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.

Keywords: antigen presentation/processing; cytokines; cytotoxic T cells; fungal; immune reconstitution; transplantation.

Figure 1

Frequency of Fungi-Reactive T Cells in Healthy Donor PBMCs Using peripheral blood from healthy donors (n = 9), we evaluated the lymphocyte responses to R. oryzae (Mucor lys), A. fumigatus (Asp lys), CMV, and EBV using IFN-γ ELISpot assays. Each symbol represents the mean number of spot-forming cells from at least two replicates containing 500,000 cells/well. Bars denote the means. *p < 0.05; **p < 0.01.

Figure 2

Optimization of Expansion Conditions (A) Comparison of the expansion of T cell products derived from three healthy donors. T cells were stimulated with the cytokines combinations IL-2/IL-7, IL-4/IL-7, IL-7/IL-15, or IL-15/IL-21. Each dot represents the mean fold expansion, and error bars denote SD. (B) Phenotype of three healthy donors expanded with the cytokines IL-2/IL-7 versus IL-4/IL-7 versus IL-7/IL-15, versus IL-15/IL-21. All subsets were gated on CD3⁺ cells, with the exception of non-T cell subsets (CD3⁻ CD56⁺ and CD3⁻ CD19⁺), which were gated on singlets from total lymphocytes. Bars represent means and SDs. (C) Antigen specificities of T cell products derived from three healthy donors as measured by IFN-γ secretion. T cells were stimulated three times with the cytokines IL-2/IL-7 versus IL-4/IL-7 versus IL-7/IL-15, versus IL-15/IL-21. Negative control denotes unpulsed APCs (monocytes alone), whereas Mucor denotes pulsed APCs (monocytes plus R. oryzae lysate). Bars indicate mean values.

Figure 3

Characterization of R. oryzae-Specific T Cells Expanded with IL-2/IL-7 (A) Mean fold expansion and SDs of antigen-specific T cells derived from healthy donors after 3 stimulations with APCs pulsed with R. oryzae lysate (n = 8). (B) Phenotype of the T cell products. All subsets are gated on CD3⁺ cells (n = 8). Bars indicate means and SDs. (C) Specificity of T cell products against R. oryzae (p < 0.01) in an ELISpot assay comparing T cell responses to unpulsed (monocytes alone) versus pulsed (monocytes + R. oryzae lysate) targets. Each symbol denotes a healthy donor (n = 8). **p < 0.01.

Figure 4

MHC Restriction of T Cells T cells recognize antigens via class II, as shown by substantial decreases in the number of IFN-γ spots when class II was blocked (unblocked versus blocked) highlighted by red borders (n = 3).

Figure 5

Cytokine Secretion of Expanded R. oryzae-Specific T Cells Cytokine responses elicited from three ex vivo expanded R. oryzae-specific T cell products were analyzed after the third stimulation using a multiplex ELISA. Bars represent means, and error bars show SDs.