Lymphocytopenia-Associated Sarcoidosis: Cd4þ T-Cell Hypo-Responsiveness to IL-2 and Lectin

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Respiratory Medicine CME 3 (2010) 113–115

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Respiratory Medicine CME

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Case Report Lymphocytopenia-associated sarcoidosis: CD4þ T-cell hypo-responsiveness to IL-2 and lectin

Nathalie Freymond a, Christophe Malcus b, Yves Pacheco a, Gilles Devouassoux a,*

a Department of Respiratory Diseases, Bat 5F, Centre Hospitalier Lyon Sud, 165 Chemin du grand Revoyet, 69495 Pierre Be´nite, France b Laboratory of Immunology, HEH, HCL, and University Claude Bernard Lyon 1, France

article info abstract

Article history: Despite classical identification of immune response impairment in sarcoidosis, such as anergy to delayed Received 21 January 2009 skin tests, the characterization of contributing mechanisms are still uncertain and infectious events Accepted 1 April 2009 complicating the course of the disease are unfrequent. Rarely, additional quantitative T-cell defects are reported in association with the disease. Although mechanisms of lymphocyte disorders are unknown, lymphocytopenia is usually revealed by an opportunistic infection. Keywords: We report the case of an 18-year-old man, with pulmonary sarcoidosis, treated by corticoids, who Sarcoidosis developed cryptococcal meningitis. A CD4þ T-cell defect was simultaneously discovered, which was not Cryptococcus neoformans Opportunistic infection influenced by corticoid arrest, persisted following infection resolution and control of sarcoidosis. In vitro CD4-lymphocytopenia experiments were performed in parallel, demonstrating a restricted CD4þ T-cell proliferation hypo- IL-2 responsiveness to both IL-2 and lectin. In addition, rhIL-2 subcutaneously administrated failed to restore Mitogenic lectin peripheral T-cell count. A multi-visceral sarcoidosis associated to CD4-lymphocytopenia is rarely reported and highly demonstrative of the related risk for an opportunistic infection development. In context, the absence of lectin and IL-2 effects on in vitro CD4þ T-cell proliferation assays and the inefficiency of in vivo rhIL-2 on T-cell count have been infrequently reported and suggested the presence of a defective intracellular signaling pathway, responsible for T cell defect. Ó 2009 Elsevier Ltd. All rights reserved.

1. Introduction We reported a case report of pulmonary sarcoidosis, associated to a severe, persistent and quantitative CD4þ T-cell defect. This Sarcoidosis is characterized by the presence of diffuse non- latter was revealed by a Cryptococcus neoformans meningitis. Our caseating granulomas, which are constituted by a peripheral ring of data, showing an in vitro hypo-responsiveness of CD4þ T-cell to IL- T- and B-cells. This local inflammation in multiple organs is coun- 2 and phytohaemagglutinin (PHA), suggested the presence of terbalanced by an anergy to delayed tuberculin skin test.1–4 This alternative impaired intracellular signaling pathways-related T-cell dual immune response has been recognized for a long time and is in sarcoidosis. This hypothesis was reinforced by the absence of in considered as an unresolved immunological paradox. Recent vivo rhIL-2 effect on CD4þ T-cell count. advances in immunology have addressed this question and pointed out a role of regulatory CD4þ CD25þ FoxP3þ T-cell.5 Although the presence of this qualitative peripheral T-cell defect 2. Materials and methods is well-established in sarcoidosis, additional effects of corticoids on T-cell-associated immune response have been reported.6–9 Alter- 2.1. Assessment of T-cell count natively, a quantitative T-lymphocytopenia is rarely described in sarcoidosis, usually revealed by an opportunistic infection.9 EDTA whole blood was lysed by Optilyse reagent (Beckman- Mechanisms of this T-cell defect remain rarely investigated and no Coulter, Miami, USA), then incubated for 15 min with anti-CD4-FITC, definitive explanations have been provided. anti-CD8-PE and anti-CD3-PC5 monoclonal antibodies (BD, Pont de Claix, France). Stained cells were analyzed on a XL-MCL flow cytometer (Beckman-Coulter, Miami, USA). Total lymphocyte count was determined by an automated hematology analyzer and T-cell * Corresponding author. Tel.: þ33 4 78 56 90 72; fax: þ33 4 78 86 33 45. subsets were calculated as follows: (% CD3 or CD4 or CD8) total E-mail address: [email protected] (G. Devouassoux). lymphocyte count.

2.2. Isolation and proliferative response of PBMC to CD4þ T-cells count of 119 per cubic millimeter) and a primary phytohaemagglutinin (PHA) and IL2 prophylaxis against Pneumocystis jiroveci was started. Flucytosine, itraconazole and corticoids were definitively stopped 9 months, 3 PBMC were isolated by Ficoll-Paque density gradient centrifu- and 5 years later respectively. Despite a persistent CD4þ lympho- gation (PAA laboratories, Pasching, Austria), then cultured (106/ml) cytopenia, no other opportunistic infection occurred. at 37 C (5% CO2), in RPMI 1640 with 25 mM HEPES, 2 mM L- To explore pathogenesis of lymphocytopenia, T-cells were first glutamine (Invitrogen life technologies, Cergy Pontoise, France), tested in vitro for IL-2R expression by flow cytometry. Spontane- 10% human heat-inactivated AB serum (EFS, Lyon, France) and ously, CD25 (IL-2R a chain) and CD132 (IL-2R g chain) were antibiotics (Sigma-Aldrich, Lyon, France) in flat-bottom 96 well expressed by 3% and 91% of CD3þ cells respectively (normal ranges plates. PBMC were stimulated either for 48 h with 10 mg/ml of PHA <2% and 85–99% respectively). In the presence of PHA, a chain was (Oxoid, Dardilly, France) or for 5 days with 20 U/ml of rhIL-2 (Roche expressed by the total CD3 and CD4 cell populations. Secondly, Applied Science, Indianapolis, USA) with or without PHA. using pulsing cultures with [3H] thymidine, T-cell proliferation was To assess lymphocyte proliferation, 10 mCi/mL of [methyl-3H]- examined in the presence of PHA and/or IL-2. PHA alone was Thymidine [2,0 Ci/mmol] (GE Healthcare Europe, Orsay, France) responsible for an increased T-cell proliferation, which peaked at was added 24 h before harvesting cells on a fiberglass filter using an 48 h (16,100 620 cpm). Addition of IL-2 was associated with automated cell harvester. Incorporated radioactivity was measured a maintained T-cell proliferation at 5 days of culture in a direct beta counter (PerkinElmer life and Analytical Sciences, (15,306 986 cpm vs 6391 389 cpm). Identical results were Courtaboeuf, France). The results are expressed as mean cpm SD observed in normal control. Finally, employing flow cytometry, we of triplicates. sought to identify proliferation assay-related T-cell subtypes. At 5 days of culture, IL-2 induced a proliferation response of both CD3þ 2.3. Staining of PBMC and flow cytometry experiments and CD8þ cells in patient (63–90% and 46–91% respectively). Similar responses were observed using PHA or PHA þ IL-2 stimulations, in Freshly isolated PBMC were stained with anti-CD4-FITC, anti- both patient and control. By contrast, PHA or PHA þ IL-2 induced CD8-FITC, anti-CD25-PE or anti-CD132-PE and anti-CD3-PECy5 weak CD4þ cell proliferation responses (17–29%) and IL-2 alone was monoclonal antibodies (BD, Pont de Claix, France) for 15 min, then responsible for a depressed CD4 response in patient (17–2%). washed and resuspended in PBS containing 0.6% formaldehyde. Finally, following the control of both sarcoidosis and crypto- After 48 h (PHA) or 5 days (rhIL-2 or rhIL-2 þ PHA) of stimulation, coccal meningitis, a rhIL-2 therapy was initiated (95,000 IU subcu- PBMC were washed with PBS and stained with monoclonal anti- taneous) weekly. After 5 months of treatment, lymphocytopenia bodies, as previously described. Typically, 100,000 to 500,000 total and CD4þ cell counts were not modified and rhIL-2 was stopped. events were acquired on flow cytometer. 3. Discussion 2.4. Observation We reported a Cryptococcus neoformans meningitis, occurring in An 18-year-old white patient, non-smoker was hospitalized context of sarcoidosis and CD4þ T-lymphocytopenia. Although for an increasing dyspnea, mediastinal lymph nodes enlargement CD4þ T-cell defect was diagnosed during an opportunistic infection and pulmonary infiltrates on chest radiograph. CT scan confirmed and after months of corticoids treatment, lymphocytopenia the presence of mediastinal lymph nodes and alveolar opacities. The appeared independent of both steroids and fungal infection. serum angiotensin-converting enzyme was augmented (193 IU/l). Besides this case report, we described additional in vitro T-cell Anergy to tuberculin skin test was present. The broncho-alveolar investigations, suggesting 1) a normal profile of IL-2R expression by lavage fluid contained 86% macrophages and 14% lymphocytes (20% T-cells, 2) a normal total T-cell proliferation response and 3) an in CD4þ cells and 80% CD8þ cells). Pathological examination of vitro restricted hypo-responsiveness of CD4þ cells to both mito- inguinal lymph node was consistent with the diagnosis of sarcoid- genic lectin PHA and rIL-2 stimulations. This latter in vitro obser- osis. Corticoids were introduced (40 mg/d, one month), then vation was in line with the absence of in vivo rhIL-2 effect on progressively decreased until 20 mg/d. Three months later, the peripheral blood CD4þ T-lymphocytopenia. patient was asymptomatic and chest x ray was normalized. Infectious diseases occurring in sarcoidosis have been previ- Seven months later, he was admitted to Emergency department ously carefully investigated, suggesting a weak frequency of with fever, headache and vomiting. Brain magnetic resonance opportunistic infections.6–8 Additionally, an exhaustive review of imagery (MRI) showed multiple abscesses. Lumbar puncture yiel- 65 opportunistic infections complicating sarcoidosis underlined ded pleocytosis (107 cells per cubic millimeter, 70% of lymphocyte) a facilitating role of corticoids (50% of cases) and the over- and a protein concentration of 1 g/l. Staining with India ink revealed representation of Cryptococcus neoformans.9 encapsulated yeasts, consistent with Cryptococcus neoformans Extra-pulmonary cryptococcal infections are mainly described meningitis. Specific antigen was detected on peripheral blood and in immuno-compromised patients and HIV-seropositive patients cerebrospinal fluid. Additionally a total lymphocyte count of are particularly exposed.10 Several lines of evidence argue for 0.430 109/l, with a CD4þ T-cells count of 94 per cubic millimeter a critical role of T-cell-mediated immunity impairment in crypto- was discovered. Tests for HIV 1 and 2 antibodies were negative and coccal diseases. Firstly, cryptococcal meningitis occurs in immunoglobulins were normal. Corticoids were stopped and profoundly immunosuppressed patients.11 Secondly, prevalence of treatment with amphotericin B (70 mg/d) and flucytosine (8 g/d) infection has declined with the introduction of antiretroviral was initiated. Clinical outcomes improved progressively, and cere- therapy.12 Finally, cryptococcal infections have been reported in bral MRI was normalized at 6 weeks, leading to switch amphoter- other situations of T-cell defect, including autoimmune disease, icin B for itraconazole (200 mg/d). Lymphocytopenia remained idiopathic lymphocytopenia and sarcoidosis.13–15 unmodified during this period. At that time, a hypercalcemia and T-cell lymphocytopenia in sarcoidosis is thought to be a rare a granulomatous medullar infiltration argued for an active disorder, usually recognized in the presence of an infectious compli- sarcoidosis and corticosteroids were reinitiated (40 mg/d), with cation.9 Since peripheral T-cell count is rarely documented, an a rapid normalization of calcium abnormalities. However, lym- underestimation of T-cell defect in sarcoidosis is expected. Sarcoid- phocytopenia was not influenced (lymphocyte count of 0.59 109/l, osis-related peripheral T-cell lymphocytopenia is classically linked to N. Freymond et al. / Respiratory Medicine CME 3 (2010) 113–115 115

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