TYPE Brief Research Report PUBLISHED 29 July 2022 DOI 10.3389/fimmu.2022.918887 Impaired antigen-specific B-cell OPEN ACCESS responses after Influenza EDITED BY Adam Wheatley, The University of Melbourne, Australia vaccination in kidney REVIEWED BY Danika Hill, transplant recipients receiving Central Clinical School, Faculty of Medicine, Nursing and Health Sciences, Monash University (MU), co-stimulation blockade Australia Rowena A Bull, with Belatacept University of, New South Wales, Australia *CORRESPONDENCE Vincent Pernin 1†, Maria Meneghini 2,3†, Alba Torija 3, Oriol Bestard obestard@vhebron.net Thomas Jouve 3,4, Arnaud Del Bello 5, Iván Sanz-Muñoz 6, † These authors have contributed Jose Maria Eiros 7, Laura Donadeu 3, Carol Polo 8, equally to this work and share Francisco Morandeira 9, Sergio Navarro 9, Cristina Masuet 10, first authorship ‡ Alexandre Favà 8, Moglie LeQuintrec 1, Nassim Kamar 4, These authors share senior authorship Elena Crespo 3‡ and Oriol Bestard 2,3*‡ SPECIALTY SECTION This article was submitted to 1 Kidney Transplant Unit, Nephrology Department. Montpellier University Hospital, Montpellier, Vaccines and Molecular Therapeutics, France, 2 Kidney transplant Unit. Nephrology Department. Vall d’Hebron Hospital Universitari, Vall a section of the journal d’Hebron Institut de Recerca (VHIR), Universitat Autònoma de Barcelona, Barcelona, Spain, Frontiers in Immunology 3 Laboratoryof Nephrology and Transplantation. Vall d’Hebron Institut de Recerca (VHIR), Universitat RECEIVED 12 April 2022 Autònoma de Barcelona, Barcelona, Spain, 4 Kidney transplant Unit, Nephrology Department. Grenoble University Hospital, Grenoble, France, 5 Centro Nacional de Gripe de Valladolid, ACCEPTED 04 July 2022 Universidad de Valladolid, Valladolid, Spain, 6 Centro Nacional de Gripe, Valladolid Universidad de PUBLISHED 29 July 2022 Valladolid, Valladolid, Spain, 7 Department of Microbiology and Parasitology, Rio Hortega University CITATION Hospital, University of Valladolid, Valladolid, Spain, 8 Kidney Transplant Unit, Nephrology Pernin V, Meneghini M, Torija A, Department, Bellvitge University Hospital, Barcelona, Spain, 9 Immunology Department, Bellvitge Jouve T, Del Bello A, Sanz-Muñoz I, University Hospital, Barcelona, Spain, 10 Department of Preventive Medicine, Bellvitge University Eiros JM, Donadeu L, Polo C, Hospital, Barcelona, Spain Morandeira F, Navarro S, Masuet C, Favà A, LeQuintrec M, Kamar N, Crespo E and Bestard O (2022) Impaired antigen-specific B-cell responses after Influenza vaccination Emerging data suggest that costimulation blockade with belatacept effectively in kidney transplant recipients controls humoral alloimmune responses. However, whether this effect may be receiving co-stimulation blockade with Belatacept. deleterious for protective anti-infectious immunity remains poorly understood. Front. Immunol. 13:918887. We performed a mechanistic exploratory study in 23 kidney transplant doi: 10.3389/fimmu.2022.918887 recipients receiving either the calcineurin-inhibitor tacrolimus (Tac, n=14) or belatacept (n=9) evaluating different cellular immune responses after influenza vaccination such as activated T follicular Helper (Tfh), plasmablasts and H1N1 hemagglutinin (HA)-specific memory B cells (HA+mBC) by flow-cytometry, and anti-influenza antibodies by hemagglutination inhibition test (HI), at baseline and days 10, 30 and 90 post-vaccination. The proportion of CD4+CD54RA- CXCR5+ Tfh was lower in belatacept than Tac patients at baseline (1.86%[1.25- 3.03] vs 4.88%[2.40-8.27], p=0.01) and remained stable post-vaccination. At M3, HA+mBc were significantly higher in Tac-treated patients (0.56%[0.32- 1.49] vs 0.27%[0.13-0.44], p=0.04) and correlated with activated Tfh numbers. When stratifying patients according to baseline HA + mBc frequencies, Frontiers in Immunology 01 frontiersin.org Pernin et al. 10.3389/fimmu.2022.918887 belatacept patients with low HA+mBC displayed significantly lower HA+mBc increases after vaccination than Tac patients (1.28[0.94-2.4] vs 2.54[1.73-5.70], p=0.04). Also, belatacept patients displayed significantly lower seroprotection rates against H1N1 at baseline than Tac-treated patients (44.4% vs 84.6%) as well as lower seroconversion rates at days 10, 30 and 90 after vaccination (50% vs 0%, 63.6% vs 0%, and 63.6% vs 0%, respectively). We show the efficacy of belatacept inhibiting T-dependent antigen-specific humoral immune responses, active immunization should be highly encouraged before starting belatacept therapy. KEYWORDS kidney transplantation, co-stimulation blockade, memory B cells, influenza vaccination, calcineurin inhibitors, T follicular helper (Tfh) cell Introduction graft survival, data are lacking on how protective humoral immune responses against infectious antigens, such as viral The calcineurin inhibitors (CNI), specifically tacrolimus, are antigen provided by vaccination, is influenced by belatacept. the main immunosuppressant agents used in organ Seasonal Influenza infection is a major public health transplantation worldwide (1). CNIs abrogates the production problem causing significant morbidity and mortality of IL2 cytokine that drives T-cell activation and proliferation by particularly among more vulnerable patients such inhibiting the activation of calcineurin induced by the immunosuppressed solid organ transplants (SOT) (10) (11), in engagement of the T-cell receptor with the peptide-MHC whom suboptimal humoral immune response seems to occur complex. More recently, the co-stimulation blockade due to chronic immunosuppression (12–14).. Notably, an early immunosuppressant Belatacept (cytotoxic T lymphocyte- expansion of Tfh cells, and especially of activated Tfh associated antigen 4 (CTLA4)-Ig; Bristol Myers Squibb), a (CD4 + CD45RA - CXCR5 + ICOS + PD1 + ) has been shown to human fusion protein which binds to surface costimulatory influence a better vaccination response (15, 16). Therefore, ligands (CD80 and C86) on antigen presenting cells (APCs) whether patients receiving belatacept may display a and thus, inhibits their interaction with CD28, consequently particularly impaired immune response after active Flu abrogating T-cell activation has been developed and can be used immunization has not been investigated yet. as an alternative of CNI in kidney transplantation (2). In this study, we investigated the dynamics of Tfh and The use of belatacept in human kidney transplantation as antigen-specific antibody responses by hemagglutination maintenance immunosuppression has shown to effectively inhibition test and B-cell immune responses in belatacept- prevent allograft rejection while improving renal function, treated kidney transplant patients compared to standard of together with a better cardio-vascular and metabolic profile as care Tacrolimus (Tac)-treated patients, after receiving a T-cell compared to a CNI based regimen, ultimately providing superior dependent booster vaccination against Influenza virus. long-term graft survival (3, 4). Notably, while higher T-cell mediated rejection (TCMR) rates have been described in belatacept-treated patients (5), a remarkable ability to inhibit Methods humoral alloimmunity by means of reduced de novo donor- specific anti-HLA antibody (DSA) formation and antibody- Study population and study design mediated rejection has also been described (6, 7). This effect is suggested to be driven by different mechanisms such as Twenty-three consecutives adult kidney transplant recipients abrogation of antigen-specific T follicular Helper (Tfh) were recruited at the kidney transplant units at Bellvitge interaction with B cells, prevention of germinal center University Hospital (Barcelona, Spain), and Toulouse University activation and a direct inhibition of donor-specific antibody- Hospital (Toulouse, France) in 2017-2018 fall periods. Patients producing plasma cells (6, 8, 9). Importantly, while modulation were included if they were scheduled to receive the influenza of these humoral alloimmune responses is beneficial to preserve vaccine, had a functioning kidney allograft for more than 1 year Frontiers in Immunology 02 frontiersin.org Pernin et al. 10.3389/fimmu.2022.918887 and were receiving maintenance immunosuppression regimen until use. PBMC were thawed, pretreated with saturating based on either the CNI tacrolimus (Tac Group) or Belatacept concentration of human aggregated immunoglobulins to block (BELA Group) (supplemental material). FcgR and then stained with the following markers: CD19 Main exclusion criteria were documented egg allergy, acute PerCPVio700 (Miltenyi Biotec), CD27 PEVio770 (Miltenyi febrile illness during the 7 days preceding vaccination, previous Biotec), IgD FITC (BD Biosciences), CD38 APCR-700 (BD allergic reaction to influenza vaccine and acute rejection in the Biosciences), CD45RA FITC (BD Biosciences), CD4 PEVio770 last 6 months. (BD Biosciences), CD185-CXCR5 PE (Biolegend), PD1 A-647 Serum and peripheral blood mononuclear cell (PBMC) (Biolegend), CD278- ICOS PerCPVio700 (Miltenyi Biotec) and samples were collected prior to (Day 0) and at 10 days, 1 H1N1-HA labeled with Alexa Fluor 647 as previously described in month and 3 months after vaccination for immune analyses. order to define the T follicular helper and the B lymphocytes The local institutional review board approved the study, and all subsets. The Live/Dead viability kit (Thermofischer, Invitrogen) was participants gave written informed consent. used to discriminate dead cells. Importantly, 10ul per million of PBMC of Alexa Fluor 647 labeled HA or human albumin was added after Live/Dead Influenza vaccines viability kit and Fc Blocker Biolegend and stored at 4° in the dark for 15 minutes before staining with the rest of antibodies All patients received a single 0.5mL intramuscular injection of according to manufacturer’s instructions. One million of PBMC the trivalent vaccine (CHIROMAS®, Seqirus, UK or VAXIGRIP®, was the minimum of cells used in each experiment. Sanofi Pasteur, France) in the deltoid muscle. These vaccines After staining, cells were acquired using the Gallios flow contain 15ug of hemagglutinin (HA) antigen from the following cytometer (Beckman Coulter). All proportions of each virus strains: A/Michigan/45/2015(H1N1)pdm09-like, A/ lymphocyte subset are expressed as a percentage of the total HongKong/4801/2014(H3N2) and B/Brisbane/60/2008. lymphocyte count. The gating strategy for Tfh and BC subsets are shown is the Supplementary Figures 1, 2 respectively together Assessment of influenza (H1N1-HA)- with representative examples for both BELA and TAC specific memory B cells patient samples. A recombinant H1N1-HA from the virus strain A/ Michigan/45/2015(H1N1)pdm09-like (eENZYME, USA) was Hemagglutination inhibition assay chemically labeled with Alexa Fluor 647 (Alexa Fluor™ NHS ester, Molecular Probes, Invitrogen) following the The analysis of the presence of haemaglutinant antibodies manufacturer’s instructions, for the detection of HA-specific against influenza H1N1 lineage was performed by Memory B cells by flow-cytometry analysis as described below. haemaglutination inhibition assay (HI), performed using the No biotinylation is necessary, as direct conjugation with the same strain contained in the 2017-2018 vaccine, as previously protein is achieved as reported on manufacturer’s instructions. described (17), according to WHO guidelines (WHO Global The degree of labeling was determined by spectrophotometry Influenza, Surveillance Network Manual for the laboratory (Supplemental material). One µg of dye was used to label 10 µg diagnosis and virological surveillance of influenza. 2011). The of HA and stored at a concentration of 30 µg/ml in PBS. test was performed in 96-microV well plates, serum samples As a negative control, Human Serum Albumin (Equipos were pre-treated with RDE (Receptor Destroying Enzyme; Medico-bioló gicos, Barcelona, Spain) labelled with Alexa Fluor Denka Seiken, Tokyo, Japan) and then adsorbed with hen red 647 with the same kit (Alexa Fluor™ NHS ester, Molecular blood cells for eliminating the unspecific inhibitors from blood. Probes, Invitrogen) was used to control the absence of unspecific Results interpretation was performed using classical European B cell interaction with the dye (Supplementary Fig 1B) in all Medicament Agency (EMA) criteria for the evaluation of vaccine patients at baseline. We did not detect specific binding to efficacy (EMA; Note for guidance on harmonization of albumin in any case. requirements for influenza vaccines (CPMP/BWP/214/96) (1997)): seroconversion was defined as either post vaccination serum titer ≥1/40 with pre-vaccination negative HI test or Flow cytometry analyses significant increase of antibody titer of at least 1 fourfold increase after vaccination. Seroprotection was defined as an Peripheral blood mononuclear cells (PBMC) were isolated by antibody titer ≥1/40 (18, 19). Geometric Mean Titers (GMT) density gradient ficoll separation and conserved in liquid nitrogen were also analyzed in different groups of patients and timepoints. Frontiers in Immunology 03 frontiersin.org Pernin et al. 10.3389/fimmu.2022.918887 Statistics Results We performed two-groups comparisons between the CNI Baseline characteristics of the and BELA groups using the Wilcoxon test for continuous study population covariates and the Fisher exact test for categorial covariates. For the temporal evolution, we considered the baseline Twenty-three kidney transplant recipients were included in differences in lymphocytes’ phenotypes using an ANCOVA this study, 9 patients were receiving Belatacept (Bela Group) and model predicting the final M3 lymphocytes subpopulations, 14 a tacrolimus-based immunosuppressive regimen (Tac group). adjusting for the baseline subpopulations and for the Patients were immunized with the trivalent influenza vaccine treatment. P-values <0.05 were considered statistically (CHIROMAS® n= 20 or VAXIGRIP® n=3). As described in significant. Analyses were performed using SPSS version 26 Table 1, there was no major difference regarding main clinical, software and the R software version 1.0.12 for the temporal demographic or immunological characteristics between all evolution analysis. Graphs were generated using GraphPad patients of the study, but Bela treated patients had a longer Prism version 8.0 software (Graphpad Software, San time since transplantation. There was no acute rejection event Diego, California). nor de novo DSA formation in any patient after vaccination TABLE 1 Main characteristics of the study population. Belatacept (n=9) Tacrolimus (n=14) p value Time after transplantation (years) 9.67 ± 4.19 3.5 ± 4.27 0.003 Recipient age (years) 60.8 ± 10.3 54.9 ± 12.3 0.25 Recipient gender (Male) (n, %) 8 (89%) 9 (64%) 0.34 Donor age (years) 60.2 ± 17.5 54.8 ± 13.4 0.51 Transplant type 0.82 Living (n, %) 1 (11%) 1 (7%) DBD/DCD (n, %) 6 (67%)/2 (22%) 11 (79%)/2 (14%) Transplant number (1st) (n, %) 7 (78%) 10 (83%) 0.48 Time on dialysis (months) 17.8 ± 18.3 34.3 ± 25 0.18 Cause of ESRD (n, %) 0.47 Glomerular diseases 4 (44%) 5 (36%) Diabetic nephropathy 0 1 (7%) Polycystic kidney disease 1 (11%) 3 (21%) Vascular/Hypertension 1 (11%) 2 (14%) Others 3 (33%) 1 (7%) Unknown 0 2 (14%) Relevant comorbidities (n, %) Diabetes 0 2 (15%) 0.49 Hypertension 6 (67%) 11 (85%) 0.61 Heart failure 1 (11%) 2 (15%) 1 Previous splenectomy 0 0 Induction treatment (n, %) 0.39 None 1 (14%) 0 Basiliximab 7 (78%) 11 (79%) rATG 1 (14%) 3 (21%) MMF dose (mg/d) 826 ± 332 881 ± 227 0.66 Prior anti-flu vaccination (n, %) 7 (78%) 12 (86%) 1 Type of vaccination (CHIROMAS) 3 (33%) 0 0.047 cPRA at time of vaccination (%) 10.2 ± 31 10.9 ± 22 0.95 DSA at time of vaccination (n, %) 0 2 (14%) 0.17 De novo DSA after vaccination (n, %) 0 0 1 Acute rejection prior to vaccination (n, %) 1 (12.5%) 2 (13%) 1 Acute rejection after vaccination (n, %) 0 0 1 eGFR at time of vaccination (mL/min/1.73m2) 54.9 ± 22 63.9 ± 15 0.34 M, male; DBD, donation after brain death; DCD, donation after cardiac death; rabbit antithymoglobulin; MMF, Mycophenolate mofetil DGF, delayed graft function; DSA, donor-specific antibodies; eGFR, estimated glomerular filtration rate; ESRD, end-stage renal disease; cPRA, calculated panel reactive antibodies. Frontiers in Immunology 04 frontiersin.org Pernin et al. 10.3389/fimmu.2022.918887 during the study follow-up. Also, none of the patients developed (Figure 3A). At day 10 after vaccination, a significant increase of a Flu infection during the study follow-up. plasmablasts in both groups was observed (0.40%[0.28-0.87] vs 0.17%[0.07-0.20], p=0.006 and 0.60%[0.28-0.89] vs 0.28%[0.16- 0.44], p=0.04, at D10 vs Baseline in the Bela and Tac groups, Kinetics of circulating Memory and respectively), and the proportion of plasmablasts became similar follicular Helper T cells after between the two groups at D10 and at M1. While after day 10 the influenza vaccination proportion of plasmablasts progressively decreased returning to baseline levels, at M3 Bela patients showed significantly lower Total lymphocyte counts were not different in both groups levels than the Tac group (0.13%[0.04-0.26] vs 0.31%[0.21- both before and after immunization (data not shown). As shown 0.57], p=0.02). in Figure 1, at baseline, the proportion of CD4+ T cells, CD4+ A moderate but significant correlation between the variation of CD45RA- memory T cells and CD4+CD45RA-CXCR5+ Tfh CD4+CD45RA-CXCR5+ICOS+PD1+ activated Tfh between Baseline cells were lower in the Bela group as compared to the Tac group to D10 (DActivated Tfh= %Activated Tfh at D10 - %Activated Tfh at (33.8%[24.9-41.6] vs 46.1%[31.5-54.7] CD4+, p=0.06; 14.7% baseline) and the percentage of plasmablasts at D10: Pearson r=0.43, [12.7-17.8] vs 26.8%[18.1-30.9] CD4+CD45RA-, p=0.002 and p=0.04 in all patients (Supplemental Figure 4). 1.86%[1.25-3.03] vs 4.88%[2.40-8.27] CD4+CD45RA-CXCR5+, p=0.01, for Bela and Tac groups respectively). After H1N1 HA-specific CD19+CD27+ memory B immunization, the proportion of CD4+ T cells, CD4 cells +CD45RA- memory T cells and CD4+CD45RA-CXCR5+ Tfh The levels of circulating HA-specific mBc (HA+CD19+CD27+) did not significantly change over time between groups as at baseline ranged between 0.11% to 3.8%, and were similar between compared to baseline. both groups (0.25% of CD19+ cells [0.19-0.86] vs 0.30%[0.20-0.68], When we focused on activated CD4+CD45RA-CXCR5+ p=0.61, in the Tac and Bela groups, respectively). Notably, while ICOS+PD1+ Tfh, at baseline, significantly lower numbers were these numbers did not differ between groups at D10 and M1 (0.50% observed in the Bela group than in the Tac group (0.05%[0.03- [0.33-1.36] vs 0.45%[0.25-1.3] at D10, p=0.71 and 0.56%[0.31-1.58] 0.07] vs 0.24%[0.16-0.48], p<0.001) (Figure 2A). Notably, while vs 0.49%[0.15-0.83] at M1, p=0.59 in Tac and Bela, respectively), a these numbers did not change at any time point after significantly higher increase of HA+CD19+CD27+ mBc in the Tac vaccination in Bela patients but in one patient, there was a group compared to Bela patients was observed at M3 (0.56%[0.32- significant increase of activated Tfh among 6/14 patients in the 1.49] vs 0.27%[0.13-0.44] respectively, p=0.04) (Figure 3B). Tac group at day 10 (Figure 2B). Activated Tfh remained To further investigate the effect of vaccination on higher in the Tac group at D10 (0.05%[0.02-0.15] vs 0.25% HA + CD19 + CD27 + mBC, we stratified patients into two [0.11-1.18], p=0.019 for Bela and Tac group respectively), M1 subgroups: High HA+CD19+CD27+ mBC or Low (0.04%[0.02-0.07] vs 0.23%[0.05-0.47] respectively, p= 0.027), HA+CD19+CD27+ mBC according to the median percentage and M3 (0.02%[0.02-0.03] vs 0.27%[0.06-0.49] respectively, of HA +CD19+ CD27+ mBc at baseline (HA+ CD19 + CD27 + p=0.004). The same feature was observed when the mBC=0.29%), suggesting the presence or absence of a percentage of activated Tfh was assessed over total Tfh cells preformed mBc response already at baseline. The expansion of (data not shown). HA+CD19+CD27+mBC after vaccination was analyzed as the ratio of HA+CD19+CD27+ mBC after immunization over the numbers of HA+CD19+CD27+mBC at baseline. As shown in Kinetics of different B-cell subsets after Figure 3C, among the Low HA+CD19+CD27+mBC group, a influenza vaccination significant higher increase of HA+ CD19 +CD27+ mBC was observed in the Tac group as compared to the Bela group Total CD19+ B cells and other B-cell subsets (Ratio HA+CD19+CD27+mBC: 2.54[1.73-5.70] vs 1.28[0.94- As illustrated in supplemental Figure 3, the percentages of 2.4] at D10, p=0.05; 2.85[1.93-3.75] vs 0.85[0.55-1.15] at M1, CD19+ B cells, CD19+CD27+ memory B cells (mBC), CD19+ p=0.09 and 2.43[1.36-7.68] vs 0.92[0.49-1.52] at M3, p=0.04 in CD27+IgD- switched mBc and CD19+CD27+IgD+ unswitched the Tac and Bela groups, respectively). Conversely, no significant mBC were similar between groups, both at baseline and at the differences were observed between groups within patients different time points after vaccination. showing High HA+CD19+CD27+ mBC (Figure 3D). Adjusting for the continuous cell subsets frequencies at baseline, Total CD19+CD27+CD38hi plasmablasts HA+CD19+CD27+ mBc at M3 was the only cell population At baseline, the percentage of CD19 + CD27 + CD38 hi associated with a treatment effect (baseline effect +0.46% p<0.001; plasmablasts was significantly lower in the Bela group than in treatment effect [Bela VS Tac]: -0.49% [p=0.028], p=ns for all the Tac group (0.17%[0.07-0.20] vs 0.28%[0.16-0.44], p=0.009) other populations). Frontiers in Immunology 05 frontiersin.org Pernin et al. 10.3389/fimmu.2022.918887 A B C FIGURE 1 Continued Frontiers in Immunology 06 frontiersin.org Pernin et al. 10.3389/fimmu.2022.918887 FIGURE 1 (Continued) Kinetics of different T-cell subsets at different time points prior and after Influenza immunization. Figure 1A. Proportion of CD4+ T cells. Figure 1B. Proportion of CD4+CD45RA- memory T cells, Figure 1C. Proportion of CD4+CD45RA-CXCR5+ Tfh cells. The proportions of CD4+ T cells, CD4+CD45RA- memory T cells and CD4+CD54RA-CXCR5+ Tfh cells are expressed as a percentage in total lymphocytes. Comparison of proportions between the two groups at each time point were analyzed using a Mann-Whitney Test. Serological responses after influenza While the inhibitory effect of belatacept on the humoral vaccination alloimmune memory response among SOT patients has been shown at the clinical, experimental, and in vitro levels its effect At baseline, despite similar previous vaccination rates in both on other humoral immune responses (such as vaccination- groups, Influenza seroprotection rate was higher in the Tac group induced immunization) among SOT patients has not been (84,6%) as compared to the Belatacept group (44,4%) (Figure 4A). reported. This inhibitory effect on allo-immunity seems After vaccination, H1N1 IgG seroprotection rate reached 100% multifactorial, e.g., preventing the cross-talk between activated already at D10 and was maintained until M3 in the Tac group, Tfh cells and B cells (6–8), ultimately preventing germinal center whereas only 66.7% of seroprevalence rate was observed in Bela activation and DSA formation. Indeed, in some autoimmune patients at M3. Accordingly, the geometric median titer (GMT) of diseases, previous studies have shown an altered humoral H1N1 IgG antibodies at baseline was also significantly higher in the immune response after influenza vaccination. In 2013, Ribeiro Tac group (74.0[44.9-122.6] vs 28.3[10.8-58.8] in the BELA group, et al. (20) reported that seroprotection was significantly reduced p=0.04, Figure 4B). Indeed, the Tac group displayed a higher after the pandemic 2009 influenza A/H1N1 vaccine in a cohort increase of GMT at the distinct time points after vaccination as of rheumatoid arthritis (RA) patients treated with the compared to BELA patients: 191.8[122.6-275.8] vs 31.8[10.8-74.1] costimulation blocker abatacept as compared to age-matched p=0.003, 218.2[137.9-336.2] vs 47.6 [15.2-123.4], p=0.008 and 236.2 RA-patients treated with methotrexate (9% vs 58%, p=0.0006). [127-396.1] vs 64.3[14.9-176.6], p=0.05, at D10, M1 and M3 in Tac Kapetanovic et al. (21) showed a seroconversion rate in only 20% and Bela, respectively) (Figure 4B). While seroconversion occurred of RA patients treated with abatacept as compared with 42% of in 50%, 63.6% and 63.6% of patients at D10, M1 and M3 patients on methotrexate and 53% on anti-TNF monotherapy respectively in the Tac group, none of the Bela patients showed after H1N1 influenza vaccine. Similarly, during recent seroconversion in the BELA group during follow-up (Figure 4C). vaccination campaign against SARS-COV2, kidney transplant Notably, and similarly to what was observed when stratifying patients receiving belatacept maintenance therapy developed patients with low/High preformed HA+CD19+CD27+ mBc, significantly inferior serological response after vaccination within serologically naïve patients (H1N1 IgG <1/40) at baseline, even if no clear effect was observed on risk of severe disease seroconversion at M3 occurred in 2/2 (100%) patients of the CNI after natural infection (22, 23). group and in none (0/3 [0%]) in the group Bela group. Likewise, Tacrolimus also reduces significantly the number of among patients with detectable H1N1 IgG titers (≥1/40) at baseline, circulating Tfh and their activation capacity (24). In this study, seroconversion at M3 was achieved in 5/9 (55.6%) Tac patients but we show that belatacept-treated patients display significantly in none of the BELA group (0/3[0%]) (Figure 4D). lower numbers of circulating Tfh cells than patients on CNI- based immunosuppression and failed to undergo a significant expansion of these activated TFH cells after vaccination, as Discussion compared to tacrolimus-treated patients. While the global percentages of mBc with distinct phenotypes (such as switched In this study, we show that the generation of a protective and unswitched mBc) and antigen specificities were similar humoral immune memory response in kidney transplant patients between treatment groups at baseline, belatacept-treated receiving a T-cell dependent booster vaccination such as that patients showed a significantly lower percentage of against Influenza virus is significantly impaired among patients plasmablasts at baseline than CNI patients. As previously receiving co-stimulation blockade with belatacept as compared to reported after influenza vaccination in healthy individuals (15, patients on a CNI-based regimen with tacrolimus. Indeed, 16), we also observed a positive correlation between the early significantly lower seroprotection rates and memory B-cell (mBc) expansion of activated Tfh cells and the increase of plasmablasts frequencies specific to influenza virus after vaccination were at D10. observed in belatacept-treated patients as compared to patients Interestingly, patients of the study had already received a on tacrolimus. previous vaccination against the Influenza virus or had been Frontiers in Immunology 07 frontiersin.org Pernin et al. 10.3389/fimmu.2022.918887 A B FIGURE 2 Comparison of the percentage of CD4+CD45RA-CXCR5-ICOS+PD1+ activated Tfh between the two study groups. Figure 2A. Baseline percentages of CD4+CD45RA-CXCR5-ICOS+PD1+activated Tfh in the two study groups. Figure 2B. Kinetics of CD4+CD45RA-CXCR5-ICOS+PD1+ activated Tfh after influenza immunization in the two study groups. The proportion of CD4+CD45RA-CXCR5-ICOS+PD1+ Tfh cells is expressed as a percentage in total lymphocytes in both figure 2A and 2B. Comparison of proportions between the two groups at each time point were analyzed using a Mann-Whitney Test. * p<0.05, ** p<0.01, ***p<0.001. naturally immunized through a viral infection in the past, therefore levels. Conversely, in patients with high preformed HA+mBC levels most patients were seroprotected already at baseline. However, at baseline, the level of HA+mBC did not significantly change after while the level of HA+mBC at baseline was variable and ranged vaccination in both groups. These results are in line with between 0.11% and 3.8% in our patients, their expansion at 3 experimental models of alloimmune sensitization in which months after vaccination was significantly lower in the BELA than belatacept seems to be especially capable to reverse a T- in the CNI group. Furthermore, belatacept seemed to particularly dependent humoral alloimmune response when the treatment is inhibit the generation of new HA+mBC as though patients with low given during the initial period after alloimmunization, whereas once HA+mBc levels at baseline showed significantly lower expansion of a complete germinal center activation has been generated, these cells at M3 than CNI patients with the same low pre-vaccine belatacept has an impact similar to that of CNIs (25, 26). Frontiers in Immunology 08 frontiersin.org Pernin et al. 10.3389/fimmu.2022.918887 A B C D FIGURE 3 Kinetics of circulating Plasmablasts and Influenza-specific B-cell subsets prior and after Influenza immunization. Figure 3A Proportion of CD19+CD27+CD38hi plasmablasts. Figure 3B. Proportion of CD19+CD27+HA+ mBc. Figure 3C. Analysis of HA+mBC expansion after immunization in patients with High preformed HA+mBc at baseline. Figure 3D. Analysis of HA+mBC expansion after immunization in patients with Low preformed HA+mBc at baseline. The proportions of CD19+CD27+CD38hi cells (Figure 2A) and CD19+CD27+H1N1+ cells (Figure 2B) are expressed as a percentage in total lymphocytes in both Figures 3A, 3B. The ratio of H1N1+CD19+CD27+ mBC at D10, M1 and M3 after immunization (Figures 3C, 3D) = % H1N1+CD19+CD27+ mBC during follow-up/% H1N1+CD19+CD27+ mBC at baseline. Comparison of proportions between the two groups at each time point were analyzed using a Mann-Whitney Test. When assessing serological response to vaccination by activation markers but we did not measure an antigen-specific means of specific HI test, we observed that even with a similar response. However, the consistent differences observed between history of previous flu vaccinations/infections, patients receiving the two groups evaluated in different cell compartments belatacept displayed inferior seroprotection at baseline as well as reinforce the biological plausibility of our data. Furthermore, lower percentage of seroconversion and antibody titers after all patients of the study were also receiving MMF as concomitant vaccination. We then stratified patients according to baseline immunosuppressant and previous reports have suggested a seroprotection, we detected a similarly poor response to deleterious effect of MMF on the generation of humoral vaccination both in patients with negative (<1/40) or positive immune responses after active immunization (12, 14, 28). (≥1/40) baseline antibody titer. Thus, although both study groups were receiving the same We acknowledge some limitations: the small sample size, the MMF doses, we cannot exclude a synergistic effect of MMF focus on the H1N1 strain (and not other hemagglutinin antigens and belatacept in impairing the humoral response, as compared contained in the vaccine) as though previous studies have to patients receiving a combination of tacrolimus and MMF. The reported that such humoral responses may differ according to main clinical difference between study group was time since the type of hemagglutinin (13, 27). It must be noted that two transplantation, being longer in BELA patients. Even if we different vaccines were used in the present study, however both cannot exclude that it might be a confounder for our results, it had the same HA and viral strains composition and no has been shown that shorter time after transplantation is a risk differences were observed in B cell nor antibody response factor for lower response to vaccination, we do not think this according to the type of vaccine. Also, for Tfh analysis we factor explains the differences observed between the two groups analyzed the kinetics of total circulating Tfh and their of treatment since the increased immunosuppressive load is Frontiers in Immunology 09 frontiersin.org Pernin et al. 10.3389/fimmu.2022.918887 A B C D FIGURE 4 Serological responses after Influenza vaccination. Figure 4A. Seroprotection rate at baseline and evolution after vaccination. Figure 4B. Kinetics of H1N1 antibody titers (GMTs) before and after vaccination. Figure 4C. Seroconversion rate after vaccination. Figure 4D. Comparison of seroconversion rates at M3 between naïve and seropositive patients prior to vaccination according to each type of immunosuppressant. commonly observed during the first 6 months after The patients/participants provided their written informed transplantation and not in such later time points as our consent to participate in this study. patient population was investigated (29). In conclusion, we showed that, unlike CNI-treated kidney transplant patients, patients on belatacept display significantly Author contributions lower circulating Tfh and plasma cell levels and have a significantly impaired humoral immune memory response EC and OB had designed the study. VP, MM, IS-M, TJ, AT, after influenza vaccination, especially those with low JME, LD performed the experiments. EC, OB, MM, CP, FM, SN, preformed antigen-specific mBC. While further investigation is CM, AF, AB and NK had enrolled patients in the study. OB, EC, warranted, our data suggest that patients considered to be VP, MM, IS-M, TJ, had participated to data analysis and wrote treated by co-stimulation blockade with belatacept are highly the manuscript. All authors contributed to the article and encouraged to receive active immunization prior to this therapy approved the submitted version. in order to increase their baseline anti-viral immune memory protection and ultimately minimize the risk of subsequent opportunistic infections. Funding This work was supported by the Instituto de Salud Carlos III Data availability statement (ISCIII) (grant numbers ICI14/00242 and PI16/01321, PI19/01710) and by the European Union’s Horizon 2020 Research and The original contributions presented in the study are included innovation program (grant agreement 754995). Also, this work in the article/supplementary material. Further inquiries can be was partly supported by the SLT002/16/00183 grant, from the directed to the corresponding author. Department of Health of the Generalitat de Catalunya by the call “Accioı́ instrumental de programes derecerca orientats en l’àmbit de la recerca i la innovacioı́ en salut.” The authors thank the Research Ethics statement Centers of Catalonia (CERCA) Programme/Generalitat de Catalunya for institutional support. OB was awarded with an The studies involving human participants were reviewed and intensification grant from the “Instituto de Salud Carlos III” approved by ethic committee of Bellvitge University Hospital. [INT19/00051]. Frontiers in Immunology 10 frontiersin.org Pernin et al. 10.3389/fimmu.2022.918887 TJ received a fellowship grant from ESOT (European Society Publisher’s note for Organ Transplantation). All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated Acknowledgments organizations, or those of the publisher, the editors and the reviewers. 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Am J Transplant (2016) 16(8):2312−23. doi: 10.1111/ajt.13761 COPYRIGHT 27. Héquet D, Pascual M, Lartey S, Pathirana RD, Bredholt G, Hoschler K, et al. © 2022 Pernin, Meneghini, Torija, Jouve, Del Bello, Sanz-Muñoz, Eiros, Humoral, T-cell and b-cell immune responses to seasonal influenza vaccine in solid Donadeu, Polo, Morandeira, Navarro, Masuet, Favà, LeQuintrec, Kamar, organ transplant recipients receiving anti-T cell therapies. Vaccine (2016) 34(31):3576 Crespo and Bestard. This is an open-access article distributed under the −83. doi: 10.1016/j.vaccine.2016.05.021 terms of the Creative Commons Attribution License (CC BY). The use, 28. Smith KG, Isbel NM, Catton MG, Leydon JA, Becker GJ, Walker RG. distribution or reproduction in other forums is permitted, provided the Suppression of the humoral immune response by mycophenolate mofetil. Nephrol original author(s) and the copyright owner(s) are credited and that the Dial Transplant (1998) 13(1):160−4. doi: 10.1093/ndt/13.1.160 original publication in this journal is cited, in accordance with accepted 29. Benotmane I, Gautier-Vargas G, Cognard N, Olagne J, Heibel F, Braun-Parvez L, academic practice. No use, distribution or reproduction is permitted et al. Low immunization rates among kidney transplant recipients who received 2 doses which does not comply with these terms. Frontiers in Immunology 12 frontiersin.org