MHC class II tetramers identify peptide-specific human CD4+ T cells proliferating in response to influenza A antigen (original) (raw)
Earlier studies have shown that peripheral blood lymphocytes from individuals with previous exposure to influenza A virus generate a class II DR-restricted T-cell proliferative response to the HA307–319 epitope (19). This conserved peptide can induce a proliferative response in a number of different DR haplotypes, including DR1, DR4, DR5, and DR7 (20).
To detect T cells specific for the HA307–319 peptide presented in the context of DR4, we synthesized class II DRA1*0101/DRB1*0401 tetramers loaded with HA307–319 peptide. We tested the specificity of the HA307–319 tetramer using a DRB1*0401-restricted human T-cell clone specific for HA307–319. As shown in Figure 1a, the clone demonstrated antigen-specific proliferation against HA307–319 in the context of DRB1*0401 expressed in a BLS-1 cell line (18). We stained the HA307–319-specific clone using DRB1*0401 tetramers loaded with the HA307–319 peptide. As shown in Figure 1b, virtually all the cells stained positive for the HA307–319 tetramer and CD4, consistent with the phenotype of the clone. As a control we also constructed a DRB1*0401 tetramer loaded with TT830-843 peptide (21). As illustrated in Figure 1c, none of the HA307–319 clonal cells stained positive for the TT830-843 tetramer.
Specificity and HLA restriction of HA307–319 tetramer. (a) Comparison with thymidine incorporation at 72 hours between the T-cell clone cultured with the DRA1*0101/DRB1*0401 transfected BLS-1 pulsed with no antigen (left bar) and 10 μg/mL of HA307–319 peptide (right bar). Error bars indicate SEM of triplicates. In b and c, clonal cells were stained with PE-labeled tetramer for 3 hours at 37°C, washed, stained with anti-CD4, washed again, and analyzed by flow cytometry. Staining with tetramer loaded with HA307–319 is shown in b, and tetramer loaded with TT830-843 peptide is shown in c. In both, cells are gated on forward and side scatter, the vertical axis shows tetramer fluorescence, and the horizontal axis shows CD4 fluorescence. Percentages shown in the margins of each panel represent the percent of total cells present in each quadrant.
We then tested the ability of the HA307–319 tetramer to detect antigen-specific T cells collected from the peripheral blood of 2 DRB1*0401 donors, including the same donor from which the HA-specific clone was derived. Nylon wool–purified T cells from peripheral blood were stained with CFSE, a fluorescent dye that stably binds cytoskeletal actin (22). CFSE-stained cells were cultured with autologous adherent cells pulsed with HA307–319 peptide, whole influenza vaccine, or TT. After 7 days of culture, HA307–319 tetramers were used to stain the cells before analysis with flow cytometry. Each time a cell divides, CFSE is apportioned equally among daughter cells, resulting in a halving of CFSE fluorescence. Therefore, the number of cell divisions can be determined by comparing the resultant CFSE fluorescence to the original fluorescence of the parent population. As shown in Figure 2, all 3 antigens induced cell proliferation as indicated by populations of cells with decreased CFSE fluorescence, shown on the horizontal axis. The vertical axis shows the fluorescence from the PE-labeled HA307–319 tetramers, and the 2 columns represent equivalent experiments done on cells from the 2 donors. As shown in the upper-left quadrants of Figure 2, a and b, the labeled tetramer clearly identified a significant number of HA307–319-specific cells in both the HA307–319 and whole influenza vaccine samples in both donors. To our knowledge, this is the first time epitope-specific T helper cells have been directly seen in a stimulation of human lymphocytes taken from the peripheral blood.
HA307–319 tetramer identification of antigen-specific cells in relation to CFSE fluorescence. Nylon wool–purified T cells, labeled with CFSE before culture with autologous adherent cells and antigen, were stained on day 7 with PE-labeled HA307–319 tetramer and analyzed subsequently by flow cytometry. Each row shows cells from a different stimulating antigen for 2 different individuals: (a) 10 μg/mL HA307–319 peptide, (b) whole influenza vaccine containing 11 μg/mL HA, (c) whole TT at a maximally stimulating dose. In all panels cells are gated on forward and side scatter, the vertical axis shows PE fluorescence of the HA307–319 tetramer, and the horizontal axis shows CFSE fluorescence over a 4-decade logarithmic scale. In addition, the horizontal axis shows the corresponding number of cell divisions, with “P” depicting the undivided parent population. This scale was calculated from the distinct CFSE fluorescence peaks produced by polyclonal stimulation with PHA and IL-2 as described in Methods. Percentages shown in the margins of each panel represent the percent of total cells present in each quadrant. The panels depict results from representative individual experiments.
Examination of the cells stimulated with HA307–319 peptide showed that in both individuals around 90% of the tetramer-binding cells were in the divided population, reflecting the specific expansion of this cohort of T cells. The observed number of cell divisions for a given CFSE fluorescence is shown along the horizontal axis for all figure parts. In donor 1, the tetramer-binding population divided an average of 6.5 times during the 7-day culture as calculated by CFSE fluorescence, whereas in donor 2 the tetramer-binding cells divided an average of 9 times. This difference in the numbers of divisions accounts for the greater number of divided cells seen in donor 2. The calculated precursor frequency of DRB1*0401 tetramer-specific cells is similar for the 2 individuals, ranging between 3 and 5 per 100,000 cells, depending on the individual experiment.
The tetramer-binding population of dividing cells comprised a distinct portion of the total dividing cells in the HA307–319 stimulated sample. In donor 1, some of the divided cells that are tetramer negative likely represent T cells specific for HA307–319 in the context of DRB1*0101 because the donor haplotype is DRB1*0401, DRB1*0101, and DR1 has been described as capable of presenting the peptide (23). In addition, background levels of proliferation of between 1% and 7% of the total cells were seen even in control samples where antigen was not added, depending on the individual (data not shown). IL-2 and other cytokines liberated by the antigen-specific HA307–319 cells would likely increase this background through increased bystander activation and proliferation.
In cells stimulated with whole influenza vaccine (Figure 2b), there was likewise a definite population of tetramer-positive cells for both individuals, shown in the upper-left quadrant. Therefore, even in the context of a vigorous and complex proliferative T-cell response to viral antigens, the specific T cells corresponding to an immunodominant epitope are readily identified. There was no tetramer labeling of cells stimulated with TT despite vigorous proliferation (Figure 2c). These results indicate that the tetramer detection method is both specific and sensitive for the population of T cells reactive toward HA307–319.
The use of tetramer staining, together with flow cytometry, to identify antigen-specific cells permits simultaneous analysis of cells using additional fluorochromes. This additional phenotypic analysis can provide important information about an antigen-specific response such as the type of T cell involved, presence of activation or other markers, and cytokine production through intracellular staining. In this study we examined surface expression levels of CD3, CD4, CD8, and CD25 to further characterize the cells identified by the HA307–319 tetramer. The data in Figure 3 is gated to show only cells identified as HA307–319-tetramer positive. Figure 3, a and b, shows that almost all the tetramer-positive cells in both the HA307–319 peptide–stimulated and whole influenza vaccine–stimulated samples are CD4+ CD8–. Similarly, Figure 3, c and d, shows that the majority of tetramer-positive cells are CD3+ CD25+ for both samples. We conclude that the DRB1*0401-HA307–319 tetramer is bound almost entirely to class II–restricted, activated, and proliferating T cells, as expected, lending further support to the specificity of the tetramer detection method.
Phenotypic characterization of HA307–319 tetramer-specific cells. Nylon wool–purified T cells were labeled with CFSE before culture with autologous adherent cells and antigen. On day 7, cells were stained with PE-labeled tetramer and then stained with combinations of fluorochrome-labeled anti-CD3, -CD4, -CD8, and -CD25 antibodies before flow-cytometry analysis. All panels are gated on tetramer-positive cells. Upper panels show CD8 versus CD4 staining in cells stimulated with (a) HA307–319 peptide, and (b) whole influenza vaccine, whereas lower panels show CD25 versus CD3 staining in cells stimulated with (c) HA307–319 peptide, and (d) whole influenza vaccine. Boundaries for positive and negative populations were determined by controls. Percentages shown in the margins of each panel represent the percent of total cells present in each quadrant. The panels depict results from a representative individual experiment.
The concomitant use of peptide-MHC class II tetramers and CFSE staining provides a powerful tool to assess and dissect antigen-specific T-cell responses. Direct identification of peptide-specific proliferation in primary cultures avoids the need for limiting dilution analysis to calculate precursor frequencies and permits simultaneous phenotypic analysis of the antigen-specific cells using flow cytometry. The present study demonstrates the immunodominance of the HA307–319 epitope in the context of the complex response against whole influenza vaccine and illustrates how tetramers can be used to directly identify immunodominant antigenic specificities. The potential for loading different peptides into the tetramers suggests multiple applications, although the stability of specific peptide-class II tetramers is likely to vary among peptides. The use of class II tetramers provides a means for understanding on a much more detailed level the immune response against infectious agents and in autoimmune disease.