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. 2006 Sep 1;108(5):1770-3.
doi: 10.1182/blood-2005-10-006536. Epub 2006 May 4.

In vitro priming and expansion of cytomegalovirus-specific Th1 and Tc1 T cells from naive cord blood lymphocytes

Kyung-Duk Park  1 , Luciana Marti, Joanne Kurtzberg, Paul Szabolcs
Affiliations

In vitro priming and expansion of cytomegalovirus-specific Th1 and Tc1 T cells from naive cord blood lymphocytes

Kyung-Duk Park et al. Blood. .

Abstract

Adoptive transfer of CMV-specific cytotoxic T cells (CTLs) expanded in vitro from memory donor T cells can reduce the incidence of CMV disease in allogeneic transplant recipients. However, this approach has been unavailable in the cord blood (CB) transplantation setting because CB T cells are antigen naive and biased toward Th2/Tc2 function. We developed a protocol to in vitro prime and expand CMV-specific CTLs from CB. T cells were primed with cytokines to trigger skewing toward Th1/Tc1 lineage before encountering monocyte and CD34+ progenitor-derived dendritic cells loaded with CMV antigen and its immune complex. CMV-pulsed cultures expanded significantly more over 4 to 6 weeks than CMV cultures despite identical cytokine milieu. T cells isolated from CMV+ cultures showed a preferential expansion of CD45RA-/RO+/CD27+ T cells compared to CMV- cultures. CMV-specific IFN-gamma- and TNF-alpha-producing CD4+ (Th1) and CD8+ (Tc1) T cells were enriched after 3 to 4 weeks and CMV-specific cytotoxicity developed 1 to 2 weeks later.

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Figures

Figure 1.
Figure 1.
CMV-IC-loaded CB DCs exhibit a potent T-cell stimulatory profile. Surface immunophenotype of day 7 Mo/DCs about 36 hours after loading with CMV lysate immune complex (CMV-IC) and subsequent maturation in PGE2 plus TNF-α. Representative of 6 independent experiments. Presented data were gated on forward/side scatter (FSC/SSC) high cellular events conforming to DCs.
Figure 2.
Figure 2.
T cells from CMV-IC-stimulated CTLs secrete Th1 and Tc1 cytokines and lyse CMV-pulsed autologous DC targets. (A) Cytokine secretion. After 4 weeks of CTL culture intracellular (ic) cytokine production (IL-2, IFN-γ, and TNF-α) was analyzed by intracellular fluorescence-activated cell sorting (FACS) staining following restimulation with CMV antigen pulsed (CMV+) or not pulsed (CMV-) DCs in the presence of brefeldin A for 8 hours. Cells in the dot plots represent CD3+ T cells gated in the FL-3 channel. The relative size of T-cell subsets within the indicated quadrants is expressed as the percentage of all CD3+ T-cell events. Results are representative of 5 independent, consecutive experiments. T cells that stained positive for the indicated fluorescent antibody are depicted in different colors. (B) Cytotoxicity. Responder T cells from CMV-IC-pulsed cultures were harvested on day 30, washed, and recultured in triplicate at graded doses, reflecting the indicated effector-target (E/T) ratios, with 5000 fresh CD34+ progenitor-derived immature DCs as targets. DC targets (5000 targets/well) were either pulsed with CMV lysate earlier as indicated or not in controls. Effector cells were added at indicated E/T ratios and cultured at 37°C in 200 μL RPMI 1640 plus 3% PHS. Appropriate controls, including targets alone and responders alone at cell doses representing each E/T ratio, were set up in parallel. Cytotoxicity was measured by fluorometric determination of released LDH in the media after 5 hours of coculture. Specific lysis was calculated after adjusting for target and effector spontaneous LDH release. Percentage specific lysis = [(experimental LDH release) - (target spontaneous LDH release) - (effector spontaneous LDH release)]/[(target total LDH release) - (target spontaneous LDH release)] × 100. Results are representative of 4 independent experiments (mean ± SD).
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