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.

Figure 1. Cytokine production by CD4+ and CD8+ T cell lines and clones in response to antigen presenting cells infected with vaccinia.

T cells lines and clones were cultured alone or in the presence of autologous LCL (background values) or vaccinia infected LCL. Supernatants were collected after 48 hours of stimulation and cytokine production was evaluated by multiplex assay as described in Materials and Methods . A. Cytokine production by VRC 19 CD4+ lines and control cultures is expressed in pg/ml. B. Normalized values resulting from the subtraction of background values from pg/ml produced by various lines and clones in response to vaccinia infected LCL are shown. C. Each value represents the stimulation index (SI) resulting from dividing the pg/ml produced in response to vaccinia infected LCL by the background values.

Figure 2. Cytokine production by vaccinia specific CD4+ T cell clones in response to vaccinia infected autologous LCL.

Clonal T cells (2.5×10⁴) were cultured alone, stimulated with PHA or in the presence of 5×10⁴ autologous LCL or vaccinia infected LCL (LCL-Vacc). Supernatants were collected after 48 hours and cytokine production was evaluated by multiplex assay. Data represents the average of the cytokine concentrations determined in the supernatant of two replicate culture wells.

Figure 3. Screening profile of clone VRC19-16 in response to the decapeptide positional scanning library.

The GM-CSF production (pg/ml) elicited by VRC19-16 T cells in response to each mixture of the decapeptide library was measured. 2.5×10⁴ VRC19-16 T cells were cultured in the presence of 5×10⁴ autologous LCL and each library mixture at a final concentration of 200 µg/ml. After 48 hours the GM-CSF production was evaluated in the culture supernatants by ELISA. Values represent average of 3 different screenings with duplicate culture wells for a total of 6 replicates. The x axis in each plot indicates the defined amino acid (one letter code) in each of the mixtures of the library. In each position, the amino acid corresponding to the amino acid present in the identified vaccinia peptide D13L-YID is indicated (*).

Figure 4. GM-CSF and TNF-α production by vaccinia specific CD4+ T cell clones in response to the identified vaccinia peptides.

Each vaccinia CD4+ T cell clone (2.5×10⁴) was stimulated for 48 hours in the presence of autologous LCL (5×10⁴) and peptide in a dose response assay at the indicated concentrations. GM-CSF and TNF-α was measured in the culture supernatants by ELISA. Values represent the average cytokine production in two independent duplicate wells and are derived from one representative experiment out of three.

Figure 5. Cytokine production by CD4+ T cell clones.

Vaccinia peptides were tested in a dose response assay with their specific T cell clone (2.5×10⁴) in the presence of autologous LCL (5×10⁴) in duplicate wells. Graphs show the cytokine production in response to 5 and 400 ng/ml of peptide (values for background wells were subtracted) at 6 hours and 48 hours post-stimulation. T cell clones were cultured in the presence of autologous LCL (background), vaccinia infected LCL (LCL-Vacc) or PHA. Cytokine production was evaluated by multiplex assay.

Figure 6. Intracellular cytokine production by vaccinia specific CD4+ T cell clones.

Each clonal T cell population was stimulated with 10 µg/ml of its specific vaccinia peptide in the presence of autologous LCL or vaccinia infected LCL for 6 hours. Brefeldin A was added to the cultures after 1 hour of initial incubation and the intracellular cytokine production was measured as described in the Material and Methods section. The percentage of CD4+ T cells producing each individual cytokine (A) or any of the 15 possible combinations of cytokines (B) is shown. The largest subset of CD4+ T cells (out of the 15 possible combinations of cytokines) for each clone in response to peptide or vaccinia infected LCL is shown as percentages above the corresponding data bars (B). Results are representative of two experiments.

Figure 7. Cytotoxic activity of vaccinia CD4+ T cell clones.

Clonal T cells were co-cultured with ⁵¹ Cr-labeled autologous LCL targets (2×10³), infected with vaccinia or seeded in the presence of vaccinia peptides at 5, 1 and 0.2 µg/ml. The cytotoxicity activity is expressed as the mean of the percent of specific lysis triggered by the cytotoxic T cell clones after 4 hours at a 30∶1 ratio. These results are representative of three experiments.

Figure 8. Determination of HLA restriction.

Cytokine production for each T cell clone was evaluated following the activation with their specific vaccinia peptides in the presence and absence of HLA blocking antibodies and isotype control anti-human IgG2a. T cells (2.5×10⁴) were seeded with autologous LCL (5×10⁴) and peptide and supernatants were collected after 48 hours of incubation. Data represents averages and standard errors of the mean of TNF-α or GM-CSF concentrations in the supernatant of two replicate culture wells in the presence of 10 µg/ml of antibody and 0.4 ng/ml of peptide, with the exception of peptide A6L-SFW that is shown at 370 ng/ml. Results are representative of two individual experiments.

Figure 9. Intracellular cytokine production by vaccinia specific T cell lines.

Vaccinia T cells lines were generated with PBMCs from vaccinated donors VRC19 and VRC47, and from unvaccinated donor TPI10 as detailed in the Material and Methods section. T cell lines were evaluated for their recognition of vaccinia virus and the vaccinia peptides by intracellular TNF-α and IFN-γ production. Data represent the percentage of CD4+ or CD8+ T cells in each line producing either of the four possible combination of cytokines in response to vaccinia infected autologous LCL and PHA (A) or to the identified vaccinia peptides (10 µg/ml) (B).