Human Bone Marrow as a Source of Multifunctional CMV-Specific CD4+ T Cells for Adoptive Cell Therapy.

Introduction Adoptive cell transfer of ex vivo primed and expanded human cytotoxic T lymphocytes (CTLs) has emerged as a promising approach to treat both infectious and malignant diseases in humans. First clinical studies have shown that transfer of Cytomegalovirus (CMV)-specific CD8+ T cells is safe and effective in reconstitution of cellular immunity against CMV disease. Efficacy of adoptive T cell therapy is limited by the numbers of CTLs in vitro and the survival and function after infusion. CD4+ T cells may enhance activity via direct or indirect effector functions (Matloubian 1994 [1]). In this study we have analysed whether bone marrow is superior to peripheral blood for expansion of CMV-specific T cells. Experimental design Paired peripheral blood and bone marrow samples were obtained from patients who underwent total hip arthroplasty. By using two different protocols T cells were expanded in the presence of IL-2 and IL-7 either from bulk culture with exposure of two different peptide pools (IE1 and pp65) or after selection via IFN-γ secretion by stimulation with pp65. CMV specific immune responses were assessed by using multiparameter flow cytometry staining cells for CD3, CD4, CD8, CCR7 and CD45RA and for the cytokines IFN-γ IL-2 and TNF at day 0 and after 10 days of in vitro expansion.

Results Similar frequencies of cytokine-producing pp65– and IE1-specific CD4+ and CD8+ T cells were found in unmanipulated paired PB and BM samples. Expansion of CMV-specific T cells from BM resulted in significantly higher frequencies of specific CD4+ T cells than from PB, whereas no difference in frequencies of CMV-specific CD8+ T cells was observed. Interestingly, significantly higher frequencies of BM pp65 and IE1-specific CD4+ T cells were multifunctional, characterized by producing simultaneously IFN-γ, TNF and IL-2 (IE1: BM mean 0.44% ± 0.16; PB mean 0.09% ± 0.05, p=0.031; pp65: BM mean 3.87% ± 2.46; PB mean 1.24% ± 0.90, p=0.031). Expansion of multi-functional CD4+ T cells from BM was observed with both the bulk and selection assay protocol. Both PB and BM CMV-specific CD4+ and CD8+ T cell lines had a predominant CD45RA-CCR7- effector memory phenotype.

Conclusions This study implicates the use of human bone marrow as a source for expansion of multifunctional CMV-specific CD4+ T cells. Recent studies in HIV and Leishmania support the crucial role of multifunctional T cells in disease control (Darrah 2007 [2]).