Clinical-scale generation of multi-specific anti-fungal T cells targeting Candida, Aspergillus and mucormycetes

Clinical-scale isolation of the total Aspergillus fumigatus–reactive T–helper cell repertoire for adoptive transfer

“…Because functionality of the final product, defined as reactivity to A. fumigatus and other Aspergillus spp. has been tested in several runs in vitro (re-stimulation assay) [36], we surmise that each product of Aspergillus-specific T cells will be more or less active against the fungus; the degree of reactivity itself may be affected by both donor and recipient and may be somewhat variable.…”

Future therapies targeted towards eliminatingCandidabiofilms and associated infections

“…There is also a single clinical report of four patients, that has stated the beneficial contribution of IFN in patients with refractory fungal infections [196]. [197]. This approach utilizes infusion of patients with dendritic cells (DCs), primed ex vivo with antigens that induce specific cytokines, in order to induce an adaptive immune response [186].…”

Stimulation with lysates of Aspergillus terreus, Candida krusei and Rhizopus oryzae maximizes cross-reactivity of anti-fungal T cells

“…Eight cultures were expanded from PBMC, four without selection and four using TNF-α immunomagnetic bead-based capture 1 week after initial stimulation. We performed the latter because cytokine capture of cells secreting IFN-γ after stimulation with fungal antigens has been used in the production of fungus-reactive T-cell products [27,29]. In our case, we used TNF-α because we found it to be released in greater amounts than IFN-γ after stimulation, as previously noted.The use of TNF-α capture for enrichment did not increase fold expansion or the absolute numbers of total or fungus-specific T cells at the end of culture, suggesting that this isolation method is not necessary for production of broadly reactive anti-fungal T cells.Three cultures generated from G-CSF-primed peripheral blood stem cell harvest from normal donors expanded at least as well as PBMC cultures and yielded a higher number of fungus-reactive T cells, possibly because of the larger number of mononuclear cells harvested permitting two dendritic cell stimulations.…”

“…Our choice to use TNF-α as the readout for fungusspecific immunity is based on our previous observation that TNF-α is the predominant Th1 cytokine produced by A fumigatus-specific T cells [22], a finding that we also noted in this study (Table II). Other groups have noted similar findings.Tramsen et al [27] showed that a higher percentage of fungus-specific T cells produce TNF-α than IFN-γ (median, 13.3% and 9.0% of cultured T cells, respectively), and Bacher et al [28] demonstrated that both TNF-α and IL-2 predominate over IFN-γ in cytokine secretion by Aspergillusspecific T cells [27,28]. We selected a combination of lysates from A terreus, C krusei and R oryzae to manufacture panfungal T-cell products.…”

“…In addition, the method of Khanna et al involves magnetic bead-based cell separation and culture with autologous feeder cells, which is complex and will be costly to implement to clinical grade. In the method by Tramsen et al [27], a combination of lysates from A fumigatus, C albicans and R oryzae was used for stimulation, followed by IFN-γ immunobead capture to isolate reactive T cells before culture in the presence of autologous feeder cells. In comparison to the method we describe, this approach involves greater complexity and cost.Whereas Tramsen et al used the most common clinically relevant fungi for cell stimulation and showed cross-reactivity to some other fungi including A niger and C tropicalis, our species choices for stimulation were made specifically for the purposes of producing a broad range of fungal recognition by the final T-cell product.…”