Cytotoxic T lymphocyte antigen-4 (CTLA-4) regulates primary and secondary peptide-specific CD4(+) T cell responses

Abstract

CTLA-4-deficient mice develop a fatal lymphoproliferative disorder, characterized by polyclonal expansion of peripheral lymphocytes. To examine the effect of restricting the CD4(+) TCR repertoire on the phenotype of CTLA-4-deficient mice and to assess the influence of CTLA-4 on peptide-specific CD4(+) T cell responses in vitro, an MHC class II-restricted T cell receptor (AND TCR) transgene was introduced into the CTLA-4(-/-) animals. The expression of the AND TCR transgene by CD4(+) T cells delays but does not prevent the lymphoproliferation in the CTLA-4(-/-) mice. The CD4(+) T cells become preferentially activated and expand. Interestingly, young AND TCR(+) CTLA-4(-/-) mice carrying a null mutation in the rag-1 gene remain healthy and the T cells maintain a naive phenotype until later in life. We demonstrate that CTLA-4 regulates the peptide-specific proliferative response generated by naive and previously activated AND TCR(+) RAG(-/-) T cells in vitro. The absence of CTLA-4 also augments the responder frequency of cytokine-secreting AND TCR(+) RAG(-/-) T cells. These results demonstrate that CTLA-4 is a key regulator of peptide-specific CD4(+) T cell responses and support the model that CTLA-4 plays a differential role in maintaining T cell homeostasis of CD4(+) vs. CD8(+) T cells.

Figure 1

CD4⁺ T cells become activated in AND TCR⁺ mice in the absence of CTLA-4. Representative flow cytometry data of the lymph node cells from AND TCR⁺ CTLA-4^−/− and littermate control animals. The expression of AND TCR transgene (A) and the phenotype of the CD4⁺ lymph node T cells (B) from an 8-week-old AND TCR⁺ CTLA-4^−/− mouse and littermate control. The absolute number of the CD4⁺ T cells is indicated.

Figure 2

Absolute cell number of CD4⁺ T cells expressing AND TCR⁺ and endogenous TCR chains increase in CTLA-4-deficient animals. Single cell suspensions were prepared from the spleen and lymph nodes and stained for CD4 and Vβ3, Vα11, and the absolute number of each subpopulation was determined. Each dot represents a single animal. AND TCR⁺ CTLA-4^{+/+, +/−}, n = 6 (⋄); CTLA-4^−/−, n = 5 (♦).

Figure 3

CD8⁺ T cells do not become activated in AND TCR⁺ mice in the absence of CTLA-4. Shown are representative flow cytometry data of the AND TCR transgene (A) and the phenotype (B) of CD8⁺ lymph node cells from AND TCR⁺ CTLA-4^−/− and littermate control mice shown in Fig. 1. The absolute number of the CD8⁺ T cells is indicated.

Figure 4

CD4⁺ AND TCR⁺ CTLA-4^−/− RAG-1^−/− T cells do not become activated in vivo. Shown are representative flow cytometry data of lymph node CD4⁺ AND TCR⁺ CTLA-4^+/− and CTLA-4^−/− T cells that are RAG-1^−/− (7 weeks old). The absolute number of AND TCR⁺ RAG^−/− T cells is indicated. The average numbers of lymph node T cells in these animals are: AND TCR⁺ CTLA-4⁺ RAG^−/−, 7.0 ± 3.8 × 10⁶, n = 17; AND TCR⁺ CTLA-4^−/− RAG^−/−, 14 ± 7.5 × 10⁶, n = 7.

Figure 5

Primary and secondary proliferative responses by AND TCR⁺ RAG^−/− T cells are greater in the absence of CTLA-4. (A) Naive AND TCR⁺ RAG^−/− CTLA-4-deficient and littermate control lymph node T cells were isolated and stimulated (2 × 10⁴ cell per well) with irradiated I-E^k-transfected L cells (2 × 10⁴ cell per well) and pigeon or moth cytochrome c (PCC_88–104 and MCC_93–103, respectively). (B) Secondary response. AND TCR⁺ RAG^−/− T cells from CTLA-4^−/− and littermate control animals were expanded in vitro as described in Methods and Materials. The cells were collected, ficolled, and restimulated with PCC_88–104 or MCC_93–103 as described above. These are representative data of at least three experiments.

Figure 6

Frequency of AND TCR⁺ T cells secreting IL-4 in response to primary and secondary PCC stimulation in vitro is increased in the absence of CTLA-4. (A) Naive AND TCR⁺ RAG^−/− T cells were stimulated in vitro with I-E^k+ B7^lo L cells plus MCC_93–103 at the indicated cell number on plates coated with anti-IL-4 antibody, as described in Methods and Materials. After incubation for 22 (a) or 36 (b) hr, the plates were developed and scored for IL-4 secretion. (B) Previously activated AND TCR⁺ RAG^−/− T cells were stimulated with MCC_93–103 (5 or 0.5 μM) or PCC_88–104 (0.5 μM) plus APC for 36 hr as described above. These are representative data of at least five experiments.