CD4 cell count and CD4/CD8 ratio increase during rituximab maintenance in granulomatosis with polyangiitis patients

Introduction

Granulomatosis with polyangiitis (GPA) is an antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) resulting in a necrotizing small to medium vessels vasculitis and a necrotizing granulomatous inflammation involving predominantly the upper and lower respiratory tract and the kidneys. GPA is the result of a complex interplay between the humoral and cellular immunity involving proteinase 3-ANCA (PR3-ANCA), neutrophils, endothelial cells, B and T cells (Kallenberg, 2011).

T cells are important in GPA as persistent activation of T cells through aberrant expression of costimulatory molecules favours the expansion of effector memory T cells and the formation of granuloma (Wilde et al., 2010). Changes in T cells in GPA patients occur during remission when circulating memory T cells are increased and naive T CD4+ cells are decreased (Abdulahad et al., 2006). Also, T cell-targeted therapies such as abatacept, alemtuzumab and gusperimus are alternative treatments to B cell depletion with rituximab (RTX) in GPA (Furuta & Jayne, 2014).

However, a subgroup of GPA patients has low CD4 cell count and inverted CD4/CD8 ratio irrespective of disease activity and the use of immunosuppressive drugs, possibly due to the recruitment of T cells into the inflamed tissue (Berden et al., 2009). It is not clear whether it is the decrease of CD4 cell count (Marinaki et al., 2006), the expansion of CD8 cell count or a combination of both (Ikeda et al., 1992; Iking-Konert et al., 2008) that is responsible for the inversion of the ratio.

RTX is a chimeric human-mouse monoclonal antibody directed against CD20 that induces rapid and sustained depletion of premature and mature B cells through antibody-dependent, complement-mediated cellular cytotoxicity and apoptosis (Leandro & de la Torre, 2009). RTX reduces auto-antibody producing precursor plasma cells, inhibits B cell interaction with auto reactive T cells and decreases the level of soluble factors secreted by B cells (Leandro & de la Torre, 2009). RTX is approved for the treatment of rheumatoid arthritis (RA) and AAV and is used off label in a large number of autoimmune conditions (Edwards & Cambridge, 2006). In AAV, RTX is used to induce (Jones et al., 2010; Stone et al., 2010) and to maintain (Guillevin et al., 2014; Smith et al., 2012; Cartin-Ceba et al., 2012; Besada, Koldingsnes & Nossent, 2013) remission through iterative infusions. Relevant side effects of RTX include late onset neutropenia (Besada, Koldingsnes & Nossent, 2012), hypogammaglobulinemia (Makatsori et al., 2014; Besada, Koldingsnes & Nossent, 2014) and an increased risk of infections (Besada, Koldingsnes & Nossent, 2013; Ram et al., 2009; Sailler et al., 2008; Gottenberg et al., 2010). In two studies, 27–44% of all severe infections during RTX treatment were either viral or fungal (Besada, Koldingsnes & Nossent, 2013; Makatsori et al., 2014), possibly related to T cell immunodeficiency.

Our study investigated the course of CD4 cell count, CD4/CD8 ratio and serum levels of total immunoglobulin (Ig) in GPA patients receiving long-term RTX treatment.

Methods

The Vasculitis Registry in Northern Norway is an observational prospective registry collecting data on disease presentation and course from patients with an established diagnosis of primary vasculitis. All patients gave informed written consent at inclusion in accordance with the declaration of Helsinki.

A total of 35 patients from the Vasculitis Registry in Northern Norway with an established diagnosis of GPA were included in the study. The study did not require formal ethical approval and conformed to the standards currently applied in Norway. Patients’ characteristics at baseline have been previously described (Besada, Koldingsnes & Nossent, 2013).

At RTX initiation, the patients (median age of 50 (14–79), 54% males) had median disease duration of 55 months (1–270). However, 86% of the patients were ANCA positive; all except one were PR3-ANCA positive. Renal, lung and orbital or/and subglottic involvement was present in 60, 63 and 57% of the patients. Median Birmingham Vasculitis Activity Score (BVAS) was 9 (0–22) at baseline. The main indication for RTX was disease relapse (80%), new disease onset (17%) and maintenance therapy (3%). Patients had received a median cumulative cyclophosphamide (CYC) dose of 14 g (0–250) prior to RTX and all had normal total Ig levels (> 6 g/l) prior to RTX.

Patients received 2 g RTX at induction (1 g twice in a fortnight) with co-administration of methylprednisolone 125 mg, paracetamol 1,000 mg and either cetirizine 10 mg or polaramine 4 mg. RTX was usually combined with a median oral daily prednisolone dose (ODPD) of 20 mg (0–60) and an immunosuppressive drug in 91% of the patients. Overall, patients received a median cumulative RTX dose of 4 g (2–6) during the first 24 months following its initiation. Respectively 49 and 40% received long-term maintenance with the 2 g annually regimen (1 g twice in a fortnight per year) or the 1 g biannually regimen (1 g every six months), while 11% received only induction.

Clinical parameters such as gender, age, erythrocyte sedimentation rate (ESR, nr: < 20 in men and < 28 mm/h in women), C-reactive protein (CRP, nr < 5 mg/l), creatinine (nr: 60–105 in men and 45–90 μmol/l in women), ANCA titers (nr < 10 IU/ml), the ODPD and the cumulative dose of CYC were recorded at baseline. Serum levels of total Ig and lymphocytes subsets were measured at RTX initiation and at 3, 6, 12, 18 and 24 months in 35, 24, 31, 34, 31 and 34 patients, respectively. CD4 and CD8 cell counts were determined by flow cytometry in blood specimens. Normal adult CD4 and CD8 cell counts for our laboratory ranged from 0.3–1.4 and 0.2–0.9 × 10⁹/l, respectively. Low CD4 cell count was defined as CD4 < 0.3 × 10⁹/l and inverted CD4/CD8 ratio when ratio < 1. Hypogammaglobulinemia was defined as serum total Ig < 6 g/l. Severe infections were defined as infections necessitating intravenous antibiotic treatment and/or hospitalisation.

Data were analysed with SPSS version 20.0 (SPSS Ltd, Chicago, IL, USA). The results are expressed in percentage for categorical variable and in median (range) for continuous variables, unless specified otherwise. Fisher’s exact test, Wilcoxon signed rank test and Man-Whitney U test were used as appropriate. Significant predictors of inversion of the CD4/CD8 ratio at 24 months determined during univariable analysis were entered in a multivariable binary logistic regression model with backward selection (p < 0.05 to enter and p < 0.10 to stay). Missing data were excluded from the statistical analyses. p-values < 0.05 were considered significant.

Results

CD4 cell count and ratio

Both the CD4 cell count and the CD4/CD8 ratio decreased initially after RTX administration, and thereafter increased gradually (Fig. 1). The CD4 cell count seemed to decrease between baseline and three months (0.36–0.28 × 10⁹/l, p = 0.166), while the ratio decreased significantly from 1.16 to 0.99 (p = 0.011). At 24 months, CD4 cell count and ratio had both significantly increased from baseline: 0.45 × 10⁹/l (p = 0.039) and 1.38 (p = 0.031), respectively.

However, while the proportion of patients with a low CD4 cell count decreased from 43% at baseline to 18% at 24 months, the proportion of patients with inverted ratio remained stable around 40% throughout the study period (Fig. 2).

Discussion

In GPA patients on long term RTX treatment, overall CD4 cell count and CD4/CD8 ratio initially decreased in the first three months and thereafter gradually increased independent of the ODPD at baseline, the CYC cumulative dose and the maintenance regimen. However in older patients, the inverted ratio at baseline remained unchanged after two years and was associated with lower levels of total Ig.

Conclusions

Our study suggests that the early decrease in CD4 after induction with RTX seemed dependent of the baseline CD4 count. Overall, CD4 cell count and CD4/CD8 ratio in GPA patients increased over time during RTX with no discernible impact of other immunosuppressive therapy. Increase in CD4 was not always followed by an increase in CD4/CD8 ratio, especially for a subgroup of older patients whom CD4/CD8 ratio remained inverted. Inverted CD4/CD8 ratio associated with lower Ig levels during maintenance with RTX.

Our study suggest that GPA patients with inverted CD4/CD8 ratio seemed to have a more profound B cell-depleting effect of RTX and a relative increase in CD8+ lymphocytes. CD4/CD8 ratio could be an important marker of a patient’s net status of immunodeficiency during RTX, since inverted CD4/CD8 ratio is a common surrogate marker of immunosenescence of impaired responses to vaccination and infections due to the loss of repertoire diversity (Blackman & Woodland, 2011).