Clinical Immunohaematology

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Maher Gandhi

Lab Head: Assoc Prof Maher Gandhi

Background
Epstein-Barr virus (EBV) is a ubiquitous herpesvirus that has infected more than 90% of us. EBV is spread via transfer of saliva. Initial infection is frequently asymptomatic, or occasionaly results in glandular fever (also known as infectious mononucleosis), which is characterised by fever, swollen neck glands and a sore throat. Infection is lifelong, with the dormant virus persisting in B cells, and is of no clinical consequence. Viral reactivation is prevented by the immune system. However, in immunosuppressed patients, such as those with HIV or those on immunosuppressive drugs, EBV can reactivate and cause malignancies, including lymphoma (a cancer of the lymphoid glands). Examples of lymphomas in which EBV is involved include some cases of Hodgkin's Lymphoma, Burkitts Lymphoma, and Post-transplantation Lymphoproliferative Disorder. In Australia, it is estimated that EBV is implicated in 150 cases of lymphoma per year. This number is far higher in developing countries.

Figure 1: Reconstitution of adoptively transferred EBV-specific allogeneic CTL in a patient with EBV-positive post-transplant lymphoproliferative disease.

Left-panel shows assays of EBV-peptide-specific cytotoxic T lymphocyte (CTL) function at 4 time-points (labelled 1-4) following combination immunotherapy. Blood was sampled at day +21, 1 hour following the fourth allogeneic CTL infusion (1), and then at days +62 (2), +194 (3), and +249 (4). EBV-EBNA3FLRGRAYGL peptide-stimulated polyclonal CTL were generated by 3 week culture, involving supplementation with interleukin-2, and weekly stimulation with irradiated recipient peptide-labelled PBMC. CTL were tested for cytotoxicity against recipient PHA blasts pre-sensitized with either EBNA3FLRGRAYGL or a no peptide (1-4NP) control. A range of effector to target (E:T) ratios were use in a standard chromium-release assay. SD was <10% at all ratios. Right-panel uses HLA class I EBV-specific pentamer analysis to show a population of in-vitro expanded CD8+ EBNA3FLRGRAYGL peptide-specific T-cells from a blood sample taken at day +249. 0.717% CD8+ EBNA3FLRGRAYGL T-cells were present within the culture. By ex-vivo analysis and in-vitro culture, CD8+ EBNA3FLRGRAYGL peptide-specific T-cells were not detectable prior to the first infusion.

Examples of lymphomas in which EBV is involved include some cases of Hodgkin's Lymphoma, Burkitts Lymphoma, and Post-transplantation Lymphoproliferative Disorder. In Australia, it is estimated that EBV is implicated in 150 cases of lymphoma per year. This number is far higher in developing countries.

At present, there is no anti-viral treatment or vaccine against EBV. It is known that restoration of EBV-specific immunity can in some cases induce tumour regression. There is therefore a high priority to develop novel immunotherapies for EBV treatment. The Clinical Immunohaematology laboratory has a broad interest in the immunobiology of the virus, with particular reference to immunotherapeutic strategies for EBV-positive lymphomas.

Figure 2. Homing of adoptively transferred EBV-specific CTL to the tumour site. Immunohistochemistry (IHC) and florescent in-situ hybridization (FISH) in left and right panels respectively demonstrating scattered CD8+ and XY cells admixed within the B-cell PTLD, taken at autopsy. The donor-CTL donor was male, and the recipient was female. The PTLD was of recipient origin.

Currently our major research interests involve viral and immune biomarkers, immuno-evasion, viral microRNA expression and optimisation of cellular immunotherapies. Success in these aims will allow us to identify patients most at risk of developing lymphoma and understand the basis by which these lymphomas develop. In turn, this may lead to new targeted therapies, including transfer of the relevant components of a healthy person's immune system into the a patient with an EBV-positive lymphoma, so as to eradicate the disease. To date, we have treated 3 patients with this approach.

Staff

Labhead:	Dr Maher Gandhi
Research Assistants:	Jamie Nourse Kimberley Jones Suman Kumar Yekollu

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Funding

We gratefully acknowledge support from the following funding bodies:
National Health and Medical Research Council
Queensland Cancer Council
Royal Australasian College of Physicians
Queensland Smart State
British Society for Haematology
Leukaemia Foundation
Atlantic Philanthropies
Griffith Medical Research College

Collaborators

Professor Dorothy Crawford (University of Edinburgh, UK): EBV-specific allogeneic CTL for EBV-positive lymphomas.
Professor Chris Goodnow (ANU, Canberra): Genetic Polymorphisms in immune thrombocytopenic purpura.
A/Prof Rajiv Khanna: EBV-specific autologous CTL for relapsed/refractory EBV-positive Hodgkin's Lymphomas (QIMR)
Professor Lyn Griffiths & A/Prof Mark Hertzberg: Biomolecular Profiling In PET/CT Directed Diffuse Large B Cell Lymphoma.

Student Projects

A variety of projects provide excellent opportunities for the involvement of Honours and PhD students. These projects are supported by a variety of different experimental approaches involving flow cytometry, immunohistochemistry, assays of immune effector function, real-time PCR, and genetic polymorphism analysis. Students in our laboratory would become familiar with all of these techniques. There will also be opportunities for successful Honours students to enrol for PhD studies and continue work in the laboratory.

Key Publications

H Tran, J Nourse, S Hall, M Green, L Griffiths, MK Gandhi. Immunodeficiency-associated Lymphomas. Blood Reviews. Accepted March 20th 2008.

MJ Hourigan, J Doecke,, PN Mollee, DS Gill, D Norris, DW Johnson, MK Gandhi. A new prognosticator for Post-transplantation lymphoproliferative disorders after renal transplantation. Br J Haematol. Accepted 07/02/2008.

H. Tran, C. Cheung, D. Gill, U. Dua, J. Nourse, R. Boyle, MK Gandhi. Methotrexate-associated mantle cell lymphoma in an elderly man with myasthenia gravis. Nature Clinical Practice Oncology. 2008 Apr;5(4):234-8. Epub 2008 Feb 19.

T Haque, GM Wilkie, MM Jones, CD Higgins, G Urquhart, P Wingate, D Burns, K McAulay, M Turner, C Bellamy, PL Amlot, D Kelly, A MacGilchrist, MK Gandhi, AJ Swerdlow, DH Crawford. Allogeneic cytotoxic T cell therapy for EBV-positive PTLD: results of a phase II multicentre clinical trial. Blood 2007; 10(4): 1123-31.

Gandhi MK, Moll G, Smith C, Dua U, Lambley E, Ramuz O, Gill D, Marlton P, Seymour JF, Khanna R. Galectin-1 mediated suppression of EBV-specific T-cell immunity in classical Hodgkin's Lymphoma. Blood. 2007; 10(4): 1326-9. IF 10.37.

D Moss, R Khanna, MK Gandhi. The Use of T-Cell Cellular Therapies in Australia. Cytotherapy 2007;9(3):222-4.

MK Gandhi, GM Wilkie, U Dua, PN Mollee, K Grimmett, T Williams, N Whitaker, D Gill and DH. Crawford. Am J Transplantation. 2007, 7(5) 1293.

AK. Nehring, U Dua, P Mollee, D Gill, K Grimmett, R Khanna, D Moss, MK Gandhi. EBV T-cell immunity despite rituximab. Brit J Haematol. 2007, 136(4), 628.

K Morris, H Tran, MK Gandhi. Staphlococcal skin infection masquerading as leukaemia cutis. BloodMed. 2006; 82.

Smith C, Cooper L, Burges M, Rist M, Webb N, Lambley E, Tellam J, Marlton P, Seymour, JK, Gandhi MK, Khanna R. Functional Reversion of Antigen-specific CD8+ T cells from Hodgkin Lymphoma Patients following stimulation with Recombinant Polyepitope and C-Cytokines. Journal of Immunology. 2006, 177 (7), 4987.

MK Gandhi, E Lambley, U Dua, S Elliott, J Duraiswamy, C Smith, D Gill, P Marlton, JF Seymour, R Khanna. Expression of LAG-3 by Tumor-infiltrating Lymphocytes is Co-incident with the Suppression of Latent Membrane Antigen-specific CD8+ T-cell Function in Hodgkin Lymphoma Patients. Blood. 2006, 108 (7) 2280-2289.

MK Gandhi and R Khanna. EBV and CLL: innocent until proven guilty. Leukemia and Lymphoma. 2006, 47(6); 779-80.

WM Wilson, U Dua, AP Grigg, MK Gandhi. Correlation of T-cell immune response with spontaneous resolution and subsequent relapse of Hodgkin Lymphoma. Leukemia and Lymphoma. 2006, 47(6) 871-6

MK Gandhi, E Lambley, J Burrows, U Dua, S Elliott, PJ Shaw, HM Prince, M Wolf, K Clarke, C Underhill, T Mills, P Mollee, D Gill, P Marlton, JF Seymour, R Khanna. Plasma Epstein-Barr virus (EBV) DNA is a biomarker for EBV-positive Hodgkin's lymphoma. Clin Cancer Res. 2006 Jan 15;12(2):460-4.

MK Gandhi and R Khanna. Viruses and Lymphoma. Pathology: Royal College of Pathology of Australasia Journal. 2005 Dec;37(6):420-33. 21. MK Gandhi. EBV-associated lymphomas. Expert Rev Anti Infect Ther. 2006 Feb;4(1):77-89.

R Khanna, D Moss, MK Gandhi. Applications of emerging immunotherapeutic strategies for EBV-associated malignancies. Nature Clinical Practice Oncology. 2005 (2):138-14.

MK Gandhi. Regulation of Clinical Research; Clin Med. Journal of the Royal College of Physicians of London. 2005 May-Jun;5(3):299.

MK Gandhi, RE Marcus. Follicular Lymphoma: Time for a Re-think? Blood Reviews. 2005, vol 19(3), 165-178.

MK Gandhi, R Khanna. Human Cytomegalovirus: Clinical Aspects, Immune Regulation and Emerging treatments. Lancet Infectious Diseases. 2004 Dec;4(12):725-38. IF 11.808.

MK Gandhi, J Tellam, R Khanna. EBV-associated Hodgkin's Lymphoma. Br J Haematol. 2004, (125), 267-81.

MK Gandhi, MR Wills, G Okecha, R Hicks, RE Marcus, JGP Sissons and AJ Carmichael. Late diversification in the clonal composition of HCMV-specific CD8+ T cells following allogeneic haematopoietic stem cell transplantation. Blood. 2003, Nov, 102, 3427-38.

MK Gandhi, MR Wills, JGP Sissons, AJ Carmichael. Human Cytomegalovirus-specific immunity following haemopoietic stem cell transplantation. Blood Reviews. Dec, 2003, 17, 259-64.

MK Gandhi,, S Lekamwasam, I Inman, S Kaptoge, L Sizer, S Love, PW Bearcroft, TP Milligan, CP Price, RE Marcus, JE Compston. Significant and persistent loss of bone mineral density in the femoral neck after haematopoietic stem cell transplantation: Long-term follow-up of a prospective study. Br J Haematol. 2003; 121(3): 462-8.

MR Wills, G Okecha, MR Weekes, MK Gandhi, JGP.Sissons and AJ Carmichael. Identification of antigen-experienced human CD8+ T cells by expression of co-stimulation and chemokine receptors: analysis of the human cytomegalovirus-specific CD8+ T cell response. Journal of Immunology. 2002, 168: 5455-5464.

MK Gandhi, W Egner, L Sizer, I Inman, M Zambon, JIO Craig, RE Marcus. Antibody responses to vaccinations given within the first two years after transplant are similar between autologous peripheral blood stem cell and bone marrow transplant recipients. Bone Marrow Transplant. 2001; 28(8): 775-781.

JA Zaia, JGP Sissons, S Riddell, JD Diamond, MR Wills, AJ Carmichael, MP Weekes, MK Gandhi, CL Rosa, M Villacres, S Lacey, S Markel, J Sun. Status of Cytomegalovirus Prevention and Treatment in 2000. ASH Educational Book, Hematology. 2000; 339-355.

S Coulthard, C Rabello, J Robson, C Howell, L Minto, P Middleton, MK Gandhi, G Jackson, H O'Brien, M Reid, A Pearson, A Hall. A comparison of molecular and enzyme-based assays for the detection of thiopurine methyltransferase mutations. Br J Haematol. 2000; 110(3):599-604.

MK Gandhi, H Jestice, MA Scott, D Bloxham, G Bass, JIO Craig, RE Marcus. A comparison of CD34+ cell selected and unselected autologous peripheral blood stem cell transplantation for multiple myeloma: a case controlled analysis. Bone Marrow Transplant. 1999; 24(5): 369-375.

MK Gandhi, H Jestice, MA Scott, D Bloxham, G Bass, RE Marcus. The minimum CD34 threshold depends on prior chemotherapy in autologous peripheral blood stem cell recipients. Bone Marrow Transplant. 1999; 23(1): 9-13.