An inverse relationship between T cell receptor affinity and antigen dose during CD4(+) T cell responses in vivo and in vitro

Abstract

Multimeric peptide/class II MHC staining reagents were synthesized and shown to bind with appropriate specificity to T cell hybridomas. A small, expanded population of T cells detected with one of these reagents in peptide-immunized C57BL/10 mice persisted for several months. This population expanded further on secondary immunization. Equating the extent of binding of this reagent to T cell receptor affinity, we saw little correlation of immunizing peptide dose to T cell receptor affinity at the peak of the primary response. However, there was an inverse relation between peptide dose and the apparent receptor affinity of the T cells that were present several months after a primary response or after a secondary stimulation either in vivo or in vitro.

Figure 1

Specific binding of peptide-IA^b staining reagents to T cell hybridomas. (A) Sequences of the antigenic peptide portion of the staining reagents 3K-IA^b/PESA, Eα-IA^b/PESA, and 2W-IA^b/PESA. (B) T cell hybridomas (3 × 10⁵) specific for 3Kp/IA^b (B3K05.06, Left) and 2Wp/IA^b (B2W01.01, Right) were incubated separately with the three peptide-IA^b/PESA-staining reagents or MCC-IE^k/PESA at 40 μg/ml in 100 μl of complete tissue culture medium for 2 hr at 37°C. Washed cells were analyzed by flow cytometry for phycoerythrin fluorescence, and histograms are shown. In Left and Right the histogram of the stain performed with the cognate-staining reagent is the dotted line and all other histograms are solid lines.

Figure 2

Direct detection of antigen-specific CD4⁺ T cells from normal C57BL/10 mice after primary immunization with 3K peptide. (A) C57BL/10 mice were immunized with CFA alone (row 1) or with 50 μg (row 2) or 0.5 μg (row 3) of 3Kp in CFA. After 8 days, a sampling of cells pooled from the draining nodes of 6–10 mice was stained with antibodies against CD4, B220, and CD44 and with the multimeric staining reagents 3K-IA^b/PESA (Left) or Eα-IA^b/PESA (Right). Peptide-IA^b/PESA vs. FITC-CD44 plots are shown for CD4⁺, B220^– cells. (B) Histograms of the events that fall in the boxed portions of the dot plots in A are shown. Histograms from mice immunized with 50 μg 3Kp, 0.5 μg 3Kp, or CFA alone are shown in solid, dotted, and dashed lines, respectively. (Upper) The percentages of CD4⁺CD44^high cells (%) from 3Kp-immunized mice that stain with 3K-IA^b/PESA are shown, as well as the mean fluorescence intensities (mfi) of the specifically staining cells. Control Eα-IA^b/PESA histograms were matched and subtracted from the 3K-IA^b/PESA histograms to calculate the percentage of specifically staining cells and their mean fluorescence intensities.

Figure 3

Expansion in the number of antigen-specific CD4 T cells detected with 3K-IA^b/PESA upon secondary challenge with 3K peptide. C57BL/10 mice were immunized with 50 μg (Upper) or 0.5 μg (Lower) 3Kp in CFA and then left alone (Left) or restimulated 9 weeks later with 100 μg (Upper Right) or 1 μg (Lower Right) 3Kp in IFA. Control mice were treated with IFA only (dotted line). Five days after treatment with IFA and peptide, cells from draining lymph nodes were pooled and aliquots were stained as described in the legend to Fig. 2 and in Materials and Methods. Staining data are shown as described in the legend to Fig. 2.

Figure 4

T cells from immunized mice that were expanded in vitro on low doses of 3K peptide stained more intensely and at higher frequency with 3K-IA^b/PESA than cells expanded on high doses of peptide. Eight days after mice were immunized with 50 or 0.5 μg 3Kp, lymph nodes were harvested and cells were cultured for 4.5 days in vitro at five different starting concentrations of 3Kp. Control Eα-IA^b/PESA histograms were matched to and subtracted from the 3K-IA^b/PESA histograms, and a normalized number of the specific, 3K-IA^b/PESA-staining events are depicted in each histogram.

Figure 5

T cell hybridomas generated on low doses of 3K peptide were far more likely to bind 3K-IA^b/PESA, and bind it strongly, than hybridomas raised on high doses of peptide. Lymphocytes from mice immunized once with 50 or 0.5 μg 3Kp in CFA were cultured for 4.5 days in vitro with 100 (Left) or 0.1 (Right) μg/ml 3Kp, respectively, and live cells were expanded in the presence of IL-2 for an additional 3 days. (A) Histograms of CD4⁺CD44^high lymphoblasts stained with 3K-IA^b/PESA (solid lines) or Eα-IA^b/PESA (dotted lines) reagents are shown. Hybridomas made from these lymphoblasts that produced IL-2 in response to C57BL/10 splenocytes and the addition of 3Kp at 25 μg/ml were scored as 3Kp/IA^b specific. (B) Collections of 3Kp/IA^b-specific T cell hybridomas raised on high doses (Left) or low doses (Right) of 3Kp were stained separately with 3K-IA^b/PESA and anti-Cβ antibody. The results are plotted as the percentage of hybridomas vs. the relative mean fluorescence intensity obtained with 3K-IA^b/PESA as a percentage of the mean fluorescence intensity obtained with anti-Cβ.