Status of Activation of Circulating Vaccine-Elicited CD8+ T Cells

Abstract

Selective blunting of the status of activation of circulating tumor-specific T cells was invoked to explain their paradoxical coexistence with unhampered tumor growth. By analogy, lack of tumor regression in the face of observable melanoma vaccine-induced T cell responses might be attributed to their status of activation. We enumerated with HLA-A*0201/peptide tetramers (tHLA) vaccine-elicited T cell precursor frequency directly in PBMC of patients with melanoma undergoing vaccination with the HLA-A*0201-associated gp100:209–217(210 M) epitope (g209-2 M). Furthermore, we tested by intracellular (IC)-FACS analysis and quantitative real-time PCR (qRT-PCR) the ability of postvaccination PBMC to produce cytokine in response to challenge with vaccine-related epitopes or vaccine-matched (HLA-A*0201) melanoma cells. Vaccine-induced enhancement of T cell precursor frequency could be detected with tHLA in PBMC from six of eight patients studied at frequencies ranging between 0.3 and 2.3% of the total CD8+ population. Stimulation with vaccine-related epitopes induced IFN-γ expression detectable by IC-FACS or qRT-PCR, respectively, in five and six of these patients. Furthermore, down-regulation of tHLA staining was noted upon cognate stimulation that could be utilized as an additional marker of T cell responsiveness. Finally, we observed in six patients an enhancement of reactivity against vaccine-matched tumor targets that was partly independent of documented vaccine-specific immune responses. A strong correlation was noted between tHLA staining of postvaccination PBMC and IFN-γ expression by the same samples upon vaccine-relevant stimulation and assessed either by IC-FACS or qRT-PCR. Thus, blunting of the status of T cell activation on itself cannot easily explain the lack of clinical responses observed with vaccination.

Biased epitope selection by recombinant vaccinia-virus (rVV)-infected mature or immature dendritic cells

“…This finding might explain our previous observation that the percent of Tcells staining with tetrameric HLA-peptide complexes and demonstrating TCR downregulation upon cognate stimulation was consistently higher than the number of intracellular IFN-g-expressing T cells. 28,32 In addition, these data suggest that the ability of DC to present Ag is not significantly limited by the decreased surface density of HLA-A*0201 molecules occurring 12-24 h after rVV-gp100 infection.…”

“…28,29 After infection with rVV and overnight incubation, the DC were used as antigenpresenting cells to stimulate a well-characterized g209-specific, HLA-A*0201-restricted CTL clone JR6C12 representative of most CTL clones derived from HLA-A*0201 melanoma patients treated at our institution. In particular, JR6C12 was originally expanded from circulating lymphocytes of a patient who had undergone active-specific immunization with g209-2M peptide administered subcutaneously in incomplete Freund's adjuvant.…”

“…In addition, we and others have previously shown that they correlate quite well with other methods of assessing Ag-specific stimulation. 16,28,29,32,52 Because of the significant reduction in HLA-A*0201 expression caused by rVV infection, we first tested the ability of rVV-infected DC to induce TCR downregulation and IFN-g expression in the g209-specific CD8+ T-cell clone JR6C12 when exogenously pulsed with gradually decreasing concentrations of relevant epitope. No significant differences were noted in the ability of iDC or mDC to induce T-cell activation, whether they had been infected or not, suggesting that the cell surface availability of functional HLA-A*0201 molecules in the conditions tested was not a major limiting factor.…”

IL-10 stimulatory effects on human NK cells explored by gene profile analysis

“…Cells were permeabilized by incubation in PBS-saponin (0.2%, Sigma, St Louis, MO, USA) in 5% milk for 6 h. 33 A donkey anti-goat secondary antibody (Molecular Probes, Eugene, OR, USA) was used for the fluorescent staining. IL-10, but not IL-2, stimulated TIA-1 protein expression in NK cells (Figure 6), confirming the genetic findings.…”

Host Absence of CCR5 Potentiates Dendritic Cell Vaccination

“…Because of the demonstrable host immune response to melanoma and the current understanding of tumor immunology, vaccine therapy is being investigated as a therapeutic strategy for melanoma (7)(8)(9)(10)(11)(12)(13). Dendritic cells (DCs) 3 are the most potent APCs, highly specialized to prime T cell-dependent Ag-specific immune responses (14 -17).…”