Characterization of Tumor-Infiltrating Lymphocyte-Derived Atypical TCRs Recognizing Breast Cancer in an MR1-Dependent Manner.

The MHC class I-related 1 (MR1) molecule is a non-polymorphic antigen-presenting molecule that presents several metabolites to MR1-restricted T cells, including mucosal-associated invariant T (MAIT) cells. MR1 ligands bind to MR1 molecules by forming a Schiff base with the K43 residue of MR1, which induces the folding of MR1 and its reach to the cell surface. An antagonistic MR1 ligand, Ac-6-FP, and the K43A mutation of MR1 are known to inhibit the responses of MR1-restricted T cells. In this study, we analyzed MR1-restricted TCRs obtained from tumor-infiltrating lymphocytes (TILs) from breast cancer patients. They responded to two breast cancer cell lines independently from microbial infection and did not respond to other cancer cell lines or normal breast cells. Interestingly, the reactivity of these TCRs was not inhibited by Ac-6-FP, while it was attenuated by the K43A mutation of MR1. Our findings suggest the existence of a novel class of MR1-restricted TCRs whose antigen is expressed in some breast cancer cells and binds to MR1 depending on the K43 residue of MR1 but without being influenced by Ac-6-FP. This work provides new insight into the physiological roles of MR1 and MR1-restricted T cells.

Candidate tumor-specific CD8+ T cell subsets identified in the malignant pleural effusion of advanced lung cancer patients by single-cell analysis.

Isolation of tumor-specific T cells and their antigen receptors (TCRs) from malignant pleural effusions (MPE) may facilitate the development of TCR-transduced adoptive cellular immunotherapy products for advanced lung cancer patients. However, the characteristics and markers of tumor-specific T-cells in MPE are largely undefined. To this end, to establish the phenotypes and antigen specificities of CD8+ T cells, we performed single-cell RNA and TCR sequencing of samples from three advanced lung cancer patients. Dimensionality reduction on a total of 4,983 CD8+ T cells revealed 10 clusters including naïve, memory, and exhausted phenotypes. We focused particularly on exhausted T cell clusters and tested their TCR reactivity against neoantigens predicted from autologous cancer cell lines. Four different TCRs specific for the same neoantigen and one orphan TCR specific for the autologous cell line were identified from one of the patients. Differential gene expression analysis in tumor-specific T cells relative to the other T cells identified CXCL13, as a candidate gene expressed by tumor-specific T cells. In addition to expressing CXCL13, tumor-specific T cells were present in a higher proportion of T cells co-expressing PDCD1(PD-1)/TNFRSF9(4-1BB). Furthermore, flow cytometric analyses in advanced lung cancer patients with MPE documented that those with high PD-1/4-1BB expression have a better prognosis in the subset of 57 adenocarcinoma patients (p = .039). These data suggest that PD-1/4-1BB co-expression might identify tumor-specific CD8+ T cells in MPE, which are associated with patients' prognosis. (233 words).

Novel chimeric antigen receptor-expressing T cells targeting the malignant mesothelioma-specific antigen sialylated HEG1.

The selection of highly specific target antigens is critical for the development of clinically efficient and safe chimeric antigen receptors (CARs). In search of diagnostic marker for malignant mesothelioma (MM), we have established SKM9-2 monoclonal antibody (mAb) which recognizes a MM-specific molecule, sialylated Protein HEG homolog 1 (HEG1), with high specificity and sensitivity. In this study, to develop a novel therapeutic approach against MM, we generated SKM9-2 mAb-derived CARs that included the CD28 (SKM-28z) or 4-1BB (SKM-BBz) costimulatory domain. SKM-28z CAR-T cells showed continuous growth and enhanced Tim-3, LAG-3, and PD-1 expression in vitro, which might be induced by tonic signaling caused by self-activation; however, these phenotypes were not observed in SKM-BBz CAR-T cells. In addition, SKM-BBz CAR-T cells exhibited slightly stronger in vitro killing activity against MM cell lines than SKM-28z CAR-T cells. More importantly, only SKM-BBz CAR-T cells, but not SKM-28z CAR-T cells, significantly inhibited tumor growth in vivo in a MM cell line xenograft mouse model. Gene expression profiling and reporter assays revealed differential signaling pathway activation; in particular, SKM-BBz CAR-T cells exhibited enhanced NF-kB signaling and reduced NFAT activation. In addition, SKM-BBz CAR-T cells showed upregulation of early memory markers, such as TCF7 and CCR7, as well as downregulation of pro-apoptotic proteins, such as BAK1 and BID, which may be associated with phenotypical and functional differences between SKM-BBz and SKM-28z CAR-T cells. In conclusion, we developed novel SKM9-2-derived CAR-T cells with the 4-1BB costimulatory domain, which could provide a promising therapeutic approach against refractory MM.

Secretory GFP reconstitution labeling of neighboring cells interrogates cell-cell interactions in metastatic niches.

Cancer cells inevitably interact with neighboring host tissue-resident cells during the process of metastatic colonization, establishing a metastatic niche to fuel their survival, growth, and invasion. However, the underlying mechanisms in the metastatic niche are yet to be fully elucidated owing to the lack of methodologies for comprehensively studying the mechanisms of cell-cell interactions in the niche. Here, we improve a split green fluorescent protein (GFP)-based genetically encoded system to develop secretory glycosylphosphatidylinositol-anchored reconstitution-activated proteins to highlight intercellular connections (sGRAPHIC) for efficient fluorescent labeling of tissue-resident cells that neighbor on and putatively interact with cancer cells in deep tissues. The sGRAPHIC system enables the isolation of metastatic niche-associated tissue-resident cells for their characterization using a single-cell RNA sequencing platform. We use this sGRAPHIC-leveraged transcriptomic platform to uncover gene expression patterns in metastatic niche-associated hepatocytes in a murine model of liver metastasis. Among the marker genes of metastatic niche-associated hepatocytes, we identify Lgals3, encoding galectin-3, as a potential pro-metastatic factor that accelerates metastatic growth and invasion.

Single-cell sequencing on CD8+ TILs revealed the nature of exhausted T cells recognizing neoantigen and cancer/testis antigen in non-small cell lung cancer.

BACKGROUND: CD8+tumor infiltrating lymphocytes (TILs) are often observed in non-small cell lung cancers (NSCLC). However, the characteristics of CD8+ TILs, especially T-cell populations specific for tumor antigens, remain poorly understood. METHODS: High throughput single-cell RNA sequencing and single-cell T-cell receptor (TCR) sequencing were performed on CD8+ TILs from three surgically-resected lung cancer specimens. Dimensional reduction for clustering was performed using Uniform Manifold Approximation and Projection. CD8+ TIL TCR specific for the cancer/testis antigen KK-LC-1 and for predicted neoantigens were investigated. Differentially-expressed gene analysis, Gene Set Enrichment Analysis (GSEA) and single sample GSEA was performed to characterize antigen-specific T cells. RESULTS: A total of 6998 CD8+ T cells was analyzed, divided into 10 clusters according to their gene expression profile. An exhausted T-cell (exhausted T (Tex)) cluster characterized by the expression of ENTPD1 (CD39), TOX, PDCD1 (PD1), HAVCR2 (TIM3) and other genes, and by T-cell oligoclonality, was identified. The Tex TCR repertoire (Tex-TCRs) contained nine different TCR clonotypes recognizing five tumor antigens including a KK-LC-1 antigen and four neoantigens. By re-clustering the tumor antigen-specific T cells (n=140), it could be seen that the individual T-cell clonotypes were present on cells at different stages of differentiation and functional states even within the same Tex cluster. Stimulating these T cells with predicted cognate peptide indicated that TCR signal strength and subsequent T-cell proliferation and cytokine production was variable but always higher for neoantigens than KK-LC-1. CONCLUSIONS: Our approach focusing on T cells with an exhausted phenotype among CD8+ TILs may facilitate the identification of tumor antigens and clarify the nature of the antigen-specific T cells to specify the promising immunotherapeutic targets in patients with NSCLC.

Contributions of the N-terminal flanking residues of an antigenic peptide from the Japanese cedar pollen allergen Cry j 1 to the T-cell activation by HLA-DP5.

Cry j 1 is a major allergen present in Japanese cedar (Cryptomeria japonica) pollens. Peptides with the core sequence of KVTVAFNQF from Cry j 1 ('pCj1') bind to HLA-DP5 and activate Th2 cells. In this study, we noticed that Ser and Lys at positions -2 and -3, respectively, in the N-terminal flanking (NF) region to pCj1 are conserved well in HLA-DP5-binding allergen peptides. A competitive binding assay showed that the double mutation of Ser(-2) and Lys(-3) to Glu [S(P-2)E/K(P-3)E] in a 13-residue Cry j 1 peptide (NF-pCj1) decreased its affinity for HLA-DP5 by about 2-fold. Similarly, this double mutation reduced, by about 2-fold, the amount of NF-pCj1 presented on the surface of mouse antigen-presenting dendritic cell line 1 (mDC1) cells stably expressing HLA-DP5. We established NF-pCj1-specific and HLA-DP5-restricted CD4+ T-cell clones from HLA-DP5 positive cedar pollinosis (CP) patients, and analyzed their IL-2 production due to the activation of mouse TG40 cells expressing the cloned T-cell receptor by the NF-pCj1-presenting mDC1 cells. The T-cell activation was actually decreased by the S(P-2)E/K(P-3)E mutation, corresponding to the reduction in the peptide presentation by this mutation. In contrast, the affinity of NF-pCj1·HLA-DP5 for the T-cell receptor was not affected by the S(P-2)E/K(P-3)E mutation, as analyzed by surface plasmon resonance. Considering the positional and side-chain differences of these NF residues from previously reported T-cell activating sequences, the mechanisms of enhanced T-cell activation by Ser(-2) and Lys(-3) of NF-pCj1 may be novel.

Selection of highly responsive T cell receptors by an analysis combining the expression of multiple markers.

The clinical success of T cell receptor (TCR) gene-transduced T (TCR-T) cell therapy is expected as one of the next-generation immunotherapies for cancer, in which the selection of TCRs with high functional avidity (high-functional TCRs) is important. One widely used approach to select high-functional TCRs is a comparison of the EC50 values of TCRs, which involves laborious experiments. Therefore, the establishment of a simpler method to select high-functional TCRs is desired. We herein attempted to establish a simple method to select high-functional TCRs based on the expression of T cell activation markers using the mouse T cell line BW5147.3 (BW). We examined relationships between the EC50 values of TCRs in interleukin-2 production and the expression levels of TCR activation markers on BW cells. In TCR-expressing BW cells stimulated with antigenic peptides, the CD69, CD137, and PD-1 expression was differentially induced by various doses of peptides. An analysis of TCRs derived from the tumor-infiltrating lymphocytes of murine melanoma and peripheral blood T cells of hepatocellular carcinoma patients treated with a peptide vaccination revealed that an analysis combining CD69, CD137, and PD-1 expression levels in BW cells stimulated with a single dose of an antigenic peptide selected high-functional TCRs with functional avidity assessed by EC50 values. Our method facilitates the section of high-functional TCRs among tumor-reacting TCRs, which will promote TCR-T cell therapy. The stimulation of BW cells expressing objective TCRs with a single dose of antigenic peptides and analysis combining the expression of CD69, CD137, and PD-1 allows us to select highly responsive TCRs.

Gene modified NK cell line as a powerful tool for evaluation of cloned TCRs for TCR-T cell therapy.

T cell receptor-engineered T cell (TCR-T) therapy is anticipated as a next generation-immunotherapy for cancer and recent advances of TCR isolation technology have enabled patient's T cells to express TCRs recognizing multiple combinations of specific peptides and human leukocyte antigens (HLA). However, evaluation processes for the TCR-induced cytotoxicity activity using primary T cells are laborious and time-consuming. In this study, we established a cell line that do not express endogenous TCRs, enabling to generate large numbers of homogeneous cells, and can measure the cytotoxic activity of the isolated TCRs. To this end, we transduced a Natural Killer (NK) cell line with human CD3 molecules and interleukin (IL)-2. The TCR expressing NK cells killed target cells as similarly to TCR-transduced primary T cells and secreted various cytokines/chemokines including IL-2. Thus, the gene-modified NK cell can be a powerful tool to rapidly and efficiently evaluate the functions of isolated TCRs.

Dissecting Naturally Arising Amino Acid Substitutions at Position L452 of SARS-CoV-2 Spike.

Mutations at spike protein L452 are recurrently observed in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern (VOC), including omicron lineages. It remains elusive how amino acid substitutions at L452 are selected in VOC. Here, we characterized all 19 possible mutations at this site and revealed that five mutants expressing the amino acids Q, K, H, M, and R gained greater fusogenicity and pseudovirus infectivity, whereas other mutants failed to maintain steady-state expression levels and/or pseudovirus infectivity. Moreover, the five mutants showed decreased sensitivity toward neutralization by vaccine-induced antisera and conferred escape from T cell recognition. Contrary to expectations, sequence data retrieved from the Global Initiative on Sharing All Influenza Data (GISAID) revealed that the naturally occurring L452 mutations were limited to Q, M, and R, all of which can arise from a single nucleotide change. Collectively, these findings highlight that the codon base change mutational barrier is a prerequisite for amino acid substitutions at L452, in addition to the phenotypic advantages of viral fitness and decreased sensitivity to host immunity. IMPORTANCE In a span of less than 3 years since the declaration of the coronavirus pandemic, numerous SARS-CoV-2 variants of concern have emerged all around the globe, fueling a surge in the number of cases and deaths that caused severe strain on the health care system. A major concern is whether viral evolution eventually promotes greater fitness advantages, transmissibility, and immune escape. In this study, we addressed the differential effect of amino acid substitutions at a frequent mutation site, L452 of SARS-CoV-2 spike, on viral antigenic and immunological profiles and demonstrated how the virus evolves to select one amino acid over the others to ensure better viral infectivity and immune evasion. Identifying such virus mutation signatures could be crucial for the preparedness of future interventions to control COVID-19.

The SARS-CoV-2 Omicron BA.1 spike G446S mutation potentiates antiviral T-cell recognition.

Although the Omicron variant of the SARS-CoV-2 virus shows resistance to neutralizing antibody, it retains susceptibility to the cellular immune response. Here we characterize vaccine-induced T cells specific for various SARS-CoV-2 variants and identified HLA-A*24:02-restricted CD8+ T cells that strongly suppress Omicron BA.1 replication in vitro. Mutagenesis analyses revealed that a G446S mutation, located just outside the N-terminus of the cognate epitope, augmented TCR recognition of this variant. In contrast, no enhanced suppression of replication is observed against cells infected with the prototype, Omicron BA.2, and Delta variants that express G446. The enhancing effect of the G446S mutation is lost when target cells are treated with inhibitors of tripeptidyl peptidase II, a protein that mediates antigen processing. These ex vivo analysis and in vitro results demonstrate that the G446S mutation in the Omicron BA.1 variant affects antigen processing/presentation and potentiates antiviral activity by vaccine-induced T cells, leading to enhanced T cell recognition towards emerging variants.

Evaluation of chimeric antigen receptor of humanized rabbit-derived T cell receptor-like antibody.

T-cell receptor (TCR)-like Abs that specifically recognize antigenic peptides presented on MHC molecules have been developed for next-generation cancer immunotherapy. Recently, we reported a rapid and efficient method to generate TCR-like Abs using a rabbit system. We humanized previously generated rabbit-derived TCR-like Abs reacting Epstein-Barr virus peptide (BRLF1p, TYPVLEEMF) in the context of HLA-A24 molecules, produced chimeric antigen receptor (CAR)-T cells, and evaluated their antitumor effects using in vitro and in vivo tumor models. Humanization of the rabbit-derived TCR-like Abs using the complementarity-determining region grafting technology maintained their specificity and affinity. We prepared a second-generation CAR using single-chain variable fragment of the humanized TCR-like Abs and then transduced them into human T cells. The CAR-T cells specifically recognized BRLF1p/MHC molecules and lysed the target cells in an antigen-specific manner in vitro. They also demonstrated antitumor activity in a mouse xenograft model. We report the generation of CAR-T cells using humanized rabbit-derived TCR-like Abs. Together with our established and efficient generation procedure for TCR-like Abs using rabbits, our platform for the clinical application of humanized rabbit-derived TCR-like Abs to CAR-T cells will help improve next-generation cancer immunotherapy.

Rapid cloning of antigen-specific T-cell receptors by leveraging the cis activation of T cells.

It is commonly understood that T cells are activated via trans interactions between antigen-specific T-cell receptors (TCRs) and antigenic peptides presented on major histocompatibility complex (MHC) molecules on antigen-presenting cells. By analysing a large number of T cells at the single-cell level on a microwell array, we show that T-cell activation can occur via cis interactions (where TCRs on the T cell interact with the antigenic peptides presented on MHC class-I molecules on the same cell), and that such cis activation can be used to detect antigen-specific T cells and clone their TCR within 4 d. We used the detection-and-cloning system to clone a tumour-antigen-specific TCR from peripheral blood mononuclear cells of healthy donors. TCR cloning by leveraging the cis activation of T cells may facilitate the development of TCR-engineered T cells for cancer therapy.

Downregulation of HLA class II is associated with relapse after allogeneic stem cell transplantation and alters recognition by antigen-specific T cells.

Genomic deletion of donor-patient-mismatched HLA alleles in leukemic cells is a major cause of relapse after allogeneic hematopoietic stem cell transplantation (HSCT). Mismatched HLA is frequently lost as an individual allele or a whole region in HLA-class I, however, it is downregulated in HLA-class II. We hypothesized that there might be a difference in T cell recognition capacity against epitopes associated with HLA-class I and HLA-class II and consequently such allogeneic immune pressure induced HLA alterations in leukemic cells. To investigate this, we conducted in vitro experiments with T cell receptor-transduced T (TCR-T) cells. The cytotoxic activity of NY-ESO-1-specific TCR-T cells exhibited similarly against K562 cells with low HLA-A*02:01 expression. However, we demonstrated that the cytokine production against low HLA-DPB1*05:01 expression line decreased gradually from the HLA expression level approximately 2-log lower than normal expressors. Using sort-purified leukemia cells before and after HSCT, we applied the next-generation sequencing, and revealed that there were several marked downregulations of HLA-class II alleles which demonstrated consistently low expression from pre-transplantation. The marked downregulation of HLA-class II may lead to decreased antigen recognition ability of antigen-specific T cells and may be one of immune evasion mechanism associated with HLA-class II downregulation.

TCR function analysis using a novel system reveals the multiple unconventional tumor-reactive T cells in human breast cancer-infiltrating lymphocytes.

Tumor-infiltrating lymphocytes (TILs) are a potent source for obtaining tumor-reactive T cell receptors (TCRs). Although comprehensive methods to analyze the TCR repertoire in TILs have been reported, the evaluation system for TCR-reactivity to endogenously expressed antigen in tumor cells remains laborious and time consuming. Consequently, very limited numbers of TCRs in TILs have been analyzed for their reactivity to tumor cells. In this study, we developed an efficient evaluation system for TCR function designated c-FIT (comprehensive functional investigation of TCRs) to analyze TCR reactivity. The c-FIT system enabled us to analyze up to 90 TCRs for their reactivity to tumor cells by a single assay within a month. Using c-FIT, we analyzed 70 TCRs of CD8+ TILs derived from two breast cancer patients and obtained 23 TCRs that reacted to tumor cells. Surprisingly, although two TCRs were HLA class I-restricted, the remaining 21 TCRs were non-HLA-restricted. Thus, c-FIT can be applied for monitoring multiple conventional and unconventional antigen-specific killer T cells in TILs, leading to the development of new designs for more effective T-cell-based immunotherapies.

NKG2D defines tumor-reacting effector CD8+ T cells within tumor microenvironment.

For successful immunotherapy for cancer, it is important to understand the immunological status of tumor antigen-specific CD8+ T cells in the tumor microenvironment during tumor progression. In this study, we monitored the behavior of B16OVA-Luc cells in mice immunized with a model tumor antigen ovalbumin (OVA). Using bioluminescence imaging, we identified the time series of OVA-specific CD8+ T-cell responses during tumor progression: initial progression, immune control, and the escape phase. As a result of analyzing the status of tumor antigen-specific CD8+ cells in those 3 different phases, we found that the expression of NKG2D defines tumor-reacting effector CD8+ T cells. NKG2D may control the fate and TOX expression of tumor-reacting CD8+ T cells, considering that NKG2D blockade in OVA-vaccinated mice delayed the growth of the B16OVA-Luc2 tumor and increased the presence of tumor-infiltrating OVA-specific CD8+ T cells.

Rapid and efficient generation of T-cell receptor-like antibodies using chip-based single-cell analysis.

Generation of TCR-like monoclonal antibodies using conventional methods is markedly laborious and inefficient. We have proposed improvements of ISAAC (chip-based Ab-secreting cell [ASC] screening method), allows comprehensive analysis of ASCs at the single-cell level to obtain TCR-like antibodies; blocking procedure enables us to avoid the detection of non-TCR-like antibodies.

Artificial T Cell Adaptor Molecule-Transduced TCR-T Cells Demonstrated Improved Proliferation Only When Transduced in a Higher Intensity.

An artificial T cell adaptor molecule (ATAM) was generated to improve persistence of T cell receptor (TCR) gene-transduced T (TCR-T) cells compared to such persistence in a preceding study. ATAMs are gene-modified CD3ζ with the intracellular domain of 4-1BB inserted in the middle of CD3ζ. NY-ESO-1 TCR-T cells transduced with an ATAM with two separated virus vectors demonstrated superior proliferation upon antigen stimulation. To further develop clinically applicable ATAM-transduced TCR-T cells, we attempted to make a single virus vector to transduce the TCR and ATAM simultaneously. Because we failed to observe improved proliferation capacity upon stimulation after one virus vector (1vv) transduction, we compared TCR-T cells transduced with 1vv and two virus vector (2vv) methods to elucidate the reason. In Jurkat reporter cells, an ATAM transduced by the 2vv method demonstrated a higher intensity than by the 1vv method, and the ATAM intensity was associated with increased nuclear factor κB (NF-κB) signals upon stimulation. In ATAM-transduced primary T cells, a transduced ATAM by the 2vv method showed higher intensity and better proliferation. ATAM-transduced TCR-T cells demonstrated improved proliferation only when the ATAM was transduced at a higher intensity. To create a simpler transduction method, we need to develop a strategy to make a higher ATAM expression to prove the efficacy of ATAM transduction in TCR-T therapy.

Identification of a Protective Leishmania Antigen Dihydrolipoyl Dehydrogenase and Its Responding CD4+ T Cells at Clonal Level.

There is currently no clinically effective vaccine against cutaneous leishmaniasis because of poor understanding of the Ags that elicit protective CD4+ T cell immunity. In this study, we identified a naturally processed peptide (DLD63-79) that is derived from Leishmania dihydrolipoyl dehydrogenase (DLD) protein. DLD is conserved in all pathogenic Leishmania species, is expressed by both the promastigote and amastigote stages of the parasite, and elicits strong CD4+ T cell responses in mice infected with L. major We generated I-Ab-DLD63-79 tetramer and identified DLD-specific CD4+ T cells at clonal level. Following L. major infection, DLD63-79-specific CD4+ T cells massively expanded and produced effector cytokines (IFN-γ and TNF). This was followed by a gradual contraction, stable maintenance following lesion resolution, and display of memory (recall) response following secondary challenge. Vaccination with rDLD protein induced strong protection in mice against virulent L. major challenge. Identification of Ags that elicit protective immunity and their responding Ag-specific T cells are critical steps necessary for developing effective vaccines and vaccination strategies against infectious agents, including protozoan parasites.

Physiologic Target, Molecular Evolution, and Pathogenic Functions of a Monoclonal Anti-Citrullinated Protein Antibody Obtained From a Patient With Rheumatoid Arthritis.

OBJECTIVE: In plasma from a patient with rheumatoid arthritis (RA), we previously isolated a human monoclonal anti-citrullinated protein antibody (ACPA), CCP-Ab1, that recognizes various citrullinated antigens. In this study, we aimed to explore the physiologic target of CCP-Ab1 and the role of molecular evolution, through affinity maturation, of this ACPA in the onset and the exacerbation of RA. METHODS: The target protein of CCP-Ab1 was identified in the plasma of a patient with RA and purified under native conditions. Germline-reverted (GL-rev) CCP-Ab1 was generated, and its reactivity was compared to that of mature CCP-Ab1. The functions of CCP-Ab1 and GL-rev CCP-Ab1 in the onset or exacerbation of autoimmune arthritis were analyzed using autoimmune arthritis-prone SKG mice. RESULTS: CCP-Ab1 bound citrullinated fibrinogen under native conditions. In cultures with GL-rev CCP-Ab1, the binding affinity to citrullinated fibrinogen was drastically reduced (P < 0.05). The elements implicated in GL-rev CCP-Ab1 binding to a citrullinated peptide, cfc1-cyc, were almost identical to those implicated in CCP-Ab1 binding. In arthritis-prone SKG mice, CCP-Ab1, but not GL-rev CCP-Ab1, induced significant exacerbation of experimental arthritis (P < 0.05). Increased production of interleukin-6, both in the joint tissue and in the serum, was observed in SKG mice treated with CCP-Ab1 compared to those treated with GL-rev CCP-Ab1 (P < 0.05). Furthermore, the immune complex formed by CCP-Ab1 and fibrinogen was detected at higher concentrations in the synovial tissue of SKG mice administered CCP-Ab1 (P < 0.05 versus control treatment groups). CONCLUSION: These data show that germline-encoded CCP-Ab1, which binds weakly to citrullinated fibrinogen, undergoes hypermutation through the activation of naive B cells by citrullinated peptides/proteins, thereby stimulating high reactivity to citrullinated fibrinogen. These findings deepen our understanding of the role of molecular evolution of ACPAs in the onset and exacerbation of RA.