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. 2001 Feb 13;98(4):1728-33.
doi: 10.1073/pnas.98.4.1728.

Dependence of lymphopenia-induced T cell proliferation on the abundance of peptide/ MHC epitopes and strength of their interaction with T cell receptors

Q Ge  1 , V P Rao, B K Cho, H N Eisen, J Chen
Affiliations

Dependence of lymphopenia-induced T cell proliferation on the abundance of peptide/ MHC epitopes and strength of their interaction with T cell receptors

Q Ge et al. Proc Natl Acad Sci U S A. .

Abstract

Factors that affect naive T cell proliferation in syngeneic lymphopenic hosts were investigated. 2C T cell receptor (TCR) transgenic T cells lacking both CD8 and CD4 survived but hardly proliferated. Proliferation of CD8(+) 2C cells was proportional to the abundance of cognate peptide/MHC complexes and was severely inhibited by injection of anti-CD8 antibody. Weakly reactive self-peptides slightly enhanced CD8(+) 2C cell proliferation whereas a potent agonist peptide promoted much more rapid proliferation, but inflammation-stimulating adjuvant had only a small effect on the rate of cell proliferation. The findings suggest that under uniform lymphopenic conditions, the widely different rates of proliferation of T cells expressing various TCR, or the same TCR in the presence or absence of CD8, reflect the strength of interaction between TCR and MHC associated with particular self-peptides.

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Figures

Figure 1
Figure 1
Analyses of surface markers of CD8+ and DN 2C T cells and their relative abundance before and after thymectomy. (A) Lymph node cells from backcrossed 2C/RAG mice were assayed for 2C TCR, CD4, and CD8. The histogram shows 2C TCR expression as detected by clonotypic antibody 1B2. The two-dimensional dot plot shows CD8 and CD4 expression by 1B2+ cells. Numbers indicate the percentages of CD4+, CD8+, and CD4−CD8− 2C cells. (B) Comparison of the cell size (forward light scatter) and indicated cell surface markers between CD8+ (thin line) and DN (bold line) 2C cells in 2C/RAG mice. Lymph node cells were assayed for 2C TCR, CD4, CD8 plus CD2, CD44, CD25, CD69 (not shown), CD5, HSA, or B220. Histograms are generated by gating on 1B2+CD8+CD4− or 1B2+CD8−CD4− cells. (C) Percentages of CD8+ and DN 2C cells in the lymph nodes of various types of mice. Lymph node cells from backcrossed (F10) and nonbackcrossed 2C/RAG mice and thymectomized 2C/RAG (F10) mice 6 or 12 weeks after surgery were assayed for 2C TCR, CD4, and CD8. CD8+ and DN 2C cells are shown as percentages of total 1B2+ cells. Each symbol represents one mouse. (D) Percentages of CD8+ and DN 2C cells in peripheral blood at different time points after thymectomy. 2C/RAG mice (F10) were thymectomized at 6–8 weeks of age. Peripheral blood leukocytes were assayed for the expression of 2C TCR, CD4, and CD8. The percentages of CD8+ and DN 2C cells are shown as a function of time for each mouse.
Figure 2
Figure 2
Requirement for CD8 for lymphopenia-induced 2C cell proliferation. (A) CFSE-labeled lymph node cells (1 × 106 CD8+1B2+) from 2C/RAG mice were transferred into RAG1−/− recipients. Seven days later, lymph node cells were recovered from recipients and analyzed for 2C TCR, CD4, CD8, and CFSE. CFSE profiles are shown for CD8+ and DN 2C cells. Percentages refer to proportion of cells that proliferated within 7 days. (B) CFSE-labeled lymph node cells from 2C/RAG mice were incubated with 10 μg/ml of anti-CD8α antibody on ice for 30 min and transferred into irradiated C3−/− recipients (1 × 106 CD8+1B2+/recipient). The recipients were injected with 500 μg of anti-CD8α immediately after transfer and again the next day. As a control, CFSE-labeled 2C cells were treated in the same way but without antibody and recipients were not injected with antibody. Lymph node cells from recipients were analyzed for 2C TCR, CD4, CD8β, and CFSE 5 days after transfer. CFSE profiles are shown for CD8β+ and DN 2C cells in anti-CD8-treated and untreated recipients. Percentages refer to proportion of cells that proliferated within 5 days. (C) CFSE-labeled lymph node cells from 2C/RAG mice were stimulated in vitro with irradiated BALB/c splenocytes in the presence of 1 × 10-7 M QL9 peptide for 3 days. Cells were analyzed for 2C TCR, CD4, CD8, and CFSE. CFSE profiles are shown for CD8+ and DN 2C cells. (D) Lymph node cells (not labeled by CFSE) were stimulated as in C. Cells were analyzed for 2C TCR, CD4, CD8, plus CD44, CD25 (not shown), CD69, or intracellular IFN-γ. Histograms of CD44, CD69, and IFN-γ expression by CD8+ and DN 2C cells are compared before (shaded) and after stimulation (bold).
Figure 3
Figure 3
Effect of pepMHC on the proliferation of 2C cells in lymphopenic recipients. (A) Comparison of proliferation of CD8+ 2C and CD8+ F5 cells in lymphopenic recipients that were either untreated or injected with cognate agonist peptides in CFA. CFSE-labeled lymph node cells (1 × 106 CD8+TCR+) from 2C/RAG or F5/RAG mice were transferred into syngeneic RAG1−/− recipients. Two days later, some of the 2C T cell recipients were challenged with 50 μg of SYRGL peptide in CFA and some of the F5 T cell recipients were challenged with 50 μg of NP68 (ASNENMDAM) peptide in CFA. Lymph node cells from recipients were analyzed for TCR, CD8, and CFSE 5 days after transfer. Proliferation of CD8+ 2C and CD8+ F5 cells are shown. Percentages refer to proportion of cells that proliferated within 5 days. Numbers in parentheses indicate the average division of cells that proliferated. (B) Comparison of CD8+ 2C cell proliferation in various lymphopenic recipients. CFSE-labeled lymph node cells from 2C/RAG mice (1 × 106 CD8+1B2+) were transferred into RAG1−/− recipients or irradiated TAP−/−, β2m−/−, or (KbDb)−/− recipients. Five days later, lymph node cells were analyzed as in A. CFSE profiles of CD8+ 2C cells from one representative experiment are shown (Left). Percentage refers to proportion of cells that proliferated within 5 days. Peripheral blood leukocytes were assayed for CD11c and Kb. Kb expression by CD11c+ cells is shown (Right). (C) Effect of peptides on Kb expression and 2C cell proliferation. CFSE-labeled lymph node cells from 2C/RAG mice (1 × 106 CD8+1B2+) were transferred into irradiated TAP−/− recipients. Recipients were either untreated or injected i.p. with 200 μg of SIINFEKL, 200 μg of dEV8, or 0.4, 2, and 50 μg of SYRGL in PBS on the day of transfer and the next day. Lymph node cells from recipients were assayed for CD11c and Kb, or 2C TCR, CD8 plus CFSE 5 days after transfer. CFSE profiles of CD8+ 2C cells are shown (Left). Percentages and numbers in parentheses are as in A. Expression of Kb on CD11c+ cells in lymph nodes is shown as histograms (Right). Percentages of positive cells are shown.
Figure 4
Figure 4
Effect of peptide and CFA on CD8+ 2C cell proliferation and activation. (A and B) CFSE-labeled lymph node cells from 2C/RAG mice (2.5 × 106 CD8+1B2+) were transferred into RAG1−/− recipients. Two days later, some recipients were injected i.p. with 50 μg of SYRGL in PBS, or s.c. with CFA alone or 20 μg of SYRGL in CFA. Five days after transfer, lymph node cells from recipients were assayed for proliferation and expression of Kb. (A) CFSE profiles of CD8+ 2C cells from one representative experiment are shown. Percentages and numbers in parentheses are as in Fig. 3A. The total number of CD8+ 2C cells recovered from lymph nodes and spleens of each recipient are shown. (B) Expression of Kb on CD11c+ cells in lymph nodes. Geometric mean of Kb fluorescent intensity is shown. (C) The experiments were carried out as in A and B, except that transferred 2C cells were not labeled with CFSE. Five days after transfer, freshly isolated lymph node cells from recipients were assayed for cytolytic activity and for 2C TCR, CD8 plus CD25, CD69, or intracellular IFN-γ. The histograms show the expression of IFN-γ by CD8+ 2C cells. Thin line, isotype control; bold line, anti-IFN-γ. The percentages of CD8+ 2C cells that express CD25 or CD69, and the percentages of specific lysis of target cells by lymph node cells from various recipients (effector/target = 5:1) are shown.
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