BAFF and CD4 T-Cells Are Major Survival Factors for Splenic Plasma Cells in B Cell Depletion Context: Implications for Autoimmune Diseases
Authors: Thai Lan-Huong , Robbins Ailsa , Le Gallou Simon , Cagnard Nicolas , Weill Jean-Claude , Reynaud Claude-Agnès , Mahevas Matthieu 
Published: November 2019

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.