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Immune Reconstitution in Paediatric Patients with Acquired Immunodeficiency

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Immune Reconstitution in Paediatric Patients with Acquired Immunodeficiency Department of Sanità pubblica e biologia cellulare University of Tor Vergata, Rome, Italy IMMUNE RECONSTITUTION IN PAEDIATRIC PATIENTS WITH ACQUIRED IMMUNODEFICIENCY Candidate: Simone Pensieroso Supervisor: Caterina Cancrini Opponent: Francesca Chiodi Rome 2007 ABSTRACT Background : Immune system plays a crucial role in defending organism from pathogens. However, immunological functions may be severely impaired by a number of disorders including acquired immunodeficiencies, possibly due to infections, chronic diseases, immunosuppressive drugs or surgeon therapies. In order to favour the immune reconstitution of patients with acquired immunodeficiency, various therapeutic treatments are constantly under investigation for both adults and paediatric patients. Aim of the study : To evaluate immune reconstitution in children with acquired immunodeficiency. Attention was focused on two groups of HIV-1 vertically infected children (subjected the first group to HAART from the third months of life, the second to a simplified treatment) and on a cohort a leukaemia children who underwent Umbilical Cord Blood Transplantation (UCBT). Materials and Methods : Immune restoration was principally evaluated in terms of lymphoproliferative response and T-cell receptor (TCR) repertoire. Immunological studies were complemented by routine clinical and laboratory analyses. Results : HIV-infected children showed a significant normalization of immune functions investigated, with a long-term maintaining of good clinical and immunological parameters. Also children who underwent UCBT showed a notable immune restoration. Conclusions : In HIV-infected children, an early application of antiretroviral treatment from the third month of life favours immune reconstitution and the application of a simplified regimen seems to permit the maintenance of good immunological results obtained during the previous successful HAART. In the context of transplanted children our data underline applicability and advantages of UBC compared to Bone marrow (BM) transplantations. Key words : Immune reconstitution, Acquired Immunodeficiency, HIV-1, HAART, Umbilical cord blood transplantation, T cell receptor, Spectratyping 3 4 IMMUNE RECONSTITUTION IN PAEDIATRIC PATIENTS WITH ACQUIRED IMMUNODEFICIENCY Simone Pensieroso Doctoral thesis from the Department of Sanità pubblica e biologia cellulare, University of Tor Vergata, Rome, Italy This thesis is based on the following articles and manuscript: 1) Palma P, Romiti ML, Cancrini C, Pensieroso S, Santucci M, Montesano C, Bernardi S, Amicosante M, Di Matteo G, Di Cesare S, Wahren B, Rossi P, Castelli-Gattinara G. Early Highly Active Antiretroviral treatment delayed over 3 months of life is associated with good clinical outcome, long-term viral control and persistent antiviral T-cell response in HIV-1 vertically infected infants. Submitted to Eur. J. of Pediatrics. 2) Palma P, Romiti ML, Cancrini C, Pensieroso S , Montesano C, Cantucci MB, Tozzi A, Bernardi S, Martino AM, Andreoni M, Amicosante M, Freda E, Rossi P & Castelli-Gattinara G. Successful simplification of protease inhibitor-based HAART with a triple nucleosides regimen in HIV-1 vertically infected children. Submitted to Antiviral therapy 3) Pensieroso S , Romiti ML, Palma P, Castelli-Gattinara G, Bernardi S, Freda E, Rossi P, Cancrini C. Switching from protease inhibitor-based HAART to a protease inhibitor-sparing regimen is associated with improved specific HIV-immune responses in HIV-infected children. AIDS. 20, 1893-1896 (2006). 4) Finocchi A, Romiti ML, Di Cesare S, Puliafito P, Pensieroso S , Rana I, Pinto R, Cancrini C, De Rossi G, Caniglia M, Rossi P. Rapid T-cell receptor CD4+ repertoire reconstitution and immune recovery in unrelated umbilical cord blood transplanted pediatric leukemia patients. J. Pediatr. Hematol. Oncol. 28, 403-411 (2006). 5 6 ABBREVIATIONS 3TC: Lamivudine Ab: Antibody ABC: Abacavir Ag: Antigen AIDS: Acquired immunodeficiency syndrome ALL: Acute lymphoblastic leukaemia AML: Acute myeloblastic leukaemia APC: Antigen presenting cell APC-Cy5 Allophycocyanin AT-2: Aldhitriol 2 ATG: Antithymocyte globulins AZT: Zidovudine BCR: B cell receptor BM: Bone marrow BU: Busulfan CCR5: CC-chemokine receptor 5 CD40L: CD40 ligand CDR: Complementary determining region CLP: Common lymphoid progenitor CMV: Cytomegalovirus Cpm: counts per minute CSA: Cyclosporine A cTEC: cortical thymic epithelial cell CTL: Cytotoxic T lymphocyte CTX: cyclophosphamide CXCR4: CXC-chemokine receptor 4 CY: Cyclophosphamide d4T Stavudine DC: Dendritic cell DC-SIGN: DC-specific ICAM3-grabbing non integrin ddI: Didanosine DN: Double negative DP: Double positive ER: Endoplasmic reticulum FasL: Fas ligand FDC: Follicular dendritic cell FISH: Fluorescence in situ hybridization 7 FITC: Fluorescein isothiocyanate FK406: Tacrolimus GC: Germinal centre G-CSF: Granulocyte Colony Stimulating Factor GM-CSF: Granulocyte/macrophage-colony stimulating factor GVHD: Graft versus host disease HAART: Highly Active Antiretroviral Therapy HIV: Human immunodeficiency virus HSCT: Haematopoietic stem cell transplantation HSV-1: Herpes virus-1 ICAM: Intercellular adhesion molecule IFN-γ: Interferon γ IL-4: Interleukin 4 IL-5: Interleukin 5 IL-6: Interleukin 6 IL-12: Interleukin 12 IVIG: Intravenous Ig LC: Langherans cell LPS: Lypopolisaccaride LPV: Lopinavir MDDC: Monocyte-derived DC MHC: Major histocompatibility complex MLR: Leucocyte mixed reaction mTEC: medullar thymic epithelial cell myDC: Myeloid dendritic cell NK: Natural killer NNRTI: Non-nucleoside reverse transcriptase inhibitor NRTI: nucleoside reverse transcriptase inhibitor PCR: Polymerase chain reaction pDCs: Plasmacytoid dendritic cell PDN: Prednisolone PE R-Phycoerythrin PHA: Phytohaemaglutinin PI: Protease Inhibitor PMA: Phorbol myristate acetate PWM: Pokeweed mitogen RAG: Recombination activating gene RT-PCR: Reverse transcriptase PCR RTV: Ritonavir SD: Standard deviation 8 SI: Stimulation index SP: Single positive TBI: Total body irradiation TCR: T cell receptor Th: T helper TLR: Toll like receptor TNF: Tumour necrosis factor TREC: TCR-excision circle UCBT: Umbilical Cord Blood Transplantation VP16: Etoposide 9 10 CONTENTS ABSTRACT.......................................................................................................3 ABBREVIATIONS ...........................................................................................7 INTRODUCTION ...........................................................................................15 The immune system .....................................................................................17 B lymphocytes..........................................................................................18 T lymphocytes..........................................................................................21 T-cell receptor (TCR)...........................................................................24 Antigen presenting cells (APCs)..............................................................27 Dendritic cells (DCs) ...............................................................................27 Major histocompatibility complex (MHC) ..........................................28 Children’ immune system ........................................................................30 Immune system disorders and immunodeficiency.......................................31 Pathogenesis of HIV ....................................................................................33 B-cell compartment..................................................................................35 T-cell compartment..................................................................................36 DCs compartment ....................................................................................37 Paediatric HIV-infection..........................................................................39 Treatment .................................................................................................39 Haematological disorders and transplantation: an other case of acquired immunodeficiency........................................................................................43 Umbilical cord blood transplantation (UCBT) ........................................45 Immune reconstitution analysis ...................................................................49 Cytofluorimetric analysis.........................................................................49 TCRBV repertoire analysis - Spectratyping ............................................49 T-cell receptor excision circles (TRECs) analysis...................................52 T-cell proliferation ...................................................................................53 AIM OF THE STUDY.....................................................................................55 DESIGN...........................................................................................................57 MATERIALS & METHODS ..........................................................................61 Patients, therapies and clinical investigations..............................................61 Study 1 .....................................................................................................61 Study 2 .....................................................................................................62 Study 3 .....................................................................................................63 HLA typing and chimerism (Study 2)..........................................................65
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