Breaking tolerance: a story of two mechanisms and one disease

Gottardo Laboratory, Vaccine and Infectious Disease Division

Rheumatoid arthritis (RA) occurs when the body’s immune system attacks its own tissue and joints. This chronic inflammatory disorder causes pain and swelling in joints, predominantly in the hands and feet. Autoantibodies, antibodies that recognize proteins in one’s body, are the hallmark of RA and include either rheumatoid factor (RF) antibodies or anti-citrullinated protein antibodies (ACPA). These autoantibodies lead to systemic inflammation and morbidity, which is supported by the efficacy of B-cell-depleting therapy for RA treatment. In normal B cell development, there are checkpoints in which self-antigen recognizing cells are deleted or inactivated (called tolerance), thus RA autoantibody producing cells must break this tolerance in order to cause pathology. In a study published in Arthritis and Rheumatology, researchers from the Gottardo lab (Vaccine and Infectious Disease Division) and colleagues looked at how these cells break tolerance. They hypothesized that the two groups (RF and ACPA) do this through distinct mechanisms.

Schematic of methodology
Schematic of methodology for B cell identification and downstream analysis.

Using an antigen-specific tetramer approach, the group isolated RF and ACPA specific cells in RA patients. These cells were rare in RA patients (0.25%) and almost undetectable in healthy donors (0.14%). Both naïve and memory cells were found in both antigen populations, suggesting an ongoing humoral response. Single-cell-RNA-Seq was performed on sorted cells as well as on tetramer negative and healthy donor cells (see figure). Using profiles produced by Single-cell-RNA-Seq, the group developed a B cell transcriptomic classifier termed BCellNet in order to predict subtypes which could be compared to classic flow cytometry subsetting. This allowed for the identification of 159 genes that could be used as predictors. By also looking at paired BCR genes, the researchers found that ACPA B cells had an elevated CDR3 (variable region where antigen binding takes place) convergence frequency, suggesting the cells undergo extensive affinity maturation compared to controls and RF populations. In addition to analyzing the sequences present in the populations, the group expressed 42 antibodies to validate antigen binding and subset usage. They found that ACPA B cells had a greater chance of being class switched and accumulating somatic mutations. This was not the case in RF cells.

The group also assessed gene expression, noting differentially expressed genes between the RA B cells and tetramer negative cells. As predicted, ACPA B cell genes supported class switching, T cell help, and downregulation of CD72 which suppressed resting B cell differentiation. In contrast, RF-specific cells upregulated expression of genes that promote an IgM response and suppressed germinal center transcription factors, supporting the low class-switched phenotype and lack of mutation accumulation. Thus, ACPA cells are more associated with germinal center responses while RF cells are associated with innate responses and rapid recall. These data suggest that multiple defective tolerance mechanisms are at work in RA and are likely a mix of environmental and genetic factors. By better understanding the breaks in tolerance, the researchers hope to better inform therapeutic strategies to target the dysregulation and thus dampen the autoreactive B cells pathogenic effect.

Lu DR, McDavid AN, Kongpachith S, Lingampalli N, Glanville J, Ju C-H, Gottardo R, Robinson WH. 2018. T cell-dependent affinity maturation and innate immune pathways differentially drive autoreactive B cell responses in rheumatoid arthritis. Arthritis Rheumatol.

This work was funded by the National Institutes of Health.

Fred Hutch/UW Cancer Consortium faculty members Raphael Gottardo contributed to this research.