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T Cell–Mediated Humoral Immune Responses to Type 3 Capsular Polysaccharide of Streptococcus Pneumoniae

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T Cell–Mediated Humoral Immune Responses to Type 3 Capsular Polysaccharide of Streptococcus Pneumoniae The Journal of Immunology T Cell–Mediated Humoral Immune Responses to Type 3 Capsular Polysaccharide of Streptococcus pneumoniae Dustin R. Middleton,1 Lina Sun,1 Amy V. Paschall, and Fikri Y. Avci Most pathogenic bacteria express surface carbohydrates called capsular polysaccharides (CPSs). CPSs are important vaccine tar- gets because they are easily accessible and recognizable by the immune system. However, CPS-specific adaptive humoral immune responses can only be achieved by the covalent conjugation of CPSs with carrier proteins to produce glycoconjugate vaccines. We previously described a mechanism by which a model glycoconjugate vaccine can activate the adaptive immune system and dem- onstrated that the mammalian CD4+ T cell repertoire contains a population of carbohydrate-specific T cells. In this study, we use glycoconjugates of type 3 Streptococcus pneumoniae CPS (Pn3P) to assess whether the carbohydrate-specific adaptive immune response exemplified in our previous study can be applied to the conjugates of this lethal pathogen. In this article, we provide evidence for the functional roles of Pn3P-specific CD4+ T cells utilizing mouse immunization schemes that induce Pn3P-specific IgG responses in a carbohydrate-specific T cell–dependent manner. The Journal of Immunology, 2017, 199: 598–603. xploiting their high antigenicity, capsular polysaccharides Gram-positive pathogen S. pneumoniae can be sorted into .90 (CPSs) have been used as main components of glycoconjugate capsular serotypes (9). Multiple studies have shown the ability of E vaccines in clinical practice worldwide in the past three de- CPS-specific Abs to provide protection from pneumococcal cades (1). Immunizations with glycoconjugates containing CPSs from challenges (10, 11). However, most CPSs are poorly immunoge- Haemophilus influenzae, Streptococcus pneumoniae,andNeisseria nic, because they cannot, in their pure form, induce T cell–dependent meningitidis have been used in preventing or controlling infectious immune responses (4, 12). Immunization with glycoconjugates, diseases caused by these bacterial pathogens (2, 3). Although as opposed to pure glycans, elicits T cell help for B cells that glycoconjugate vaccines have provided great health benefits in produce high-affinity IgG Abs to the CPS component of the controlling bacterial diseases, glycoconjugate construction has vaccine and induces memory B and T cell development (4, 12). often been a random process of empirically linking two molecules Since the introduction of the first pneumococcal conjugate vac- (carbohydrate and protein) with minimum consideration of their cine, PCV7, the incidence rate of pneumococcal disease has been mechanism of action (4), resulting in poorly characterized, hetero- reduced significantly (11). The current pneumococcal conjugate geneous, and variably immunogenic glycoconjugate vaccines (1, 5). vaccine is the 13-valent Prevnar13, encompassing CPSs from 13 Demystifying T cell activation mechanisms of glycoconjugate of the most prevalent serotypes of S. pneumoniae (11). The model vaccines is a key step toward designing new-generation vaccines. conjugate vaccine used in this study is in fact a component of the We recently demonstrated a mechanism through which uptake of a existing 13-valent pneumococcal conjugate vaccine. S. pneumoniae glycoconjugate vaccine by APCs results in the presentation of a car- remains among the most lethal infectious agents despite the bohydrate epitope by the MHC class II, thus stimulating carbohydrate- availability of global vaccination programs (11, 13). The type 3 specific CD4+ T cells (Tcarbs) (6–8). strain in particular is among the most virulent strains. Despite In this study, we use model glycoconjugates of type 3 S. pneumoniae current vaccination programs, morbidity of the type 3 strain CPS (Pn3P) to examine whether the carbohydrate-specific adap- remains high, raising questions regarding the efficacy of this tive immune responses exemplified in our previous findings apply vaccine (14). The knowledge gained in this study may have to other carbohydrate Ags used in glycoconjugate vaccines. The implications in producing a highly protective knowledge-based pneumococcal vaccine. Department of Biochemistry and Molecular Biology, Center for Molecular Medi- cine and Complex Carbohydrate Research Center, University of Georgia, Athens, Materials and Methods GA 30602 Mice 1D.R.M. and L.S. are cofirst authors. Eight-week-old female BALB/c mice were obtained from Taconic Bio- ORCIDs: 0000-0001-8973-9116 (D.R.M.); 0000-0001-9144-7534 (A.V.P.); 0000- sciences (Hudson, NY) and housed in the Coverdell Rodent Vivarium at the 0002-1902-7532 (F.Y.A.). University of Georgia. Mice were kept in microisolator cages and handled Received for publication January 6, 2017. Accepted for publication May 6, 2017. under a laminar flow hood. All mouse experiments were in compliance with This work was supported by National Institutes of Health Grants AI-115451-01 and the University of Georgia Institutional Animal Care and Use Committee 1R01AI123383-01A1. under the approved animal use protocol A2013 12-011-Y1-A0. Address correspondence and reprint requests to Prof. Fikri Y. Avci, Department of Ags Biochemistry and Molecular Biology, Center for Molecular Medicine and Complex Carbohydrate Research Center, University of Georgia, 315 Riverbend Road, Athens, Purified Pn3P powder was obtained from American Type Cell Collection GA 30602. E-mail address: [email protected] (catalog number 172-X). Pn3P was reduced to an average m.w. of 100 kDa The online version of this article contains supplemental material. by hydrolysis with 0.3 M trifluoroacetic acid. Pn3P was conjugated to either Abbreviations used in this article: CPS, capsular polysaccharide; KLH, keyhole OVA (A7641; Sigma) or keyhole limpet hemocyanin (KLH; 374805; limpet hemocyanin; Pn3P, type 3 S. pneumoniae CPS; Tcarb, carbohydrate-specific Calbiochem) through reductive amination as previously described (6, 7). CD4+ T cell. Pn3P conjugates were isolated from unconjugated components by size ex- clusion chromatography (Supplemental Fig. 1). A combination of phenol Copyright Ó 2017 by The American Association of Immunologists, Inc. 0022-1767/17/$30.00 sulfuric acid and BCA assays using Pn3P and carrier proteins for standard www.jimmunol.org/cgi/doi/10.4049/jimmunol.1700026 The Journal of Immunology 599 curve generation confirmed that the conjugates consisted of 45–55% protein Percent viability was calculated as each duplicate reaction subtracted from and 45–55% Pn3P (7). the mean values obtained for control samples (naive serum, 100% survival). Immunizations Abs and flow cytometry Groups of BALB/c mice were immunized i.p. on days 0 and 14 with 5 mgof The following fluorophore-conjugated Abs used in flow cytometry detection Ag in PBS mixed with 2% alhydrogel (vac-alu-50; Invivogen) in a 3:1 were purchased from BioLegend: anti-mouse CD3ε (clone145-2C11), anti- ratio. For T cell assays, groups of BALB/c mice were immunized with Ags mouse CD4 (clone GK1.5), and anti-mouse CD69 (clone H1.2F3). CFSE emulsified in Freund’s adjuvant (Thermo Scientific) by s.c. injection. was purchased from Sigma. After surface staining of cell suspensions at 4˚C, Where indicated, mixtures consisted of quantities consistent with conju- samples were analyzed on CyAn (Beckman Coulter). Data were analyzed gate ratios. using FlowJo software (Tree Star). Adoptive transfers ELISPOT assay Groups of donor BALB/c mice were primed and boosted with 5 mgofPn3P Groups of BALB/c mice were immunized with 10 mg of Pn3P-OVA emul- or 10 mg of Pn3P-KLH s.c. at 3-wk intervals. Mice were sacrificed 5 d after sified in Freund’s adjuvant (Thermo Scientific) by s.c. injection. CD4+ T cells boost. CD4+ T cells were isolated from spleen and lymph nodes of Pn3P or were isolated from lymph nodes of Pn3P-OVA–immunized mice and stim- Pn3P-KLH primed mice, and negatively selected using mouse CD4+ ulated in vitro in the presence of irradiated APCs pulsed with indicated Ags. T lymphocyte enrichment magnetic beads (558131; BD Biosciences). After 24-h stimulation, IL-2 production was detected by ELISPOT assay Splenic B cells from Pn3P-KLH primed mice were isolated using mouse using mouse ELISPOT reagent set (Cellular Technology) according to the B lymphocyte enrichment magnetic beads (557792; BD Biosciences). Iso- manufacturer’s protocol. The ELISPOT samples were analyzed by Immuno- lation of a given cell type was confirmed by flow cytometry. CD4+ Tcells Spot S6 analyzer using the ImmunoCapture 6.3 and ImmunoSpot 5.0 Pro DC (1 3 107) from either Pn3P- or Pn3P-KLH–immunized donors and B cells software (Cellular Technology). (1 3 107) from Pn3P-KLH–immunized mice were adoptively transferred to recipient mice through tail-vein injections in PBS. The recipient mice were Generation of Pn3P-specific T cell hybridomas then immunized with Pn3P-KLH or Pn3P-OVA 1 d after adoptive transfer. T cell hybridomas were generated as previously described (16). In brief, Detection of Pn3P-specific serum Abs BALB/c mice were immunized with Pn3P-KLH emulsified in Freund’s adjuvant (Thermo Scientific). Primary CD4+ T cells obtained from lymph Mice were bled from the tail vein on days 14 and 21 during the prime-boost node of immunized mice were restimulated in vitro with APCs that pulsed immunization experiments. Mice used in the adoptive transfer experiment with Pn3P-KLH. Activated CD4+ T cells were fused with BW5147 T cell were bled 3 d after booster immunization (4 d after transfer). Pn3P-specific lymphoma cells at a ratio of 1:1. The T cell hybrids were selected in Abs in serum were detected by ELISA in 96-well plates coated with 2.5 mg/ml DMEM containing hypoxanthine-aminopterin-thymidine and screened Pn3P-HSA conjugate. Four immune sera per group were used in all ELISA for CD3 and CD4 expression. Ag specificity was screened by activating the experiments. T hybridoma cells with appropriate Ags in the presence of APCs. Culture supernatant was collected after 24-h stimulation to test IL-2 production by Opsonophagocytic killing assay ELISA assay using paired rat mAbs for mouse IL-2 (BioLegend).
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