A New Method for Reactivating and Expanding T Cells Specific for Rhizopus oryzae

Abstract

Mucormycosis is responsible for an increasing proportion of deaths after allogeneic bone marrow transplantation. Because this disease is associated with severe immunodeficiency and has shown resistance to even the newest antifungal agents, we determined the feasibility of reactivating and expanding Rhizopus oryzae-specific T cells for use as adoptive immunotherapy in transplant recipients. R. oryzae extract-pulsed monocytes were used to stimulate peripheral blood mononuclear cells from healthy donors, in the presence of different cytokine combinations. The generated R. oryzae-specific T cell products were phenotyped after the third stimulation and further characterized by the use of antibodies that block class I/II molecules, as well as pattern recognition receptors. Despite the very low frequency of R. oryzae-specific T cells of healthy donors, we found that stimulation with interleukin-2 (IL-2)/IL-7 cytokine combination could expand these rare cells. The expanded populations included 17%–83% CD4+ T cells that were specific for R. oryzae antigens. Besides interferon-γ (IFN-γ), these cells secreted IL-5, IL-10, IL-13, and tumor necrosis factor alpha (TNF-α), and recognized fungal antigens presented by HLA-II molecules rather than through nonspecific signaling. The method described herein is robust and reproducible, and could be used to generate adequate quantities of activated R. oryzae-specific T cells for clinical testing of safety and antifungal efficacy in patients with mucormycosis.

Mucormycosis in children with haematological malignancies is a salvageable disease: a report from the Israeli Study Group of Childhood Leukemia

“…In this cohort, 26 patients underwent two or more debridement procedures or extended surgical resections (range 2–14), some with considerable long‐term sequelae. Recent studies have raised hopes for innovative alternative treatment strategies for mucormycosis (Armstrong‐James et al , ; Castillo et al , ) Currently, surgical debridement is strongly recommended for rhinocerebral and soft‐tissue mucormycosis according to the ECIL‐6 guidelines and it is advised for case‐by‐case consideration for pulmonary and disseminated mucormycosis (Tissot et al , ).…”

Pathogenicity patterns of mucormycosis: epidemiology, interaction with immune cells and virulence factors

“…84 Treating T- inactivated cells with IL-2, IL-7, or both cytokines enhances the production of Mucorales-specific T cells and their cytokines IL-5, IL-10, IL-13, and also stimulates the production of CD4+ T cells that are a specific Mucorales antigens. 85 This new immune therapeutic method is a promising way to increase the response of T cells against mucormycosis. 85…”

Host Immune Defense upon Fungal Infections with Mucorales: Pathogen-Immune Cell Interactions as Drivers of Inflammatory Responses

“…Moreover, in the patients with hematological malignancies, T cells produced a characteristic cytokine profile with IL-10 secreted during the initial stages of the infection, followed by IFN-γ and IL-4 production at later time points [ 38 ]. On the contrary, another study showed that T cells from healthy donors have a low reactivity to R. oryzae and low IFN-γ production [ 37 ]. These discrepancies may rely on the methods used for the evaluation of specific-T cell activity.…”

“…Recently, Castillo et al found that the treatment of T cells with a combination of IL-2/IL-7 induce a proper expansion of R. oryzae -specific T cells with a strong production of IL-13, IL-5, TNFα, and IL-10. Therefore, the authors considered the expansion of rare Mucorales-specific T cells from healthy donors to be used for potential adoptive immunotherapy [ 37 ]. Page et al evaluated Mucorales-specific T cells and cytokine profiles in healthy donors as biomarkers of environmental mold exposure.…”

“…Furthermore, these cytokines increased the percentage of PMN-mediated hyphal damage to R. oryzae , R. microsporus , and L. corymbifera [ 30 ]. Moreover, IL-2 in combination with IL-7 induced expansion of R. oryzae -specific T cells in healthy donors [ 37 ]. These findings support initiatives for cytokine-mediated adoptive immunotherapy, which could either boost different cellular-types of the innate immune response or promote the expansion of Mucorales-specific T cells.…”