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Gaundar S.S.,University of Sydney | Clancy L.,University of Sydney | Clancy L.,Sydney Cellular Therapies Laboratory | Blyth E.,University of Sydney | And 2 more authors.
Cytotherapy | Year: 2012

Background and aims. Aspergillus fumigatus infections are the leading cause of invasive fungal infection-related deaths in stem cell transplant patients, and may be amenable to correction with adoptive immunotherapy providing T lymphocytes specific for A. fumigatus. However, a clinically usable source of antigen and a reliable procedure for the generation of large numbers of Aspergillus-specific T lymphocytes to clinical-grade standards is not available. Methods. An environmental strain of A. fumigatus (WMAfES) was isolated and cultured using materials and reagents suitable for clinical manufacture. Water-soluble lysate from germinated conidia of WMAfES was used as the antigen source. Peripheral blood mononuclear cells were stimulated with antigen-pulsed autologous dendritic cells on days 0 and 7. Cells were expanded with a cocktail of interleukin (IL)-2, IL-7 and IL-15 from days 7 to 21. Results. We obtained a mean 32.8-fold increase in cell numbers over 21 days of culture (n 8). Resultant cultures were predominantly effector and central memory CD4 T cells, which produced T-helper (h)1 and Th17 cytokines when restimulated with A. fumigatus antigen derived from environmental or clinically isolated A. fumigatus. Cultured cells exhibited a high level of specific expansion and chemokine production when restimulated. Moreover, cultured cells cross-reacted with antigens from other fungi, including Penicillium, Candida albicans and other non-fumigatus Aspergillus species. Conclusions. We describe a simple, robust, reproducible and clinically applicable procedure using a clinically appropriate antigen preparation for the expansion of polyfunctional A. fumigatus-specific T cells from normal donors of varying HLA types. © 2012 Informa Healthcare.

Bishop D.C.,University of Sydney | Johnston A.J.,University of Sydney | Antonenas V.,Sydney Cellular Therapies Laboratory | Gottlieb D.J.,University of Sydney | Gottlieb D.J.,Sydney Cellular Therapies Laboratory
Internal Medicine Journal | Year: 2014

Elderly patients with acute myeloid leukaemia (AML) have a poor prognosis with standard chemotherapy. Two elderly AML patients treated with infusion of family-derived partially human leukocyte antigen (HLA)-matched peripheral blood stem cells following each cycle of chemotherapy entered morphological complete remission without graft versus host disease or major toxicity. Our results support this as a non-toxic approach for inducing a graft versus leukaemia effect in patients not suitable for allogeneic transplantation. Additional resources required for donor assessment and harvest may be reduced by using banked partially HLA-matched mononuclear cells from unrelated donors. © 2014 Royal Australasian College of Physicians.

Deo S.S.,Westmead Millennium Institute for Medical Research | Deo S.S.,University of Sydney | Virassamy B.,Westmead Millennium Institute for Medical Research | Halliday C.,Center for Infectious Diseases and Microbiology | And 11 more authors.
Cytotherapy | Year: 2016

Background aims. Invasive fungal diseases caused by filamentous fungi and yeasts are significant causes of morbidity and mortality in immunosuppressed hematology patients. We previously published a method to expand Aspergillus fumigatus-specific T cells for clinical cell therapy. In the present study, we investigated expansion of T cells specific for other fungal pathogens and creation of a broadly reactive panfungal T-cell product. Methods. Fungal strains selected were those frequently observed in the clinical hematology setting and included Aspergillus, Candida, Fusarium, Rhizopus and Lomentospora/Scedosporium. Four T-cell cultures specific to each fungus were established. We selected lysates of Aspergillus terreus, Candida krusei and Rhizopus oryzae to expand panfungal T cells. Allelic restriction of anti-fungal activity was determined through the use of specific major histocompatibility complex class II-blocking antibodies. Results.Individual T-cell cultures specific to each fungus could be expanded in vitro, generating predominantly CD4+ T cells of which 8% to 20% were fungus-specific. We successfully expanded panfungal T cells from the peripheral blood (n = 8) and granulocyte-colony-stimulating factor-primed stem cell products (n = 3) of normal donors by using a combination of lysates from Aspergillus terreus, Candida krusei and Rhizopus oryzae. Anti-fungal activity was mediated through human leukocyte antigen (HLA)-DR alleles and was maintained when antigen-presenting cells from partially HLA-DRB1-matched donors were used to stimulate T cells. Conclusions. We demonstrate a method to manufacture panfungal T-cell products with specificity against a range of clinical fungal pathogens by use of the blood and stem cells of healthy donors as the starting material. The safety and efficacy of these products will need to be tested clinically. © 2015 International Society for Cellular Therapy.

Gaundar S.S.,University of Sydney | Blyth E.,University of Sydney | Clancy L.,University of Sydney | Clancy L.,Sydney Cellular Therapies Laboratory | And 5 more authors.
Cytotherapy | Year: 2012

Background aims. Influenza viruses cause potentially fatal respiratory infections in stem cell transplant patients. Specific T cells provide long-lived host adaptive immunity to influenza viruses, and the potential for generating such cells for clinical use was investigated. Methods. The inactivated influenza vaccine (Fluvax) approved for human use was used as the antigen source. Monocyte-derived dendritic cells pulsed with Fluvax were used to stimulate autologous peripheral blood mononuclear cells (PBMC) on days 0 and 7. Cells were expanded with interleukin (IL)-2 from day 7 onwards. Cell numbers and phenotype were assessed on day 21. The presence of influenza virus-specific cells was assessed by cytokine production and proliferative responses following restimulation with influenza antigens. Results. Over 21 days of culture, a mean fold increase of 26.3 in cell number was observed (n = 7). Cultures were predominantly effector and central memory CD4+ cells, and expressed a phenotype characteristic of activated antigen-specific cells capable of B-cell helper function. Cytotoxic CD4+ and CD8+ cells specific for influenza and a high percentage of CD4+ cells specific for each of three influenza viruses targeted by Fluvax (H1N1, H3N2 and Brisbane viruses) were generated. In addition, T cells expanded when restimulated with antigens derived from influenza viruses. Conclusions. We have demonstrated a clinically usable method for producing influenza virus-specific T cells that yield high numbers of highly reactive CD4+ cells suitable for adoptive immunotherapy. We propose that reconstructing host immunity through adoptive transfer of influenza virus-specific T cells will reduce the frequency of influenza-related deaths in the period of severe immune suppression that follows stem cell transplantation. © 2012 Informa Healthcare.

Macesic N.,Royal Melbourne Hospital | Langsford D.,Royal Melbourne Hospital | Nicholls K.,Royal Melbourne Hospital | Hughes P.,Royal Melbourne Hospital | And 9 more authors.
American Journal of Transplantation | Year: 2015

Cytomegalovirus (CMV) is a significant cause of morbidity, mortality and graft loss in solid organ transplantation (SOT). Treatment options for ganciclovir-resistant CMV are limited. We describe a case of ganciclovir-resistant CMV disease in a renal transplant recipient manifested by thrombotic microangiopathy-associated glomerulopathy. Adoptive T cell immunotherapy using CMV-specific T cells from a donor bank was used as salvage therapy. This report is a proof-of-concept of the clinical and logistical feasibility of this therapy in SOT recipients. Copyright © 2015 The American Society of Transplantation and the American Society of Transplant Surgeons.

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