Immune monitoring in humans after manipulation by B cell depletion and immunization

The overall goal of this thesis was to study the influence of immunomodulatory drugs on the human immune system. The approach was bi-directional; ex vivo studies were performed after B cell depletion with rituximab treatment in patients with SLE and RA, and an in vivo assay was established to study effects of drugs on immune responsiveness.

To study immunological consequences of B cell depletion by rituximab in treatment resistant patients with SLE and RA we collected blood samples before and after treatment. Our analysis was focused on time points related to absence and return of B cells after depletion. In patients with SLE, changes of cellular phenotypes were analyzed by flow cytometry and changes in antibody levels in serum by ELISA. We observed an increase of activated T cells as well as an increase of regulatory T cells after B cell depletion. There was a selective decrease of antibodies in serum. IgM and IgE decreased rapidly after B cells depletion as well as the disease activity associated autoantibodies against dsDNA and C1q. BAFF and APRIL levels were measured by ELISA in serum of both SLE and RA patients. Levels of BAFF increased in all patients after rituximab therapy. In patients with SLE, the levels of APRIL decreased after B cell depletion, and in patients with RA we observed heterogeneous changes.

To set up an assay to measure individual immune responsiveness, we used immunizations as a tool to trigger an immune response. Healthy volunteers were immunized with four weeks interval with either influenza vaccine, three or four times (n=11) or with tetanus toxoid four times (n=7). Blood samples were collected before and at four occasions between days 4 to 10 after each immunization. Humoral immunity to the influenza vaccine and tetanus toxoid was measured by ELISPOT (circulating B cells) and ELISA (antibodies). Cellular reactivity to the influenza vaccine was measured by FASCIA (proliferation) and Bio-Plex suspension array system (cytokines). Comparing the response to influenza and tetanus, the first immunization with influenza vaccine resulted in ten times more B cells secreting antigen-specific antibodies compared to immunization with tetanus toxoid. The second and third influenza immunizations induced comparable B cell immune responses, while there was a very low or no response to tetanus toxoid. Antibody titers and T cell responses only increased significantly after the first immunization. Thus measuring B cell responses by ELISPOT to repeated immunizations with influenza vaccine provide a method to evaluate immune responsiveness. Tetanus toxoid was not a suitable antigen trigger for such studies.

In conclusion, we observed an effect on both the humoral and the cellular immune systems after rituximab-induced B cell depletion in patients with SLE. Further on, levels of BAFF increased after the therapy in patients with SLE and RA, while APRIL decreased in SLE only. We have set up an assay in which reproducible immune responses can be measured after immunization 2 and 3 with influenza vaccine. Such an assay allows us in the future to estimate the influence of immunomodulatory drugs on the immune responsiveness in single patients.

List of scientific papers

I. Vallerskog T, Gunnarsson I, Widhe M, Risselada A, Klareskog L, van Vollenhoven R, Malmstrom V, Trollmo C. (2007). "Treatment with rituximab affects both the cellular and the humoral arm of the immune system in patients with SLE." Clin Immunol 122(1): 62-74
https://pubmed.ncbi.nlm.nih.gov/17046329

II. Vallerskog T, Heimburger M, Gunnarsson I, Zhou W, Wahren-Herlenius M, Trollmo C, Malmstrom V. (2006). "Differential effects on BAFF and APRIL levels in rituximab-treated patients with systemic lupus erythematosus and rheumatoid arthritis." Arthritis Res Ther 8(6): R167
https://pubmed.ncbi.nlm.nih.gov/17092341

III. Vallerskog T, Gaines H, Feldman A, Culbert E, Klareskog L, Malmström V, Trollmo C. (2007). "Serial re-challenge with influenza vaccine as a tool to study individual immune responses." (Manuscript)

IV. Vallerskog T, Klareskog L, Culbert E, Trollmo C. (2007). "Multiple immunizations with tetanus toxoid are not suitable to measure reproducible immune responsiveness." (Manuscript)