Background/Purpose: The “Targeting Immune Responses for Prevention of RA” (TIP-RA) collaboration studies individuals at high risk for developing rheumatoid arthritis (RA) because of serum anti-citrullinated protein antibody (ACPA) positivity in absence of arthritis at baseline, and is focused on defining how they transition from at-risk to classifiable disease. One potential mechanism is through the development, expansion and class-switching of B cells expressing pathogenic ACPA. Previous studies identified blood plasmablasts expressing ACPA in pre-clinical and established RA. Individuals enrolled in the TIP-RA cohort were analyzed by antibody repertoire sequencing, serum ACPA profiling, and serum cytokine analysis to identify features associated with progression to RA.

Methods: Cytokine array and ACPA array profiling were performed on serum samples from CCP- controls (n=156), (n-78), RA converters (n=3), and seropositive early-RA (n = 32). RNA was extracted from PAXgene whole blood tubes from CCP- (n=170), CCP3+ at risk (n=85), RA converters (n=3) and early RA individuals (n=41). Purified RNA was used for paired-end sequencing of immunoglobulin heavy-chain and run on a HiSeq 2500. Bulk IgH data was de-multiplexed with adapter sequences trimmed using MIGEC. Overlapping paired end reads were merged using MiTools. MIXCR was used to align sequencing reads to germline VDJ genes of B cell receptors obtained from IMGT, and subsequently group clones. VDJ gene family usage was analyzed using Alakazam (Immcantation).

Results: ACPA array analysis demonstrated development of ACPA targeting citrullinated fibrinogen and histone 2B as well as native vimentin as associated with transition to clinical arthritis.  Cytokine profiling revealed increased levels of multiple pro-inflammatory cytokines and chemokines in the blood. Immunoglobulin heavy chain sequencing identified persistent clones (based on shared V and J gene usage, identical CDR3 lengths, and >60% amino acid homology) that underwent both expansion and class switching to IgA and IgG. Specifically, we identified clones encoding IgH-V3-23/IgHJ4 and IgHV1-2/IgHJ4 that switched and expanded from IgM pre-conversion to IgA and IgG isotypes post-conversion to RA (Figure 1).

Conclusion: Asymptomatic anti-CCP+ individuals that converted to RA exhibited increased levels of blood cytokines and ACPA. These individuals also exhibited expression, expansion and class-switching of B cell clones encoding IgH-V3-23/IgHJ4 and IgHV1-2/IgHJ4, antibody V genes previously identified as being expressed by RA plasmablasts and encoding anti-citrullinated protein antibodies (ACPA).  IgH-V3-23/IgHJ4 and IgHV1-2/IgHJ4 may encode ACPA that promote the transition to clinical RA, and further investigation is needed to fully define their citrullinated antigen targets and potential role in promoting the transition to clinical RA.

Figure 1 edit

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/antibody-repertoire-sequencing-antigen-array-analysis-and-cytokine-profiling-of-blood-from-individuals-at-high-risk-for-ra-reveals-candidate-immunoglobulin-v-genes-acpa-and-cytokines-that-may-prom/