Immune reconstitution 20 years after treatment with alemtuzumab in a rheumatoid arthritis cohort: implications for lymphocyte depleting therapies

Patients and controls

Patients who were treated with alemtuzumab (CAMPATH-1H) for RA between 1991 and 1994 were identified from the study database. Cumulative alemtuzumab doses were documented for each individual; of note, these doses were comparatively lower than those seen with other more widely used biological therapies. Mortality data for this cohort were collected from 9 March 2006 (end date of our previous study) until 1 January 2015 from the National Health Service Central Registry. Morbidity information was collected on all living and consenting patients by either interview or review of clinical case notes. Special attention was given to episodes of severe infection, cancers and autoimmunity. Age and sex matched patients with RA of similar disease duration, who had not received alemtuzumab, were identified locally in Cambridge, UK. Research was performed in compliance with the Declaration of Helsinki and the International Conference on Harmonisation Good Clinical Practice. Ethical approval for the study was provided by Scotland A Research Ethics Committee (REC 10/MRE00/68).

Clinical parameters

Clinical and serological parameters were obtained from all patients attending interview. Markers of disease activity included erythrocyte sedimentation rate (ESR), C-reactive protein (CRP), tender joint count (TJC), swollen joint count (SJC), patient-reported visual analogue scale (VAS), disease activity score in 28 joints (DAS-28)-ESR and patient health assessment questionnaire (HAQ). Anti-cyclic citrullinated peptide (anti-CCP) antibody and rheumatoid factor (RF) IgM titres and serum immunoglobulins (IgG, IgA, IgM), serum electrophoresis and lymphocyte count were analysed in the clinical laboratories of Addenbrooke’s Hospital, Cambridge, according to routine clinical practice and national standards.

Vaccine responses

Subjects were offered vaccination with: 0.5 ml influenza vaccine (Pfizer Ltd, Sandwich, UK), a split, inactivated influenza vaccine containing antigens equivalent to A/California/7/2009 (H1N1) pdm09-derived strain (NYMC X-181), A/Victoria/361/2011 (H3N2)-derived strain (IVR-165) and B/Wisconsin/1/2010–like strain (NYMC BX-39) derived from B/Hubei- B/ Wujiagang/158/2009; 0.5 ml Pneumovax II (Sanofi Pasteur MSD Ltd, Maidenhead, UK), a vaccine containing 25 μg of each of the following 23 pneumococcal polysaccharide serotypes: 1, 2, 3, 4, 5, 6B, 7 F, 8, 9 N, 9 V, 10A, 11A, 12 F, 14, 15B, 17 F, 18C, 19 F, 19A, 20, 22 F, 23 F, 33 F; and 0.5 ml Revaxis (Sanofi Pasteur MSD Ltd, Maidenhead, UK) containing 2 IU purified diphtheria toxoid, 20 IU purified tetanus toxoid and inactivated poliomyelitis virus types 1-3.

Vaccine responses were assessed in serum obtained at baseline and 4 weeks post vaccination. Analyses were performed at the Respiratory Virus Unit, Health Protection Agency, London UK (influenza), the Vaccine Evaluation Unit, Public Health England, Manchester (pneumococcus, diphtheria and tetanus) and Public Heath England, Enteric Virus Unit, London (poliovirus). Satisfactory response to pneumococcal vaccine was defined as a doubling (or greater) in antibody concentrations to 6 or more of 12 pneumococcal serotypes (1, 3, 4, 5, 6B, 7 F, 9 V, 14, 19A, 19 F, 23 F and 18C). Tetanus and diphtheria seroprotection was achieved when IgG titres were >1.0 IU/ml. These were booster vaccines and some patients had residual seroprotection pre-administration. These patients were excluded from subsequent seroconversion analysis, which was defined as when vaccination achieved new seroprotection.

For poliovirus, neutralizing antibodies were quantified, with seroprotection with titres ≥1:8 and seroconversion following ≥4 fold increase in titres. For influenza, HAI assays were performed, with seroprotection when titres were >1:40 and seroconversion when post-vaccination titre increased by ≥3 fold. The seroconversion factor was the mean rise in geometric mean titres (GMT) post vaccination (recommended ≥2) and seroconversion rate was the percentage of vaccinees with an increase in haemagglutination inhibition (HAI) titre ≥4 fold following vaccination (recommended >30%). Some patients had annual influenza vaccines prior to recruitment into the study. These individuals were not vaccinated again but anti-influenza titres were measured in baseline serum. Any adverse effects were collected 4 weeks post vaccination.

Serum cytokines

At the initial visit cytokines (IL-15, IL-7, IFN-γ, IL-10, IL-12, IL-13, IL1-beta, IL-2, IL-4, IL-6, IL-8, TNF-alpha and granulocyte macrophage colony stimulating factor (GM-CSF)) were measured in serum by MSD technology (Meso Scale Discovery, MD, USA) as per established protocol.

Immunophenotyping

Peripheral blood lymphocytes were immunophenotyped by multicolour flow cytometry using the following antibodies: anti-CD3 Pacific Blue, anti-CD56 FITC, Anti-CD27 PE, anti-CD28 APC, anti-CD1d PE, anti-CD19 APC, anti-CD27 V450, anti-CD38 PerCP-Cy5.5 (all from BD Biosciences, San Jose, CA, USA) and anti-CD45RA PerCP-Cy5.5, anti-CD62L PE-Cy7, anti-CD5 PE-Cy7, anti-CD24 APC-eFluor 780 and anti-CD4 APC-eFluor 780 (from eBioscience, Inc. San Diego, CA, USA). Staining was performed on whole blood using BD FACS Lysing Solution (BD Biosciences) as per the manufacturer’s instructions. A minimum of 250,000 events were acquired for T cell panels and 500,000 events for B cell panels to ensure adequate capture of rare populations. Subsequent detailed analysis of lymphocyte sub-populations was performed on the gated lymphocyte population using FlowJo (Treestar, Inc., OR, USA). Absolute counts for the different lymphocyte populations were calculated per litre of blood, based on haematology laboratory reported total lymphocyte count.

Statistical analysis

Statistical analysis was performed using the Mann-Whitney U test, Wilcoxon signed rank test and linear regression using Prism 4.0 (GraphPad Software, Inc., La Jolla, CA, USA). P values <0.05 were considered significant.

Demographics

Briefly, the median age of the entire alemtuzumab cohort was 68 (range 45–81) years, 69% were female, the median disease duration was 31 years (range 23–43) and the median alemtuzumab cumulative dose was 62 mg (range 2–400 mg). Patients who underwent vaccination and peripheral blood analysis had a median age of 65 years (range 45–81), 77% of them were female, the median disease duration was 31 years (range 23–40), the median DAS-28 was 3.44 (range 1.38–5.33) and the median alemtuzumab cumulative dose was 60 mg (range 18–400 mg). The median age in the controls was 61.5 years (range 52–79), 87.5% were female, the median disease duration was 24.5 years (range 20–41) and the median DAS-28 was 3.90 (range 2.03–5.75). There was no significant difference in age, sex, disease duration, SJC, TJC or DAS-28 between the cohorts that underwent vaccination. The alemtuzumab cohort had a higher HAQ score (median 2.25 compared with 1.69 in controls), CRP (median 7 compared with <5 in controls) and ESR (median 34 compared with 11.5 in controls).

Nine of the alemtuzumab cohort were taking conventional synthetic disease-modifying anti-rheumatic drugs (csDMARDs) only, three were receiving biological DMARD (bDMARD) monotherapy and one was receiving concurrent bDMARDs and csDMARDs. One of these patients had recently received rituximab prior to being switched to their current therapy (abatacept) and was therefore excluded from B cell analysis. No further patients in either the control or alemtuzumab cohorts had previously received rituximab. In the control cohort four patients were taking csDMARDs only, three were receiving both csDMARDs and bDMARDs and one was receiving bDMARD monotherapy.

There was no significant difference in immunoglobulin titres (IgG, IgA and IgM) between the alemtuzumab and established RA control cohort. RF titres were performed in all patients, and anti-CCP antibody titres were obtained in eight patients taking alemtuzumab and five controls. The median RF titre inr the alemtuzumab cohort was 0 IU, (range 0–67 IU) and in the controls the median was 39 IU (range 0–275) (p = 0.06). The median anti-CCP value in alemtuzumab patients was 0.55 U/ml (range 0–340) and in controls it was 215 U/ml (range 1.2–340) (p = 0.039).

Mortality and morbidity

Morbidity in the 13 alemtuzumab patients (interview and case note review) focused on new medical diagnoses, particularly malignancy, episodes of severe infection and autoimmunity. Two patients had a new diagnosis of malignancy, both of which were skin cancers with a background of Bowen’s disease. Two had new autoimmune conditions - hyperthyroidism and coeliac disease. The remaining new diagnoses were osteoporosis, osteoarthritis, vitamin D deficiency, cardiac arrhythmia and ischaemic heart disease. There were no reports of severe infection.

Alemtuzumab patients have persistent circulating lymphocyte abnormalities

We previously reported that at a mean follow up of 12 years after treatment, our alemtuzumab cohort remained profoundly lymphopenic [1, 3]. While our current analysis (Table 6) suggests that compared with age and sex matched disease controls, overall lymphopenia had now resolved (p = 0.7001), specific differences remained. CD4⁺ and CD8⁺ central memory subsets remained low (p = 0.0360 and p = 0.0274 respectively) and we noted B cell lymphopenia (p = 0.0041), even after excluding a patient who had recently received rituximab. This predominantly reflects reduced naïve B cells (p = 0.0041) but a previously highlighted deficiency of CD5⁺CD19 B cells (p = 0.0175) persists. There is also a significant reduction in CD19⁺CD24^hiCD38^hi transitional B cells (Fig. 1a). However other previously noted abnormalities had normalised; in particular naïve and effector memory T cell populations and NK cells were not different to controls.

Cell count, median and range (×109/L)				Percentage of lymphocyte population		Previous cell count, median and range (×109/L)
Lymphocyte subpopulation	ALEM (n = 9*)	CON (n = 8)	P valuea	ALEM (n = 9*)	CON (n = 8)	ALEM cohort 2012 data (n = 20)	ALEM cohort 2001 data (n = 40)
Total lymphocytes	0.93 (0.41 − 3.1)	1.125 (0.34 − 2.38)	0.7001	n/a	n/a	1.15 (0.3 − 2.9)	n/a
CD4+ T cells	0.37 (0.13 − 0.94)	0.48 (0.13 − 0.65)	0.4406	30.5 (11.5 − 46.7)	34.9 (13.3 − 43.7)	0.55 (0.12 − 1.94)	0.000185
CD4+ naïve T cells	0.23 (0.01 − 0.61)	0.21 (0.07 − 0.29)	1	20.3 (1.23 − 30.5)	13.3 (4.15 − 20.6)	0.09 (0.01 − 0.65)	n/a
CD4+ total memory T cells	0.13 (0.11–0.34)	0.26 (0.06 − 0.56)	0.2359	12.52 (7.26 − 18.93)	18.5 (9.3 − 29.74)	0.37 (0.10 − 0.73)	n/a
CD4+ central memory T cells	0.08 (0.05 − 0.16)	0.2 (0.03 − 0.42)	0.0360	7.24 (4.74 − 13.28)	12.82 (6.73 − 22.45)	0.11 (0.02 − 0.07)	n/a
CD4+ effector memory T cells	0.05 (0.02–0.17)	0.06 (0.02 − 0.14)	0.9626	4.37 (1.81 − 7.58)	3.78 (2.42 − 7.56)	0.26 (0.07 − 0.55)	n/a
CD8+ T cells	0.10 (0.04 − 0.72)	0.11 (0.05 − 0.42)	0.9626	7.27 (4.95 − 31.9)	9.04 (3.96 − 22.7)	0.25 (0.02 − 0.78)	0.00009
CD8+ naïve T cells	0.05 (0.01 − 0.14)	0.02 (0.01 − 0.08)	0.2766	3.32 (0.46 − 7.27)	1.57 (0.64 − 5.94)	0.05 (0.001 − 0.17)	n/a
CD8+ total memory T cells	0.02 (0.07 − 0.13)	0.06 (0.01 − 0.19)	0.0592	1.82 (0.72 − 4.41)	3.97 (0.62 − 0.55)	0.12 (0.01 − 0.41)	n/a
CD8+ central memory T cells	0.01 (0.003 − 0.01)	0.02 (0.005 − 0.11)	0.0274	0.69 (0.26 − 1.33)	1.84 (0.34 − 6.01)	0.02 (0.003 − 0.12)	n/a
CD8+ effector memory T cells	0.01 (0.007–0.07)	0.03 (0.007 − 0.1)	0.1672	1.51 (0.45 − 3.2)	1.94 (0.31 − 5.72)	0.07 (0.01 − 0.37)	n/a
B cells	0.01 (0.01 − 0.05)	0.09 (0.02 − 0.2)	0.0041	1.7 (1.12 − 4.15)	8.96 (4.64 − 17.5)	0.08 (0.02 − 0.26)	0.000115
Naïve B cells	0.01 (0.001–0.02)	0.08 (0.7 − 0.14)	0.0041	1.18 (0.71 − 2.69)	5.47 (3.62 − 16.4)	0.06 (0.01 − 0.23)	n/a
Memory B cells	0.006 (0.002 − 0.02)	0.01 (0.008–0.03)	0.1282	0.56 (0.37 − 0.86)	1.19 (0.22 − 3.91)	0.02 (0.002 − 0.19)	n/a
CD5+ B cells	0.001 (0.001 − 0.005)	0.03 (0.001 − 0.08)	0.0175	0.28 (0.084 − 0.94)	2.22 (0.33 − 3.48)	0.005 (0.0009 − 0.03)	n/a
CD19+CD24hiCD38hi B cells	0.001 (0.00005 − 0.002)	0.009 (0.0008 − 0.038)	0.003	0.061 (0.014 − 0.42)	0.70 (0.51 − 2.31)	n/a	n/a
NK cells	0.1 (0.02 − 0.17)	0.07 (0.02 − 0.10)	0.4234	10.5 (3.3 − 19.2)	8.35 (4.84 − 13.3)	0.06 (0.01 − 0.2)	n/a
NK T cells	0.01 (0.0006–0.13)	0.009 (0.002 − 0.02)	0.6730	0.76 (0.12 − 4.34)	0.73 (0.52 − 1.27)	0.05 (0.003–0.27)	n/a

Median values and ranges are displayed for both absolute number and percentage of lymphocyte population. ^a P value for alemtuzumab versus established rheumatoid arthritis cell count absolute number); values in italics are significant. Gating strategies were as follows: CD4⁺ T cells: CD3⁺CD4⁺; CD4⁺ naïve T cells: CD3⁺CD4⁺CD45RA⁺CD62L⁺; CD4⁺ total memory T cells: CD3⁺CD4⁺CD45RA^-; CD4⁺ central memory T cells: CD3⁺CD4⁺CD45RA^-CD62L⁺; CD4⁺ effector memory T cells: CD3⁺CD4⁺CD45RA^-CD62L^-; CD8⁺ T cells: CD3⁺CD4^-, CD8⁺ naïve T cells: CD3⁺CD4^-CD45RA⁺CD62L⁺; CD8⁺ total memory T cells: CD3⁺CD4^-CD45RA^-; CD8⁺ central memory T cells: CD3⁺CD4^-CD45RA^-CD62L⁺; CD4⁺ effector memory T cells: CD3⁺CD4^-CD45RA^-CD62L^-; B cells: CD19⁺; Naïve B cells: CD19⁺CD27^-; Memory B cells: CD19⁺CD27⁺; CD5⁺ B cells: CD19⁺CD5⁺; CD19⁺CD24^hiCD38^hi B cells: CD19⁺CD24^hiCD38^hi; NK Cells: CD3^-CD56⁺, NK T Cells: CD3⁺CD56⁺. For comparison previous analyses (years 2001 and 2012) of lymphocyte subpopulations of the alemtuzumab cohort are included. *Single patient had rituximab within the preceding month and was excluded from B-cell analyses. NK natural killer. n/a not available

Seropositive patients with RA have persistently reduced RF titres following alemtuzumab therapy

We reviewed RF titres as documented in the medical notes at the time of alemtuzumab administration. Of our current alemtuzumab cohort only two patients had positive RF at the time of alemtuzumab therapy, but these values were now reduced compared to baseline values 20 years earlier (432 → 20 IU/ml and 57 → 25 IU/ml). We therefore looked at data from eight additional (deceased) RF-positive patients who had received alemtuzumab, and compared their baseline RF titres with titres published at their last follow up [2]. Whilst not being statistically significant (p = 0.084), there was a reduction in RF titres 12 years after alemtuzumab treatment (Fig. 1b).

Vaccine responses in patients receiving alemtuzumab and in controls

All patients who attended interview were offered vaccination, dependent on their vaccination status at the time. Four patients on alemtuzumab and three controls received influenza vaccine, the remainder having already received seasonal influenza vaccine prior to recruitment. For these latter patients we assessed the seroprotection rate only. Seven patients on alemtuzumab and six controls received pneumococcus vaccination, the others having been vaccinated within the last 5 years (n = 2) or having declined vaccination (n = 2). Six patients on alemtuzumab and four control patients received the combined diphtheria, tetanus and polio vaccine. There were no significant adverse events following any vaccination.

Cytokines

There were significantly higher serum IFN-γ (p < 0.0001) and IL-15 (p = 0.019) levels detected in the alemtuzumab cohort compared with controls (Fig. 2a). There was a significant inverse association between serum IL-15 and CD4⁺ total memory and central memory T cells (p = 0.034 and p = 0.037), see Fig. 2b and c. A similar trend for CD8⁺ total memory and central memory T cells was also seen albeit not significant (p > 0.05). There were no significant associations detected in the effector memory compartments.