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Prigione, Ignazia PhD*; Castagnola, Elio MD; Imberti, Luisa MD; Gambini, Claudio MD§; Gradoni, Luigi PhD; Dianzani, Umberto MD; Ramenghi, Ugo MD**; Giacopelli, Francesca PhD††; Moretta, Alessandro MD‡‡; Moretta, Lorenzo MD§§; Plebani, Alessandro MD¶¶; Fischer, Alain MD∥∥; Pistoia, Vito MD*

The Pediatric Infectious Disease Journal: February 2009 - Volume 28 - Issue 2 - p 161-163
doi: 10.1097/INF.0b013e318185513d
Brief Reports

An adolescent with idiopathic CD4+ lymphocytopenia suffered from 4 visceral leishmaniasis relapses despite appropriate treatment. CD8 lymphocytopenia and abnormal expansion of TCRαβ+, CD4, CD8 cells were consistently detected together with reduced export of mature T cells from thymus. This novel form of idiopathic CD4+ lymphocytopenia may predispose to multiple visceral leishmaniasis relapses.

From the *Laboratory of Oncology and †Unit of Infectious Diseases, G.Gaslini Scientific Institute, Genova Italy; ‡Department of Laboratory Diagnostics, Terzo Servizio Analisi, Spedali Civili of Brescia, Brescia, Italy; §Laboratory of Pathology, G.Gaslini Scientific Institute, Genova, Italy; ¶Unit of Vector-borne Diseases and International Health, MIPI Department, Istituto Superiore di Sanità, Rome, Italy; ∥Department of Medical Sciences, University of Eastern Piedmont, Novara, Italy; **Department of Pediatrics, University of Turin, Torino, Italy; ††Laboratory of Molecular Genetics, G.Gaslini Scientific Institute, Genova, Italy; ‡‡Department of Experimental Medicine and Centro di Eccellenza per le Ricerche Biomediche, University of Genoa, Genova, Italy; §§Laboratory of Experimental and Clinical Immunology, G.Gaslini Institute and Centro di Eccellenza per le Ricerche Biomediche, University of Genoa, Genova; ¶¶Department of Pediatrics, “Angelo Nocivelli” Institute of Molecular Medicine, University of Brescia, Brescia, Italy; and ∥∥Unité d'Immunologie et Hématologie Pédiatrique, Hôpital Necker-Enfants Malades, Paris, France.

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Accepted for publication July 1, 2008.

Supported by grants from Ministero della Salute, Progetti di Ricerca Corrente 2006.

Ignazia Prigione, PhD, and Elio Castagnola, MD, are shared first authors.

Address for correspondence: Ignazia Prigione, PhD, Laboratory of Oncology, G.Gaslini Institute, Largo G.Gaslini 5, 16148 Genova, Italy. E-mail:

Idiopathic CD4+ T lymphocytopenia (ICL) is defined as a persistent CD4+ T cell count lower than 300 cells/μL in the absence of HIV infection or other known causes of immunodeficiency.1 ICL may be accompanied by CD8, T, B, or NK lymphocytopenia, thus mimicking other immunodeficiencies. ICL patients suffer from opportunistic infections caused by intracellular pathogens.2

Visceral leishmaniasis (VL) is caused by Leishmania infantum in the Mediterranean region.3 The majority of infected people never develop disease and symptomatic infection in immunocompetent individuals is eradicated by liposomal amphotericin B.4 Multiple VL relapses are exceedingly rare and associated with immunodeficiency.5 The first case of ICL associated with relapsing VL has been published recently,6 but detailed immunologic studies were not performed.

Here we report an Italian adolescent suffering from multiple VL relapses in the absence of other opportunistic infections. Immunologic and molecular investigations revealed a novel form of ICL characterized by previously unrecognized abnormalities.

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Peripheral blood and serum samples were obtained following informed consent according to the Helsinki declaration. Peripheral blood mononuclear cells (PBMNC) were isolated by Ficoll-Hypaque density gradients.7 Lymph node cell suspension was prepared by mechanical disaggregation (Medimachine-DakoCytomation Denmark A/S, Glostrup, Denmark). Cells were stained with fluorochrome-conjugate mAbs (BD-Biosciences, San Diego, CA) and analyzed by flow cytometry (FACSCalibur, BD, Cellquest Software).7 Absolute cell counts (cells/μL) were obtained by multiplying the percentage of antibody stained cells by total lymphocyte counts. Intracellular cytokine staining was performed using anti-IFN-γ-FITC and anti-IL-4-PE mAbs (Caltag, Burlingame, CA), as reported.7

Formalin-fixed, paraffin-embedded lymph node tissue sections were stained with hematoxylin-eosin for histology and immunoperoxidase for immunohistochemistry; mAbs were from Dako, Glostrup, Denmark.

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Case Report.

D.V. was admitted to the G.Gaslini Children's Hospital in 2003 at the age of 11. She had been diagnosed with VL in June 2002 and had received liposomal amphotericin B (3 mg/kg for 6 doses).4 The patient relapsed 6 months later and was treated with the same drug (3 mg/kg for 10 doses) with apparent benefit.

Upon admission, the patient was asymptomatic, in good clinical condition, and with normal growth according to WHO Child Growth Standards for age and sex. Lymphocytopenia (500 cells/μL) and a serum M component (IgG λ) were detected. The patient had severe CD4 lymphocytopenia, CD8 lymphocytopenia, and abnormally expanded CD3+, TCRαβ+, CD4, CD8 cells (Fig. 1, panel A), all of which were detected over a long term follow-up (Fig. 1, panel B). Retrospective analyses showed that lymphocytopenia dated back to 1996. At that time, no clinical and laboratory evidence for VL was detected and the patient was discharged without any treatment.



In 2005, the patient presented increasing asthenia and was readmitted for evaluation. She had mild anemia (hemoglobin 11 g/dL), anti-leishmania antibody titer was 1:2560 (immunofluorescence assay), and the serum M component was still present. A new VL relapse was suspected and liposomal amphotericin B, 3 mg/kg for 10 doses followed by 1 weekly dose for 4 weeks, was administered. Clinical conditions improved and antileishmania antibody titer decreased to 1:640. At that time the presence of an underlying immunodeficiency was suspected. HIV infection was excluded because of the absence of specific antibodies and viral RNA in serum and other causes were investigated (see below for immunologic studies).

In February 2006, the patient presented enlarged cervical lymph nodes and Leishmania was detected in a lymph node biopsy, but not in a bone marrow aspirate. Lymph node architecture was altered by huge plasmablastic and histiocytic infiltration of the paracortical area (Fig. 1, panel C1). Some histiocytes were infected with Leishmania parasites (Fig. 1, panel C1, inset). The histiocytic (CD68+)/plasmablastic (CD138+) infiltrate compressed and obliterated the majority of lymphoid follicles (Fig. 1, panels C2–C4). CD3+ T cells had a perifollicular distribution, while CD20+ B cells were observed in the follicles (Fig. 1, panel C5 and C6). The lymph node CD4/CD8 ratio (Fig. 2, panel A; was 0.5:1, while it ranged from 3:1 to 5:1 in reactive lymph nodes from 10 age-matched patients. Most T cells expressed HLA-DR antigens (not shown) pointing to their activated state. CD19+, CD20, CD138+ plasmablasts were detected by flow cytometry (Fig. 2, panel A; Antileishmania antibody titer was 1:5120. Liposomal amphotericin B (3 mg/kg for 10 doses followed by 1 weekly dose for 4 weeks) was administered in combination with allopurinol (15 mg/kg/daily for 2 months).

In July 2007 the patient was in good clinical conditions without lymphadenopathy. Antileishmania antibody titer was 1:1280.

In April 2008 the patient presented enlargement of tonsils and adenoids and Leishmania was still detected by histologic studies. She is still receiving a further course of liposomal amphotericin B, with apparent improvement.

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Immunologic Studies.

T cell receptor excision circle8 number was lower in the patient than in age-matched control PBMNC (Fig. 2, panel B;, pointing to defective thymic output of recently rearranged TCRαβ T cells.

DNA sequence of the Th-POK gene,9 a regulator of CD4 T cell differentiation, and the JAK-3 gene,10 mutated in T- B+ severe combined immune deficiency, was normal.

CD3+, TCRαβ+, CD4, CD8 cell expansion represents a hallmark of the autoimmune lymphoproliferative syndrome.11 However, apoptotic response of patient peripheral blood T cell blasts to an agonistic CD95 monoclonal antibody was normal, as serum level of soluble CD95. DNA sequence of FAS, Caspase 10 11, XIAP 12, and TACI 13 genes, all related to autoimmune lymphoproliferative syndrome or immunodeficiencies was normal.

Freshly isolated CD4+, CD8+, and CD4 CD8 TCRαβ+ T cells produced similar amounts of IFN-γ and IL-4 (not shown). Seventy-two hour proliferation of patient PBMNC to polyclonal stimuli was reduced versus that of PBMNC from age-matched controls. T cell blasts expanded after 6–7 days contained both CD4+ and CD8+, but not CD4, CD8, CD3+, TCRαβ+ cells (not shown).

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Our unique ICL patient displayed multiple VL relapses associated with abnormal and persistent expansion of CD3+, TCRαβ+, CD4, CD8 cells and reduced output of T cells from thymus. Both the latter features may be related to altered T cell differentiation leading to reduced production of mature T cells and release of early thymocytes in the circulation.

Notably, abnormally expanded CD3+, TCRαβ+, and CD4 CD8 cells were (1) consistently detected in the peripheral blood, but were absent from Leishmania infected lymph node, and (2) did not grow in vitro after polyclonal stimulation. Taken together these findings may suggest that CD3+, TCRαβ+, and CD4 CD8 cells were destined to die by apoptosis before gaining access to secondary lymphoid organs in vivo or growing in vitro. Genetic and functional studies ruled out that this patient suffered from other congenital immunodeficiencies.9–13

Peripheral blood CD4 lymphocytopenia reached values as low as 20 cells/μL, mimicking advanced HIV infection. In our case, susceptibility to Leishmania infection might be related to host genetic factors, because other opportunistic infections were never observed.

The cytokine profile of CD4+ and CD8+ T cells, as well as of the abnormal CD3+, TCRαβ+, CD4, and CD8 cells, was similar to that detected in immunocompetent VL patients.3 Thus, CD4 lymphocytopenia per se rather than functional T cell defects was likely the major factor responsible for the failure to clear Leishmania.

Lymph node infection by Leishmania is rare and has been reported only in some immunocompromised patients.14 The presence of Leishmania in the lymph node from our patient was associated with a strong inflammatory reaction characterized by plasmablastic and histiocytic infiltrates that altered tissue architecture. Leishmania lymph node localization may represent a mechanism of immune escape, especially in the setting of profound CD4 lymphocytopenia.

We do not know whether Leishmania infection was the trigger of this peculiar immunologic status (eg, as EBV for XLP) or rather the consequence of a still unknown congenital defect. In any case, this novel form of ICL should be considered in diagnostic work-up of patients with multiple VL relapses.

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The authors thank Paola Bocca and Dr. Fabio Morandi for the help in flow cytometry experiments, and Chiara Bernardini for excellent secretarial assistance.

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idiopathic CD4+ lymphocytopenia; relapsing visceral leishmaniasis; Immune deficiency

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