Cells and Reduced CD247 Expression Immunodeficiency With

A Novel Thymoma-Associated Immunodeficiency with Increased Naive T Cells and Reduced CD247 Expression

This information is current as Petros Christopoulos, Elaine P. Dopfer, Miroslav of October 1, 2021. Malkovsky, Philipp R. Esser, Hans-Eckart Schaefer, Alexander Marx, Sylvia Kock, Nicole Rupp, Myriam R. Lorenz, Klaus Schwarz, Jan Harder, Stefan F. Martin, Martin Werner, Christian Bogdan, Wolfgang W. A. Schamel and Paul Fisch

J Immunol 2015; 194:3045-3053; Prepublished online 2 Downloaded from March 2015; doi: 10.4049/jimmunol.1402805 http://www.jimmunol.org/content/194/7/3045 http://www.jimmunol.org/

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2015 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

A Novel Thymoma-Associated Immunodeﬁciency with Increased Naive T Cells and Reduced CD247 Expression

Petros Christopoulos,* Elaine P. Dopfer,†,‡,x Miroslav Malkovsky,{ Philipp R. Esser,‖ Hans-Eckart Schaefer,* Alexander Marx,# Sylvia Kock,* Nicole Rupp,* Myriam R. Lorenz,** Klaus Schwarz,**,†† Jan Harder,‡‡,1 Stefan F. Martin,‖ Martin Werner,* Christian Bogdan,xx Wolfgang W. A. Schamel,†,‡,x and Paul Fisch*

The mechanisms underlying thymoma-associated immunodeﬁciency are largely unknown, and the signiﬁcance of increased blood gd Τ cells often remains elusive. In this study we address these questions based on an index patient with thymoma, chronic visceral leishmaniasis, myasthenia gravis, and a marked increase of rare gd T cell subsets in the peripheral blood. This patient showed cutaneous anergy, even though he had normal numbers of peripheral blood total lymphocytes as well as CD4+ and CD8+ T cells.

Despite his chronic infection, analyses of immunophenotypes and spectratyping of his lymphocytes revealed an unusual accumu- Downloaded from lation of naive gd and ab T cells, suggesting a generalized T cell activation defect. Functional studies in vitro demonstrated substantially diminished IL-2 and IFN-g production following TCR stimulation of his “untouched” naive CD4+ T cells. Biochem- ical analysis revealed that his gd and ab T cells carried an altered TCR complex with reduced amounts of the z-chain (CD247). No mutations were found in the CD247 gene that encodes the homodimeric z protein. The diminished presence of CD247 and increased numbers of gd T cells were also observed in thymocyte populations obtained from three other thymoma patients. Thus, our findings describe a novel type of a clinically relevant acquired T cell immunodeficiency in thymoma patients that is distinct http://www.jimmunol.org/ from Good’s syndrome. Its characteristics are an accumulation of CD247-deficient, hyporresponsive naive gd and ab T cells and an increased susceptibility to infections. The Journal of Immunology, 2015, 194: 3045–3053.

hymomas are rare neoplasms of the thymic epithelium immunodeficiency (Good’s syndrome) (3), an absolute lympho- with an annual incidence of one to five per million. Diverse cytosis, or a relative increase in circulating naive T cells (4, 5). T immunologic abnormalities can complicate these tumors Additionally, an isolated T cell immunodeficiency with unknown through mechanisms that remain poorly understood. Common pathogenesis has been described that may be more frequent than by guest on October 1, 2021 examples are autoimmune manifestations, such as myasthenia classical Good’s syndrome (6–8). gravis and giant cell myocarditis (1, 2), combined B and T cell In healthy adults peripheral blood gd T cells comprise ,5% of the circulating CD3+ lymphocytes and belong primarily . *Department of Pathology, University of Freiburg Medical Center, 79106 Freiburg, ( 70%) to the Vg9Vd2 subtype (9, 10). Their TCR consists of Germany; †Department of Molecular Immunology, BIOSS Centre for Biological aTCRg and a TCRd chain, instead of the TCRab heterodimer Signalling Studies, Faculty of Biology, University of Freiburg Medical Center, expressed by ab T cells, and the signal-transducing CD3- 79106 Freiburg, Germany; ‡Center for Chronic Immunodeficiency CCI, University x ε of Freiburg Medical Center, 79106 Freiburg, Germany; Max-Planck-Institute of g/-d/- chains and z (CD247) molecules. Vg9Vd2 T cells rec- Immunobiology and Epigenetics, 79108 Freiburg, Germany; {Department of Medical ognize widely distributed Ags such as bacterial and synthetic Microbiology and Immunology, University of Wisconsin School of Medicine and ‖ phosphoantigens and alkylamines in a TCR-dependent but Public Health, Madison, WI 53726; Department of Dermatology and Venereology (Allergy Research Group), University of Freiburg Medical Center, 79106 Freiburg, MHC-unrestricted manner (11–13). Increased levels of blood Germany; #Institute of Pathology, University Medical Center Mannheim, 68167 Vg9Vd2 T cells have been reported in infections, such as Mannheim, Germany; **Institute for Transfusion Medicine, University Ulm, 89081 Ulm, Germany; ††Institute for Clinical Transfusion Medicine and Immunogenetics leishmaniasis, malaria, and tuberculosis, as well as following Ulm, German Red Cross Blood Service, Baden-Wurttemberg–Hessen,€ 89081 Ulm, bone marrow transplantation (9, 14). Less commonly seen are Germany; ‡‡Department of Gastroenterology, University of Freiburg Medical Center, xx increases in circulating Vd1 cells, which have mostly been 79106 Freiburg, Germany; and Institute of Clinical Microbiology, Immunology and Hygiene, Friedrich Alexander University Erlangen–Nuremberg and University described in viral infections such as CMV and HIV (9, 15). Vd1 Hospital Erlangen, 91054 Erlangen, Germany cells normally reside within epithelia as a part of immuno- 1Current address: Department of Medicine II, Gastroenterology, Hegau-Bodensee- surveillance measures against microbes and tumors. Common Klinikum, Singen, Germany. effector functions of both gd T cell subsets include cytotoxic- Received for publication November 5, 2014. Accepted for publication February 2, ity, cytokine production, and immunomodulation (9). Although 2015. their activities may occasionally appear to be redundant in the This work was supported by Deutsche Forschungsgemeinschaft Grants CRC620- normal immune system, their function could become important B6, CRC620-Z2, CRC643-A5, and EXC294 (to the BIOSS Centre for Biological Signalling Studies), Jose´ Carreras Leuka¨mie Stiftung Grant DJCLS R 05/01, and by in cases of immunodeficiency as implied by the observations of European Union Seventh Framework Programme 2007–2013 (SYBILLA project). increased numbers of gd T cells in patients with primary im- Address correspondence and reprint requests to Prof. Paul Fisch, Department of mune defects (16, 17). In this study we describe a novel T cell Pathology, University of Freiburg Medical Center, Breisacher Strasse 115a, 79106 immunodeficiency, in a patient with thymoma, chronic sys- Freiburg, Germany. E-mail address: paul.fi[email protected] temic leishmaniasis, and myasthenia gravis, characterized by The online version of this article contains supplemental material. normal numbers of PBLs containing expanded rare gd Tcell Copyright Ó 2015 by The American Association of Immunologists, Inc. 0022-1767/15/$25.00 subsets. www.jimmunol.org/cgi/doi/10.4049/jimmunol.1402805 3046 A NOVEL TYPE OF T CELL IMMUNODEFICIENCY IN THYMOMA

Materials and Methods quality of the cell purifications was confirmed by flow cytometry (purity . Case report 85%, data not shown). The purified cells were stimulated at a concentration of 105/well with 250 ng/ml PHA-P or with plate-coated anti-CD3 A previously healthy 35-y-old Sicilian salesman, designated patient B, (BioCoat 96-well plates; Becton Dickinson, Heidelberg, Germany) alone presented with a 2-mo history of fever and night sweats. Workup revealed or with additional soluble anti-CD28 at a concentration of 2 mg/ml for generalized lymphadenopathy with a mediastinal mass, hepatospleno- 48 h. Measurements of human IL-2 and IFN-g were performed by ELISA megaly, and pericardial and right pleural effusions. Immunological tests (BD OptEIA kits) using the manufacturer’s protocols. demonstrated cutaneous anergy against all seven recall Ags in the Multitest Me´rieux (Clostridium tetani and Corynebacterium diphtheria toxoids, T cell proliferation assay to Leishmania Ags tuberculin, Ags from Streptococcus group C, Proteus mirabilis, Candida gd T cells were purified from fresh PBMCs of patient B with MACS albicans, and Trichophyton mentagrophytes), normal numbers of total + 2 + + + microbeads, after which the purified gd and gd (ab ) T cell fractions circulating lymphocytes and CD4 (840/ml) and CD8 (528/ml) T cells, an were each cultured in flat-bottom 96-well plates in complete medium at expansion of gd T lymphocytes (30% of T cells), marked NK cell cyto- 5 3 104 (gd+)or105 (ab+) responding cells/well, with autologous irradiated penia (64/ml), and a polyclonal hypergammaglobulinemia (IgM 3.25 g/l PBMCs as APCs (105/well) and Leishmania donovani Ags in various [normal values 0.40–2.30], IgG 31.50 g/l [7.00–16.00], and IgA 4.46 g/l concentrations. L. infantum belongs to the L. donovani species complex [0.70–4.00]). No Abs against HIV or Toxoplasma gondii were detectable. (20). Controls were freshly isolated PBMCs from two healthy control Biopsies of cervical lymph nodes, bone marrow, and skin revealed abun- donors (N1, N2) that were assayed in parallel without cell separation. After dant Leishman–Donovan bodies inside macrophages, which expanded 72 h the wells were pulsed overnight with tritiated thymidine (1 mCi/well), dramatically during the following 2 y (Supplemental Figs. 1, 2). The harvested, and counted on a TopCount microplate scintillation counter complete absence of epithelioid cell transformation at all times was striking. (PerkinElmer, Rodgau-Jugesheim,€ Germany). Electron microscopy of the patient’s lymph nodes (Supplemental Fig. 2) confirmed massive infestation by a subspecies of Leishmania, which was Immunophenotyping identified as Leishmania infantum through culturing, PCR with parasite species determination via restriction fragment length polymorphism ana- Standard techniques of direct and indirect immunofluorescence in flow Downloaded from lyses, and serology (18, 19). Although fever, lymphadenopathy, and orga- cytometry were used with mAbs reactive against the epitopes indicated. The nomegaly rapidly subsided during and after treatment with liposomal Abs were anti-Vd1 (clones A13 and R9-12-6-2), anti-Vd2 (clone BB3), amphotericin B, the mediastinal mass remained unchanged and the patient anti-Vd3 (clone P8.6B1), anti-Vg9 (clone TigA), anti-Vg8 (clone R4.5.1), developed diplopia and muscle weakness. Serum acetylcholine receptor anti-Vg5 (clone 56.3), and anti-Vg4 (clone 23D12), of which the clones Abs were diagnostic for myasthenia gravis, and a biopsy of the mediastinal BB3 and A13 were gifts from L. Moretta and A. Moretta (Genoa, Italy), mass revealed a type B2 thymoma, with invasion of the pericardium and the the clone 23D12 was a gift from D. Wesch and D. Kabelitz (Kiel, Germany), right pleura. Steroids and pyridostigmine were administered, as well as six and the other mAbs were gifts from M. Bonneville (Nantes, France). The http://www.jimmunol.org/ cycles of VIP-E chemotherapy (etoposide, ifosfamide, cisplatin, and epi- fluorescence-labeled anti-TCRgd mAb(clone515)aswellasmAbsreactive rubicin) followed by tumor resection. A histologically confirmed pleural against CD3, CD4, CD27, CD45, and CD45RA were purchased from relapse of the thymoma 1 y later was treated with two additional VIP-E Beckman Coulter (Krefeld, Germany). courses and radiotherapy. Thereafter, the thymoma remained stable, but the patient continued to develop relapses of his leishmaniasis, despite normal Spectratyping PBLs (1850–3400/ml) and CD4+ and CD8+ T cell counts. During his TCR b-, g-, and d-chain spectratyping was performed as described (17, periods of exacerbated leishmaniasis, low numbers of NK cells (,100/ml) 21). were observed, which rose to .150–200/ml after longer remission intervals. He also suffered from approximately four acute respiratory tract infections Western blotting per year, but no chronic infections other than the visceral leishmaniasis. The leishmaniasis relapses were successfully treated with liposomal amphoter- Freshly isolated PBMCs or PHA-P and IL-2–expanded PBMCs from pa- by guest on October 1, 2021 icin B, pentavalent antimony, INF-g, or oral miltefosine. Approximately 9 y tient B and healthy donors were lysed in buffer containing 1% digitonin after the initial diagnosis, the patient developed heart failure and died of (22). The gd and ab TCRs were sequentially affinity purified. In the first cardiogenic shock a few months later. At autopsy, giant cell myocarditis and immunoprecipitation, 2 mg anti-TCRd1 mAb and protein A/G–coupled lymphocytic myositis were noted. Leishmania amastigotes were still de- Sepharose (GE Healthcare, Freiburg, Germany) were used. Then, residual tectable in some of the bone marrow macrophages, although the thymoma Ab was cleared from the lysate by overnight incubation with excess protein had not progressed. A/G–coupled Sepharose. Subsequently, a second purification step with 2 mg TCRb-chain–specific mAb Jovi1 (Abcam, Cambridge, U.K.) and Blood samples and cell culture protein A/G–coupled Sepharose was performed. Purified TCRs were washed three times with buffer containing 0.1% digitonin, subjected to Following informed consent we obtained many blood samples from patient reducing SDS-PAGE, and visualized by Western blotting using anti-CD3ε B and from healthy volunteer donors during an interval of .7 y. PBMCs were (M20, Santa Cruz Biotechnology), anti-CD3d (M20d, Santa Cruz Bio- isolated on Ficoll gradients (Lymphoprep; Progen Biotechnik, Heidelberg, technology), and anti-CD247 Abs (449, rabbit polyclonal). The 449 serum Germany). Additionally, we used cryopreserved samples of thymocytes was produced as described (23). A similar analysis was performed for the from three other patients with World Health Organization type B2 thymoma CD3ε and CD247 chains after immunoprecipitation of the TCR with and myasthenia gravis. Purified ab and gd T cell subsets were cultured in OKT3 mAb in cryopreserved thymocytes from thymomas and normal IMDM (Life Technologies/Invitrogen, Karlsruhe, Germany) supplemented thymi or in MACS-purified naive and memory T cells from healthy donors. with heat-inactivated 10% human AB serum (PAN-Biotech, Aidenbach, Germany) as well as L-glutamine, penicillin, and streptomycin (all from Sequencing of the CD247 gene Life Technologies) (“complete medium”). Coding sequences and exon/intron boundaries of the genomic CD247 CFSE proliferation assay (CD3Z) gene were amplified using the Taq polymerase system (Qiagen). PCR products were sequenced directly using the BigDye Terminator v3.1 For the CFSE proliferation assay, freshly obtained PBMCs from patient B cycle sequencing kit on an ABI Prism 3130XL genetic analyzer (Life and healthy controls were labeled with CFSE (5 mM CFSE in complete Technologies, Darmstadt, Germany). The primer sequences are given in medium at 37˚C for 2 min, followed by washing in cold complete medium) Supplemental Table I. and stimulated in complete medium with PHA-P (250 ng/ml; Oxoid, Wesel, Germany) and IL-2 (100 IU/ml; Roche, Mannheim, Germany) in a round-bottom 96-well plate (5 3 104/well). At 48 and 120 h cells were Results harvested and analyzed by flow cytometry by gating on DAPI2 ab and gd Expansion of naive gd and ab T cells in the peripheral blood T cells. of patient B Cytokine ELISA In repeated measurements, patient B had up to 30% or 340–680/ml circulating gd T cells (normally ,5% of circulating T cells or For measuring cytokine release we used cryopreserved PBMCs from patient , ∼ B and healthy controls that were thawed followed by purification of neg- 100/ml) with a subset composition of 60% Vd1-, 22% Vd2-, atively selected (“untouched”) naive CD4+ TCRab T cells using magnetic and 18% Vd3-expressing cells. Similarly, analysis of TCRg var- microbeads (MACS; Miltenyi Biotec, Bergisch Gladbach, Germany). The iable chain expression using a set of commercially unavailable The Journal of Immunology 3047 mAbs demonstrated a polyclonal gd T cell expansion (20% Vg9-, expression of HLA-DR (,4%) on the cell surface, no expression 24% Vg8-, 20% Vg5-, and 28% Vg4-expressing cells, data not of NK inhibitory receptors, such as the NKG2A-CD94 hetero- shown). Most of patient B’s gd and ab T cells showed a naive, dimer, LIR1 (CD85j), and various killer Ig receptors, with no nonactivated phenotype (76 and 80% CD45RA+CD27+, Fig. 1A, detectable NK-like cytotoxicity against K562 target cells in 51Cr- 1B), in contrast to healthy donors (,50% CD45RA+CD27+), as release assays (data not shown). By spectratyping, the expanded also described by others (24). His gd T cells displayed very low gd T cells featured a highly unusual, almost Gaussian distribution Downloaded from http://www.jimmunol.org/ by guest on October 1, 2021

FIGURE 1. Expansion of naive gd and ab T cells in patient B. (A) Naive (CD45RA+CD27+) and memory subsets among gd T cells from patient B and a healthy donor (N1). (B) Naive (CD45RA+CD27+) and memory subsets among ab T cells from patient B and a healthy donor (N1). (C) TCRd spectratyping of fresh lymphocytes from patient B and two healthy donors (N1, N2). The red peaks represent m.w. standards. 3048 A NOVEL TYPE OF T CELL IMMUNODEFICIENCY IN THYMOMA of the CDR3 lengths across the complete Vg and Vd repertoire, in contrast to healthy donors (Fig. 1C and data not shown). Similarly, analysis of the TCRb variable gene repertoire by spectratyping showed only minimal distortions of the Gaussian CDR3 length distributions (Supplemental Fig. 3). This very unusual Gaussian-type distribution of the gd T cell repertoire was reproducible in six blood samples obtained from the patient during a 5-y period.

Naive T cells are functionally impaired whereas in vivo–primed ab T cells react against Leishmania Ags The clinically observed immunodeﬁciency with cutaneous anergy and multiple relapses of leishmaniasis in patient B, as well as his naive T cell repertoire and phenotype, suggested a generalized defect of T cell function. Thus, we compared the ability of untouched naive CD4+ T cells from this patient to produce cytokines after polyclonal stimulation ex vivo compared with those from healthy donors. Production of IL-2 and IFN-g was signiﬁcantly impaired after a 48-h ex vivo stimulation with PHA and plastic- Downloaded from coated anti-CD3 mAb (Fig. 2). Upon additional costimulation by soluble anti-CD28 mAb, the patient’s cells showed normal production of IL-2, the main cytokine produced by naive T cells, but still showed suboptimal IFN-g responses. T cell proliferation in response to strong stimulators during longer periods of incubation appeared normal. Following 5 d of stimulation by PHA and IL-2, +

FIGURE 2. Cytokine production of puriﬁed untouched naive CD4 cells http://www.jimmunol.org/ patient B’s CFSE-labeled PBMCs showed upregulation of HLA- from patient B and healthy donors. (A) The concentrations of IL-2 pro- DR and vigorous proliferation of the ab and gd T cell subsets duced by untouched naive CD4+ cells from patient B and two healthy + + (including the CD45RA CD27 fractions) similar to correspond- donors after 48 h of stimulation with PHA-P (open bars) and coated anti- ing responses in healthy donors (Fig. 3A, 3B and not shown CD3 mAb with (black bars) or without (gray bars) soluble anti-CD28 mAb data). Freshly obtained T cells from patient B mediated immune measured in the cell culture supernatants. (B) The concentrations of IFN-g + responses to Leishmania Ags in vitro with secondary type kinetics. produced by untouched naive CD4 cells from patient B and two healthy There was considerable tritiated thymidine incorporation on day 3 donors after 48 h of stimulation with PHA-P with (shaded bars) or without after stimulation, whereas unprimed normal donors showed much (open bars) IL-2 as well as with coated anti-CD3 with (black bars) or without (gray bars) soluble anti-CD28 measured in the cell culture

milder proliferative responses, which was compatible with the by guest on October 1, 2021 supernatants. The values following stimulation with medium alone were presence of unprimed cells (Fig. 3C). Proliferation of patient B’s negligible (between 0 and 1 pg/ml). Assays were performed in triplicates PBMCs to Leishmania Ags in vitro was exclusively mediated by and statistical comparisons were with the Student t test, with error bars his ab T cells. These results suggest a defect in TCR-dependent depicting the SEM. activation of naive T cells, which could be partially reversed by stronger stimulatory signals or longer in vitro incubations, whereas in vivo–primed ab T cells responded to Leishmania Ags glycosylation, was also detected in the patient’s ab T cells in vitro quite effectively. (Fig. 4B, lane 1). All alterations of gd and ab TCR-CD3 com- Biochemical analyses of the TCR from patient B and additional ponents were restored following T cell activation by PHA-P and thymoma patients IL-2 treatment for 5 d (Fig. 4A, 4B, lanes 4–6). This T cell activation defect might be caused by an aberrant We next examined whether this CD247 defect might be at- TCR complex, which normally has a TCRabCD3εgεdzz or tributable to the predominantly naive phenotype of the cells from TCRgdCD3εgεdzz stoichiometry (25, 26). To test this possibility, patient B. When comparing the amount of z (CD247) protein ε we isolated the TCR complexes and quantified the amount of relative to the amount of CD3 protein in the TCR complexes copurified CD3 subunits by Western blotting separately for precipitated from naive and memory T cells of healthy donors, TCRgd and TCRab (see Fig. 5A, 5B). Larger amounts of CD3ε we found no apparent differences (Fig. 4C). Thus, reduction of and CD3d were obtained with anti-TCRgd immunopurification CD247 within the TCR complexes of naive T cells from patient from lymphocytes of patient B (Fig. 4A, lanes 1 and 4) than from B could be a consequence of the thymoma. Indeed, we found healthy donors (lanes 2, 3, 5, and 6), which reflects the higher that the TCR complexes of thymocytes isolated from an ad- percentage of gd T cells in patient B. Remarkably, the TCRgd ditional three thymoma patients contained less CD247 than did complex from the PBMC preparations of patient B contained less thymocytes from three normal thymuses (Fig. 5A). Moreover, z (CD247) protein than did the controls relative to the amount of the thymomas contained more thymocytes expressing the copurified CD3ε protein (lanes 1–3). Additionally, PBMC-derived TCRgd than the normal thymi, as determined by flow cytom- CD3d from the controls was detected in two bands (closed and etry (Fig. 5B). open arrows, lanes 2 and 3), whereas only the slower migrating, higher m.w. band of CD3d was present in the patient’s gd T cells Sequencing of the CD247 gene from patient B (open arrow, lane 1). Because these bands most likely represent Following isolation of genomic DNA from purified gd T cells from different CD3d glycosylation isoforms, practically all gd T cells patient B, the eight exons with and without 10 bp intronic se- of patient B contained a highly glycosylated CD3d isoform. quence of the CD247 gene (encoding the z-chain [CD247]) were Importantly, the CD247 association defect, but not the peculiar sequenced. No mutations were detected. The Journal of Immunology 3049 Downloaded from http://www.jimmunol.org/ by guest on October 1, 2021

FIGURE 3. Ex vivo proliferation of gd and ab T cells from patient B after polyclonal stimulation and exposure to Leishmania Ags. (A) Proliferation (CFSE dilution) of gd T cells from patient B and a healthy donor (N1) after 5 d of ex vivo stimulation with PHA-P plus IL-2 by ﬂow cytometry, gated on 2 DAPI cells. (B) Proliferation (CFSE dilution) of ab T cells from patient B and a healthy donor (N1) in the same experiment as (Figure legend continues) 3050 A NOVEL TYPE OF T CELL IMMUNODEFICIENCY IN THYMOMA

FIGURE 4. TCR subunit analysis of primary and expanded gd and ab T cells from patient B and healthy donors. (A) Western blotting of the CD3ε, CD3d, and CD247 chains contained in immunoprecipitated gd TCRs from freshly isolated (PBMC) or PHA- and IL-2–expanded PBMCs (expan.) of patient B (Pt B) and healthy donors (N1 and N2). The open and closed arrows indicate two different glycosylation forms of CD3d in the gd TCR. This experiment was repeated three times. (B) Western blotting of the CD3ε, CD3d, and CD247 chains contained in immunoprecipitated TCRab from freshly isolated (PBMC) or PHA- and IL-2–expanded PBMCs (expan.) of patient B (Pt B) and healthy donors (N1 and N2). (C) Western blotting of the CD3ε and CD247 chains contained in immunoprecipitated TCRs from puriﬁed naive and memory Downloaded from T cells from two healthy donors.

Discussion microorganisms. A lack of exposure is supported by the fact that http://www.jimmunol.org/ Thymoma patients can develop clinically overt immune deficiency, the patient had no Abs against T. gondii and, therefore, belonged but the underlying pathophysiology is largely unknown (3). A to the ∼60–70% seronegative persons in his age group that do not relative increase in circulating naive T cells in thymoma patients harbor persistent Toxoplasma parasites. has been described, but the mechanism and clinical significance In vitro functional studies confirmed the presence of a T cell remain unknown (4). In this study, we address these questions activation defect, because this patient’s untouched naive CD4+ based on observations in a patient with thymoma, chronic leish- T cells showed markedly diminished IFN-g and IL-2 production maniasis, and an expansion of rare gd T cell subsets in the pe- following stimulation by PHA or anti-CD3, in comparison with ripheral blood in the presence of normal numbers of circulating cells from healthy donors (Fig. 2). However, unselected fresh lymphocytes as well as CD4 and CD8 T cells. Clinically, the di- PBMCs from patient B proliferated quite strongly following lon- by guest on October 1, 2021 agnosis of immunodeficiency in this patient was based on cutane- ger polyclonal stimulations by PHA and IL-2 in vitro (Fig. 3A, ous anergy and the fulminant clinical presentation of leishmaniasis 3B). Additionally, his unselected PBMCs showed at least partially with multiple relapses in the absence of immunosuppressive ther- effective secondary-type responses to Leishmania Ags in vitro apy. Histomorphological evidence for immunodeficiency was that (Fig. 3C). However, one cannot conclude that these memory ab this patient’s leishmania-infected macrophages completely lacked T cell responses to Leishmania were entirely normal, because no signs of epithelioid cell transformation in all biopsy specimens immune competent control patients with previous exposure to and at autopsy. Because such activated macrophages typically Leishmania Ags were available for comparison. In fact, these appear in Th cell–induced granulomas, this absence of epithelioid secondary responses to Leishmania might represent an “old” cell transformation in the infected lymph nodes of our patient memory response that was generated when patient B was still documents a Th cell defect. Despite his chronic infection, most gd thymoma-free. It is also unlikely that the observed functional and ab T cells of this patient displayed a naive phenotype (Fig. abnormalities of naive ab T cells in this patient were due to the 1A, 1B) and Gaussian spectratypes (Fig. 1C, Supplemental Fig. 3), naive gd T cell population, because negatively purified helper ab which suggested a generalized defect in T cell activation. The T cells displayed defective cytokine production after stimulation low NK cell numbers fluctuating with disease activity could be in vitro (Fig. 2) and because the strongest proliferative responses explained as a reaction to leishmaniasis (27). The clinical obser- to Leishmania Ags were observed with unseparated patient B vation that the patient only suffered from relapses of visceral PBMCs (Fig. 3C). Taken together, these data suggest a mild defect leishmaniasis, but did not develop persistent or recurrent infec- of lymphocyte activation in patient B as the basis for his immu- tions with other pathogens, might indicate impairment of his im- nodeficiency, mainly involving lymphokine production. Notably, munosurveillance against only certain intracellular microbes. He IFN-g plays a crucial role in the defense against leishmania and the presumably became infected with Leishmania in Sicily or while other intracellular pathogens (28). traveling to Africa as a salesman. Nevertheless, it is possible that In search of a biochemical correlate of this defect, we analyzed had he been exposed to other intracellular pathogens such as the TCR complex of lymphocytes from patient B and found re- Mycobacteria spp., Salmonella spp., T. gondii, Cryptococcus duced amounts of CD247 in his gd and ab T cells (Fig. 4). The neoformans,orHistoplasma capsulatum, he might have also CD247 molecule, the subunit that is added last to the TCR during developed chronic clinically significant infections with these assembly (29), is a critical signal transduction component that is

described above. (C) Proliferation ([3H]thymidine incorporation) of gd and ab T cells from patient B and two healthy donors (N1, N2) after 72 h of exposure to increasing concentrations of Leishmania Ags. Assays were performed in quadruplicates, with error bars depicting the SEs of the mean. The Journal of Immunology 3051

FIGURE 5. TCR subunit and flow cytometric analysis of thymocytes from thymoma patients and healthy donors. (A) Western blotting of the CD3ε and CD247 chains from the thymocytes of three patients with World Health Organization type B2 thymomas and myasthenia gravis (Pt1–3, other than Pt B) and three healthy donors (N1–3). The inten- sities of the precipitated CD3ε and CD247 subunits were quantified using the LI-COR Odyssey system, and the CD247/CD3ε ratios were compared using the Student t test, with error bars depicting the SEs of the mean. This experiment was repeated with similar results. (B) Cryopreserved thymocytes from the same three thymomas and normal thymi were analyzed by flow cytometry to quantify percentages of gd+ T cells within the CD3+ T cells (Student t Downloaded from test, with the bars depicting mean values). http://www.jimmunol.org/ massively phosphorylated upon Ag recognition, and its down- thymoma tissues compared with normal thymic grafts (44). A regulation results in impairment of TCR signaling (30). Such thymic origin for the peripheral gd T cells in patient B is directly a defect has been described as a consequence of chronic inflam- implied by their naive phenotype, the Gaussian-like (naive) CDR3 mation in patients with cancers, infections, autoimmune disorders lengths distributions, and because they mostly expressed rare (31–34), including systemic lupus erythematosus (35, 36), or in variable Vg and Vd genes such as Vg4, Vg5, and Vg8, combined patients undergoing hemodialysis (37), but not in naive T cells, with Vd1 and Vd3 (detected by mAbs) and Vd4, Vd5, and Vd6 such as the lymphocytes of patient B. The naive phenotype by (detected by spectratyping, data not shown). These rare gd T cell itself cannot explain the reduced CD247 chain content in subsets are normally replenished through thymic T cell develop- by guest on October 1, 2021 lymphocytes from patient B because naive and memory T cells ment, in contrast to the more common Vg9Vd2 T cells, which from healthy donors contained similar amounts of CD247 (Fig generally expand after activation in the periphery (24, 45). 4C), confirming results from earlier studies (38). Besides, the Impairment of T cell function with thymoma is commonly reduction of CD247 in patient B’s lymphocytes was associated described as Good’s syndrome, together with hypogammaglobu- with a higher degree of glycosylation of the TCRgd CD3d com- linemia and reduced or absent B cells (3, 46). However, our ponent (25). All TCR alterations in patient B were restored fol- patient with hypergammaglobulinemia and normal numbers of lowing prolonged T cell activation by PHA-P and IL-2 (Fig. 4A, B cells clearly did not have Good’s syndrome, which typically 4B), which parallels the restoration of CD247 expression in manifests itself by susceptibility to encapsulated bacteria, as well tumor-infiltrating lymphocytes by IL-2 (39). Thus, these abnor- as opportunistic viral and fungal infections. Extrapolating from malities were most likely acquired rather than genetically deter- the literature, an isolated T cell immunodeficiency appears to be mined (40), as also indicated by the lack of mutations in the even more common in thymoma patients (6–8) than classical CD247 (CD3Z) gene of patient B. Good’s syndrome (46). Our findings extend the observation that A causal relationship of the CD247 defect with thymomas is patients with thymoma have increased percentages of naive T cells strongly suggested by the observation that the reduction of CD247 (4, 47), and they also suggest that the diminished presence of expression was present in thymocytes from other thymoma CD247 in the TCR complex in thymoma patients may lead to patients, but not in normal thymocytes (Fig. 5A). Thus, the CD247 T cell immunodeficiency and opportunistic infections with intra- abnormality is not just a special characteristic of our patient cellular pathogens, such as Cryptococcus (8), tuberculosis (48), secondary to the Leishmania infection, but it seems to represent and salmonellosis (49). Similar to our patient B, the clinical a more general mechanism of thymoma-related immune dysreg- presentation in each of these case reports was dominated by ulation, which may underlie the functional T cell defects in other a single infection. patients with thymomas as well (7, 8). INF-g, TNF, myeloid How can the expansion of naive gd T cells, the presence of suppressor cells, and decreased levels of L-arginine have been functionally impaired naive ab T cells, and the CD247 defect be reported to downregulate CD247 (30, 31, 41–43). Thus, the reconciled? As shown above, the reduced CD247 content is not microenvironemnt in some thymomas could contribute to the typical for normal naive T cells, but it appears to be related to observed CD247 dysregulation. The accumulation of rare naive gd T cell development in patients with thymomas. This CD247 ab- T cells in the blood of patient B is very likely attributable to his normality may lead to the accumulation of naive T cells due to the thymoma. We found that gd T cells were also increased in thy- inability of these cells to reach the activation threshold and attain mocytes from other thymoma patients as well (Fig. 5B), whereas the memory state under physiological conditions of stimulation. others described a preferential development of human gd T cells The naive phenotype in patient B persisted for years despite in humanized SCID mice following transplantation of human multiple reactivations of leishmaniasis, which is compatible with 3052 A NOVEL TYPE OF T CELL IMMUNODEFICIENCY IN THYMOMA the observations of increased circulating naive T cells in thymoma 12. Vavassori, S., A. Kumar, G. S. Wan, G. S. Ramanjaneyulu, M. Cavallari, S. El Daker, T. Beddoe, A. Theodossis, N. K. Williams, E. Gostick, et al. 2013. patients and in patients with paraneoplastic myasthenia gravis (4, Butyrophilin 3A1 binds phosphorylated antigens and stimulates human gd 47). Strong TCR signals during intrathymic T cell maturation at T cells. Nat. Immunol. 14: 908–916. the double-negative stage are thought to promote gd Tcelldif- 13. Fisch, P., M. Malkovsky, S. Kovats, E. Sturm, E. Braakman, B. S. Klein, S. D. Voss, L. W. Morrissey, R. DeMars, W. J. Welch, et al. 1990. Recognition by ferentiation (50, 51), whereas excessive TCR stimulation of human V gamma 9/V delta 2 T cells of a GroEL homolog on Daudi Burkitt’s mature T cells induces the CD247 defect in patients with chronic lymphoma cells. Science 250: 1269–1273. immune activation (30, 31). Thus, the neoplastic thymic epi- 14. van der Harst, D., A. Brand, S. A. van Luxemburg-Heijs, Y. M. Kooij-Winkelaar, F. E. Zwaan, and F. Koning. 1991. 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current taxonomy. Proc. Natl. Acad. Sci. USA 104: 9375–9380. http://www.jimmunol.org/ ficiency and immune dysregulation of clinical significance. 21. Pannetier, C., J. P. Levraud, A. Lim, J. Even, and P. Kourilsky. 1998. The Immunoscope approach for the analysis of T-cell repertoires. In The Antigen T-Cell Receptor: Selected Protocols and Applications. J. R. Oksenberg, ed. Acknowledgments Chapman & Hall, New York, p. 287–325. We thank Joachim Clos for the gifts of L. donovani Ags, Lorenzo and 22. Minguet, S., M. Swamy, B. Alarcoń, I. F. Luescher, and W. W. Schamel. 2007. Alessandro Moretta, Daniela Wesch, and Dieter Kabelitz as well as Marc Full activation of the T cell receptor requires both clustering and conformational changes at CD3. Immunity 26: 43–54. Bonneville for many anti-TCRgd Abs. We also thank Daniela Bukatz and 23. San Jose´, E., A. G. Sahuquillo, R. Bragado, and B. Alarcoń. 1998. Assembly of Sabine Glatzel for expert technical assistance, Marie Follo for carefully the TCR/CD3 complex: CD3 ε/d and CD3 ε/g dimers associate indistinctly with reading the manuscript, Annette Schmitt-Gra¨ff for helpful discussions, both TCR a and TCR b chains. Evidence for a double TCR heterodimer model. Alexandros Spyridonidis for taking care of the patient and clinical support Eur. J. Immunol. 28: 12–21.

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