Splenic and Circulating Human T Follicular Helper Cell Regulation By B Cell Depleting Therapy during Immune Thrombocytopenia

Introduction

T follicular helper cells, defined as CD3⁺CD4⁺CXCR5⁺ICOS⁺PD-1⁺, support B cell differentiation, proliferation and class switch recombination, through CD40/CD40L interaction and secretion of IL-21. Furthermore, studies in experimental models have brought evidence for the involvement of B cells in TFH development and survival. In mice, cognate B cells participate to TFH stimulation by presenting antigen but also through the ICOS-L/ICOS and CD40/CD40L signaling pathways. As a result, B cell depletion leads to a decrease in TFH within secondary lymphoid organs. B cell depleting therapy such as rituximab (RTX), are increasingly used to treat B cell-mediated auto-immune diseases, notably immune thrombocytopenia (ITP), despite its off-label use. Circulating TFH expansion and the production of CXCL13, a chemokine produced by TFH, are reversed following RTX during various autoimmune diseases such as type 1 diabetes and vasculitis. In humans, data concerning the effect RTX on TFH within lymphoid organs are scarce. As both RTX and splenectomy are part of the treatment of ITP, we aimed to assess the effect of B cell depletion on TFH in humans, both in the spleen and in blood.

Methods

Forty-three primary chronic ITP patients were studied. Blood analyses were performed on 22 patients and the spleens of 23 patients were analyzed. All patients gave an informed consent in accordance with the declaration of Helsinki. The study was approved by the institutional review board and the ethics committee. Flow cytometry (FCM), quantitative polymerase chain reaction (qPCR) and immunohistochemistry (IHC) were performed. Data are given by median [interquartile range]. Mann-Whitney, Wilcoxon matched-pairs test or Fisher's exact test were used as appropriate. P<.05 was considered significant.

Results

When compared to patients not treated with RTX, B cell depletion leads to a decrease in splenic TFH (2% [1.4-3.2] vs. .1% [.05-.23], P=.001). In line with this result, total CD4⁺ splenic T cells showed a decreased expression of CXCR5 (8.4 fold change (FC) [4.6-13] vs. 1.9 [1.5-5.3], P=.02), CXCL13 (214 FC [57-418] vs. 29 [12-80], P=.02) and IL-21 (13 [7-17] vs. 3.6 [2-5], P=.01) as measured by qPCR.

To address whether RTX could directly target TFH, total splenocytes of RTX-untreated patients were cultured with RTX in vitro. B cell percentage diminished by half after18 hours and represented only 10% of the pretreatment percentage at day 3. Conversely, TFH frequency progressively decreased overtime, representing around 60% of TFH before treatment at day 3, thus arguing against a direct effect of RTX on TFH.

Circulating TFH were measured in 16 patients, before and 3 months after RTX treatment. A slight but significant decrease in their frequency was observed after treatment, most particularly in responder patients (.1 [.04-.22] vs. .04 [.02-.08], P=.04). Of note, before treatment, circulating TFH percentage tend to be higher in responders when compared to non-responders, suggesting that TFH could represent a predictive response marker. To confirm this result, we measured CXCL13 in the serum of patients, before and after RTX treatment. A slight but significant decrease in CXCL13 after RTX treatment (111 [82-440] vs. 81 ng/mL [31-173], P=.01) was observed.

To determine predictive factors of response to RTX, age, sex, disease duration, serum CXCL13 levels and the percentages of TFH and B cell subsets were compared between responders and non-responders. None of these markers was associated with response to RTX. However, TFH percentages before treatment tended to be higher in patients who responded to treatment (.1 [.04-.22] vs. .04 [.03-.11]): 70% of responders displayed a TFH frequency above .04% before treatment whereas only 28% of non-responders did.

Conclusion

Our data support a direct role of B cells in the survival of TFH within secondary lymphoid organs, particularly in the spleen during ITP. Interestingly, circulating TFH frequency tends to be higher in patients who will respond to RTX compared to non-responders. If confirmed on larger studies, circulating TFH percentage could help to determine whose patients will benefit from RTX treatment.