Recent studies have shown that CD4+ T cell help is required for the generation of memory CD8+ T cells that can proliferate and differentiate into effector cells on Ag restimulation. The importance of help for primary CD8+ T cell responses remains controversial. It has been suggested that help is not required for the initial proliferation and differentiation of CD8+ T cells in vivo and that classical models of helper-dependent responses describe impaired secondary responses to Ag in vitro. We have measured primary CD8+ T cell responses to peptide-pulsed dendritic cells in mice by cytokine ELISPOT and tetramer staining. No responses were detected in the absence of help, either when normal dendritic cells were injected into MHC II-deficient mice or when MHC II-deficient dendritic cells were injected into normal mice. Thus, the primary in vivo CD8+ T cell response depends absolutely on help from CD4+ T cells in our experimental system.

Our previous work has demonstrated that human follicular lymphoma (FL) infiltrating T cells are anergic, in part due to suppression by regulatory T cells. In this study, we identify pericellular adenosine, interacting with T cell-associated G protein-coupled A2A/B adenosine receptors (AR), as contributing to FL T cell hyporesponsiveness. In a subset of FL patient samples, treatment of lymph node mononuclear cells (LNMC) with specific A2A/B AR antagonists results in an increase in IFN-γ or IL-2 secretion upon anti-CD3/CD28 Ab stimulation, as compared with that seen without inhibitors. In contrast, treatment with an A1 AR antagonist had no effect on cytokine secretion. As the rate limiting step for adenosine generation from pericellular ATP is the ecto-ATPase CD39, we next show that inhibition of CD39 activity using the inhibitor ARL 67156 partially overcomes T cell hyporesponsiveness in a subset of patient samples. Phenotypic characterization of LNMC demonstrates populations of CD39-expressing CD4+ and CD8+ T cells, which are overrepresented in FL as compared with that seen in normal or reactive nodes, or normal peripheral blood. Thirty percent of the FL CD4+CD39+ T cells coexpress CD25high and FOXP3 (consistent with regulatory T cells). Finally, FL or normal LNMC hydrolyze ATP in vitro, in a dose- and time-dependent fashion, with the rate of ATP consumption being associated with the degree of CD39+ T cell infiltration. Together, these results support the finding that the ATP-ectonucleotidase-adenosine system mediates T cell anergy in a human tumor. In addition, these studies suggest that the A2A/B AR as well as CD39 are novel pharmacological targets for augmenting cancer immunotherapy.

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Human peripheral blood dendritic cells (PBDC) are a rare population comprised of several distinctive subsets. Analysis of these cells has been hindered by their low frequency. In this study, we report a novel direct ex vivo 11-color flow cytometric assay that combines subset identification with analysis of activation status and endocytic ability of three major PBDC subsets (CD1c+CD11c+ “MDC1,” CD141+CD11c+ “MDC2,” and CD303+CD11c− “PDC”) within a single platform. This method eliminates the need for DC enrichment, isolation, or prolonged culture. Human peripheral blood mononuclear cells (PBMC) from healthy donors are incubated with FITC-dextran directly ex vivo, prior to cell surface staining with various markers. As expected, PBDC identified by this assay express low levels of CD40 and CD86 directly ex vivo, and significantly upregulate expression of these molecules upon stimulation with toll-like receptor ligands LPS and CpG oligonucleotides. In addition, PDC internalize FITC-labeled dextran poorly in comparison to MDC1 and MDC2 subsets. Specificity of FITC-dextran endocytosis is further verified by imaging flow cytometry. Furthermore, the combination of surface markers used in this assay reveals a previously unreported CD4+CD11c+CD303−CD1c-CD141− cell population. Taken together, this assay is a rapid and cost-effective method that avoids manipulation of PBDC while providing direct ex vivo high-dimensional flow cytometry data for PBDC studies.

Integrated Raman and angular-scattering microscopy (IRAM) is a multimodal platform capable of noninvasively probing both the chemistry and morphology of a single cell without prior labeling. Using this system, we are able to detect activation-dependent changes in the Raman and elastic-scattering signals from CD8+ T cells stimulated with either Staphylococcal enterotoxin B (SEB) or phorbol myristate acetate (PMA). In both cases, results obtained from the IRAM instrument correlate well with results obtained from traditional fluorescence-based flow cytometry for paired samples. SEB-mediated activation was distinguished from resting state in CD8+ T cells by an increase in the number and mean size of small ( approximately 500-nm) elastic scatterers as well as a decrease in Raman bands, indicating changes in nuclear content. PMA-mediated activation induced a different profile in CD8+ T cells from SEB, showing a similar increase in small elastic scatterers but a different Raman change, with elevation of cellular protein and lipid bands. These results suggest the potential of this multimodal, label-free optical technique for studying processes in single cells.