ORIGINAL RESEARCH published: 08 October 2021 doi: 10.3389/fimmu.2021.760546 Role of B Cell Profile for Predicting Secondary Autoimmunity in Patients Treated With Alemtuzumab Paulette Esperanza Walo-Delgado 1, Enric Monreal 2, Silvia Medina 1, Ester Quintana 3, Susana Sainz de la Maza 2, José Ignacio Fernández-Velasco 1, Paloma Lapuente 1, Manuel Comabella 4, Lluis Ramió-Torrentà 3, Xavier Montalban 4, Luciana Midaglia 4, Noelia Villarrubia 1, Angela Carrasco-Sayalero 1, Eulalia Rodrı́guez-Martı́n 1, Ernesto Roldán 1, José Meca-Lallana 5, Roberto Alvarez-Lafuente 6, Jaime Masjuan 2, Edited by: Lucienne Costa-Frossard 2 and Luisa Maria Villar 1* Jorge Correale, Fundación Para la Lucha Contra las 1 Department of Immunology, Ramón y Cajal University Hospital, Instituto Ramón y Cajal de Investigación Sanitaria (IRYCIS), Enfermedades Neurológicas de la Red Española de Esclerosis Múltiple (REEM), Madrid, Spain, 2 Department of Neurology, Ramón y Cajal University Hospital, Infancia (FLENI), Argentina IRYCIS, Red Española de Esclerosis Múltiple (REEM), Madrid, Spain, 3 Neuroimmunology and Multiple Sclerosis Unit, Reviewed by: Neurology Department, Neurodegeneration and Neuroinflammation Research Group, Biomedical Research Institute (IDIBGI), Hans-Peter Hartung, Red Española de Esclerosis Múltiple (REEM), Girona, Spain, 4 Servei de Neurologia-Neuroimmunologia, Centre d’Esclerosi Heinrich Heine University of Múltiple de Catalunya (Cemcat), Institut de Recerca Vall d’Hebron (VHIR), Hospital Universitari Vall d’Hebron, Universitat Düsseldorf, Germany Autònoma de Barcelona, Red Española de Esclerosis Múltiple (REEM), Barcelona, Spain, 5 Department of Neurology, Virgen Wendy Gilmore, de la Arrixaca University Hospital, Murcia, Spain, 6 Grupo de Investigación de Factores Ambientales en Enfermedades University of Southern California, Degenerativas, Instituto de Investigación Sanitaria del Hospital Clı´nico San Carlos, Hospital Clı´nico San Carlos, Red Española United States de Esclerosis Múltiple (REEM), Madrid, Spain *Correspondence: Luisa Maria Villar Objective: To explore if baseline blood lymphocyte profile could identify relapsing luisamaria.villar@salud.madrid.org remitting multiple sclerosis (RRMS) patients at higher risk of developing secondary Specialty section: autoimmune adverse events (AIAEs) after alemtuzumab treatment. This article was submitted to Multiple Sclerosis Methods: Multicenter prospective study including 57 RRMS patients treated with and Neuroimmunology, alemtuzumab followed for 3.25 [3.5-4.21] years, (median [interquartile range]). Blood a section of the journal samples were collected at baseline, and leukocyte subsets determined by flow cytometry. Frontiers in Immunology We had additional samples one year after the first cycle of alemtuzumab treatment in Received: 18 August 2021 Accepted: 21 September 2021 39 cases. Published: 08 October 2021 Results: Twenty-two patients (38.6%) developed AIAEs during follow-up. They had Citation: Walo-Delgado PE, Monreal E, higher B-cell percentages at baseline (p=0.0014), being differences mainly due to Medina S, Qiuntana E, plasmablasts/plasma cells (PB/PC, p=0.0011). Those with no AIAEs had higher Sainz de la Maza S, percentages of CD4+ T cells (p=0.013), mainly due to terminally differentiated (TD) Fernández-Velasco JI, Lapuente P, Comabella M, Ramió-Torrentà L, (p=0.034) and effector memory (EM) (p=0.031) phenotypes. AIAEs- patients also Montalban X, Midaglia L, showed higher values of TNF-alpha-producing CD8+ T cells (p=0.029). The percentage Villarrubia N, Carrasco-Sayalero A, Rodrı´guez-Martı´n E, Roldán E, of PB/PC was the best variable to differentiate both groups of patients. Baseline values Meca-Lallana J, Alvarez-Lafuente R, >0.10% closely associated with higher AIAE risk (Odds ratio [OR]: 5.91, 95% CI: 1.83- Masjuan J, Costa-Frossard L 19.10, p=0.004). When excluding the 12 patients with natalizumab, which decreases and Villar LM (2021) Role of B Cell Profile for Predicting blood PB/PC percentages, being the last treatment before alemtuzumab, baseline PB/PC Secondary Autoimmunity in Patients >0.1% even predicted more accurately the risk of AIAEs (OR: 11.67, 95% CI: 2.62-51.89, Treated With Alemtuzumab. Front. Immunol. 12:760546. p=0.0007). The AIAEs+ group continued having high percentages of PB/PC after a year of doi: 10.3389/fimmu.2021.760546 alemtuzumab treatment (p=0.0058). Frontiers in Immunology | www.frontiersin.org 1 October 2021 | Volume 12 | Article 760546 Walo-Delgado et al. Predicting Secondary Autoimmunity to Alemtuzumab Conclusions: A PB/PC percentage <0.1% at baseline identifies MS patients at low risk of secondary autoimmunity during alemtuzumab treatment. Keywords: multiple sclerosis, side effects, autoimmunity, disease modifying treatments, alemtuzumab, biomarkers, B cells INTRODUCTION We aimed to explore if the blood lymphocyte profile before alemtuzumab treatment initiation and after a year of treatment Alemtuzumab (Lemtrada®; Sanofi, Paris, France) is a humanized could identify MS patients at high risk of AIAEs. monoclonal antibody approved for the treatment of relapsing- remitting multiple sclerosis (RRMS). It is administered as two annual courses and proved to be efficacious for patients with MATERIALS AND METHODS highly inflammatory disease, resulting in prolonged remission periods (1). Study Design Alemtuzumab targets CD52, a molecule expressed at high levels This was a multicenter prospective longitudinal exploratory by T and B lymphocytes (2). It causes a selective depletion of these study including 59 patients diagnosed with RRMS according to cells via antibody and complement dependent cytotoxicity and 2010 McDonald criteria (11) who were initiating treatment with apoptosis (3). T and B-cell repopulation begins within weeks, alemtuzumab. Patients were consecutively recruited in five with a distinctive pattern; B cells undergo faster repopulation, Spanish hospitals. while T cells remain depleted longer (4). Repopulation associates with increases of regulatory CD56bright natural killer (NK) cells (2), Patients and regulatory T cells and with a reduction of the production of Patients were followed for 3.55 [3.25-4.21] years (median [25%- pro-inflammatory cytokines (5). This can explain the long duration 75% interquartile range (IQR)]) after alemtuzumab treatment of clinical effects in the absence of continuous treatment. As a initiation. All patients received two courses of alemtuzumab (12 counterpart, frequent adverse events associated with alemtuzumab mg/d IV on five consecutive days at baseline and 12 mg/day IV include infusion-associated reactions, infections, and mainly on three consecutive days 12 month later). Five patients received autoimmune adverse events (AIAEs) (6, 7). The most frequent an additional course for presenting new clinical relapses after the AIAEs are those involving the thyroid gland, observed in about 38% second course. Clinical and demographic data of patients at of patients treated with alemtuzumab (8). Other secondary AIAEs study inclusion are described in Table 1. initially described in the clinical trials included immune thrombocytopenia and nephropathies. In the post-marketing Patient Follow-Up setting, new ones have also been reported (9, 10). These side Patients were evaluated every three months to assess the effects, although mild in most cases, limit the use of this drug. appearance of new AIAEs, the occurrence of new relapses and Therefore, reliable biomarkers predicting patient individual risk for the Expanded Disability Status Scale (EDSS) score. MRI scans developing autoimmunity, and hence, guide patient selection for were performed at baseline and yearly after treatment initiation. this particular therapy, would be of great clinical importance. Analytical tests, including blood count, serum analyses of renal TABLE 1 | Clinical and demographic features of patients. Variable Patients (n = 59) Sex (F/M) 39/20 Age at disease onset [years] – median (IQR) 28 (23-33.6) Age at treatment onset [years] – median (IQR) 37 (30-44) Disease duration [years] – median (IQR) 7.0 (3.0-11.5) EDSS score at treatment onset – median (IQR) 2.75 (1.5-3.63) Annualized relapse rate in the two previous years – median (IQR) 0.83 (0.63-1.75) Previous disease modifying treatments (Yes/No) 46/13 Number of previous treatments - median (range) 2 (0-6) Last treatment (Number of patients) None 14 IFN-beta/GA/Teriflunomide/DMF 19 Fingolimod 13 Natalizumab 13 Time of follow-up since alemtuzumab treatment onset [years] – median (IQR) 3.55 (3.25-4.21) Alemtuzumab curses (Number of patients) Two courses 54 Three courses 5 n, number of patients; F, Female; M, male; EDSS, Expanded Disability Status Scale; IQR, 25%-75% interquartile range; IFN, Interferon; GA, Glatiramer acetate; DMF, Dimethyl fumarate. Frontiers in Immunology | www.frontiersin.org 2 October 2021 | Volume 12 | Article 760546 Walo-Delgado et al. Predicting Secondary Autoimmunity to Alemtuzumab and thyroid function were performed monthly, while the tests were used to explore continuous variables. ROC curves were presence of anti-glomerular basement membrane and anti- used to establish cut-off values and Fisher’s exact tests to analyze thyroid antibodies (including anti-thyroid peroxidase, anti- categorical variables. p values below 0.05 were considered thyroglobulin and anti-thyroid-stimulating hormone receptor) as significant. was tested every three months. The missing data were not imputed since the proportion was Patients were trained to recognize symptoms suggestive of less than 10% in all variables and we can assume that they were AIAEs early, and promptly inform the treating physician. AIAEs missing completely at random. were defined as the appearance of any autoimmune events during follow-up, with special attention paid to thyroid-associated events, Standard Protocol Approvals, immune thrombocytopenia and autoimmune nephropathy. Registrations, and Patient Consents The study was approved by the ethics committee of the Ramó n y Sample Collection Cajal University Hospital. All patients signed a written informed Blood samples were collected before initiating alemtuzumab and consent form before inclusion. in 39 cases, one year after. Peripheral blood mononuclear cells (PBMCs) were obtained from heparinized whole blood (20 mL) by Ficoll density gradient centrifugation (Abbott) and RESULTS cryopreserved in aliquots of 5x10 (6) cells until studied. Aditionally, fresh blood was collected in an EDTA tube to Fifty-nine RRMS patients initiating alemtuzumab were included. explore total cell counts in a Coulter counter. To avoid bias due to previous autoimmune diseases, one patient was excluded for presenting previous autoimmune pathology. Monoclonal Antibodies One patient was lost during follow-up. Fifty-seven RRMS Leukocyte subpopulations were assessed using the following patients were finally studied. Twenty-two (38.6%) experienced monoclonal antibodies: CD3-PerCP, CD3-BV421, CD4-APC- AIAEs during follow-up. Twenty-one of them presented H7, CD8-FITC, CD8-APC-H7, CD14-APC-H7, CD19-PE-Cy7, autoimmune thyroid alterations, and the remaining one CD20-FITC, CD24-FITC, CD25-PE-Cy7, CD27-PE, CD38- developed autoimmune thrombocytopenia. No significant APC-H7, CD45-V500, CD45RO-APC, CD56-APC, CD127- differences were found in clinical and demographic BV421 and CCR7-PE. Intracellular cytokines were stained characteristics at baseline or during follow-up between patients using the next panel: Interleukin (IL)-10-PE, Interferon (IFN)- showing AIAEs (AIAEs+) and patients who did not develop gamma-FITC, Granulocyte-Macrophage Colony Stimulating AIAEs (AIAEs-) (Table 2). Factor (GM-CSF)-PE, Tumor necrosis factor (TNF)-alpha- PerCP-Cy5.5 and IL-17A -APC. All monoclonal antibodies Flow Cytometry Analyses were purchased from BD Biosciences, except IL-17A-APC, We studied leukocyte profiles at baseline. Results are shown in from R&D Systems. Tables 3 and 4. When exploring innate immunity, represented by monocytes and NK cells, no significant differences in cellular Flow Cytometry Analyses percentages were observed between AIAEs+ and AIAEs- To study surface antigens, cells were stained with the respective patients. We did not find differences either in regulatory T and monoclonal antibodies during 30 min at 4°C in the dark, washed NK as well as in IL-10 producing B and T cells. By contrast, there with saline and analyzed in a FACSCanto II flow cytometer (BD was an increase in the percentages of CD4+ T cells (p=0.012) in Biosciences), as described before (12). AIAEs- group, mainly due to effector memory (EM, p=0.031) For intracytoplasmic cytokine detection, cells were stimulated and terminally differentiated (TD, p=0.034) subsets. In addition, with phorbol-12-myristate-13-acetate (PMA, Merck) and AIAEs-patients showed an augment of CD8 T cells producing Ionomycin (Merck), in presence of Brefeldin A (GolgiPlug, BD TNF-alpha (p=0.03). However, the highest differences between Biosciences) and Monensin (GolgiStop, BD Biosciences), and AIAEs+ and AIAEs- patients was found in B cells (Figure 2). then incubated 4 h at 37°C in 5% CO2 atmosphere. For the Those acquiring AIAEs showed higher percentages of B cells analysis of IL-10 producing B cells, PBMC were incubated prior (p=0.0014), being differences mainly due to plasmablasts/plasma to stimulation with CpG oligonucleotide (InvivoGen) for 20 h. cells (PB/PC, p=0.0011) and to a lesser extent to naïve cells After incubation, surface markers were stained, then the cellular (p=0.013). No significant differences were found in other B cell membrane permeabilized with Cytofix/Cytoperm Kit (BD subsets or in cytokine producing B cells. Biosciences), and incubated with intracellular antibodies, We measured absolute cell counts in all lymphocyte subsets following the same protocol previously described (12). associated with autoimmunity. The relative augment of PB/PC Cells were analyzed using FACSDiva Software V.8.0 (BD present in AIAEs+ patients was also found when exploring total Biosciences). A minimum amount of 5x10 (4) events were cell numbers (p=0.024). No significant differences in absolute cell acquired. Gating strategy is defined in Figure 1. numbers were found in any other cell subset (Supplementary Table 1). Statistical Analysis Finally, we explored if changes observed at baseline remained Data were analyzed using the Prism 8.0 (GraphPad Software) after a year of alemtuzumab treatment. We studied PBMCs and Stata 14 (StataCorp) statistical packages. Mann–Whitney U obtained before administering the second cycle of the drug in Frontiers in Immunology | www.frontiersin.org 3 October 2021 | Volume 12 | Article 760546 Walo-Delgado et al. Predicting Secondary Autoimmunity to Alemtuzumab FIGURE 1 | Gating strategy showing representative images for flow cytometry analysis. Peripheral blood mononuclear cells (PBMC) were first gated for excluding debris and apoptotic cells (gate P1) and duplets (gate P2). Lymphocytes were recognized by CD45 staining and low SSC (gate Lymph). Monocytes were recognized as CD14++ cells with intermediate SSC (gate Mon). B cells were detected by the expression of CD19 (gate B lymph) and subdivided by CD27 staining into memory cells: CD27+ (gate Mem B), naïve: CD27- (gate Naïve), plasmablasts/plasma cells: CD27hi, CD38hi (gate PB/PC), and transitional B cells: CD27-, CD38hi, CD24hi, (gate Trans B). Total CD4+ T cells were identified as CD3+ CD8- T cells (gate CD4), and CD8+ T cells were gated as CD3+ CD8++ cells (gate CD8). CD4 and CD8 T cells were classified as naïve: CCR7+ CD45RO− (gate Naïve), central memory: CCR7+ CD45RO+ (gate CM), effector memory: CCR7− CD45RO+ (gate EM), and terminally differentiated: CCR7− CD45RO− (gate TD). CD4+ T regulator cells were identified as CD25hi and CD127-/low (gate Treg). NK cells were identified based on their CD56 expression intensity and CD3 co-expression into CD56+ CD3- (gate NK), CD56+ CD3+ (gate NKT), and CD56++ CD3- (gate CD56++). FSC-A= Forward Scatter-Area; FSC-H= Forward Scatter-Height; SSC-A= Side Scatter-Area. 39 of the patients studied at baseline (13 of AIAEs+ group and 26 plasmablasts (13, 14). We found that five of the eight AIAEs of the AIAEs- one). Results are shown in Table 5. PB/PC patients who presented a percentages of PB/PC at baseline <0,1%, remained high in AIAEs+ patients a year after administering received natalizumab as the last treatment prior to alemtuzumab. the first alemtuzumab cycle (p= 0.0058). Representative In fact, PB/PC percentages did not differ in patients previously examples are shown in Figure 3. No differences were observed treated with natalizumab who showed or not AIAEs [AIAEs+ in other cell subsets at this point. median (IQR)=0.07 (0.06-0.09); AIAEs- median (IQR)=0.05 Our data suggest that the percentages of PB/PC at baseline (0.05-0.07), p=ns]. We explored the effect of the previous could be a good tool to identify patients with low risk of treatment with natalizumab in other leukocyte subsets, and did autoimmunity when treated with alemtuzumab. To address this not find any significant changes from our results with the issue we performed a ROC curve analysis [area under the curve whole cohort. (AUC)=0.75, p=0.001; sensitivity=77.1, specificity=63.6] and As a consequence, we performed a new ROC curve analysis established a cut-off value of 0.10. Fourteen out of 22 patients on the PB/PC percentages excluding patients who received presenting AIAEs but only eight of the 35 AIAEs- patients had a natalizumab as the last treatment before alemtuzumab. percentage of PB/PC higher than 0.10 [Odds ratio (OR) 5.91, 95% This improved the results (AUC 0.80, p=0.0007; CI 1.83-19.10, p=0.004, Figure 4A]. To improve the sensitivity of sensitivity=75.0, specificity=82.4). The best cut-off value was the assay, we next explored if the heterogeneity found in AIAEs again 0.1. Results of the new analysis clearly improved those group could be related to previous treatment, since it has been obtained with the whole cohort (OR=11.67 95% CI 2.62-51.89, described that natalizumab diminishes the concentration of blood p=0.0007, Figure 4B). Frontiers in Immunology | www.frontiersin.org 4 October 2021 | Volume 12 | Article 760546 Walo-Delgado et al. Predicting Secondary Autoimmunity to Alemtuzumab TABLE 2 | Clinical and demographic characteristics of patients classified according to the appearance of autoimmune adverse events. Variable AIAEs+ (n = 22) AIAEs- (n = 35) p value Sex (F/M). 16/6 21/14 ns Age at disease onset [years] – median (IQR). 26.5 (23-33) 30 (25-36) ns Age at alemtuzumab treatment onset [years] – median (IQR) 36 (31-39) 37 (30-47) ns Disease duration [years] – median (IQR) 7.0 (1.0-12.0) 7 (3-11.2) ns EDSS score at treatment onset – median (IQR). 2.00 (1.5-4.0) 3.0 (2.0-3.5) ns Relapse rate in the two previous years – median (IQR) 0.86 (0.51-2.75) 0.78 (0.64-1.61) ns Time of follow-up since alemtuzumab treatment onset [years] – median (IQR) 3.78 (3.41-4.59) 3.47 (3.18-3.99) ns Number of previous treatments - median (range) 2.5 (0-5) 2.0 (0-6) ns Prior treatment (Number of patients) ns None (n=14) 7 7 IFN-beta/GA/Teriflunomide/DMF (n=19) 3 16 Fingolimod (n=12) 7 5 Natalizumab (n=12) 5 7 n, number of patients; AIAEs, Autoimmune adverse events (n=22, consisting of 21 patients with autoimmune thyroiditis, and one patient with autoimmune thrombocytopenia); F, Female; M, male; IQR=25%-75% interquartile range; ns, not significant; EDSS, Expanded Disability Status Scale; IFN, Interferon-beta; GA, Glatiramer acetate; DMF, Dimethyl fumarate. p-values were obtained using Mann–Whitney U tests for continuous variables and Fisher’s exact tests for categorical variables. DISCUSSION On the other hand, high serum levels of IL-21, a cytokine promoting T and B-cell differentiation and antibody production, The search for biomarkers predicting the appearance of new AIAEs were proposed as predictors of secondary autoimmunity after after alemtuzumab treatment is of the utmost importance, since alemtuzumab treatment (17–19). However ulterior analyses these side effects limit the use of this drug, which has proven to be limited its use as a biomarker since there is some overlapping very efficacious in patients with highly active MS (1). between patients who develop AAIEs and those who not, and The data obtained in the pivotal phase III trials suggested that detection tests currently available could not distinguish the pattern of T- and B-cell depletion and repopulation following accurately between both groups of patients (20). Likewise, pre- alemtuzumab could influence the appearance of secondary treatment presence of serum anti-thyroid autoantibodies autoimmunity (15). However, repopulation kinetics of the associated with increased risk for clinical onset of thyroid peripheral lymphocyte subsets are comparable between autoimmunity after alemtuzumab treatment (21), but this patients with or without AIAEs (16). biomarker would only benefit a small group of patients. TABLE 3 | Percentages of peripheral blood mononuclear cells at baseline. AIAEs+, n = 22 median (IQR) AIAEs-, n = 35 median (IQR) p value Innate immune cells NK CD56dim 15.0 (9.58-20.3) 12.2 (8.51-17.7) ns NKT 2.67 (1.58-3.99) 3.30 (1.84-5.80) ns Monocytes 11.3 (8.44-20.1) 13.2 (8.33-21.8) ns Regulatory cells Regulatory T cells 2.08 (1.66–3.34) 2.33 (1.50-3.40) ns NK CD56 bright 0.97 (0.76-1.35) 0.97 (0.55-1.13) ns CD4 IL-10+ 0.17 (0.10-0.31) 0.14 (0.10-0.20) ns CD8 IL-10+ 0.11 (0.04-0.20) 0.11 (0.06-0.26) ns CD19 IL-10+ 0.19 (0.09-0.30) 0.23 (0.10-0.35) ns T cells Total T cells 47.4 (39.7-56.3) 53.3 (48.8-63.8) 0.020 Total CD4+ T cells 32.4 (26.4-39.9) 41.8 (34.9-45.3) 0.013 CD4+ Naïve 17.9 (12.7-25.6) 18.9 (10.3-28.5) ns CD4+ CM 8.56 (7.0-10.2) 9.42 (7.76-12.5) ns CD4+ EM 3.83 (3.01-6.64) 7.21 (3.40-11.2) 0.031 CD4+ TD 1.05 (0.70-1.54) 1.60 (0.93-2.20) 0.034 Total CD8+ T cells 10.4 (8.14-14.3) 9.19 (6.75-17.1) ns CD8+ Naïve 5.10 (2.93-7.62) 3.27 (1.80-5.60) ns CD8+ CM 0.44 (0.30-0.81) 0.31 (0.24-0.65) ns CD8+ EM 2.22 (1.58-3.35) 1.93 (1.15-3.10) ns CD8+ TD 2.59 (1.45-3.22) 2.40 (0.96-6.40) ns B cells Total B cells 7.95 (5.10-13.1) 5.79 (3.90-6.66) 0.0014 Transitional B cells 0.09 (0.03-0.20) 0.04 (0.03-0.10) ns Naïve 4.10 (2.44-9.94) 2.46 (1.65-3.90) 0.013 Memory 2.11 (1.30-3.21) 1.60 (1.25-2.97) ns Plasmablasts/Plasma cells 0.13 (0.08-0.23) 0.07 (0.05-0.10) 0.0011 AIAEs, Autoimmune adverse events; IQR, 25%-75% interquartile range; ns, not significant; NK, Natural Killer cells; NKT, Natural Killer T cells; CM, central memory; EM, effector memory; TD, terminally differentiated. Percentages were calculated over total peripheral blood mononuclear cells. p-values were obtained using Mann–Whitney U test. Frontiers in Immunology | www.frontiersin.org 5 October 2021 | Volume 12 | Article 760546 Walo-Delgado et al. Predicting Secondary Autoimmunity to Alemtuzumab TABLE 4 | Percentages of T and B cells producing pro-inflammatory cytokines at baseline. AIAEs+, n = 22 median (IQR) AIAEs-, n = 35 median (IQR) p value CD4+ T cells CD4+ TNF-alpha+ 13.2 (9.57-17.1) 16.9 (12.9-22.1) ns CD4+ GM-CSF+ 1.30 (0.79-1.66) 1.37 (0.97-2.47) ns CD4+ IFN-gamma+ 2.60 (2.08-4.50) 3.88 (2.27-5.50) ns CD4+ IL-17+ 0.17 (0.09-0.31) 0.12 (0.09-0.20) ns CD8+ T cells CD8+ TNF-alpha+ 3.39 (1.98-5.36) 5.42 (3.15-8.02) 0.029 CD8+ GM-CSF+ 0.59 (0.30-1.01) 0.74 (0.50-1.07) ns CD8+ IFN-gamma+ 2.48 (1.47-4.05) 3.11 (1.78-4.86) ns CD8+ IL-17+ 0.06 (0.03-0.10) 0.09 (0.04-0.10) ns B cells TNF-alpha+ 1.70 (1.10-2.99) 2.42 (1.33-5.54) ns GM-CSF+ 0.32 (0.18-0.58) 0.30 (0.20-0.40) ns AIAEs, Autoimmune adverse events; IQR, 25%-75% interquartile range; ns, not significant; IL, Interleukin; TNF, Tumor necrosis factor; GM-CSF, Granulocyte-Macrophage Colony Stimulating Factor; IFN, Interferon. Percentages were calculated over total peripheral blood mononuclear cells. p-values were obtained using Mann–Whitney U test. MS is a heterogeneous disease with different immunological differentiated and effector memory cells, and also lower values mechanisms playing a main role in the inflammatory response of CD8+ T cells producing TNF-alpha. (22). Our group focused on studying the immune cell profile The highest differences were found on B cells, particularly in before alemtuzumab treatment, assessing both the absolute counts plasmablasts/plasma cells (PB/PC), which were significantly higher and percentages of the peripheral blood mononuclear cells. at baseline on patients who later developed autoimmunity. These Patients who later developed autoimmunity presented some effector cells, which produce immunoglobulins at a high rate, were subtle differences at baseline on T cells, including lower implicated in autoimmune responses in MS, where some blood percentages of CD4+ T cells, particularly of terminally plasmablasts are autoreactive and recognize brain gray matter A B C D E F FIGURE 2 | Percentages of cell subsets classified according to the presence or absence of adverse autoimmune events (AIAEs). Percentages of total B cells (A, D), naïve B cells (B, E) and plasmablasts (C, F) at baseline (A–C) and at 12 months of alemtuzumab treatment onset (D–F) in 22 patients who developed AIAEs (+) and 35 who did not (-). All percentages are relative to total peripheral blood mononuclear cells. Median values and 25%-75% interquartile range are shown in plots. p- value were obtained using Mann–Whitney U test. ns, not significant. Frontiers in Immunology | www.frontiersin.org 6 October 2021 | Volume 12 | Article 760546 Walo-Delgado et al. Predicting Secondary Autoimmunity to Alemtuzumab TABLE 5 | Percentages of cell subsets one year after first cycle of treatment in patients with secondary autoimmunity who had associations with baseline subsets. AIAEs+, n = 13 median (IQR) AIAEs-, n = 26 median (IQR) p value T cells CD4 T cells 22.0 (17.5-27.4) 18.9 (15.0-21.3) ns CD4 EM 2.55 (2.00-3.67) 4.15 (2.08-6.08) ns CD4 TD 0.70 (0.39-1.19) 0.90 (0.60-1.45) ns CD8+ TNF-alpha 2.30 (1.92-7.64) 2.63 (1.15-3.17) ns B cells Total B cells 13.4 (5.06-18.2) 10.3 (6.00-14.7) ns Naïve 9.25 (3.14-16.8) 8.29 (4.37-12.6) ns Plasmablasts/plasma cells 0.17 (0.09-0.28) 0.09 (0.05-0.10) 0.0058 AIAEs, Autoimmune adverse events; EM, effector memory; IQR, 25%-75% interquartile range; ns, not significant; TD, terminally differentiated. Percentages were calculated over total peripheral blood mononuclear cells. p-values were obtained using Mann–Whitney U test. antigens, what produces and perpetuates an autoimmune response poor expression of CD52, the target molecule of alemtuzumab, in directed toward neurons (23). plasmablasts (25) and mostly in antibody secreting cells (26), which When exploring the ability of these cells to predict secondary may minimize the effect of this drug in these B cell subsets. autoimmunity at baseline, we found that this was not the case in Naïve B cell repopulation is completed about six months after patients treated previously with natalizumab, who mostly presented administrating alemtuzumab, while that of CD8+ T cells lasts for a lower percentage of plasmablasts. This agrees with previous data a year and reconstitution of memory B cells and of CD4+ T cells showing that natalizumab and no other disease modifying drugs and may last more than two years (6, 15). Additionally, the ratios diminish the percentages of plasmablasts in blood (14, 15, 24). between activated and regulatory cells seem to decrease in We also observed that those patients who presented at baseline a patients treated with alemtuzumab during reconstitution (27). lymphocyte profile associated with a high probability of developing This may contribute to the long quality responses reached after AIAE, one year after the first cycle of treatment continued the administration of this drug and to the delay in the onset of presenting high percentages of PB/PC. This may be due to the secondary autoimmunity (6–8). However, when repopulation is A B C D FIGURE 3 | Representative dot plots showing plasmablasts/plasma cells percentages from a AIAEs+ and AIAEs- patient at baseline and after one year of treatment. Percentages of plasmablasts/plasma cells from a AIAEs+ patient at baseline (A) and after a one year of treatment (B) and from a AIAEs- patient at baseline (C) and after one year of treatment (D). Percentages are relative to total peripheral blood mononuclear cells. Frontiers in Immunology | www.frontiersin.org 7 October 2021 | Volume 12 | Article 760546 Walo-Delgado et al. Predicting Secondary Autoimmunity to Alemtuzumab patients receiving Natalizumab as the last drug before Alemtuzumab initiation. A DATA AVAILABILITY STATEMENT The original contributions presented in the study are included in the article/Supplementary Material. Further inquiries can be directed to the corresponding author. ETHICS STATEMENT The studies involving human participants were reviewed and approved by Ethics committee of the Ramó n y Cajal University B Hospital. The patients/participants provided their written informed consent to participate in this study. AUTHOR CONTRIBUTIONS PEWD played a major role in performing experiments, acquisition and analysis of data and drafted the manuscript; EM contributed to statistical analyses and data adquisition. SM, JIFV, PL and NV collected samples and performed flow cytometry experiments; ER and ER-M supervised flow cytometry studies. ACS performed auto-antibody quantification; SSM, MC, EQ, LR-T, XM, LM, JM-L, RA-L, LCF, and JM visited MS patients, contributed to patient FIGURE 4 | Number of patients with (+) and without (-) autoimmune adverse inclusion and collected clinical data; LMV designed and events (AIAEs) classified according to plasmablasts/plasma cells percentages supervised the study and revised the draft. All authors revised before alemtuzumab treatment initiation. (A) Analysis performed in all patients included in the study (n=57); (B) Analysis performed excluding patients who the manuscript and approved the final version. received natalizumab as the last treatment before alemtuzumab (n=45). AIAES, autoimmune adverse events; PB/PC, plasmablasts/plasma cells; N, number; OR, odds ratio; CI, confidence interval. ROC curves were used to establish cut-off values. p-value was obtained using Fisher’s exact tests. FUNDING This work was supported by grants from Red Española de Esclerosis Mú ltiple (REEM) (RD16/0015/0001; RD16/0015/ completed, the proportion of regulatory T, B and NK cells 0004; RD16/0015/0006; RD16/0015/0013) and PI18/00572 decreases in patients treated with alemtuzumab (27). The integrated in the Plan Estatal I+D+I and co-funded by ISCIII- coincidence of lower values of regulatory cells with high Subdirecció n General de Evaluació n and Fondo Europeo de numbers of plasmablasts may increase the risk of developing Desarrollo Regional (FEDER, “Una manera de hacer Europa”). other antibody-mediated autoimmune diseases, as those mainly occurring after alemtuzumab treatment (8). This was observed in our series, where baseline percentages of plasmablasts > 0.10 clearly predicted a higher risk of secondary autoimmunity after ACKNOWLEDGMENTS alemtuzumab. These data suggest that the immunological mechanisms predominating in individual patients not only Authors acknowledge AI Pé rez Macı́as and S Ortega for their influence the response to treatment in MS (28) but can have a excellent technical support. clear influence on the side effects. Although these findings should be validated in larger cohorts with longer follow-up periods, our results suggest that to assess SUPPLEMENTARY MATERIAL baseline percentages of plasmablasts could be a useful tool to identify MS patients at high risk of autoimmunity upon The Supplementary Material for this article can be found online alemtuzumab treatment, with the limitation of previous at: https://www.frontiersin.org/articles/10.3389/fimmu.2021. treatment, since PB/PC percentages should not be assessed in 760546/full#supplementary-material Frontiers in Immunology | www.frontiersin.org 8 October 2021 | Volume 12 | Article 760546 Walo-Delgado et al. Predicting Secondary Autoimmunity to Alemtuzumab REFERENCES 17. Costelloe L, Jones J, Coles A. Secondary Autoimmune Diseases Following Alemtuzumab Therapy for Multiple Sclerosis. Expert Rev Neurother (2012) 12 1. Cohen JA, Coles AJ, Arnold DL, Confavreux C, Fox EJ, Hartung HP, et al. 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MC has Scler (2018) 24(10):1317–27. doi: 10.1177/1352458517717088 received compensation for consulting services and speaking honoraria from 13. Cuculiza Henriksen A, Ammitzbøll C, Petersen ER, McWilliam O, Sellebjerg Bayer Schering Pharma, Merk Serono, Biogen-Idec, Teva Pharmaceuticals, F, von Essen MR, et al. Natalizumab Differentially Affects Plasmablasts and B Sanofi-Aventis, Genzyme, and Novartis. LR-T has received speaking honoraria Cells in Multiple Sclerosis. Mult Scler Relat Disord (2021) 52:102987. and travel expenses for scientific meetings and has participated in advisory boards doi: 10.1016/j.msard.2021.102987 in the past years with Bayer Schering Pharma, Biogen, EMD Merck Serono, Sanofi 14. Kemmerer CL, Pernpeintner V, Ruschil C, Abdelhak A, Scholl M, Ziemann U, Genzyme, Novartis, Sanofi-Aventis, Teva Phramaceuticals, Almirall and Roche. et al. Differential Effects of Disease Modifying Drugs on Peripheral Blood B XM has received speaking honoraria and travel expenses for participation in Cell Subsets: A Cross Sectional Study in Multiple Sclerosis Patients Treated scientific meetings, has been a steering committee member of clinical trials or With Interferon-b, Glatiramer Acetate, Dimethyl Fumarate, Fingolimod or participated in advisory boards of clinical trials in the past years with Actelion, Natalizumab. PloS One (2020) 15(7):e0235449. doi: 10.1371/ Alexion, Bayer, Biogen, Celgene, EMD Serono, Genzyme, Immunic, Medday, journal.pone.0235449 Merck KGaA, Darmstadt Germany, Mylan, Nervgen, Novartis, Roche, Sanofi- 15. Baker D, Herrod SS, Alvarez-Gonzalez C, Giovannoni G, Schmierer K. Genzyme, Teva MSIF and NMSS. LM has received travel funding from Genzyme, Interpreting Lymphocyte Reconstitution Data From the Pivotal Phase 3 Roche, Biogen Idec and Novartis, and personal fee for lectures from Roche. JM-L Trials of Alemtuzumab. JAMA Neurol (2017) 74(8):961–9. doi: 10.1001/ received grants and consulting or speaking fees from Almirall, Biogen, Celgene, jamaneurol.2017.0676 Genzyme, Merck, Novartis, Roche and Teva. RA-L reports grants and personal 16. Wiendl H, Carraro M, Comi G, Izquierdo G, Kim HJ, Sharrack B, et al. fees from Merck Serono, personal fees and non-financial support from Biogen Lymphocyte Pharmacodynamics Are Not Associated With Autoimmunity or IDEC, grants, personal fees and non-financial support from Novartis Efficacy After Alemtuzumab. Neurol Neuroimmunol Neuroinflamm (2019) 7 Pharmaceuticals S.A., grants and personal fees from Genzyme, non-financial (1):e635. doi: 10.1212/NXI.0000000000000635 support from TEVA Pharma, S.L. LC-F received speaker fees, travel support, and/ Frontiers in Immunology | www.frontiersin.org 9 October 2021 | Volume 12 | Article 760546 Walo-Delgado et al. Predicting Secondary Autoimmunity to Alemtuzumab or served on advisory boards by Biogen, Sanofi, Merck, Bayer, Novartis, Roche, this article, or claim that may be made by its manufacturer, is not guaranteed or Teva, Celgene, Ipsen, Biopas, Almirall. LV received research grants, travel support endorsed by the publisher. or honoraria for speaking engagements from Biogen, Merck, Novartis, Roche, Sanofi-Genzyme and Bristol-Myers. Copyright © 2021 Walo-Delgado, Monreal, Medina, Quintana, Sainz de la Maza, Fernań dez-Velasco, Lapuente, Comabella, Ramio-́ Torrentà, Montalban, Midaglia, The remaining authors declare that the research was conducted in the absence of ́ Villarrubia, Carrasco-Sayalero, Rodrı guez-Martı ́ Rolda n n, ́ , Meca-Lallana, any commercial or financial relationships that could be construed as a potential Alvarez-Lafuente, Masjuan, Costa-Frossard and Villar. 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