High-throughput sequencing of IgG B-cell receptors reveals frequent usage of the rearranged IGHV4-28/IGHJ4 gene in primary immune thrombocytopenia

Primary immune thrombocytopenia (ITP) is an acquired form of thrombocytopenia caused by IgG anti-platelet autoantibodies and represents an organ-specific autoimmune disorder. Although the glycoprotein (GP) IIb/IIIa and GPIb/IX have been shown to be targets for autoantibodies, the antigen specificity of autoantibodies is not fully elucidated. To identify the characteristics of IgG B-cell receptor (BCR) repertoires in ITP, we took advantage of adaptor-ligation PCR and high-throughput DNA sequencing methods for analyzing the clone-based repertoires of IgG-expressing peripheral blood B cells. A total of 2,009,943 in-frame and 315,469 unique reads for IGH (immunoglobulin heavy) were obtained from twenty blood samples. Comparison of the IGHV repertoires between patients and controls revealed an increased usage of IGHV4-28 in ITP patients. One hundred eighty-six distinct IGHV4-28-carrying sequences were identified in ITP patients and the majority of these clones used an IGHJ4 segment. The IGHV4-28/IGHJ4-carrying B-cell clones were found in all ITP patients. Oligoclonal expansions of IGHV4-28/IGHJ4-carrying B cells were accompanied by multiple related clones with single amino substitution in the CDR3 region suggesting somatic hypermutation. Taken together, the expansion of IGHV4-28/IGHJ4-carrying IgG-expressing B cells in ITP may be the result of certain antigenic pressure and may provide a clue for the immune pathophysiology of ITP

31st Annual Meeting and Associated Programs of the Society for Immunotherapy of Cancer (SITC 2016) : part two

Background The immunological escape of tumors represents one of the main ob- stacles to the treatment of malignancies. The blockade of PD-1 or CTLA-4 receptors represented a milestone in the history of immunotherapy. However, immune checkpoint inhibitors seem to be effective in specific cohorts of patients. It has been proposed that their efficacy relies on the presence of an immunological response. Thus, we hypothesized that disruption of the PD-L1/PD-1 axis would synergize with our oncolytic vaccine platform PeptiCRAd. Methods We used murine B16OVA in vivo tumor models and flow cytometry analysis to investigate the immunological background. Results First, we found that high-burden B16OVA tumors were refractory to combination immunotherapy. However, with a more aggressive schedule, tumors with a lower burden were more susceptible to the combination of PeptiCRAd and PD-L1 blockade. The therapy signifi- cantly increased the median survival of mice (Fig. 7). Interestingly, the reduced growth of contralaterally injected B16F10 cells sug- gested the presence of a long lasting immunological memory also against non-targeted antigens. Concerning the functional state of tumor infiltrating lymphocytes (TILs), we found that all the immune therapies would enhance the percentage of activated (PD-1pos TIM- 3neg) T lymphocytes and reduce the amount of exhausted (PD-1pos TIM-3pos) cells compared to placebo. As expected, we found that PeptiCRAd monotherapy could increase the number of antigen spe- cific CD8+ T cells compared to other treatments. However, only the combination with PD-L1 blockade could significantly increase the ra- tio between activated and exhausted pentamer positive cells (p= 0.0058), suggesting that by disrupting the PD-1/PD-L1 axis we could decrease the amount of dysfunctional antigen specific T cells. We ob- served that the anatomical location deeply influenced the state of CD4+ and CD8+ T lymphocytes. In fact, TIM-3 expression was in- creased by 2 fold on TILs compared to splenic and lymphoid T cells. In the CD8+ compartment, the expression of PD-1 on the surface seemed to be restricted to the tumor micro-environment, while CD4 + T cells had a high expression of PD-1 also in lymphoid organs. Interestingly, we found that the levels of PD-1 were significantly higher on CD8+ T cells than on CD4+ T cells into the tumor micro- environment (p < 0.0001). Conclusions In conclusion, we demonstrated that the efficacy of immune check- point inhibitors might be strongly enhanced by their combination with cancer vaccines. PeptiCRAd was able to increase the number of antigen-specific T cells and PD-L1 blockade prevented their exhaus- tion, resulting in long-lasting immunological memory and increased median survival

Additional file 1: of A new high-throughput sequencing method for determining diversity and similarity of T cell receptor (TCR) α and β repertoires and identifying potential new invariant TCR α chains

Table S1. Age, gender and chronic illness of 20 healthy individuals. Table S2. Numbers of unique reads, reads and nucleotides in TRA reads obtained from PBMCs of 20 healthy individuals. Table S3. Numbers of unique reads, reads and nucleotides in TRB reads obtained from PBMCs of 20 healthy individuals. Table S4. Percentage of mismatched nucleotides in in-frame and out-of-frame TCR sequences. Table S5. Occurrence frequency of out-of-frame unique sequence reads in TRA and TRB. Table S6. Percentage frequency of shared TRA reads between all pairs of individuals. Table S7. Percentage frequency of shared TRB reads between all pairs of individuals. Figure S1. Correlation of gene usage of TRAV, TRAJ, TRBV and TRBJ between healthy individuals. Figure S2. Concordance correlation coefficient in TRAV, TRAJ, TRBV and TRBJ. Figure S3. Comparison of TCR usages between in-frame and out-of-frame reads sequences. Figure S4. Diversity indices of in-frame and out-of-frame TRA and TRB. Figure S5. Correlation of TCR diversity with age. Figure S6. Correlation of TCR usage from a published FACS data with AL-PCR and Multiplex PCR. (DOCX 548 kb

Type IVb Hypersensitivity Reaction in the Novel Murine Model of Palladium–Induced Intraoral Allergic Contact Mucositis

Palladium (Pd) is a component of several alloy types that are widely used in our environment, including several dental alloy types that cause adverse reactions such as hypersensitivity in the oral mucosa. However, the pathological mechanism of intraoral Pd allergies remains unclear because its animal model in the oral mucosa has not been established. In this study, we established a novel murine model of Pd–induced allergies in the oral mucosa, and explored the immune response of cytokine profiles and T cell diversity in terms of the T cell receptor. The Pd–induced allergy mouse was generated by two sensitizations with PdCl2, plus a lipopolysaccharide solution into the postauricular skin followed by a single Pd challenge of the buccal mucosa. Significant swelling and pathological features were histologically evident at five days after the challenge, and CD4–positive T cells producing high levels of T helper 2 type cytokines had accumulated in the allergic oral mucosa. Characterization of the T cell receptor repertoire in Palladium allergic mice indicated that Pd–specific T cell populations were limited in V and J genes but were diverse at the clonal level. Our model demonstrated that a Pd–specific T cell population with Th2 type response tendencies may be involved in the Pd–induced intraoral metal contact allergy

Different Somatic Hypermutation Levels among Antibody Subclasses Disclosed by a New Next-Generation Sequencing-Based Antibody Repertoire Analysis

A diverse antibody repertoire is primarily generated by the rearrangement of V, D, and J genes and subsequent somatic hypermutation (SHM). Class-switch recombination (CSR) produces various isotypes and subclasses with different functional properties. Although antibody isotypes and subclasses are considered to be produced by both direct and sequential CSR, it is still not fully understood how SHMs accumulate during the process in which antibody subclasses are generated. Here, we developed a new next-generation sequencing (NGS)-based antibody repertoire analysis capable of identifying all antibody isotype and subclass genes and used it to examine the peripheral blood mononuclear cells of 12 healthy individuals. Using a total of 5,480,040 sequences, we compared percentage frequency of variable (V), junctional (J) sequence, and a combination of V and J, diversity, length, and amino acid compositions of CDR3, SHM, and shared clones in the IgM, IgD, IgG3, IgG1, IgG2, IgG4, IgA1, IgE, and IgA2 genes. The usage and diversity were similar among the immunoglobulin (Ig) subclasses. Clonally related sequences sharing identical V, D, J, and CDR3 amino acid sequences were frequently found within multiple Ig subclasses, especially between IgG1 and IgG2 or IgA1 and IgA2. SHM occurred most frequently in IgG4, while IgG3 genes were the least mutated among all IgG subclasses. The shared clones had almost the same SHM levels among Ig subclasses, while subclass-specific clones had different levels of SHM dependent on the genomic location. Given the sequential CSR, these results suggest that CSR occurs sequentially over multiple subclasses in the order corresponding to the genomic location of IGHCs, but CSR is likely to occur more quickly than SHMs accumulate within Ig genes under physiological conditions. NGS-based antibody repertoire analysis should provide critical information on how various antibodies are generated in the immune system

Type IVb Hypersensitivity Reaction in the Novel Murine Model of Palladium&ndash;Induced Intraoral Allergic Contact Mucositis

Palladium (Pd) is a component of several alloy types that are widely used in our environment, including several dental alloy types that cause adverse reactions such as hypersensitivity in the oral mucosa. However, the pathological mechanism of intraoral Pd allergies remains unclear because its animal model in the oral mucosa has not been established. In this study, we established a novel murine model of Pd&ndash;induced allergies in the oral mucosa, and explored the immune response of cytokine profiles and T cell diversity in terms of the T cell receptor. The Pd&ndash;induced allergy mouse was generated by two sensitizations with PdCl2, plus a lipopolysaccharide solution into the postauricular skin followed by a single Pd challenge of the buccal mucosa. Significant swelling and pathological features were histologically evident at five days after the challenge, and CD4&ndash;positive T cells producing high levels of T helper 2 type cytokines had accumulated in the allergic oral mucosa. Characterization of the T cell receptor repertoire in Palladium allergic mice indicated that Pd&ndash;specific T cell populations were limited in V and J genes but were diverse at the clonal level. Our model demonstrated that a Pd&ndash;specific T cell population with Th2 type response tendencies may be involved in the Pd&ndash;induced intraoral metal contact allergy

Fexofenadine Suppresses Delayed-Type Hypersensitivity in the Murine Model of Palladium Allergy

Palladium is frequently used in dental materials, and sometimes causes metal allergy. It has been suggested that the immune response by palladium-specific T cells may be responsible for the pathogenesis of delayed-type hypersensitivity in study of palladium allergic model mice. In the clinical setting, glucocorticoids and antihistamine drugs are commonly used for treatment of contact dermatitis. However, the precise mechanism of immune suppression in palladium allergy remains unknown. We investigated inhibition of the immune response in palladium allergic mice by administration of prednisolone as a glucocorticoid and fexofenadine hydrochloride as an antihistamine. Compared with glucocorticoids, fexofenadine hydrochloride significantly suppressed the number of T cells by interfering with the development of antigen-presenting cells from the sensitization phase. Our results suggest that antihistamine has a beneficial effect on the treatment of palladium allergy compared to glucocorticoids

Cross-Reactivity of Intraoral Allergic Contact Mucositis in the Nickel-Sensitized Ear Model of Metal Allergy

Cross-reactivity of metal allergies can make metal allergy treatment complicated because the background of immune response in cross-reactions remains unknown. In clinical settings, cross-reactivity among several metals has been suspected. However, the precise mechanism of immune response in cross-reactivity is unclear. Two sensitizations with nickel, palladium, and chromium plus lipopolysaccharide solution into the postauricular skin were followed by a single nickel, palladium, and chromium challenge of the oral mucosa to generate the intraoral metal contact allergy mouse model. Results showed that the infiltrating T cells in nickel-sensitized, palladium- or chromium-challenged mice expressed CD8+ cells, cytotoxic granules, and inflammation-related cytokines. Thus, nickel ear sensitization can cause cross-reactive intraoral metal allergy

Characterization of Metal-Specific T-Cells in Inflamed Oral Mucosa in a Novel Murine Model of Chromium-Induced Allergic Contact Dermatitis

The element chromium (Cr) is a component of several types of alloys found in the environment, or utilized in dentistry, that may cause intraoral metal contact allergy. However, the pathological mechanism of intraoral Cr allergy remains unclear because there is no established animal model of Cr allergy in the oral mucosa. In this study, we established a novel murine model of Cr-induced intraoral metal contact allergy and elucidated the immune response in terms of cytokine profiles and T-cell receptor repertoire. Two sensitizations with Cr plus lipopolysaccharide solution into the postauricular skin were followed by a single Cr challenge of the oral mucosa to generate the intraoral metal contact allergy model. Histological examination revealed that CD3+ T-cells had infiltrated the allergic oral mucosa one day after exposure to the allergen. The increase in T-cell markers and cytokines in allergic oral mucosa was also confirmed via quantitative PCR analysis. We detected Cr-specific T-cells bearing TRAV12D-1-TRAJ22 and natural killer (NK) T-cells in the oral mucosa and lymph nodes. Our model demonstrated that Cr-specific T-cells and potent NKT-cell activation may be involved in the immune responses of Cr-induced intraoral metal contact allergy