GM-CSF Production Allows the Identification of Immunoprevalent Antigens Recognized by Human CD4+ T Cells Following Smallpox Vaccination

Abstract

The threat of bioterrorism with smallpox and the broad use of vaccinia vectors for other vaccines have led to the resurgence in the study of vaccinia immunological memory. The importance of the role of CD4+ T cells in the control of vaccinia infection is well known. However, more CD8+ than CD4+ T cell epitopes recognized by human subjects immunized with vaccinia virus have been reported. This could be, in part, due to the fact that most of the studies that have identified human CD4+ specific protein-derived fragments or peptides have used IFN-γ production to evaluate vaccinia specific T cell responses. Based on these findings, we reasoned that analyzing a large panel of cytokines would permit us to generate a more complete analysis of the CD4 T cell responses. The results presented provide clear evidence that TNF-α is an excellent readout of vaccinia specificity and that other cytokines such as GM-CSF can be used to evaluate the reactivity of CD4+ T cells in response to vaccinia antigens. Furthermore, using these cytokines as readout of vaccinia specificity, we present the identification of novel peptides from immunoprevalent vaccinia proteins recognized by CD4+ T cells derived from smallpox vaccinated human subjects. In conclusion, we describe a “T cell–driven” methodology that can be implemented to determine the specificity of the T cell response upon vaccination or infection. Together, the single pathogen in vitro stimulation, the selection of CD4+ T cells specific to the pathogen by limiting dilution, the evaluation of pathogen specificity by detecting multiple cytokines, and the screening of the clones with synthetic combinatorial libraries, constitutes a novel and valuable approach for the elucidation of human CD4+ T cell specificity in response to large pathogens.

HTS by NMR of Combinatorial Libraries: A Fragment-Based Approach to Ligand Discovery

“…The scanning approach has been extensively used and validated in the identification of antigen-specific and protease-specific synthetic peptides from screening of large positional scanned libraries (Judkowski et al, 2011; Lim and Craik, 2009; Lustgarten et al, 2006; Pinilla et al, 1999; Reddy et al, 2011; Sospedra et al, 2010). However, unlike these previous positional scanning assay studies, which depend mostly on either fluorescence or absorbance readouts (Diamond, 2007; Lim and Craik, 2009), using protein-based NMR to perform positional scanning does not require specific knowledge of protein function for assay establishment.…”

Cytokine Production but Lack of Proliferation in Peripheral Blood Mononuclear Cells from Chronic Chagas' Disease Cardiomyopathy Patients in Response to T. cruzi Ribosomal P Proteins

“…In particular, GM-CSF was included because not only its production has been associated to antigen mediated activation of T cells by us and others but also, the threshold of antigen requirement for its production is lower than for other cytokines as TNF-α, IL-4 or IFN-γ [52]–[54].…”

Cytokine Production Associated with Smallpox Vaccine Responses

“…GM-CSF is produced by T cells (mainly CD4 + T cells) in response to vaccinia infection, and its production requires very low antigen concentrations in comparison to the concentrations necessary to stimulate the production of other cytokines during vaccinia infection. Additionally, the concentration of GM-CSF had a higher fold increase from 6- to 48-h post infection than the other cytokines evaluated in this study (IL-13, IL-2, IL-5, IFN-γ and TNF-α) [27]. These results imply that GM-CSF may play a protective role in the early stages of the immune response to VV infection.…”

“…It has also been shown to be upregulated in atopic dermatitis skin cells and play a role in increased viral replication in VV-infected keratinocytes in immunocompromised patients, including those with atopic dermatitis [65,96,97]. IL-13 production by vaccinia-specific T cells following VV infection was observed in the in vitro study carried out by Judkowski et al [27]. …”