Monoclonal paraprotein influences baseline B-cell repertoire diversity and perturbates influenza vaccination-induced B-cell response
2015; Elsevier BV; Volume: 43; Issue: 6 Linguagem: Inglês
10.1016/j.exphem.2015.02.005
ISSN1873-2399
AutoresSarah M. Tete, David Kipling, Johanna Westra, Aalzen de Haan, Marc Bijl, Deborah K. Dunn–Walters, Surinder S. Sahota, Nicolaas A. Bos,
Tópico(s)Multiple Myeloma Research and Treatments
Resumo•Monoclonal expansion in monoclonal gammopathy of undetermined significance influences the B-cell repertoire diversity.•Oligoclonality of B cells is present in 50% of monoclonal gammopathy of undetermined significance.•Vaccination-induced B-cell repertoire perturbations correlate with monoclonal protein concentrations. Monoclonal gammopathy of undetermined significance (MGUS) arises from a clonal expansion of plasma cells in the bone marrow, secreting monoclonal (M) paraprotein. It is associated with increased susceptibility to infections, which may reflect altered B-cell repertoire. To investigate this, we examined the immunoglobulin (Ig) M, IgG, and IgA B-cell repertoire diversity in MGUS at baseline and after influenza vaccination (n = 16) in comparison with healthy controls (HCs; n = 16). The Complementary Determining Region 3 region of the immunoglobulin heavy chain variable region gene was amplified and B-cell spectratypes analyzed by high-resolution electrophoresis. Spectratype Gaussian distribution, kurtosis, and skewness were quantified to measure repertoire shifts. Both HC and MGUS baseline spectratypes show interindividual variability that is more pronounced in the IGHG and IGHA repertoires. Overall, baseline B-cell repertoire is more altered in MGUS, with oligoclonality observed in 50% (p = 0.01). Postvaccination, significant differences emerged in MGUS in relation to M-protein levels. High M-protein concentration is associated with a more oligoclonal IgG and IgA response at day 7 postvaccination, and, in contrast to HCs, vaccination also induced significant perturbations in the MGUS IgM repertoire at day 7 (p = 0.005). Monoclonal expansion in MGUS thus has an effect on the baseline B-cell repertoire and influences the recruited repertoire upon vaccination. Monoclonal gammopathy of undetermined significance (MGUS) arises from a clonal expansion of plasma cells in the bone marrow, secreting monoclonal (M) paraprotein. It is associated with increased susceptibility to infections, which may reflect altered B-cell repertoire. To investigate this, we examined the immunoglobulin (Ig) M, IgG, and IgA B-cell repertoire diversity in MGUS at baseline and after influenza vaccination (n = 16) in comparison with healthy controls (HCs; n = 16). The Complementary Determining Region 3 region of the immunoglobulin heavy chain variable region gene was amplified and B-cell spectratypes analyzed by high-resolution electrophoresis. Spectratype Gaussian distribution, kurtosis, and skewness were quantified to measure repertoire shifts. Both HC and MGUS baseline spectratypes show interindividual variability that is more pronounced in the IGHG and IGHA repertoires. Overall, baseline B-cell repertoire is more altered in MGUS, with oligoclonality observed in 50% (p = 0.01). Postvaccination, significant differences emerged in MGUS in relation to M-protein levels. High M-protein concentration is associated with a more oligoclonal IgG and IgA response at day 7 postvaccination, and, in contrast to HCs, vaccination also induced significant perturbations in the MGUS IgM repertoire at day 7 (p = 0.005). Monoclonal expansion in MGUS thus has an effect on the baseline B-cell repertoire and influences the recruited repertoire upon vaccination. Aging is associated with changes in the functioning of the immune system that manifest as poor responses to infections and vaccination [1Bernstein E. Kaye D. Abrutyn E. Gross P. Dorfman M. Murasko D.M. Immune response to influenza vaccination in a large healthy elderly population.Vaccine. 1999; 17: 82-94Crossref PubMed Scopus (155) Google Scholar, 2Gardner E.M. Bernstein E.D. Dran S. et al.Characterization of antibody responses to annual influenza vaccination over four years in a healthy elderly population.Vaccine. 2001; 19: 4610-4617Crossref PubMed Scopus (52) Google Scholar, 3Murasko D.M. Bernstein E.D. Gardner E.M. et al.Role of humoral and cell-mediated immunity in protection from influenza disease after immunization of healthy elderly.Exp Gerontol. 2002; 37: 427-439Crossref PubMed Scopus (192) Google Scholar]. Since antibody responses are the mainstay of protection against many infectious agents, recent investigations have focused on understanding amelioration of the humoral responses in aging, including the underlying B-cell repertoire shifts [4Wang C. Liu Y. Xu L.T. et al.Effects of aging, cytomegalovirus infection, and EBV infection on human B cell repertoires.J Immunol. 2014; 192: 603-611Crossref PubMed Scopus (128) Google Scholar, 5Shi Y. Yamazaki T. Okubo Y. Uehara Y. Sugane K. Agematsu K. Regulation of aged humoral immune defense against pneumococcal bacteria by IgM memory B cell.J Immunol. 2005; 175: 3262-3267Crossref PubMed Scopus (171) Google Scholar]. During aging, the emergence of a nonmalignant condition like monoclonal gammopathy of undetermined significance (MGUS) in the elderly may further alter this susceptibility to infection or response to vaccination. MGUS is a condition that is generally identified surreptitiously and arises from monoclonal expansion of plasma cells in the bone marrow, with potential to transform to full malignancy such as multiple myeloma [6Landgren O. Kyle R.A. Pfeiffer R.M. et al.Monoclonal gammopathy of undetermined significance (MGUS) consistently precedes multiple myeloma: A prospective study.Blood. 2009; 113: 5412-5417Crossref PubMed Scopus (745) Google Scholar, 7Blade J. Rosinol L. Cibeira M.T. de Larrea C.F. Pathogenesis and progression of monoclonal gammopathy of undetermined significance.Leukemia. 2008; 22: 1651-1657Crossref PubMed Scopus (79) Google Scholar]. In MGUS, the expansion of plasma cells results in the secretion of elevated levels of monoclonal protein (M-protein), which can increase to reflect a further expansion [8Kyle R.A. Therneau T.M. Rajkumar S.V. et al.Prevalence of monoclonal gammopathy of undetermined significance.N Engl J Med. 2006; 354: 1362-1369Crossref PubMed Scopus (940) Google Scholar, 9Kyle R.A. Durie B.G.M. Rajkumar S.V. et al.Monoclonal gammopathy of undetermined significance (MGUS) and smoldering (asymptomatic) multiple myeloma: IMWG consensus perspectives risk factors for progression and guidelines for monitoring and management.Leukemia. 2010; 24: 1121-1127Crossref PubMed Scopus (584) Google Scholar]. The condition is associated with hypogammaglobulinaemia in 20%–28% of cases [10Kyle R.A. Remstein E.D. Therneau T.M. et al.Clinical course and prognosis of smoldering (asymptomatic) multiple myeloma.N Engl J Med. 2007; 356: 2582-2590Crossref PubMed Scopus (593) Google Scholar, 11Zemble R.M. Takach P.A. 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The risk of bacteremia in patients with monoclonal gammopathy of undetermined significance.Eur J Haematol. 1998; 61: 140-144Crossref PubMed Scopus (27) Google Scholar]. Furthermore, high monoclonal protein at diagnosis of MGUS has also been shown to be associated with higher risks of developing infections. These include pneumonia, influenza, and herpes zoster infections [13Gregersen H. Madsen K.M. Sorensen H.T. Schonheyder H.C. Ibsen J.S. Dahlerup J.F. The risk of bacteremia in patients with monoclonal gammopathy of undetermined significance.Eur J Haematol. 1998; 61: 140-144Crossref PubMed Scopus (27) Google Scholar]. Besides the increased risk of infections, significantly depressed levels of background antibodies to a number of common infectious antigens have been reported, notably to staphylococcal, pneumococcal, varicella zoster, and fungal antigens such as candida and aspergillus [14Kristinsson S.Y. Tang M. Pfeiffer R.M. et al.Monoclonal gammopathy of undetermined significance and risk of infections: a population-based study.Haematologica. 2012; 16: 854-858Crossref Scopus (76) Google Scholar]. Responses to vaccination in MGUS remain poorly understood. Among infectious agents, influenza is a significant morbidity factor in aging individuals, and influenza vaccination is recommended in this high-risk group [15European Centre for Disease Prevention and Control. ECDC guidance: priority risk groups for influenza vaccination. European Centre for Disease Prevention and Control web site. 2008. Available at: http://www.ecdc.europa.eu/en/publications/Publications/Forms/ECDC_DispForm.aspx?ID=315. Accessed February 10, 2012.Google Scholar]. Influenza vaccination has an efficacy of 70% in healthy young adults [16Demicheli V. Di Pietrantonj C. Jefferson T. Rivetti A. Rivetti D. Vaccines for preventing influenza in healthy adults.Cochrane Database Syst Rev. 2007; : CD001269PubMed Google Scholar], but the efficacy is decreased in the normal elderly population [17Jefferson T. Rivetti D. Rivetti A. Rudin M. Di Pietrantonj C. Demicheli V. Efficacy and effectiveness of influenza vaccines in elderly people: a systematic review.Lancet. 2005; 366: 1165-1174Abstract Full Text Full Text PDF PubMed Scopus (515) Google Scholar]. Hemaglutination-inhibiting antibody responses, a universally established correlate of protection for influenza, are reduced in MGUS and correlate with serum influenza-specific immunoglobulin (Ig) G titers [18Tete S.M. Newling M. Westra J. et al.Hampered influenza-specific IgG B cell responses whereas IgM and IgA responses are maintained in monoclonal gammopathy of undetermined significance.Hematology and Leukemia. 2014; 2: 5-12Crossref Google Scholar]. In MGUS specifically, we have recently shown that the humoral immune response to influenza vaccination negatively correlates with the size of the M-protein [18Tete S.M. Newling M. Westra J. et al.Hampered influenza-specific IgG B cell responses whereas IgM and IgA responses are maintained in monoclonal gammopathy of undetermined significance.Hematology and Leukemia. 2014; 2: 5-12Crossref Google Scholar, 19Tete S.M. Wilting K.R. Horst G. et al.IgG antibody and TH1 immune responses to influenza vaccination negatively correlate with M-protein burden in monoclonal gammopathy of undetermined significance.Hematology and Leukemia. 2013; 1: 3-10Crossref Google Scholar]. Patients with MGUS and with high M-protein levels elicited poor responses compared with age-matched controls, and this implies that high M-protein is a marker of underlying B-cell dysregulation resulting in the poor immune response [20Rajewsky K. Clonal selection and learning in the antibody system.Nature. 1996; 381: 751-758Crossref PubMed Scopus (1374) Google Scholar]. There is therefore a clear need to understand the nature of this immune dysregulation in MGUS patients and to develop strategies to counter their elevated susceptibility to infections and decreased response to vaccination. B-cell dysregulation impacts on the B-cell repertoire and diversity can be mapped by analyzing the immunoglobulin variable region (IGV) gene repertoire [21Wu Y.C. Kipling D. Leong H.S. Martin V. Ademokun A.A. Dunn-Walters D.K. High-throughput immunoglobulin repertoire analysis distinguishes between human IgM memory and switched memory B-cell populations.Blood. 2010; 116: 1070-1078Crossref PubMed Scopus (217) Google Scholar]. The Complementary Determining Region 3 (CDR3) covers the junctional region where the three segments (IGHV-IGHD-IGHJ) are joined [22Wrammert J. Koutsonanos D. Li G. et al.Broadly cross-reactive antibodies dominate the human B cell response against 2009 pandemic H1N1 influenza virus infection.J Exp Med. 2011; 208: 181-193Crossref PubMed Scopus (624) Google Scholar], forming the core antigen-binding site in an antibody molecule [21Wu Y.C. Kipling D. Leong H.S. Martin V. Ademokun A.A. Dunn-Walters D.K. High-throughput immunoglobulin repertoire analysis distinguishes between human IgM memory and switched memory B-cell populations.Blood. 2010; 116: 1070-1078Crossref PubMed Scopus (217) Google Scholar]. Diversity of CDR3 is therefore a critical measure of functional diversity of any B-cell repertoire. In a normal, diverse B-cell population, a large number of clones derived from random V(D)J rearrangements result in a Gaussian distribution of CDR3 sizes. Where repertoire is perturbed, as with biases in repertoire usage or clonal expansions, the CDR3 distribution is altered and can be assayed by spectratyping [23Foreman A.L. Lemercier B. Lim A. et al.VH gene usage and CDR3 analysis of B cell receptor in the peripheral blood of patients with PBC.Autoimmunity. 2008; 41: 80-86Crossref PubMed Scopus (12) Google Scholar, 24Baranzini S.E. Jeong M.C. Butunoi C. Murray R.S. Bernard C.C. Oksenberg J.R. B cell repertoire diversity and clonal expansion in multiple sclerosis brain lesions.J Immunol. 1999; 163: 5133-5144PubMed Google Scholar]. Spectratyping has been used to show that the B-cell repertoire is compromised in the elderly. Aging is associated with oligoclonal expansions of cells and a collapse in the diversity of the B-cell repertoire [25Gibson K.L. Wu Y.C. Barnett Y. et al.B-cell diversity decreases in old age and is correlated with poor health status.Aging Cell. 2009; 8: 18-25Crossref PubMed Scopus (289) Google Scholar] that correlates with a general phenotype of frailty [26Ademokun A. Wu Y.C. Martin V. et al.Vaccination-induced changes in human B-cell repertoire and pneumococcal IgM and IgA antibody at different ages.Aging Cell. 2011; 10: 922-930Crossref PubMed Scopus (95) Google Scholar]. Vaccination has also been shown to induce changes in the B-cell repertoire. However, these changes are attenuated in aging, and resolution of the response differs. Importantly, the IgM and IgA responses were impaired in the elderly and challenge resulted in smaller CDR3 sizes [25Gibson K.L. Wu Y.C. Barnett Y. et al.B-cell diversity decreases in old age and is correlated with poor health status.Aging Cell. 2009; 8: 18-25Crossref PubMed Scopus (289) Google Scholar]. The onset of a monoclonal expansion like MGUS in the elderly may further alter repertoire diversity, and thus far, to our knowledge, this has not been evaluated. To address this, we sought to delineate the baseline B-cell repertoire in MGUS and humoral response to vaccination against influenza. We hypothesized that monoclonal plasma cell expansions in MGUS located in the bone marrow may further alter the aging humoral response. To test this hypothesis, we analyzed the B-cell repertoire using spectratype resolution of the CDR3 region of IGHV genes and modeled data to assess diversity in MGUS patients before and after influenza vaccination. Monoclonal gammopathy of undetermined significance patients from the University Medical Centre Groningen hematology department and age-matched healthy controls were recruited during the 2010–2011 influenza vaccination season. The MGUS patients fulfilled the standard diagnostic criteria for MGUS: serum M protein levels <30 g/L, clonal plasma cells in bone marrow <10%, and no myeloma-related dysfunction or other B-cell proliferative diseases. All participants gave written, informed consent in accordance with the Declaration of Helsinki. The institutional medical ethics committee of University Medical Centre Groningen approved the study. All participants received influenza vaccination (Influvac 2010/2011, Solvay Pharmaceuticals, Weesp, Netherlands), and blood samples were collected before the vaccination as well as 7 days and 28 days postvaccination. Peripheral blood mononuclear cells (PBMCs) were isolated from the blood by density gradient centrifugation of CPT Vacutainer tubes (BD, Franklin Lane, NJ). We then froze PBMCs in Roswell Park Memorial Institute 1640 medium (Cambrex Bioscience, Verviers, Belgium) supplemented with 10% human pooled serum, 50 μg/mL gentamicin (Gibco, Paisley, United Kingdom) and 10% dimethylsulfoxide (Merck, Darmstadt, Germany). We stored PBMCs in liquid nitrogen until further use. Total DNA and RNA were extracted using the Qiagen Allprep (Germantown, MD) DNA/RNA mini kit according to the manufacturer's specifications. First-strand cDNA was synthesized from RNA following standard protocols using Superscript-III (Invitrogen, Waltham, MA) in a reverse transcriptase system (Promega, Madison, WI). cDNA was used for the amplification of the IGH gene CDR3 region using a polymerase chain reaction with primers specific for the constant regions Cα (GGAAGAAGCCCTGGACCAGGC), Cμ (CAGGAGACGAGGGGGAA), or Cγ (CACCGTCACCGGTTCGG), in combination with Fw3Fam (ACACGGCTGTGTATTACTGT). We added 5 μL of each reaction sample to 1.5 μL Tamra 350 size standard in formamide and ran it on the ABI 3730 xl capillary sequencer. We analyzed CDR3 spectratype data and visualized peak profiles using Genescan software (Applied Biosystems, Carlsbad, CA). Data were then exported to Excel (Microsoft, Redmons, WA), and frequency distributions of the CDR3 lengths were obtained. Excel files were then imported into the R statistical programming environment (R Development Core Team, 2011, R foundation, Vienna, Austria), and the raw peak data were normalized before spectratype curves were generated. A reference Gaussian distribution was generated and repertoire diversity assessed by comparing the mean CDR3 size to the reference Gaussian distribution (CGD). To account for the shape of the spectratype distribution, kurtosis and skewness were measured for each spectratype distribution [27Collette A. Cazenave P. Pied S. Six A. New methods and software tools for high throughput CDR3 spectratyping. Application to T lymphocyte repertoire modifications during experimental malaria.J Immunol Methods. 2003; 278: 105-116Crossref PubMed Scopus (23) Google Scholar, 28Matsumoto Y. Yoon W.K. Jee Y. et al.Complementarity-determining region 3 spectratyping analysis of the TCR repertoire in multiple sclerosis.J Immunol. 2003; 170: 4846-4853Crossref PubMed Scopus (41) Google Scholar, 29Peggs K.S. Verfuerth S. Pizzey A. et al.Reconstitution of T-cell repertoire after autologous stem cell transplantation: Influence of CD34 selection and cytomegalovirus infection.Biol Blood Marrow Transplant. 2003; 9: 198-205Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar]. (Detailed methods are given in Supplementary Methods, online only, available at www.exphem.org.) Statistical analysis was performed using Graphpad Prism 5.0 (Graphpad, La Jolla, USA). Mann-Whitney U tests, Wilcoxon signed rank tests, and χ2 tests for categorical data were used as appropriate. For correlations between different measurements, Pearson's correlation analysis was used. We considered p values <0.05 to indicate statistical significance. A total of 16 MGUS patients and 16 age-matched HCs were included in this study (Table 1). Our MGUS cohort exhibited considerable heterogeneity in terms of the type and size of M protein as well as duration of the condition. The concentration of M protein varied from unquantifiable levels to 24.8 g/L. We detected M-protein by immunofixation, but it was unquantifiable in seven patients by serum protein electrophoresis because the concentration was very low or the M-protein migrated in the β fraction. A concentration of 0.01 g/L was therefore assigned to this subset of MGUS patients. The study demographics are shown in Table 1.Table 1Baseline characteristics of MGUS patients and controlsHealthy controls (n = 16)MGUS (n = 16)pAge (y): median (range)63 (49–82)64 (48–81)NSSex: n male/n female11/511/5NSVaccination 2009/2010: n (%)14 (87.5)16 (100)NSDuration of MGUS, years: median (range)NA4 (1–19.2)Monoclonal protein size, g/L: median (range)NA6 (0.01–24.8)Monoclonal protein isotype IgG: n (%)NA8 (50) IgA: n (%)NA4 (25) IgM: n (%)NA4 (25)NA = not applicable; NS = not significant. Open table in a new tab NA = not applicable; NS = not significant. Spectratyping profiles can map polyclonality in a normal healthy immune humoral response arising from a homeostatic B-cell repertoire and accurately identify deviant repertoires that are altered by specific conditions. There are various alterations in the spectratype profiles that can be seen in a deviant repertoire. These alterations affect the shape and distribution of the spectratypes and can be modeled by CGD, kurtosis, and skewness. Figure 1A shows representative spectratypes, and the metrics measured from four MGUS patients at day 0, along with a comparative analysis of samples from four healthy controls, is shown in Figure 1B. In healthy controls, a polyclonal distribution of the CDR3 spectratypes was observed, although interindividual variability occurred, as shown by the variations in CGD, kurtosis, and skewness. The B-cell repertoire was variable in MGUS, with some showing polyclonal distribution of the CDR3 spectratypes similar to the healthy controls. Nonetheless, loss in diversity, indicated by a few dominant expansions in the CDR3 sizes, was observed in a subset of MGUS patients. These oligoclonal spectratypes (CGD < 0.6) were observed in the MGUS baseline repertoire at higher frequency than in healthy controls. Fifty percent of the MGUS patients had oligoclonal spectratype profiles for at least one of the three IGHV immunoglobulin gene repertoires, in comparison with only one (6%) healthy control (p = 0.01). A spectratype distribution was considered to be normal (approximately symmetric) if the skewness was between −0.5 and 0.5. However, a moderately skewed distribution had skewness between −1 and −0.5 or between 0.5 and 1, and a highly skewed distribution had skewness values less than −1 or greater than 1 [30Bulmer M.G. Principles of Statistics. Dover Publications, New York1979Google Scholar]. Based on this interpretation, three MGUS immunoglobulin heavy chain IgM (IGHM) spectratypes showed moderate skewness compared with two HC samples, and five MGUS immunoglobulin heavy chain IgA (IGHA) spectratypes were moderately skewed, in comparison with three in HCs. In IGHG spectratypes, we observed increased skewness in MGUS, with three MGUS IGHG samples moderately skewed and two highly skewed, in comparison with none in HCs, in which all spectratypes were approximately symmetric. Interindividual variability in the HCs' B-cell repertoires was more pronounced in the IGHG (p = 0.001) and IGHA (p = 0.036) spectratypes than in IGHM. Of the healthy controls, 69% had IGHM CGD values of 0.9 or higher, indicating repertoire complexity as depicted by the large number of differently sized CDR3 (Table 2).Table 2Samples matching Gaussian distribution; CGD ≥ 0.9HCMGUSIGHM: n (%)11/16 (68.8)9/16 (56)IGHG: n (%)6/16 (37.5)2/16 (12.5)aSignificantly lower MGUS samples matching CGD than IGHM samples; p < 0.05.IGHA: n (%)7/16 (43.8)3/16 (18.8)aSignificantly lower MGUS samples matching CGD than IGHM samples; p < 0.05.a Significantly lower MGUS samples matching CGD than IGHM samples; p < 0.05. Open table in a new tab Furthermore, we investigated the influence of immunoparesis on the immunoglobulin repertoire. Baseline levels of total IgG in MGUS had a mean of 7.7 g/L (range = 4.4–12.5). Of the 16 MGUS patients, 5 (31%) had immunoparesis (IgG < 6 g/L). In HCs, the mean was 11.0 g/L (range = 6.9–14.7). However, immunoparesis did not correlate with the CGD index. We subsequently investigated whether age was associated with a less diverse repertoire in HCs and MGUS. No age-related changes were observed in the IGHM, IGHG, or IGHA spectratypes of the healthy controls. However, age was associated with a decrease in MGUS baseline IGHG CGD index, regardless of the monoclonal protein isotype, with a Pearson's r of −0.55 (p = 0.04; Fig. 1C). Following encounter with antigen, specific B cells expand, and this leads to the change in baseline repertoire distribution (Figs. 2A and 2B). There was a pronounced shift in the shapes of the spectratypes at day 7 that then returned to the baseline shape at day 28. Some profiles in the MGUS patients indicated a diverse repertoire at day 0. However, in response to vaccination, there are marked differences in the spectratypes between patients (Fig. 2B). Perturbations in the repertoire evidenced by oligoclonal CDR3 profiles and lower CGD were seen in some patients at day 7 that did not return to baseline values at day 28 but remained oligoclonal. In three MGUS patients, a perturbed repertoire with a dominant CDR3 length was seen at day 7 in response to vaccination. However, on day 28, the spectratypes remained oligoclonal but with a different pattern; a different sized dominant peak was seen. Interestingly, distortions in some of the MGUS spectratype profiles that could be related to the monoclonal expansion of B cells in MGUS were observed in three of the patients. In these patients, the spectratype profiles had a major CDR3 length that is dominant in the repertoire, demonstrated as a peak expansion. The major CDR3 length dominated the profiles at all three time points, before and after vaccination (Fig. 2C). Notably, the dominant CDR3 length was seen in the isotype of the spectratype profile that matched the isotype of the M-protein from the MGUS patient. For example, the peak expansion was observed on IGHG spectratypes for an IgG MGUS patient. Influenza vaccination resulted in change in CGD index of the spectratypes that were mostly in the IGHG and IGHA isotypes but not in IGHM in the healthy controls. In MGUS patients, we observed a trend toward having a lower baseline IGHG repertoire diversity (p = 0.07) in comparison with healthy controls (Fig. 2D). Nonetheless, a significant change in the CGD similar to that of healthy controls was observed for IGHG (p = 0.05) and IGHA (p = 0.03) spectratypes on day 7 postvaccination. In contrast to healthy controls, there is significant repertoire perturbation in the MGUS IGHM spectratypes at day 7 (p = 0.005), with the CGD returning to baseline values at day 28. This resulted in significantly lower IGHM CGD values for MGUS at day 7 in comparison with healthy controls (p = 0.006). To investigate whether influenza vaccination has an influence on the immunoglobulin IGHV gene repertoire selection, we looked at the change in mean CDR3 sizes. No change in the CDR3 size was observed in healthy controls following vaccination for both IgM and IgG. However, there was a significant increase in the mean CDR3 length at day 7 in the IgA repertoire (p = 0.05) that then returned to smaller length at day 28, as seen at baseline. There is evidence of repertoire bias toward use of longer CDR3 length in the IgG and IgA repertoires, but not in IgM, for MGUS patients in response to vaccination (Fig. 2E). The mean CDR3 length at day 7 in MGUS was significantly higher than in healthy controls (p = 0.008) for IgG. Monoclonal protein levels significantly correlated with CGD at day 7 in the IgG and IgA repertoires (Fig. 2F). Higher M-protein concentration was associated with more oligoclonal response at day 7 in the MGUS IGHA repertoire, with CGD as low as 0.1, indicating expansions in a few distinct CDR3 sizes (r2 = −0.51; p = 0.04). This notion that a few CDR3 sizes expand in response to vaccination is supported by the observation that leptokurtosis was significantly correlated with M-protein at day 7 for the IGHA spectratypes (r2 = 0.66, p = 0.005). Additionally, CGD at day 7 was negatively correlated with M-protein concentration for the IGHG spectratypes (r2 = −0.66, p = 0.005) but not for IGHM. Further stratification of the MGUS patients into two groups based on M-protein concentration showed that M-protein > 10 g/L (n = 7) was associated with a significantly lower CGD at day 7 postvaccination for both IGHA (p = 0.0164) and IGHG (p = 0.05) in comparison with MGUS, with M-protein concentration <10 g/L. Furthermore, we investigated whether a less diverse repertoire could predict a worse response to vaccination. Immunoglobulin G responses specific to H1N1 were established in the same MGUS cohort as for this study and were recently reported separately [19Tete S.M. Wilting K.R. Horst G. et al.IgG antibody and TH1 immune responses to influenza vaccination negatively correlate with M-protein burden in monoclonal gammopathy of undetermined significance.Hematology and Leukemia. 2013; 1: 3-10Crossref Google Scholar]. We therefore looked for correlation between the spectratype metrics and H1N1 influenza-specific IgG responses. No correlations were observed for the healthy controls. However, lower CGD at day 7 in the IGHM repertoire was associated with lower H1N1 influenza-specific IgG responses at day 28 in MGUS (r2 = 0.55; p = 0.02). Those with higher serum IgG response showed less perturbation in the IGHM repertoire following vaccination (Fig. 3). In this study, we investigated the B-cell repertoire in MGUS by looking at CDR3 spectratypes at baseline and examined the effect of vaccination on the IGHM, IGHG, and IGHA CDR3 spectratypes. Naïve B-cell repertoires show a characteristic Gaussian distribution of CDR3 length diversity, reflecting polyclonality of the B cells. Deviation from Gaussian distribution is considered to be a sign of oligoclonal expansions. There were interindividual variations in the spectratype metrics in all three isotypes, which were more prominent
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