p21^Cip1 induced by n-butyrate was maintained in anergic T helper type 1 (Th1) cells. The Th1 cells were stimulated with keyhole limpet haemocyanin (day 0). n-Butyrate was added to some of the Th1 cells on day 1 (D₁). Total cell lysates were prepared daily during the 6-day primary culture and Western blot analysis of p21^Cip1 (a) and other cell-cycle proteins (b) was performed. Because n-butyrate was added to the cultures at the end of day 1, there is a common day 1 (D₁) lane for the two groups. As there was insufficient material, the D₁ level of some proteins could not be included. Densitometric analysis of the p21^Cip1 levels was performed and the are results presented as per cent adjusted volume (PAV). Day 6 levels of p21^Cip1 were studied in four separate experiments with consistent results.

p21^Cip1 did not preferentially interact with cyclin-dependent kinases (cdk) in anergic T helper type 1 (Th1) cells. The Th1 cells were incubated in primary cultures with antigen ± n-butyrate. Following 6 days in primary cultures, viable Th1 cells were isolated and then restimulated with antigen for 36 hr. Following restimulation, whole cell lysates were prepared, and cdk2, cdk4 and cdk6 were immunoprecipitated from the lysates. The immunoprecipitated proteins (IP) and the proteins remaining in the lysate supernatant (SN) were separated by sodium dodecyl sulphate–polyacrylamide gel electrophoresis and then immunoblotted with antibodies specific for p21^Cip1 and the respective cdk.

p21^Cip1 interacted with phosphorylated c-Jun N-terminal kinase (p-JNK) and p-c-jun in anergic T helper type 1 (Th1) cells in reciprocal immunoprecipitations. Th1 cells from primary cultures stimulated with antigen in the absence (lane 1) or presence (lane 2) of n-butyrate were restimulated with anti-CD3 and anti-CD28 antibody-coated beads for 2 hr before lysis. p-JNK (a) and p-c-jun (b) were immunoprecipitated from lysates, separated by sodium dodecyl sulphate–polyacrylamide gel electrophoresis and then immunoblotted with antibodies specific for p21^Cip1, p27^Kip1and p-JNK.

n-Butyrate induced anergy in T helper type 1 (Th1) cells. Th1 cells were stimulated with keyhole limpet haemocyanin (KLH) in the presence or absence of n-butyrate in primary cultures. (a) Following 24 hr in the primary cultures, proliferation was measured as a function of [³H]thymidine incorporation. (b) Following 6 days in primary cultures, Th1 cells were isolated, and along with previously untreated Th1 cells, were stimulated in equivalent numbers for 24 hr with KLH or with interleukin-2 (IL-2) -containing conditioned medium. Proliferation results are presented as mean ± standard error. This experiment was repeated nine times with similar results. Asterisk indicates statistical difference between anergic and control Th1 cells. (c) Following 6 days in primary cultures, Th1 cells were isolated and were stimulated in equivalent numbers for 24 hr with anti-CD3 and anti-CD28 antibody-coated beads at a 1 : 4 bead to cell ratio or with IL-2-containing conditioned medium. (d) Following 6 days in primary cultures, Th1 cells stimulated with antigen ±n-butyrate were restimulated with anti-CD3 and anti-CD28 antibody-coated beads at a 1 : 1, 1 : 2 or 1 : 4 bead to cell ratio for 24 hr. Culture supernatants were collected and IL-2 and interferon-γ levels were measured.

p21^Cip1 was detected in both cytoplasmic and nuclear fractions of anergic T helper type 1 (Th1) cells. The Th1 cells were incubated in primary cultures with antigen ± n-butyrate. Following 6 days in primary cultures, viable cells were isolated and cell lysates were prepared. Western blot analysis of p21^Cip1 and U1 SnRNP 70 was performed. This experiment has been repeated once with similar results.

p21^Cip1 preferentially interacted with phosphorylated c-Jun N-terminla kinase (p-JNK) and p-c-jun in anergic T helper type 1 (Th1) cells. (a) The Th1 cells were stimulated with anti-CD3 and anti-CD28 antibody-coated beads for 0–24 hr, lysed at different time-points, separated by sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE)and immunoblotted with antibody specific for p21^Cip1. (b) The Th1 cells were stimulated with antigen in the presence or absence of n-butyrate in primary cultures. Viable control or anergic Th1 cells were then isolated. Control Th1 cells were stimulated for 24 hr with anti-CD3 and anti-CD28 antibody-coated beads (lane 1). febs_Half of the anergic Th1 cells were lysed at the end of primary cultures (lane 2) and the other half were restimulated with anti-CD3 and anti-CD28 antibody-coated beads for 2 hr before lysis (lane 3). Cell lysates were prepared from the three different groups and p21^Cip1 was immunoprecipitated from the lysates. The immunoprecipitated p21^cip1 as well as the remaining lysate supernatant (SN) were separated on SDS–PAGE and then immunoblotted with antibodies specific for p21^Cip1, cdk2, PCNA, JNK, p38, p-JNK and p-c-jun. This experiment was repeated once with similar results.

c-Fos and c-jun activity was inhibited in the anergic T helper type 1 (Th1) cells. (a) Th1 cells were stimulated with antigen ± n-butyrate in primary cultures. The Th1 cells were then restimulated using anti-CD3 and anti-CD28 antibody-coated beads for 2 hr for c-fos assay and 1 hr for c-jun assay. Nuclear lysates were prepared from restimulated Th1 cells as well as from unstimulated resting Th1 cells (group R). c-fos and c-jun activity was measured using TransAM Transcription Factor Activity Assay kits. This experiment was performed twice with consistent results. Statistical differences between control and anergic Th1 cells are indicated as **P< 0·01 or *P < 0·05. (b) Th1 cells were stimulated with immobilized anti-CD3 and anti-CD28 antibodies in the presence or absence of 5 μm SP600125 for 24 hr. Proliferation was measured by [³H]thymidine incorporation. This experiment has been repeated twice with similar results. Asterisk indicates statistical difference differences between Th1 cells incubated in the presence or absence of SP600125.