58 - Developing T cell based immunotherapies for mucormycosis post HSCT

Abstract

Mucormycosis (most of which are caused by Rhizopus oryzae) following allogeneic hematopoietic stem cell transplantation (HSCT), is an increasing problem due to the resistance of these fungi to available anti-fungal agents. Mortality rates remain as high as 90%. Because mucormycosis is an opportunistic infection associated with severe immune deficiency, including T cell dysfunction, following HSCT, we evaluated whether allogeneic Mucorales-specific T cells could be manufactured as a novel therapeutic to protect high risk patients after allogeneic HSCT. We used monocytes pulsed with a Rhizopus oryzae extract in an attempt to stimulate Mucorales-specific T cells from healthy donors and patients with invasive mucormycosis in the presence of different cytokine combinations. Despite the ubiquitous nature of the pathogen, we were unable to elicit readily observable responses to R. oryzae from healthy donor T cells (n = 9). Our next goal was to evaluate whether Mucorales-specific T cells could be expanded in vitro from healthy donors. A combination of IL2 and IL7 yielded the best expansion and specificity, compared with combinations involving IL4 and IL7, IL7 and IL15, and IL 15 and IL21. After three stimulations, R. oryzae-specific T-cells expanded up to 50-fold comprising a mean of 50% CD4+ T cells (range 16.6–82.9%), a mean of 33.26% CD8+ T cells (range 9.1–70.8%), and the majority of T cells with effector memory phenotypes (mean of 77.98% CD45RO+CD45RA− range 7.2–97.7%, as compared with a mean of 3.275% (range 0–6.7%) naïve CD45RA+ CCR7+ cells. The mean frequency of expanded R.oryzae-reactive T-cells as determined by Elispot assay was 314 IFNg SFC/1 × 105 T-cells (range 15–1057) compared to a mean of 20 IFNg SFC/1 × 105 T-cells against irrelevant antigens (n = 8, range 0–94)). R.oryzae-specific T cells (n = 3) also secreted IL-13, IL-10, IL-5, and TNF-α in response to antigen. In conclusion, using R. oryzae as a representative Mucorales pathogen, we describe a method to elicit Mucorales-specific T cells. Such approach might be valuable as adjunct immunotherapy in patients with mucormycosis post allogeneic HSCT.