Molecular Immunoprofiling the T Cell Repertoire after Rituximab Administration Reveals Frequent Oligoclonality Albeit with Different Patterns Depending on the Clinical Context
Authors: Papalexandri Apostolia , Iskas Michail , Stalika Evangelia , Karypidou Maria , Tachynopoulou Barbara , Kotta Konstantina , Zerva Panagiota , Kanellis George , Sakellari Ioanna , Stavroyianni Niki , Anagnostopoulos Achilles , Papadaki Theodora , Stamatopoulos Kostas 
Published: October 2019

Abstract
In patients with B lymphoid malignancies, depletion of B cells by Rituximab, an anti-CD20 humanized monoclonal antibody, can induce changes in the subset composition, activation and function of T cells. The spectrum of resultant immune-mediated sequelae encompasses organ-specific manifestations (e.g. pneumonitis, gastritis) as well as Rituximab-associated late onset neutropenia (R-LON). Although the pathogenesis of these clinical syndromes is not fully elucidated, evidence suggests that at least a fraction of cases may develop in a setting of expanded cytotoxic T cell populations with a large granular lymphocyte (LGL) phenotype (CD3+CD8+CD57+). Similar cytotoxic T cell expansions can be observed after Rituximab administration in other clinical settings e.g. allogeneic transplantation (allo-HCT), where selective restriction of the T-cell receptor (TR) gene repertoire is probably driven by multifactorial mechanisms. Here, we sought to obtain more insight into this phenomenon by molecular immunoprofiling of the TR gene repertoire in two groups of patients who received Rituximab: (i) Group A: patients (n=10) with chronic lymphocytic leukemia (CLL) treated with fludarabine-cyclophosphamide-rituximab (FCR); and, Group B: patients (n=14) who underwent allo-HCT for hematologic malignancies and received Rituximab either as pre-emptive treatment for EBV reactivation or against refractory cGvHD. Each patient included in the study received a mean of 6 cycles of Rituximab (range, 1-14). TR repertoire analysis was performed 11-88 (median, 36) and 5-24 months (median, 5) after the first Rituximab administration in Group A and Group B, respectively. TR beta gene rearrangements were PCR amplified on genomic DNA isolated from bone marrow samples using the BIOMED2 protocol and subjected to classic subcloning/Sanger sequencing. Sequence data were analyzed using the IMGT/V-QUEST tool. A total of 579 productive TRBV-TRBD-TRBJ rearrangements were analysed, 291 for Group A, 288 for Group B (6-91/case, median=20). Among the 46 TRBV functional genes identified, 3 accounted for >25% of cases in both groups: (i) TRBV27 (13% in both Groups A and B); ii) TRBV19-1 (13% in group A, 7% in group B); and, (iii) TRBV6-1 (7% and 6%, respectively). Clusters of identical (>=2) rearrangements corresponding to clonotypes were identified in all patients. Oligoclonality with immunodominant clonotypes (>12% of the repertoire) accounting for over 30% of the analyzed sequences was more frequent in Group A (7/10 cases) versus Group B (5/10 cases); however, larger clonotype expansions were seen in group B. Longitudinal analysis was performed in 3 patients with oligoclonality, 1 from group A and 2 from Group B: in the Group A patient, immunodominant clonotypes disappeared, while both patients in Group B retained the oligoclonal repertoire. Lymphocyte subpopulation analysis by flow cytometry was performed in 6 patients of each group. T-LGL proliferations (defined as CD4/CD8 abnormal ratio and CD3+CD8+CD57+ >30%) were found more often in Group B (3/6 cases in Group A versus 6/6 in Group B). They were related to oligoclonal TR gene repertoire in Group A (3/3) but not in Group B patients (3/6 cases). However, true expansions could be considered only in group B patients, since CD8+ lymphocytes >1.0*109/l were seen in all 6 Group B versus only 1/6 group A cases. Self-limiting R-LON was observed in 8 patients (4 in each group), but no association of oligoclonality to R-LON could be found. In conclusion, we report frequent development of oligoclonal T cell populations after Rituximab treatment in two different clinical contexts. The Groups analyzed differed with respect to the extent of oligoclonality, suggesting that the precise clinical setting determines the amplitude of TR repertoire skewing after Ritximab. Sustained oligoclonal cytotoxic expansions were recognised more often among allo-HCT patients, presenting with a highly restricted TR gene repertoire and likely reflecting strong antigenic stimulation by viruses and/or cGVHD aggravated by T cell imbalances induced by Rituximab.

Disclosures
Stamatopoulos: Gilead: Consultancy, Honoraria, Research Funding; Abbvie: Honoraria, Other: Travel expenses; Novartis: Honoraria, Research Funding; Janssen: Honoraria, Other: Travel expenses, Research Funding.