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Vedolizumab as a successful treatment of CTLA-4–associated autoimmune enterocolitis

2016; Elsevier BV; Volume: 139; Issue: 3 Linguagem: Inglês

10.1016/j.jaci.2016.08.042

1097-6825

Alexander A. Navarini, Petr Hrúz, Christoph T. Berger, Tie Zheng Hou, Charlotte Schwab, Annemarie Gabrysch, Rebecca Higgins, Natalie Frede, Barbara-Christina Padberg Sgier, Olle Kämpe, Anne‐Valérie Burgener, Florian Marquardsen, Fabian Baldin, Marc B. Bigler, Anne Kistner, Annaïse Jauch, Olivier Bignucolo, B. Meyer, Fabian Meienberg, Matthias Mehling, Lukas T. Jeker, Ingmar Heijnen, Thomas Daikeler, Jan‐Olaf Gebbers, Bodo Grimbacher, David M. Sansom, Raphael Jeker, Christoph Hess, Mike Recher,

Blood groups and transfusion

In 2007, a 39-year-old white male (unique patient identifier [UPI]: CTLA-4_AAA.II.1) presented with chronic, noninfectious diarrhea. The patient's history was noticeable for adrenal insufficiency diagnosed in 1991. In 2013, his diarrhea worsened, resulting in weight loss of more than 20 kg and severe dehydration. Prednisolone (1 mg/kg of body weight given for several weeks) was entirely ineffective. Macroscopic enterocolitis was seen, corresponding histologically to extensive infiltration with CD3+ T cells in cryptal areas (Fig E1, A, in this article's Online Repository at www.jacionline.org). Enterocytes showed enhanced positivity for Ki-67, indicating augmented proliferation (Fig E1, B). Complete absence of mucus-producing goblet cells was observed in colon and small intestine (data not shown). At that time, hypogammaglobulinemia (IgG, 4.4 g/L [normal, 7-16 g/L]; IgA, 0.53 g/L [normal, 0.7-4 g/L]) was first noticed, whereas serum IgM level was within normal range. On a computed tomography scan, no evidence for malignancy or lymphoproliferation was found and lung morphology was normal. Intravenous immunoglobulin (IVIG) substitution (0.5 g/kg body weight per month, given for 4 months) had no effect on diarrhea and the patient required intravenous fluids repeatedly. In 2014, the patient developed severe hyporegenerative anemia. Bone marrow biopsy revealed isolated yet almost complete absence of erythropoietic cells (data not shown), and the diagnosis of pure red cell aplasia was established. Parvovirus was tested negative by PCR. In May 2014, while the patient was still on IVIG treatment, an immunologic workup was performed (Table I). B-cell counts were low (2% of lymphocytes, Table I). Analysis of B-cell subpopulations revealed normal relative differentiation into marginal zone-like (IgD+CD27+, 27% of all B cells) and class-switched memory (IgD–CD27+, 15% of all B cells) subsets. In contrast, the proportion of CD21low B cells was clearly elevated (28% of B cells)—a finding associated with granulomas and splenomegaly in patients with CVID.1Rakhmanov M. Keller B. Gutenberger S. Foerster C. Hoenig M. Driessen G. et al.Circulating CD21low B cells in common variable immunodeficiency resemble tissue homing, innate-like B cells.Proc Natl Acad Sci U S A. 2009; 106: 13451-13456Crossref PubMed Scopus (245) Google Scholar Within the T-cell fraction, regulatory T cells (defined as both CD3+CD4+CD127lowCD25high or CD3+CD4+CD45RAnegFOXP3high; Fig 1) were normal or even enhanced in numbers, whereas the proportions of central-memory and effector-memory CD4+ and CD8+ T cells were comparable to those in healthy control (Fig 1, B). T-cell–mediated colitis has recently been described as a prominent feature in patients with heterozygous mutations in cytotoxic T-lymphocyte–associated protein 4 (CTLA-4), a negative regulator of T-cell–mediated immune responses.2Kuehn H.S. Ouyang W. Lo B. Deenick E.K. Niemela J.E. Avery D.T. et al.Immune dysregulation in human subjects with heterozygous germline mutations in CTLA4.Science. 2014; 345: 1623-1627Crossref PubMed Scopus (607) Google Scholar, 3Schubert D. Bode C. Kenefeck R. Hou T.Z. Wing J.B. Kennedy A. et al.Autosomal dominant immune dysregulation syndrome in humans with CTLA4 mutations.Nat Med. 2014; 20: 1410-1416Crossref PubMed Scopus (589) Google Scholar Colitis is also commonly induced in patients with melanoma treated with ipilimumab, an anti–CTLA-4 antibody.4Quirk S.K. Shure A.K. Agrawal D.K. Immune-mediated adverse events of anticytotoxic T lymphocyte-associated antigen 4 antibody therapy in metastatic melanoma.Transl Res. 2015; 166: 412-424Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar, 5Bertrand A. Kostine M. Barnetche T. Truchetet M.E. Schaeverbeke T. Immune related adverse events associated with anti-CTLA-4 antibodies: systematic review and meta-analysis.BMC Med. 2015; 13: 211Crossref PubMed Scopus (471) Google Scholar The DNA of the patient was analyzed by whole-exome sequencing, which indeed identified a heterozygous missense mutation in the CTLA4 gene at cDNA position 257 (c.C257T), resulting in an alanine to valine substitution at position 86 (p.A86V) (a graphic representation of the mutation is shown in Fig E2 in this article's Online Repository at www.jacionline.org). The alanine at this position is highly conserved across various species (see Fig E3 in this article's Online Repository at www.jacionline.org) and the mutation was predicted to have a deleterious consequence (Combined Annotation Dependent Depletion [CADD] score, 24.2; PolyPhen1 "probably damaging"). The other rare nonsynonymous allelic variants found in primary immunodeficiency genes (adapted from Picard et al6Picard C. Al-Herz W. Bousfiha A. Casanova J.L. Chatila T. Conley M.E. et al.Primary immunodeficiency diseases: an update on the classification from the International Union of Immunological Societies Expert Committee for Primary Immunodeficiency 2015.J Clin Immunol. 2015; 35: 696-726Crossref PubMed Scopus (172) Google Scholar) were unlikely to explain the patient's clinical phenotype (see Table E1 in this article's Online Repository at www.jacionline.org). At the protein level, CTLA-4 expression on regulatory T (Treg) cells was low compared with control, both in the absence or following in vitro stimulation of Treg cells (Fig 1, C and D) with mean fluorescent intensity reductions similar to what was published in patients with CTLA-4 deficiency.3Schubert D. Bode C. Kenefeck R. Hou T.Z. Wing J.B. Kennedy A. et al.Autosomal dominant immune dysregulation syndrome in humans with CTLA4 mutations.Nat Med. 2014; 20: 1410-1416Crossref PubMed Scopus (589) Google Scholar To address CTLA-4 function, a previously published transendocytosis assay was performed measuring the CTLA-4–driven capacity to transendocytose a CD80-green fluorescent protein fusion protein.3Schubert D. Bode C. Kenefeck R. Hou T.Z. Wing J.B. Kennedy A. et al.Autosomal dominant immune dysregulation syndrome in humans with CTLA4 mutations.Nat Med. 2014; 20: 1410-1416Crossref PubMed Scopus (589) Google Scholar CTLA-4–mediated transendocytosis was clearly reduced in patient-derived CD4+ T cells (Fig 2, E).Table IHematologic and immunologic laboratory results of the patientCell populationPatient valueNormal valuesRBC (cells/μL)4.34 G/L4.5-6.3 G/LWBC (cells/mL)8.09 G/L3.5-10 G/LANC (cells/mL)5.8 G/L1.3-6.7 G/LPlatelet count (cells/mL)354 G/L150-450 G/LLymphocytes absolute (cells/mL)1.254 G/L0.9-3.3 G/LLymphocyte subpopulationsCD3+ (cells/μL) and (%)1331/μL (86%)742-2750/μL (55%-86%)CD3+CD4+ T cells (cells/μL) and (%)565/μL (36%)404-1612/μL (33%-58%)CD3+CD8+ T cells (cells/μL) and (%)728/μL (46%)220-1129/μL (13%-39%)CD19+ (cells/μL) and (%)24/μL (2%)80-616/μL (5%-22%)CD56+CD16+ (cells/μL) and (%)190/μL (12%)84-724/μL (5%-26%)B-cell subpopulationsIgD+CD27− (cells/μL) and (%) out of CD19+7/μL (27.2% of CD19)66-228/μL (25.1%-92.4%)IgD−CD27+ (cells/μL) and (%) out of CD19+4/μL (15.2% of CD19)8-102/μL (2.4%-32.6%)CD21lowCD38− B cells (cells/μL) and (%)7/μL (28.6% of CD19)1-12/μL (0.5%-4.7%)ANC, Absolute neutrophil count; RBC, red blood count; WBC, white blood cells.Values outside the normal range are given in boldface. Open table in a new tab ANC, Absolute neutrophil count; RBC, red blood count; WBC, white blood cells. Values outside the normal range are given in boldface. With the clinical condition of the patient unchanged, at this time, treatment with vedolizumab was started. Vedolizumab is an α4β7 integrin-specific humanized mAb that inhibits binding of this gut-homing integrin to mucosal MAdCAM-1, while leaving the binding to the vascular adhesion protein VCAM-1 intact. Vedolizumab has recently been approved for the treatment of inflammatory bowel disease refractory to TNF-α blockade.7Feagan B.G. Rutgeerts P. Sands B.E. Hanauer S. Colombel J.F. Sandborn W.J. et al.Vedolizumab as induction and maintenance therapy for ulcerative colitis.N Engl J Med. 2013; 369: 699-710Crossref PubMed Scopus (1579) Google Scholar After 3 infusions at standard dose, diarrhea was markedly reduced and the patient gradually regained body weight. Diarrhea had completely resolved 3 months after starting treatment with vedolizumab. Currently, 18 months after initiating vedolizumab, the patient is back at work with no abdominal complaints. In a control endoscopy, normal colonic mucosa was seen. Vedolizumab was well tolerated and no infectious complications occurred. Vedolizumab had no impact on the pure red cell aplasia, and cyclosporine was started 7 months after starting vedolizumab treatment at 2 × 100 mg/d, and later reduced to 75 mg/d. One and a half months later, hemoglobin level raised from 78 g/L to 127 g/L, coinciding with a 20-fold relative increase in reticulocytes. The histopathology and the adult onset of the colitis matches the description reported in other patients with CTLA-4 deficiency.2Kuehn H.S. Ouyang W. Lo B. Deenick E.K. Niemela J.E. Avery D.T. et al.Immune dysregulation in human subjects with heterozygous germline mutations in CTLA4.Science. 2014; 345: 1623-1627Crossref PubMed Scopus (607) Google Scholar, 3Schubert D. Bode C. Kenefeck R. Hou T.Z. Wing J.B. Kennedy A. et al.Autosomal dominant immune dysregulation syndrome in humans with CTLA4 mutations.Nat Med. 2014; 20: 1410-1416Crossref PubMed Scopus (589) Google Scholar However, pure red cell aplasia has not been previously linked to CTLA-4 deficiency. Cyclosporine A induces remission in roughly 70% of patients with acquired pure red cell aplasia.8Sawada K. Fujishima N. Hirokawa M. Acquired pure red cell aplasia: updated review of treatment.Br J Haematol. 2008; 142: 505-514Crossref PubMed Scopus (147) Google Scholar We report here for the first time that it also can successfully induce remission in CTLA-4–associated pure red cell aplasia. Adrenalitis resulting in adrenal insufficiency has rarely been described in ipilimumab- treated patients, whereas hypophysitis is a much more common side effect, occurring in 10% to 15% of patients treated with this mAb.5Bertrand A. Kostine M. Barnetche T. Truchetet M.E. Schaeverbeke T. Immune related adverse events associated with anti-CTLA-4 antibodies: systematic review and meta-analysis.BMC Med. 2015; 13: 211Crossref PubMed Scopus (471) Google Scholar The most important novelty of this case study is the reporting of the efficacy of vedolizumab in the treatment of CTLA-4–associated colitis. Published evidence shows that vedolizumab has a good safety profile.9Hagan M. Cross R.K. Safety of vedolizumab in the treatment of Crohn's disease and ulcerative colitis.Expert Opin Drug Saf. 2015; 14: 1473-1479Crossref PubMed Scopus (11) Google Scholar No cases of progressive multifocal leucencephalopathy, a major side effect of other integrin-blocking antibodies such as natalizumab, have been reported in randomized clinical trials.9Hagan M. Cross R.K. Safety of vedolizumab in the treatment of Crohn's disease and ulcerative colitis.Expert Opin Drug Saf. 2015; 14: 1473-1479Crossref PubMed Scopus (11) Google Scholar TNF-α–blocking antibodies have been successfully used to treat antiipilimumab-induced colitis in patients with melanoma.10Marthey L. Mateus C. Mussini C. Nachury M. Nancey S. Grange F. et al.Cancer immunotherapy with anti-CTLA-4 monoclonal antibodies induces an inflammatory bowel disease.J Crohn's Colitis. 2016; 10: 395-401Crossref PubMed Scopus (209) Google Scholar However, avoiding TNF-α blockade in highly autoimmunity-prone patients with primary immunodeficiency—such as individuals with CTLA-4 deficiency—is desirable because blocking TNF-α per se can promote autoimmunity.11De Rycke L. Kruithof E. Van Damme N. Hoffman I.E. Van den Bossche N. Van den Bosch F. et al.Antinuclear antibodies following infliximab treatment in patients with rheumatoid arthritis or spondylarthropathy.Arthritis Rheum. 2003; 48: 1015-1023Crossref PubMed Scopus (190) Google Scholar Other immunosuppressive drugs may worsen hypogammaglobulinemia associated with CTLA-4 deficiency and, notably, high-dose prednisolone was ineffective in our patient. Steroid- refractory colitis has also previously been described in CTLA-4 deficiency,2Kuehn H.S. Ouyang W. Lo B. Deenick E.K. Niemela J.E. Avery D.T. et al.Immune dysregulation in human subjects with heterozygous germline mutations in CTLA4.Science. 2014; 345: 1623-1627Crossref PubMed Scopus (607) Google Scholar underlining the need for effective therapies in this setting. In summary, we describe a patient with a heterozygous CTLA4 mutation, associated with low CTLA-4 expression and function of Treg cells, clinically associated with adrenal insufficiency, pure red cell aplasia, and severe T-cell–mediated enterocolitis. The latter was successfully treated with vedolizumab, without apparent side effects. The clinical usefulness of vedolizumab should be assessed further in enterocolitis associated with genetic or drug-induced functional CTLA-4 deficiency. Ipilimumab was a gift from Heinz Läubli. Following informed consent, the patient was included into a prospective cohort of patients with primary immunodeficiency/immune dysregulation that was ethically approved (EKNZ 2015-187) according to Swiss law. Immunohistochemistry was performed using the avidin-biotin-peroxidase-complex method. The antibodies used were directed against CD3 (clone PS1, Leica) and Ki67 (clone SP6, Cell Marque). The following antibodies from Biolegend (San Diego, Calif) were used for surface staining of specific lymphocyte subsets: CD27 (clone O323), CD25 (clone BC96), CD45RO (clone UCHL1), CD4 (clone A161A1), CD3 (clone UCHT1), CD8 (clone SK1), CD127 (clone A019D5), CD19 (clone HIB19), CTLA-4 (clone L3D10). Genetic sequencing was performed following informed consent. DNA was extracted from cultured T-cell blasts and sheared, followed by pull-down of coding sequences, adapter ligation, and massively parallel sequencing on Illumina HiSeq 2000 appliances at Functional Genomics Center (Zurich, Switzerland). Read lengths of 2 × 100 bp were produced, aiming for average target sequence coverage of more than 60× and generating more than 20 reads for 90% of the Gencode exome. The raw sequence reads were quality controlled and aligned to the reference sequence, genotypes were called with Genome Analysis Toolkit,E1McKenna A. Hanna M. Banks E. Sivachenko A. Cibulskis K. Kernytsky A. et al.The genome analysis toolkit: a mapReduce framework for analyzing next-generation DNA sequencing data.Genome Res. 2010; 20: 1297-1303Crossref PubMed Scopus (14770) Google Scholar and variants were annotated with the position of nucleotide change with respect to coding genes. Results were filtered according to a list of known primary immunodeficiency genes.E2Picard C. Al-Herz W. Bousfiha A. Casanova J.L. Chatila T. Conley M.E. et al.Primary immunodeficiency diseases: an update on the classification from the International Union of Immunological Societies Expert Committee for Primary Immunodeficiency 2015.J Clin Immunol. 2015; 35: 696-726Crossref PubMed Scopus (515) Google Scholar Alleles giving rise to nonsynonymous amino acid substitutions, aberrant splicing, or protein truncation events were filtered for functional impact on the basis of PolyPhen2E3Adzhubei I.A. Schmidt S. Peshkin L. Ramensky V.E. Gerasimova A. Bork P. et al.A method and server for predicting damaging missense mutations.Nat Methods. 2010; 7: 248-249Crossref PubMed Scopus (9288) Google Scholar, E4Adzhubei I. Jordan D.M. Sunyaev S.R. Predicting functional effect of human missense mutations using PolyPhen-2.Curr Protoc Hum Genet. 2013; (Jan, Chapter 7: Unit 7.20)Crossref PubMed Scopus (2065) Google Scholar and CADDE5Kircher M. Witten D.M. Jain P. O'Roak B.J. Cooper G.M. Shendure J. et al.A general framework for estimating the relative pathogenicity of human genetic variants.Nat Genet. 2014; 46: 310-315Crossref PubMed Scopus (3675) Google Scholar scores, on a minor allele frequency of less than 0.001 in public databases (1000 Genomes,E6Abecasis G.R. Altshuler D. Auton A. Brooks L.D. Durbin R.M. Gibbs R.A. et al.A map of human genome variation from population-scale sequencing.Nature. 2010; 467: 1061-1073Crossref PubMed Scopus (5937) Google Scholar NHLBI GO Exome Sequencing Project,E7Exome Variant Server. 2015.Google Scholarand Exome Aggregation Consortium ExACE8Exome Aggregation Consortium (ExAC). 2015.Google Scholar) and our in-house database of more than 2700 exomes. PBMCs were isolated from fresh blood of control or patient by density centrifugation. CD4+ T cells were purified from PBMCs by negative selection using human CD4+ T-cell kit (Stemcell Technologies, Vancouver, British Columbia, Canada). CD4+ T cells were cultured in the absence or presence of CD3/CD28 Beads (Invitrogen, Waltham, Mass) in RMPI with 10% FBS culture media for 16 hours. Cells were then surface stained using anti-CD4 Alexa Fluor 700 (clone RPA-T4, BD) and anti-CD45RA PerCP-Cy5.5 (clone HI100, eBioscience, San Diego, Calif) at 4°C for 30 minutes. For intracellular staining, cells were then washed, fixed/permeabilized using FoxP3 staining buffer (eBioscience), and stained by anti–CTLA-4 PE (clone BN13, BD Biosciences, San Jose, Calif) and anti-FoxP3 APC (clone 236A-E7, eBioscience). Cells were washed and analyzed by BD FACS LSRII and FlowJo (LLC, Ashland, Ore) software. The transendocytosis assay was performed as previously published.E9Qureshi O.S. Zheng Y. Nakamura K. Attridge K. Manzotti C. Schmidt E.M. et al.Trans-endocytosis of CD80 and CD86: a molecular basis for the cell-extrinsic function of CTLA-4.Science. 2011; 332: 600-603Crossref PubMed Scopus (1078) Google Scholar Briefly, CD4+ T cells were isolated from frozen PBMCs using CD4 T-cell isolation kit (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany) and cultured 1:1 with CD80-green fluorescent protein–expressing Chinese hamster ovary (CHO) cells or control CHO cells upon stimulation with CD3/CD28 dynabeads (Thermofisher, Waltham, Mass) for 16 hours at 37°C in RPMI containing 10% FCS and 1% penicillin-streptomycin. Stimulation was used in a ratio of 1:2 beads per T cell. Bafilomycin was added to the coculture (20 nM). Anti-CTLA4 was used in 2.5 μg per well as indicated. T cells were labeled with antihuman CD4 PerCP-Cy5.5, CD45RO PE-Cy7, FoxP3 PE (eBioscience), and CTLA-4 BV 421 (BD Bioscience). Intracellular staining was performed after fixation and permeabilization using FoxP3 Fix/Perm Set (eBioscience). The NMR solution structure (PDB code 1AH1)E10Metzler W.J. Bajorath J. Fenderson W. Shaw S.Y. Constantine K.L. Naemura J. et al.Solution structure of human CTLA-4 and delineation of a CD80/CD86 binding site conserved in CD28.Nat Struct Biol. 1997; 4: 527-531Crossref PubMed Scopus (110) Google Scholar was used to construct the molecular representations, using the software VMD, version 1.9.1, developed by the National Institutes of Health Center for Biomolecular Modelling and Bioinformatics.E11Humphrey W. Dalke A. Schulten K. VMD—visual molecular dynamics.J Molec Graph. 1996; 14: 33-38Crossref PubMed Scopus (38418) Google Scholar The mutation was performed using the VMD plug in MUTATOR.Fig E23D reconstruction of wild-type and A86V variant CTLA-4.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig E3The alanine at position 86 of human CTLA-4 is highly conserved.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Table E1Next-generation sequencing results from the patient-derived DNAChr.BasepairRef.Alt.ZygosityGene symbolNucleotideAmino acidrsIDESPExAC1KGCADD- PHREDPolyPhen1PolyPhen2SIFT score2204735456CTHETCTLA4c.C257Tp.A86VRS3760387960.00010.00000.000224.210.9780.22247168856CGHETTTC7Ac.C176Gp.P59RRS201805434NA0.00450.0010NA0.0280.0080.5633281504CAHETTAPBPc.G175Tp.D59YRS455837370.00550.00510.003426.210.99606109796653GAHETZBTB24c.C1237Tp.R413CRS1496908230.00050.00030.0002350.9990.82809311975GAHETDOCK8c.G346Ap.V116MRS1434616440.00090.00090.000219.640.9940.7630.121KG, 1000 Genomes Project; Alt., alternative sequence; CADD, Combined Annotation Dependent Depletion; Chr., chromosome; ESP, NHLBI Grand Opportunity Exome Sequencing Project; ExAC, Exome Aggregation Consortium; HET, heterozygous; PolyPhen, Polymorphism Phenotyping; Ref., reference sequence; rsID, single nucleotide polymorphism database identifier; SIFT, Sorting Intolerant from Tolerant.Information regarding the CTLA-4 variant is shown in boldface. Open table in a new tab 1KG, 1000 Genomes Project; Alt., alternative sequence; CADD, Combined Annotation Dependent Depletion; Chr., chromosome; ESP, NHLBI Grand Opportunity Exome Sequencing Project; ExAC, Exome Aggregation Consortium; HET, heterozygous; PolyPhen, Polymorphism Phenotyping; Ref., reference sequence; rsID, single nucleotide polymorphism database identifier; SIFT, Sorting Intolerant from Tolerant. Information regarding the CTLA-4 variant is shown in boldface.