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The pathogenic role of B cells in immune thrombocytopenia (ITP) has justified the therapeutic use of anti-CD20 antibodies such as rituximab (RTX). However, 60% of ITP patients do not respond to RTX. To decipher the mechanisms implicated in the failure of RTX, and because the spleen plays a well-recognized role in ITP pathogenesis, 12 spleens from ITP patients who had been nonresponders to RTX therapy were compared with 11 spleens from RTX-untreated ITP patients and 9 controls. We here demonstrate that in RTX-nonresponder ITP patients, preferential Th1 and Tc1 T lymphocyte polarizations occur, associated with an increase in splenic effector memory CD8(+) T-cell frequency. Moreover, in the RTX- nonresponder patient group, the CD8(+) T-cell repertoire displays a restricted pattern. In the blood, the phenotype of CD8(+) T cells before and after RTX treatment is not modified in responders or nonresponders. Altogether, these results demonstrate for the first time an activation of splenic CD8(+) T cells in ITP patients who did not respond to RTX and suggest their involvement in platelet destruction in these patients.

Immune thrombocytopenia (ITP) is an autoimmune disease with a complex pathogenesis. As in many B cell–related autoimmune diseases, rituximab (RTX) has been shown to increase platelet counts in some ITP patients. From an immunologic standpoint, the mode of action of RTX and the reasons underlying its limited efficacy have yet to be elucidated. Because splenectomy is a cornerstone treatment of ITP, the immune effect of RTX on this major secondary lymphoid organ was investigated in 18 spleens removed from ITP patients who were treated or not with RTX. Spleens from ITP individuals had follicular hyperplasia consistent with secondary follicles. RTX therapy resulted in complete B-cell depletion in the blood and a significant reduction in splenic B cells, but these patients did not achieve remission. Moreover, whereas the percentage of circulating regulatory T cells (Tregs) was similar to that in controls, splenic Tregs were reduced in ITP patients. Interestingly, the ratio of proinflammatory Th1 cells to suppressive Tregs was increased in the spleens of patients who failed RTX therapy. These results indicate that although B cells are involved in ITP pathogenesis, RTX-induced total B-cell depletion is not correlated with its therapeutic effects, which suggests additional immune-mediated mechanisms of action of this drug.

Antiplatelet-antibody-producing B cells play a key role in immune thrombocytopenia (ITP) pathogenesis; however, little is known about T-cell dysregulations that support B-cell differentiation. During the past decade, T follicular helper cells (TFHs) have been characterized as the main T-cell subset within secondary lymphoid organs that promotes B-cell differentiation leading to antibody class-switch recombination and secretion. Herein, we characterized TFHs within the spleen of 8 controls and 13 ITP patients. We show that human splenic TFHs are the main producers of interleukin (IL)-21, express CD40 ligand (CD154), and are located within the germinal center of secondary follicles. Compared with controls, splenic TFH frequency is higher in ITP patients and correlates with germinal center and plasma cell percentages that are also increased. In vitro, IL-21 stimulation combined with an anti-CD40 agonist antibody led to the differentiation of splenic B cells into plasma cells and to the secretion of antiplatelet antibodies in ITP patients. Overall, these results point out the involvement of TFH in ITP pathophysiology and the potential interest of IL-21 and CD40 as therapeutic targets in ITP.

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 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. Disclosures Off Label Use: Off label use of Rituxan.

Abstract 623 Background: Different mechanisms are involved in the pathogenesis of immune thrombocytopenia (ITP). Platelet production is impaired as result of an immune response directed against megakaryocytes and an inappropriate level of thrombopoietin. Furthermore, platelets targeted by autoantibodies against their membrane glycoproteins are cleared by the reticuloendothelial system, mainly in the spleen. Thus, treatments targeting the autoimmune humoral response, notably rituximab (off-label use), have been proposed. However, the one-year response rate after rituximab (RTX) is not greater than 40% and the mechanisms involved in its failure remain to be defined. CD8 T cells which have been demonstrated to be involved in platelet destruction both in a mouse ITP model and in humans may play a part in the inefficiency of RTX. Purpose: To compare the splenic CD8 T cell response of ITP patients who have been refractory to RTX with untreated ITP patients Methods: The spleens of 23 primary ITP patients were assessed: 12 patients previously treated with RTX without improvement were examined as RTX refractory patients and 11 patients who have not received RTX were referred as untreated patients. Splenectomy was performed at a median of 7.1 months after RTX administration. Nine post-traumatic spleens were used as controls. Flow cytometry analysis was performed on CD8+ splenocytes to determine the expression of activation marker (HLA-DR), memory T cell markers (CD27, CD28), chemokine receptor (CCR7), adhesion molecule (CD62L) and intracellular cytotoxic proteins (granzyme B, perforin). Lineage commitment of T cells was determined after stimulation for 4 hours with PMA and ionomycin in presence of brefeldin-A, by intracellular staining of interferon-γ (Tc1, Th1), IL-4 (Tc2, Th2) and IL-17 (Tc17, Th17). Results are expressed by median and [interquartile range]. Statistical analysis was performed using non parametric tests (Kruskal-Wallis and Mann-Whitney) to compare the different groups. Results: After RTX infusion, B cells represented only 1.4% [0.3–3.4] in total splenic lymphocytes compared to about 35% in controls and untreated patients. No alteration in the CD8/CD4 ratio was observed after RTX. Nevertheless, expression of HLA-DR and granzyme B by splenic CD8 T cells was increased in RTX refractory patients when compared to untreated patients: 60% [50.6–68.9] vs. 41.8% [33.7–54.2] (p=0.02) and 50.3% [32.2–72.3] vs. 23.9% [14.4–27.7] (p=0.006), respectively. Upon stimulation, interferon-γ expression was significantly higher in CD3+CD8+ (68% [59.1–81.6]) but also in CD3+CD8− (32.5% [24.8–44]) in RTX refractory patients compared to untreated patients. The percentage of CCR7−CD62L− and CD27−CD28− among CD8+ T cells was increased in RTX refractory patients compared to untreated patients, respectively 85.4%[77.1–93.2] vs. 66.5%[56.2–73.2] (p=0.001) and 33.2% [23.3–67.1] vs. 13.4%[11.6–24.9] (p=0.04). Conclusions: Our results show that splenic CD8 T cells from RTX refractory patients express more HLA-DR, granzyme B and interferon-γ, whereas CCR7, CD62L, CD27 and CD28 are lower in comparison to untreated patients. This phenotype is consistent with effector T cells localizing in the red pulp as they lack CCR7 expression, and presumably playing a main role in splenic platelet destruction. Our results strongly support that platelet destruction is preferentially mediated by CD8 T cells rather than by the humoral response in some ITP patients, which may explain their unresponsiveness to rituximab. Disclosures: Off Label Use: Rituximab used during Immune Thrombocytopenia.