Abstract
The use of monoclonal anti-CD20 antibody (Rituximab) has greatly improved the treatment of B-cell mediated autoimmune diseases, albeit with variable outcomes. Our previous data in humans suggested that Rituximab induced paradoxically the settlement of splenic long-lived plasma cells (LLPC) in the context of 2 autoimmune cytopenia, immune thrombocytopenia and warm autoimmune hemolytic anemia (1) (2). The presence of splenic autoreactive LLPC explained the failure of Rituximab treatment. To investigate whether this mechanism could have a general relevance and decipher the cellular and molecular mechanism of this process, we used both non auto-immune and auto-immune mouse models.
We have taken advantage of the knock-in transgenic mouse model AID-CreERT2-EYFP, which allows the irreversible expression of EYFP in B cells engaged in a germinal center-dependent immune response after tamoxifen regimen, to follow plasma cells (PC) at different times of immunization by sheep red blood cells, and upon anti-CD20 regimen (clone 18B12, Biogen Idec), in the spleen and bone marrow (3).
By using a set of diagnostic genes that allowed us to distinguish short-lived and long-lived plasma cells, we compared the transcriptional program by multiplex PCR of EYFP+ B220- PC from controls and anti-CD20 treated mice, immunized and analyzed at the same time, corresponding to the nadir of B-cell depletion. While splenic PC of untreated mice displayed an intermediate profile between short-lived and long-lived plasma cells, splenic PC from anti-CD20 treated mice composed a homogeneous population that displayed a more mature program, similar to the one of natural long-lived bone marrow PC. The absolute number of splenic EYFP+ B220- did not change upon anti-CD20 treatment indicating that B-cell depletion promoted PC differentiation rather than a long-lived PC selection. We identified BAFF (B-cell activating factor) as a major player of this process. Indeed, as described in human spleens, we observed that BAFF level was increased in the supernatants of splenocytes after B-cell depletion. Above all, combination of anti-CD20 and anti-BAFF (clone 10F4, GSK) antibodies dramatically reduced the number of splenic EYFP+B220- LLPC (decrease >5 fold compared with anti-CD20 and control groups, P < 0.001). Targeting BAFF had no major impact on protective long-lived bone marrow PC as IgG1 level in the sera remained unchanged after combination therapy. We identified neutrophils as the main source of BAFF production in the spleen. Finally, CD4+ T-cells also appeared to play a key role in context of B-cell depletion for supporting plasma cell survival in the spleen as they appeared to closely interact with EYFP+ plasma cells by confocal microscopy. Moreover, their depletion (clones YTS 191.1 or GK 1.5, Bioxcell) in vivo induced a significant decrease in the number of splenic LLPCs (decrease > 2 fold compared with anti-CD20 group, P < 0.05). To assess whether B-cell depletion could also modify the splenic plasma cell program in an auto-immune context characterized by an ongoing immune response, we used NZB/NZW mice that spontaneously develop a disease closely resembling human systemic lupus. In line with our previous findings, anti-CD20 treatment also promoted the differentiation of LLPC in the spleen of the NZB/NZW model, while a treatment combining anti-CD20 with anti-BAFF induced a marked reduction in total PC numbers(decrease > 3 fold compared with anti-CD20 group, P < 0.05).
In conclusion, the process of PC maturation upon anti-CD20 treatment appeared to be a general mechanism, both in non auto-immune and auto-immune models. We identified BAFF and CD4+ T-cells as key factors in the splenic environment responsible for the emergence of such LLPC. Finally, our results suggest that interfering with the plasma cell survival niche with monoclonal anti-BAFF antibody at the time of B-cell depletion might greatly improve the response rate in B-cell mediated auto-immune cytopenia.
(1) Mahevas M, et al, Journal of Clinical Investigation , 2013
(2) Mahevas M, et al, Journal of Autoimmunity, 2015
(3) Dogan I, et al, Nature Immunology, 2009
Disclosures
No relevant conflicts of interest to declare.
Targeting autoreactive plasma cells in autoimmunity: a new treatment approach combining plasma cell and B cell depletion