, 2011. T.R.P. was supported by a fellowship from the Danish Agency for Science, Technology and Innovation. Address correspondence and reprint requests to Dr. Peter S. Andersen, Symphogen A/S, Elektrovej Building 375, 2800 Lyngby, Denmark. E-mail address: [email protected] The online version of this article contains supplemental material. Abbreviations used in this article: IMGT, International ImMunoGeneTics; MSD, multiplicative standard deviation; RU, response unit; TT, tetanus toxoid; TT1, tetanus toxoid Ab repertoire donor 1; TT2, tetanus toxoid Ab repertoire donor 2. Copyright Ó 2011 by The American Association of Immunologists, Inc. 0022-1767/11/$16.00 www.jimmunol.org/cgi/doi/10.4049/jimmunol.1000928 the known germline gene fragments, and originate from up to 50 clonally different rearrangements (7, 11) pointing to Ab repertoires being limited to ∼100 clonally different Abs (7). However, it is unresolved how the diversity of B cell repertoires develops in response to repeated exposures to the same Ag as is often the case in prophylactic vaccination of adults. Do repertoires converge to be dominated by a few B cell clones? Do they maintain their clonal composition or are they dynamic and constantly recruiting new B cells? Also, to what extent can somatic hypermutations be introduced to increase repertoire diversity? And finally, to what extent do the diversification processes influence the physical limits for affinity maturation? Kinetic boundaries for maturation of Ab affinity have been proposed on theoretical grounds (12, 13). By considering physiochemical limits for association rates (kon) and the rate of receptor internalization upon Ag binding as limiting dissociation-rate (koff) maturation, Foote and Eisen (12) predicted a maximum natural kon in the range 105 to 106 M21 s21 and minimal natural koff in the range 1023 to 1024 s21 leading to a natural affinity ceiling with equilibrium dissociation constants (KD) of the order 1028 to 10210 M. Supporting this, an average affinity of 2.3 3 1029 M was reported for Abs isolated from two human donors after a single vaccination with tetanus toxoid (7). This was ∼5-fold higher than the average from an influenzavaccinated donor (8) and two orders of magnitude higher than that of Abs isolated from a donor receiving an HBV booster vaccination (10). Combined, these results clearly point to a progressive (and Ag-dependent) development of Ab responses. To determine the aforementioned limits for Ab repertoire diversification and affinity maturation, Ab gene repertoires were prepared and characterized from two individuals after three repeated challenges with a tetanus toxoid (TT). TT was chosen as model Ag, as it is one of the most immunogenic vaccines used in man and known to induce a strong, long-lasting, and presumably unbiased Ab response. Thus, any observed boundaries in the Ab repertoires are likely to be independent of the Ag but inherent to the adaptive immune system. A representative panel of Abs specific for TT was isolated after each boost resulting in a total of 594 unique Abs estimated to originate from at least 195 different naive B cell Downloaded from http://www.jimmunol.org/ by guest on June 14, 2017 The immune system is known to generate a diverse panel of high-affinity Abs by adaptively improving the recognition of pathogens during ongoing immune responses. In this study, we report the biological limits for Ag-driven affinity maturation and repertoire diversification by analyzing Ab repertoires in two adult volunteers after each of three consecutive booster vaccinations with tetanus toxoid. Maturation of on-rates and off-rates occurred independently, indicating a kinetically controlled affinity maturation process. The third vaccination induced no significant changes in the distribution of somatic mutations and binding rate constants implying that the limits for affinity maturation and repertoire diversification had been reached. These fully matured Ab repertoires remained similar in size, genetically diverse, and dynamic. Somatic mutations and kinetic rate constants showed normal and log-normal distribution profiles, respectively. Mean values can therefore be considered as biological constants defining the observed boundaries. At physiological temperature, affinity maturation peaked at kon = 1.6 3 104 M21 s21 and koff = 1.7 3 1024 s21 leading to a maximum mean affinity of KD = 1.0 3 1029 M. At ambient temperature, the average affinity increased to KD = 3.4 3 10210 M mainly due to slower off-rates. This experimentally determined set of constants can be used as a benchmark for analysis of the maturation level of human Abs and Ab responses. The Journal of Immunology, 2011, 187: 000–000. 2 LIMITS FOR HUMAN Ab RESPONSES progenitors. We hereby present a comparative genetic and functional analysis of these Abs leading to experimental estimates of the natural limits for Ab repertoire diversification and affinity maturation in humans. Materials and Methods Donors and vaccination Donors were vaccinated in parallel with a commercially available TT vaccine (State Serum Institute, Copenhagen, Denmark). The three vaccinations were done over a 5-y period separated first by 3.5 y and subsequently by 1.5 y. Blood was donated 6 to 7 d after each vaccination. The project was approved by the regional Ethical Committee in Copenhagen, Denmark. Informed consent was obtained from each of the donors prior to each vaccination. Generation and analysis of Ab repertoires Affinities (KD), on-rates (kon), and off-rates (koff) were determined by surface plasmon resonance analysis on a Biacore 2000 (GE Healthcare/ Biacore, Uppsala, Sweden) or a Proteon XPR36 (Bio-Rad). Mass transport limitation was minimized by measuring at low ligand densities and relatively high flow rates. Briefly, TT was immobilized at low ligand densities on CM5 chips (Biacore) or GLC chips (Bio-Rad), resulting in maximum response unit (RU) values of ∼100 RU or less when binding Fab fragments. Fab fragments were diluted to 96 or 48 nM, and rate constants were measured by injection of at least four serial or parallel (depending on apparatus setup) 2-fold dilutions at 50 ml/min. Purified Fab fragments were diluted in running buffer (10 mM HEPES pH 7.4, 0.15 M NaCl, 3 mM EDTA, 0.005% surfactant P20 on Biacore 2000 and PBS pH 7.2, 0.005% Tween 20 on Proteon XPR36 as recommended by each manufacturer) and passed over the chip at 50 ml/min. All data included in this study fitted well to a single exponential 1:1 interaction model after referencing with a blank. All measurements were conducted at 25˚C or 37˚C. Association was measured for 5 min and dissociation for 20–30 min. However, for interactions with very slow off-rates (below 1 3 1024 s21), dissociation was measured for longer periods of time, up to 16 h, depending on the off-rate. Global analysis of the serially diluted analyte responses was performed with either BIAevaluation or Proteon software 2.0. Statistical analyses All statistics were performed using SAS JMP version 7.0 or 8.0. Logtransformed experimental data were fitted to the standard equation for a normal distribution: ! 1 ðx 2 lÞ2 fðxÞ ¼ pffiffiffiffiffiffiffiffiffi exp 2 : 2s2 2ps Estimates of the geometric mean and the multiplicative SDs describing the normal distribution were obtained using the following equations: n 1 x ¼ 10∧ + ½log10ðxi Þ n i¼1 1 0 x i2 1=2 1 nh C i + log10 S ¼ 10 @ A: x n 2 1 i¼1 ∧B Results Ab responses were followed in two human volunteers subjected to three consecutive boosts with the standard TT vaccine. Vaccinations were separated by at least 1.5 y, and blood was drawn for analysis 6 d after each challenge, at which time point the circulating Ab-secreting cells specific for TT are known to peak (16, 17). Using the Symplex technology (5), Ab repertoires were prepared from circulating plasma blasts separately after each challenge. Cognate pairs of VH and VL genes were covalently linked from single plasma blasts by PCR leading to repertoires representing 400– 2400 different plasma blasts (Table I). Ab genes from 2100 to 3600 bacterial clones from each of the repertoires were expressed Table I. Results of Symplex repertoire cloning Donor TT1 TT1 TT1 TT2 TT2 TT2 Total Boost Number Estimated Number of Isolated Ab-Producing Cellsa Number of Bacterial Clones Screened Number of TTSpecific Clones Number of Clones Sequenced Number of Unique V Domain Amino Acid Sequences Estimated Number of Clonotypes V1 V2 V3 V1 V2 V3 400 1,700 2,400 400 1,600 1,400 7,900 3,600 2,100 2,600 3,400 3,500 3,600 18,800 338 427 372 187 95 104 1,523 169 155 176 102 82 83 767 120 108 154 84 69 59 594 30b 50 67 42b 36 29 195 a Number of single cells where PCR products were obtained were estimated by gel electrophoresis of a representative set of single-cell RT-PCR samples (from 10 to 20% of the total). Unique V genes were identified based on the deduced amino acid sequences from DNA sequencing. Abs were assigned to the same clonotype if they shared VH, JH, VK, and JK family segments, H chain CDR3 lengths, and a consensus sequence in CDR3 for both H and L chains. b The estimated number of clonotypes from Poulsen et al. (7) (TT1-V1 and TT2-V1) were adjusted from 29 and 40 to 30 and 42, respectively, due to a slightly stricter clonotype definition. Downloaded from http://www.jimmunol.org/ by guest on June 14, 2017 Repertoires from the first and the second vaccinations were prepared as previously described (5, 7). For the third vaccination, the procedure for sorting single B cells had been optimized to include a marker for CD27. For all repertoires, single plasma blasts were applied directly into microtiter plates for the Symplex PCR procedure. Briefly, VH and LCk genes were coamplified in a single PCR reaction. VH and LCk genes were linked and amplified further in subsequent nested PCR reactions using flanking primers containing appropriate restriction sites. Cloning and screening of the first set of repertoires were reported previously (7). Ab V gene pairs were cloned into an Escherichia coli Fab expression vector, and single bacterial colonies were grown in 96-well plates where Fab expression was induced with 0.1 mM isopropyl b-D-1-thiogalactopyranoside (5, 7), after which supernatants were screened for TT reactivity by ELISA. Repertoires from the second and third vaccination were cloned into a mammalian Fab expression vector (14), and single Ab clones were expressed by transient transfection of HEK293 cells in 384-well plates. Supernatants were harvested and screened for TT reactivity. Screening using the two expression systems worked equally well and yielded a compatible frequency of hits with similar genetic diversity indicating no significant screening bias associated with the expression system (data not shown). A compatible number of TT-positive clones were selected for DNA sequencing of variable genes (Table I; GenBank accession numbers: H chains, JN110463– JN111216; L chains, JN111217–111975; http://www.ncbi.nlm.nih.gov/ genbank/). The deduced VH-Vk amino acid sequences were aligned and grouped into clusters according to sequence homology. For each group, the V-D-J usage and location of somatic mutations were determined by alignment with germline sequences using the International ImMunoGeneTics (IMGT, http://www.imgt.org) sequence directory (15). Ab clones were selected for kinetic characterization based mainly on representation of the genetic differences within the repertoire as well as on homology to Abs from previous repertoires from the same donor to follow the affinity maturation progress within a single genetic cluster. Fab fragments of the first set of repertoires were produced in up to 1-l E. coli cultures including induction of expression with 0.1 mM isopropyl b-D-1-thiogalactopyranoside overnight. Fab fragments from the second and third set of repertoires were produced in 50–200 ml cultures of HEK293 cells transiently transfected with Ab DNA. Fab fragments were purified using a mini spin column kit with Protein L beads (Thermo Fischer Scientific). The concentration of purified Fab fragments was determined by an indirect immunoassay. Surface plasmon resonance The Journal of Immunology as Fab fragments and screened for TT binding, and a comparable number of positive clones were picked for DNA sequencing and functional characterization. As previously described (7), V and J gene segment usage and somatic mutations were determined by aligning VH and VL gene sequences to the IMGT database (15). The amino acid sequences of 594 unique TT-specific Ab clones were analyzed and estimated to originate from at least 195 different naive B cell progenitors (termed clonotypes). The entire Ab panel was highly diverse and contained germline gene segments from all the major families of VH, JH, VK, and JK except for the rarely found VK6 (see Supplemental Table 1a and 1b). Despite repeated Ag challenge and consequently consecutive rounds of somatic mutations and affinity selection, the overall number of somatic mutations present in each repertoire remained constant for each donor (Fig. 1A–C). Thus, the maximum tolerated level of somatic mutations appeared to have been reached. Statistical analysis of the combined distribution of amino acid so- matic mutations in all six repertoires indicate a normal distribution around clearly defined mean values of 13.2, 7.3, and 20.5 for VH, VL, and the combined repertoire, respectively. Previous reports on somatic mutations (8, 18–21) fall within the presently observed limits, which can thus be regarded representative for a fully matured response. The number of unique clonotypes identified in each repertoire varied between 29 and 67 (Table I), and the maximum likelihood estimates of the overall repertoire sizes were determined as previously reported (7, 22) (Fig. 1D). It appears that Ag-induced human IgH-VK repertoires do not exceed of the order 100 clonotypes (7). As the Ag is not considered to impose any restrictions to the observed responses, the observed limitation in repertoire sizes is likely to be intrinsic to the immune system when responding to large, structurally complex Ags (5, 11, 21, 23). Approximately two thirds of the clonotypes in the TT Ab donor 2 repertoires (TT2) were found associated with only a single Downloaded from http://www.jimmunol.org/ by guest on June 14, 2017 FIGURE 1. Statistical analysis of the frequency of somatic amino acid mutations in V gene segments and repertoire size. A–C, The number of somatic mutations for H chains (A), L chains (B), and heavy and light pair combined (C) are plotted for each repertoire (indicated below). Quantile box plots show distributions of the full data set containing all six repertoires. The box plots show the maximum, minimum, and median values, the 25th and 75th interquartile values, and the 90% and 10% percentiles. The diamonds mark the mean with a 95% confidence interval. To the far right, distributions are shown as histograms including a superimposed fitted normal distribution profile. “n” indicates the number of unique amino acid sequences in each data set. D, Analysis of repertoire sizes. The darker shade (bottom) represents the number of clonotypes observed in at least two of the repertoires from each donor. The lighter shade (top) represents the clonotypes that are uniquely associated with a single repertoire. Diamonds illustrate statistical maximum likelihood estimates of repertoire sizes derived statistically from the total number of unique sequences and the number of clonotypes from each repertoire (7, 22). 3 4 LIMITS FOR HUMAN Ab RESPONSES vaccination event, and this fraction appeared to increase for TT Ab donor 1 repertoires (TT1) as vaccinations progressed (Fig. 1D). Furthermore, several clonotypes unique to the TT1 repertoires carried no somatic mutations, notably after the second booster vaccination. Together, this suggests a constant influx of new clonotypes from the naive repertoire to an already existing repertoire. Therefore, despite repeated exposure, Ab responses against complex Ags remain unrestricted and dynamic rather than converge Table II. Biological constants for affinity maturation at different temperatures Temperature (˚C) 25 37 Parameters kon (M21 s21) x*, geometric mean 68% upper CI (x*·s*) 68% lower CI (x*/s*) s*, MSDc x*, geometric mean 68% upper CI (x*·s*) 68% lower CI (x*/s*) s*, MSDc 2.0 3 10 1.2 3 106 3.1 3 104 6.4 1.6 3 105 7.6 3 105 3.5 3 104 4.6 5 koff (s21) 25 7.0 3 10 2.6 3 1024 1.9 3 1025 3.7 1.7 3 1024 5.5 3 1024 5.0 3 1025 3.3 KD (M)a 210 3.4 3 10 2.9 3 1029 3.9 3 10211 8.6 1.0 3 1029 7.6 3 1029 1.3 3 10210 7.6 t1/2 (min21)b 165 619 44 3.7 70 232 21 3.3 Values were obtained by fitting the standard equation for a normal distribution to log10 transformed experimentally determined data sets. MSD, multiplicative standard deviation. a KD was calculated as koff/kon. b t1/2 was calculated as ln(2)/(koff/60). c MSDs are unit-less. CI, confidence interval. Downloaded from http://www.jimmunol.org/ by guest on June 14, 2017 FIGURE 2. Limits for affinity maturation measured at 25˚C. A–C, Experimentally determined kon (A), koff (B), and calculated KD (C) values are plotted for each repertoire as indicated. Medians are shown as black bars. A two-way ANOVA was conducted for each data set (on-rates, off-rates, and affinities) to test for differences between repertoires. The arrow indicates that the off-rate of this particular Ab was too slow to measure accurately using surface plasmon resonance. Hence, this data point only indicates an upper limit for the off-rate and affinity. D–F, Statistical analysis of the distribution of binding constants. Distributions of log10 transformed kon (D), koff (E), and calculated KD (F) plotted as normal quantile plots (upper panels) and histograms (lower panels). Data from all six repertoires were combined in the analysis of on-rate, and data from the last two vaccinations were combined for the analysis of the distribution of off-rates and affinities. The log10 transformed data were fitted by the normal distribution to identify the limit for affinity maturation on kinetic constants. The horizontal line in the normal quantile plots represents the mean, and the curved dotted lines are 95% confidence intervals. The Journal of Immunology toward a few clonotypes with optimal binding properties, as it has been reported for hapten-driven responses (24–27). To determine the progression in affinity maturation, kon and koff were determined at 25˚C for a genetically diverse set of 19–35 Abs from each repertoire (Fig. 2A–C). The differences between the six repertoires in distributions of kon values were not statistically significant, indicating that the ceiling for maturation of kon had already been reached prior to the first booster vaccinations of this study. This is in accordance with the Danish child vaccination program, which involves at least four tetanus immunizations. To quantify the limit for affinity maturation of kon, koff, and KD, selected data sets were combined and analyzed statistically (Fig. 2D–F, Table II). The joint distribution of kon appeared log-normal with a geometric mean of 2.0 3 105 M-1 s21. For both donors, 5 repertoires from the first vaccination had significantly faster average koff (TT1: p = 0.001; TT2: p , 0.001) indicating that the limit for maturation of koff was first reached after the second vaccination. Thus, to quantify the limit for affinity maturation of koff, the four data sets from the last two vaccinations were combined for statistical analysis. They showed a clear log-normal distribution with a geometric mean at 7.0 3 1025 s21. As a consequence of the relatively dispersed and fully matured kon, differences between repertoires were insignificant for KD. The combined average KD from the second and third vaccinations was determined to be 3.4 3 10210 M. To determine the limits for affinity maturation at physiologically relevant temperature, the rate constants were determined at 37˚C for a representative set of 50 Abs from the last two vaccinations Downloaded from http://www.jimmunol.org/ by guest on June 14, 2017 FIGURE 3. Limits for affinity maturation measured at 37˚C. A–C, Binding constants kon (A), koff (B), and calculated KD (C) were determined from a set of 50 Abs evenly selected from the four repertoires of the last two booster vaccinations. D–F, Statistical analysis of the distribution of binding constants measured at physiological temperature as presented in Fig. 2. The superimposed lines indicate the normal distribution determined at 37˚C (solid) and 25˚C (dotted). 6 from both donors (Fig. 3A–C). As observed for measurements at ambient temperature, data were log-normally distributed around clearly defined mean values (Fig. 3D–F). The geometric mean for kon was not affected by the change in temperature. In contrast, the koff geometric mean value increased 2.4-fold to 1.7 3 1024 s21, which corresponds with a drop in average interaction t1/2 from 165 min to 70 min when binding at physiological temperature. Combined, this gave a 3.0-fold change in the mean KD leading to an average affinity of 1.0 3 1029 M (Table II). Discussion after the second booster vaccination. The observed separate maturation of kinetic rate constants supports that maturation of Ab responses is kinetically rather than thermodynamically controlled (2, 3, 29) implying, as proposed by Foote and Eisen (12), that clonal selection by on-rates and off-rates occurs through independent mechanisms. Furthermore, the identified kinetic mean constants at physiological temperature are in accordance with the proposed theoretical ranges (12) although the relatively long mean half-life at physiological temperature of 70 min suggests that apart from Ag internalization, with a t1/2 of 8 min, additional factors such as competition for Ag in germinal centers (30, 31) could influence the affinity maturation process. We hereby report the experimental determination of the limits for Ab affinity maturation and repertoire diversification in single individuals in response to repeated vaccinations. The reported biological constants can be used to evaluate the maturation level of Ab repertoires, polyclonal sera, and mAbs in particular and are hence of value in basic immunological science as well as in Ab drug discovery and vaccine development. Acknowledgments We thank Lars S. Nielsen, Per-Johan Meijer, Klaus Koefoed, Mette Thorn, and Johan Lantto for advice and critical discussions. Disclosures All authors are employees (with equity interests) or former employees of Symphogen. References 1. Eisen, H. N., and G. W. Siskind. 1964. Variations in affinities of antibodies during the immune response. Biochemistry 3: 996–1008. 2. Foote, J., and C. Milstein. 1991. Kinetic maturation of an immune response. Nature 352: 530–532. 3. Roost, H. P., M. F. Bachmann, A. Haag, U. Kalinke, V. Pliska, H. Hengartner, and R. M. Zinkernagel. 1995. Early high-affinity neutralizing anti-viral IgG responses without further overall improvements of affinity. Proc. Natl. Acad. Sci. USA 92: 1257–1261. 4. 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Helms, et al. 2008. Rapid cloning of highaffinity human monoclonal antibodies against influenza virus. Nature 453: 667–671. 9. Tiller, T., E. Meffre, S. Yurasov, M. Tsuiji, M. C. Nussenzweig, and H. Wardemann. 2008. Efficient generation of monoclonal antibodies from single human B cells by single cell RT-PCR and expression vector cloning. J. Immunol. Methods 329: 112–124. 10. Jin, A., T. Ozawa, K. Tajiri, T. Obata, S. Kondo, K. Kinoshita, S. Kadowaki, K. Takahashi, T. Sugiyama, H. Kishi, and A. Muraguchi. 2009. A rapid and efficient single-cell manipulation method for screening antigen-specific antibodysecreting cells from human peripheral blood. Nat. Med. 15: 1088–1092. 11. Scheid, J. F., H. Mouquet, N. Feldhahn, M. S. Seaman, K. Velinzon, J. Pietzsch, R. G. Ott, R. M. Anthony, H. Zebroski, A. Hurley, A. Phogat, et al. 2009. Broad diversity of neutralizing antibodies isolated from memory B cells in HIVinfected individuals. Nature 458: 636–640. 12. Foote, J., and H. N. Eisen. 1995. Kinetic and affinity limits on antibodies produced during immune responses. Proc. Natl. Acad. Sci. USA 92: 1254–1256. 13. Batista, F. D., and M. S. Neuberger. 1998. Affinity dependence of the B cell response to antigen: a threshold, a ceiling, and the importance of off-rate. Immunity 8: 751–759. 14. Meijer, P. J., L. S. Nielsen, J. Lantto, and A. Jensen. 2009. Human antibody repertoires. Methods Mol. Biol. 525: 261–277. 15. Lefranc, M.-P. 2009. IMGT Repertoire 2. Proteins and alleles (IG and TR). Available at: http://www.imgt.org/IMGTrepertoire/Proteins/. Accessed: June 3, 2009. 16. Kodo, H., R. P. Gale, and A. Saxon. 1984. Antibody synthesis by bone marrow cells in vitro following primary and booster tetanus toxoid immunization in humans. J. Clin. Invest. 73: 1377–1384. Downloaded from http://www.jimmunol.org/ by guest on June 14, 2017 To determine the natural boundaries for maturation of human Abs in response to Ag, individual Ab repertoires were made from two human donors after each of three booster vaccinations with TT. Repertoires were characterized at the clonal level to make a comprehensive genetic and functional analysis of evolving Ab responses against a well-defined Ag. As previously reported for the repertoires from the first vaccinations (7), Abs were generally extensively mutated, and within each repertoire it was not possible to observe any correlation between binding strength and level of somatic mutations indicative of highly matured responses. Furthermore, the average level of somatic mutations did not change in the course of this study demonstrating that the maximum tolerated level of somatic mutations had already been reached during the preceding routine vaccination program. The L chain VK genes accumulated on average 7.7 and 6.8 aa transitions and H chain VH genes accumulated on average 14.9 and 11.5 aa transitions for donor TT1 and TT2, respectively. The difference in mutational level of VH repertoires between donors is significant (p , 0.01) and can be explained by allelic variations in VH gene segments relative to the IMGT reference database (15). Nevertheless, as the accumulation of somatic mutations occurs irrespective of the particular Ag in use, the peak average of 20 aa transitions per variable domain appears to represent an average measure for the maximal level of degeneracy tolerated by an evolving Ab repertoire. Ab repertoires remained genetically dynamic and diverse in response to the repeated exposures to the TT vaccine. Approximately two thirds of the B cell clonotypes were uniquely associated with a single vaccination event. This parallels previous observations in hapten responses (24) and supports a compartmentalization of the B cell response in oligoclonal germinal centers (25, 27). The most likely immunological significance of this dynamic behavior is that it provides an efficient countermeasure to the constant genetic evolution of pathogenic microorganisms. Compartmentalization would also explain the apparent restrictions in repertoire size of the order 100 clonotypes with the limiting factor being the actual number of germinal centers formed during an immune response. For all repertoires, the distribution of binding constants fitted well to log-normal distribution profiles with clearly defined medians and unit-less multiplicative standard deviations (MSDs) ranging from 3.7 to 8.6. Compared with log-normal distributions in other biological systems, where MSDs usually range from 1.1 to 3.2 (28), Ab repertoires can be considered strongly skewed toward log-normal behavior. 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