A natural immunological adjuvant enhances T cell clonal expansion through a CD28-dependent, interleukin (IL)-2-independent mechanism

Abstract

The adoptive transfer of naive CD4(+) T cell receptor (TCR) transgenic T cells was used to investigate the mechanisms by which the adjuvant lipopolysaccharide (LPS) enhance T cell clonal expansion in vivo. Subcutaneous administration of soluble antigen (Ag) resulted in rapid and transient accumulation of the Ag-specific T cells in the draining lymph nodes (LNs), which was preceded by the production of interleukin (IL)-2. CD28-deficient, Ag-specific T cells produced only small amounts of IL-2 in response to soluble Ag and did not accumulate in the LN to the same extent as wild-type T cells. Injection of Ag and LPS, a natural immunological adjuvant, enhanced IL-2 production and LN accumulation of wild-type, Ag-specific T cells but had no significant effect on CD28-deficient, Ag-specific T cells. Therefore, CD28 is critical for Ag-driven IL-2 production and T cell proliferation in vivo, and is essential for the LPS-mediated enhancement of these events. However, enhancement of IL-2 production could not explain the LPS-dependent increase of T cell accumulation because IL-2-deficient, Ag-specific T cells accumulated to a greater extent in the LN than wild-type T cells in response to Ag plus LPS. These results indicate that adjuvants improve T cell proliferation in vivo via a CD28-dependent signal that can operate in the absence of IL-2.

Figure 1

LPS increases CD4⁺, KJ1-26⁺ clonal expansion. The total number of CD4⁺, KJ1-26⁺ cells was measured in the draining LNs (axillary, brachial, inguinal, and cervical) after s.c. injection of 2 mg OVA without (open circles) or with LPS (filled circles). One representative experiment of 15 is shown.

Figure 2

Ag-specific CD4⁺ T cells express IL-2 mRNA and protein after Ag injection. (A) Adoptively transferred mice were either uninjected (−) or injected subcutaneously with soluble OVA (+). Peripheral LNs were harvested 6 h after Ag injection. Total RNA was obtained from unfractionated LN cells (LN Cells), from cells that were depleted of DO11.10 T cells by KJ1-26 mAb-coated magnetic beads (KJ1-26 depleted), and from magnetic bead-enriched KJ1-26⁺ cells (Beads). IL-2 and HPRT RT-PCR products were detected by Southern blot and autoradiographic techniques. (B) BALB/c mice were injected with 2.5 × 10⁶ DO11.10 T cells as described in the Materials and Methods section. FACS^® profiles for brachial, axillary, and inguinal LN cells from normal (left) or adoptive transfer mice (right) stained with Cy-Chrome–labeled anti-CD4 mAb and FITC-labeled KJ1-26 mAb are shown. (C) Cells in B, right were further stained with PE-labeled anti–IL-2 mAb. Histograms represent PE-channel fluorescence of stained LN cells recovered from uninjected (histogram 2) or OVA-injected animals (histograms 1, 3, and 4). Histogram 1, CD4⁺, KJ1-26⁺ cells stained with PE-labeled isotype matched mAb (rat IgG2a); histogram 2, CD4⁺, KJ1-26⁺ cells from an uninjected mouse stained with PE-labeled anti–IL-2 mAb; histograms 3 and 4, CD4⁺, KJ1-26⁺ and CD4⁺, KJ1-26⁻ cells stained with PE-labeled anti–IL-2 mAb. (D) The kinetics of IL-2 mRNA (open circles) and protein (filled circles) expression after s.c. injection of 2 mg OVA. Data from three separate experiments on the kinetics of IL-2 mRNA expression were pooled. IL-2 mRNA was detected by RT-PCR and quantified as described in Materials and Methods. Each point represents the mean from 4–10 mice ± SEM. IL-2 protein expression was measured as described in B and C. One representative experiment of four is shown. Each point represents the mean of two mice ± range.

Figure 3

Effect of LPS on IL-2 protein production by Ag-specific T cells. 2 mg of OVA were injected without (OVA) or with LPS (OVA/LPS) 10 h before killing. LN cells were stained for IL-2 directly (A) or incubated in vitro for 2.5 h (B). (A) intracellular IL-2 protein was measured in the CD4⁺, KJ1-26⁺ population by three-color flow cytometry as described in Fig 2, B–D. (B) IL-2 protein secreted into the supernatants during the ex vivo culture was measured by ELISA. Each column represents two mice and is shown as the mean ± range. No IL-2 could be detected in the culture supernatants of LN cells recovered from animals that were transferred with DO11.10 TCR transgenic T cells, but did not receive OVA. The figure is representative of two independent experiments.

Figure 3

Effect of LPS on IL-2 protein production by Ag-specific T cells. 2 mg of OVA were injected without (OVA) or with LPS (OVA/LPS) 10 h before killing. LN cells were stained for IL-2 directly (A) or incubated in vitro for 2.5 h (B). (A) intracellular IL-2 protein was measured in the CD4⁺, KJ1-26⁺ population by three-color flow cytometry as described in Fig 2, B–D. (B) IL-2 protein secreted into the supernatants during the ex vivo culture was measured by ELISA. Each column represents two mice and is shown as the mean ± range. No IL-2 could be detected in the culture supernatants of LN cells recovered from animals that were transferred with DO11.10 TCR transgenic T cells, but did not receive OVA. The figure is representative of two independent experiments.

Figure 4

Kinetics of blastogenesis of Ag-specific T cells is normal in the absence of CD28 costimulation, but IL-2 production and Ag-specific T cell expansion are decreased and are not potentiated by LPS. Mice were adoptively transferred with wild-type (filled symbols) or CD28-deficient (open symbols) DO11.10 cells and injected subcutaneously at different times with OVA with (triangles) or without LPS (circles). Data shown in B–D were obtained from one experiment. Data shown in panel A covering earlier time points were from a separate experiment. C shows the kinetics of blastogenesis as determined by the percent increase in forward light scatter (size) of CD4⁺, KJ1-26⁺ cells from injected mice as compared with CD4⁺, KJ1-26⁺ cells from uninjected mice. Each point in A–C represents the mean ± SD for 2–4 mice per group. D shows the fold increase in the number of Ag-specific T cells on day 3 after Ag injection, calculated as a ratio of the total number of Ag-specific T cells present in the draining LNs from Ag-injected animals divided by the total number of Ag-specific T cells present in the same LNs in uninjected animals. Each column represents the mean of data obtained from four mice ± SD. This experiment is representative of four independent experiments.

Figure 4

Kinetics of blastogenesis of Ag-specific T cells is normal in the absence of CD28 costimulation, but IL-2 production and Ag-specific T cell expansion are decreased and are not potentiated by LPS. Mice were adoptively transferred with wild-type (filled symbols) or CD28-deficient (open symbols) DO11.10 cells and injected subcutaneously at different times with OVA with (triangles) or without LPS (circles). Data shown in B–D were obtained from one experiment. Data shown in panel A covering earlier time points were from a separate experiment. C shows the kinetics of blastogenesis as determined by the percent increase in forward light scatter (size) of CD4⁺, KJ1-26⁺ cells from injected mice as compared with CD4⁺, KJ1-26⁺ cells from uninjected mice. Each point in A–C represents the mean ± SD for 2–4 mice per group. D shows the fold increase in the number of Ag-specific T cells on day 3 after Ag injection, calculated as a ratio of the total number of Ag-specific T cells present in the draining LNs from Ag-injected animals divided by the total number of Ag-specific T cells present in the same LNs in uninjected animals. Each column represents the mean of data obtained from four mice ± SD. This experiment is representative of four independent experiments.

Figure 4

Kinetics of blastogenesis of Ag-specific T cells is normal in the absence of CD28 costimulation, but IL-2 production and Ag-specific T cell expansion are decreased and are not potentiated by LPS. Mice were adoptively transferred with wild-type (filled symbols) or CD28-deficient (open symbols) DO11.10 cells and injected subcutaneously at different times with OVA with (triangles) or without LPS (circles). Data shown in B–D were obtained from one experiment. Data shown in panel A covering earlier time points were from a separate experiment. C shows the kinetics of blastogenesis as determined by the percent increase in forward light scatter (size) of CD4⁺, KJ1-26⁺ cells from injected mice as compared with CD4⁺, KJ1-26⁺ cells from uninjected mice. Each point in A–C represents the mean ± SD for 2–4 mice per group. D shows the fold increase in the number of Ag-specific T cells on day 3 after Ag injection, calculated as a ratio of the total number of Ag-specific T cells present in the draining LNs from Ag-injected animals divided by the total number of Ag-specific T cells present in the same LNs in uninjected animals. Each column represents the mean of data obtained from four mice ± SD. This experiment is representative of four independent experiments.

Figure 4

Kinetics of blastogenesis of Ag-specific T cells is normal in the absence of CD28 costimulation, but IL-2 production and Ag-specific T cell expansion are decreased and are not potentiated by LPS. Mice were adoptively transferred with wild-type (filled symbols) or CD28-deficient (open symbols) DO11.10 cells and injected subcutaneously at different times with OVA with (triangles) or without LPS (circles). Data shown in B–D were obtained from one experiment. Data shown in panel A covering earlier time points were from a separate experiment. C shows the kinetics of blastogenesis as determined by the percent increase in forward light scatter (size) of CD4⁺, KJ1-26⁺ cells from injected mice as compared with CD4⁺, KJ1-26⁺ cells from uninjected mice. Each point in A–C represents the mean ± SD for 2–4 mice per group. D shows the fold increase in the number of Ag-specific T cells on day 3 after Ag injection, calculated as a ratio of the total number of Ag-specific T cells present in the draining LNs from Ag-injected animals divided by the total number of Ag-specific T cells present in the same LNs in uninjected animals. Each column represents the mean of data obtained from four mice ± SD. This experiment is representative of four independent experiments.

Figure 5

OVA-specific IL-2– deficient T cells have defective proliferative responses in vitro, but not in vivo, and are resistant to cell death. (A) Cells recovered from mice adoptively transferred with wild-type (filled circles) or IL-2–deficient (open circles) DO11.10 T cells were incubated in vitro in the presence of 0.5 μM OVA peptide 323-339, for the indicated number of days. At each time point replicate wells were harvested and the cells were counted and stained for CD4 and KJ1-26. Mean fold increase ± SD of the total number of transgenic T cells is shown. (B) Mice adoptively transferred with wild-type (open symbols) or IL-2–deficient (filled symbols) DO11.10 T cells were immunized with OVA in the absence or in the presence of 150 μg of LPS. The total number of CD4⁺, KJ1-26⁺ T cells was assessed as described in Fig. 1. Each point on the graph represents the mean ± SE for five mice per group. (C) LN cells recovered from mice adoptively transferred with wild-type (open symbols) or IL-2–deficient (filled symbols) DO11.10 T cells that were injected in vivo with either OVA (circles) or with OVA/LPS (triangles) 3 d before the start of ex vivo culture in the absence of any additional Ag stimulation. The cells were stained at the indicated times for CD4 and KJ1-26 and analyzed by flow cytometry. The mean percentage of initial viable input ± SD of two animals per group is shown.

Figure 5

OVA-specific IL-2– deficient T cells have defective proliferative responses in vitro, but not in vivo, and are resistant to cell death. (A) Cells recovered from mice adoptively transferred with wild-type (filled circles) or IL-2–deficient (open circles) DO11.10 T cells were incubated in vitro in the presence of 0.5 μM OVA peptide 323-339, for the indicated number of days. At each time point replicate wells were harvested and the cells were counted and stained for CD4 and KJ1-26. Mean fold increase ± SD of the total number of transgenic T cells is shown. (B) Mice adoptively transferred with wild-type (open symbols) or IL-2–deficient (filled symbols) DO11.10 T cells were immunized with OVA in the absence or in the presence of 150 μg of LPS. The total number of CD4⁺, KJ1-26⁺ T cells was assessed as described in Fig. 1. Each point on the graph represents the mean ± SE for five mice per group. (C) LN cells recovered from mice adoptively transferred with wild-type (open symbols) or IL-2–deficient (filled symbols) DO11.10 T cells that were injected in vivo with either OVA (circles) or with OVA/LPS (triangles) 3 d before the start of ex vivo culture in the absence of any additional Ag stimulation. The cells were stained at the indicated times for CD4 and KJ1-26 and analyzed by flow cytometry. The mean percentage of initial viable input ± SD of two animals per group is shown.

Figure 5

OVA-specific IL-2– deficient T cells have defective proliferative responses in vitro, but not in vivo, and are resistant to cell death. (A) Cells recovered from mice adoptively transferred with wild-type (filled circles) or IL-2–deficient (open circles) DO11.10 T cells were incubated in vitro in the presence of 0.5 μM OVA peptide 323-339, for the indicated number of days. At each time point replicate wells were harvested and the cells were counted and stained for CD4 and KJ1-26. Mean fold increase ± SD of the total number of transgenic T cells is shown. (B) Mice adoptively transferred with wild-type (open symbols) or IL-2–deficient (filled symbols) DO11.10 T cells were immunized with OVA in the absence or in the presence of 150 μg of LPS. The total number of CD4⁺, KJ1-26⁺ T cells was assessed as described in Fig. 1. Each point on the graph represents the mean ± SE for five mice per group. (C) LN cells recovered from mice adoptively transferred with wild-type (open symbols) or IL-2–deficient (filled symbols) DO11.10 T cells that were injected in vivo with either OVA (circles) or with OVA/LPS (triangles) 3 d before the start of ex vivo culture in the absence of any additional Ag stimulation. The cells were stained at the indicated times for CD4 and KJ1-26 and analyzed by flow cytometry. The mean percentage of initial viable input ± SD of two animals per group is shown.

Figure 6

LPS enhances clonal expansion of naive and memory Ag-specific T cells with and without IL-2 production. CD4⁺ T cells from wild-type or IL-2–deficient DO11.10 transgenic mice were sorted into CD45RB^high and CD45RB^low cells and injected into normal BALB/c mice. The recipients were injected with 2 mg of OVA and 150 μg of LPS, or 2 mg of OVA alone, or left uninjected. (A) The histogram in the upper panel shows CD45RB staining of CD4⁺ T cells from the IL-2–deficient DO11.10 transgenic donor before sorting. The histograms in the lower panel show equal numbers of sorted CD45RB^high and CD45RB^low IL-2–deficient CD4⁺ T cells just before the adoptive transfer. (B) Total numbers of CD4⁺, KJ1-26⁺ cells present in the draining LNs on day 3 after Ag injection are shown. Each column represents the mean ± SD with two mice per group. Since both CD45RB^high and CD45RB^low cells were obtained from the same sort and were transferred into the same number of recipients, this figure also shows the relative contributions of naive and memory T cells to clonal expansion in the unsorted adoptive transfer experiments. (C) As the baseline numbers of CD4⁺, KJ1-26⁺ T cells in the LNs of uninjected mice were different for each group, clonal expansion was also represented as fold increase in CD4⁺, KJ1-26⁺ cells. The figure represents one of two experiments with similar results.

Figure 7

In vivo neutralization of IL-2 does not affect T cell clonal expansion and increases T cell persistence. Mice adoptively transferred with wild-type DO11.10 T cells were treated with anti–IL-2 mAb S4B6 or rat IgG as described in Materials and Methods and injected subcutaneously with 2 mg of soluble OVA. The number of CD4⁺, KJ1-26⁺ cells present in the draining LNs was determined 3 and 6 d after Ag injection. Each point represents the mean ± SE for five mice per group. The anti–IL-2 mAb treatment did not affect the numbers of CD4⁺, KJ1-26⁺ T cells on day 3 in two other independent experiments. (Filled circles) OVA s.c. + rat IgG; (open circles) OVA s.c. + anti–IL-2; (filled squares) T only.

Figure 7

In vivo neutralization of IL-2 does not affect T cell clonal expansion and increases T cell persistence. Mice adoptively transferred with wild-type DO11.10 T cells were treated with anti–IL-2 mAb S4B6 or rat IgG as described in Materials and Methods and injected subcutaneously with 2 mg of soluble OVA. The number of CD4⁺, KJ1-26⁺ cells present in the draining LNs was determined 3 and 6 d after Ag injection. Each point represents the mean ± SE for five mice per group. The anti–IL-2 mAb treatment did not affect the numbers of CD4⁺, KJ1-26⁺ T cells on day 3 in two other independent experiments. (Filled circles) OVA s.c. + rat IgG; (open circles) OVA s.c. + anti–IL-2; (filled squares) T only.

Figure 8

Expansion of Ag-specific T cells after injection of Ag with LPS is augmented in mice treated with anti–IL-2 mAb. Mice adoptively transferred with wild-type DO11.10 T cells were treated with anti–IL-2 mAb S4B6 or rat IgG and injected subcutaneously with 2 mg OVA and 150 μg LPS. The number of CD4⁺, KJ1-26⁺ cells present in the draining LNs was determined on day 6. Each bar represents the mean ± SE with five mice per group.