Bystander activation of cytotoxic T cells: studies on the mechanism and evaluation of in vivo significance in a transgenic mouse model

Abstract

Bystander activation, i.e., activation of T cells specific for an antigen X during an immune response against antigen Y may occur during viral infections. However, the low frequency of bystander-activated T cells has rendered it difficult to define the mechanisms and possible in vivo relevance of this nonspecific activation. This study uses transgenic mice expressing a major histocompatibility complex class I-restricted TCR specific for glycoprotein peptide 33-41 of lymphocytic choriomeningitis virus (LCMV) to overcome this limitation. CD8+ T cells from specific pathogen-free maintained, unimmunized "naive" TCR transgenic mice can differentiate into LCMV-specific cytolytic effector CTL during infections with vaccinia virus or Listeria monocytogenes in vivo or mixed lymphocyte culture in vitro. We show that in these model situations (a) nonspecifically activated CTL are able to confer antiviral protection in vivo, (b) bystander activation is largely independent of the expression of a second T cell receptor of different specificity, (c) bystander activation is not mediated by a broadly cross-reactive TCR, but rather by cytokines, (d) bystander activation can be mediated by cytokines such as IL-2, but not alpha/beta-IFN in vitro; (e) bystander activation is, overall, a rare event, occuring in vivo in roughly 1 in 200 of the LCMV-specific CTL during infection of TCR transgenic mice with vaccinia virus; (f) bystander activation does not have a significant functional impact on nontransgenic CTL memory under the conditions tested; and (g) even in the TCR transgenic situation, where unphysiologically high numbers of T cells of a single specificity are present, bystander activation is not sufficient to cause clinically manifest autoimmune disease in a transgenic mouse model of diabetes. We conclude that although bystander activation via cytokines may generate cytolytically active CTL from naive precursors, quantitative considerations suggest that this is usually not of major biological consequence.

Figure 1

Activation of LCMV-specific TCR transgenic T cells by unrelated pathogens in vivo and by allo-antigens in vitro. (a–e) C57BL/6 mice (closed circles) and TCR transgenic mice from line 327 (open circles) were infected intravenously with 2 × 10⁶ PFU vaccinia WR (a–c) or with 5 × 10³ CFU Listeria monocytogenes (d and e). 6 and 5 d later, respectively, splenic cytotoxic activities were tested on MC57G target cells infected with vaccinia WR (a), labeled with the LCMV peptide gp33 (b and d), or left unlabeled (c and e). (f–h) Spleen cells from C57BL/6 (closed circles) and TCR trangenic mice of line 327 (both H-2^b; open circles) were stimulated in a mixed lymphocyte reaction on irradiated spleen cells from BALB/c mice (H-2^d). 5 d later, cytotoxic activity of the MLC cultures was tested on P815 (H-2^d) (f), MC57G (g), or MC57G (H-2^b) (h) cells pulsed with LCMV gp33. Spontaneous release in all assays was <24%. All experiments were performed at least two times.

Figure 2

Antiviral protection in vivo induced by bystander activation of cytotoxic T cells. C57BL/6 mice were infected with 10⁴ PFU LCMVWE intravenously, and 10 h after infection, adoptively transfused with the indicated numbers of spleen cells from naive (groups 1 and 3), vaccinia infected (groups 2 and 4), or LCMV-infected (groups 5 and 6) C57BL/6 or TCR transgenic mice (line 327). 18 h after adoptive transfer, virus titers were determined in the spleens of recipient mice. One of three similar experiments is shown.

Figure 3

Functional analysis of bystander-activated LCMV-specific memory CTL with no evidence of enhancement of cytolytic CTL activity assessed in vitro and in vivo. (a) Primary ex vivo cytolytic activity of C57BL/6 mice immunized with 200 PFU LCMV-WE 50 d previously (open circles) or immunized with LCMV and boosted with 2 × 10⁶ PFU vaccinia WR 6 d before the assay (closed circles) was analyzed in a 5-h and 15-h ⁵¹Cr-release assay on EL-4 target cells labeled with LCMV gp33. Spontaneous release was 28%. (b) C57BL/6 mice were infected with 10⁴ PFU LCMV-WE intravenously, and 10 h after infection, adoptively transfused with the indicated numbers of spleen cells from naive (group 1), LCMV infected 50 d previously (group 2), LCMV infected and boosted with vaccinia virus 6 d before the assay (group 3), or acutely LCMV infected (group 4) C57BL/6 mice. 18 h after adoptive transfer, virus titers were determined in the spleens of recipient mice. (c) Primary ex vivo cytolytic activity of C57BL/6 mice immunized with 200 PFU LCMV-WE 150 d previously (open circles) or immunized with LCMV and injected with 200 μg poly IC 3 and 1 d before the assay (closed circles) was analyzed in a 15-h ⁵¹Cr-release assay on EL-4 target cells labeled with LCMV gp33. Spontaneous release was 28%.

Figure 4

Quantitative analysis of vaccinia virus–induced LCMVspecific TCR transgenic CTL. (a) TCR transgenic mice from line 327 (closed circles), line 318 (closed triangles), as well as C57BL/6 mice adoptively transfused with 10⁸ (closed squares) or 2 × 10⁷ (open squares) spleen cells from mice of line 318 were infected with 2 × 10⁶ PFU vaccinia WR. 6 d later, LCMV-specific splenic cytotoxicity was assessed in a 15-h ⁵¹Cr-release assay on EL-4 target cells pulsed with LCMV gp33 or unlabeled. Spontaneous release was 29%. One of two similar experiments is shown. (b) C57BL/6 mice (closed circles) were infected with 200 PFU LCMV-WE and TCR transgenic mice from line 327 (open circles) with 2 × 10⁶ PFU vaccinia WR. 8 and 6 d after infection, respectively, splenic cytotoxic activity was tested in a 15-h ⁵¹Cr-release assay on EL-4 target cells pulsed with LCMV gp33. Spontaneous release was 31%. One of two similar experiments is shown.

Figure 5

LCMV-specific cytotoxicity generated by vaccinia infection of TCR transgenic mice cannot be blocked by vaccinia virus–infected target cells. (a) TCR transgenic mice (line 327) were infected with 2 × 10⁶ PFU vaccinia WR, and 6 d later, splenic cytotoxic activity was tested on ⁵¹Cr-labeled “hot” MC57G cells infected with LCMV-WE at a fixed effector/ target ratio of 100:1. Nonradiolabeled “cold” MC57G cells uninfected (closed bars), or infected with LCMV-WE (open bars) or with vaccinia WR (hatched bars) were added at the indicated hot/ cold target ratio. (b) Spleen cells from TCR transgenic mice (line 327; H-2^b) were stimulated in an MLC with irradiated BALB/c (H-2^d) spleen cells for 5 d and used as effectors in a cold target competition assay as outlined above. MC57G cells (H-2^b), either uninfected (closed bars), or infected with LCMV-WE (open bars) or P815 (H-2^d) cells (hatched bars) were used as cold targets. Spontaneous release was <23% in both assays. Each experiment was repeated three times.

Figure 6

Nonspecific activation of TCR transgenic LCMV-specific CTL during a mixed lymphocyte reaction is mediated by cytokines and not a cross-reactive TCR. (a–c) Spleen cells from C57BL/6 (open circles), TCR transgenic line 327 (closed circles), TCR × RAG −/− mice (closed circles), and spleen cells from TCR × RAG −/− mice supplemented with 10% C57BL/6 (closed triangles) spleen cells (all H-2^b) were used as responder cells in a mixed lymphocyte reaction on BALB/c (H-2^d) stimulator cells. (d–f) Spleen cells from TCR × RAG −/− mice were cultured in the absence of stimulator cells either in supernatant of C57BL/6 versus BALB/c MLC (closed triangles) or in medium supplemented with 500 U/ml of IL-2 on days 1 and 4 of culture (closed circles). ⁵¹Cr-labeled P815 cells (H-2^d) (a, c), or EL-4 cells (H-2^b) labeled with gp33 (b, d) or unlabeled (c, f   ) were used as target cells.

Figure 7

No role for α/β-IFN in bystander activation of naive LCMVspecific TCR transgenic CTL. (a–c) Spleen cells from TCR transgenic mice (line 318) lacking the receptor for α/β-IFN (open circles) and heterozygous littermates (closed circles) (both H-2^b) were used as responder cells in a mixed lymphocyte reaction on BALB/c (H-2^d) stimulators. After 5 d of culture, cytotoxic activity was tested on MC57G (H-2^b) target cells either unlabeled (c) or labeled with LCMV gp33 (b) and on P815 (H-2^d) cells (a). Spontaneous release was <25%. One of two similar experiments is shown. (d–f) TCR transgenic mice (line 327) were injected with 200 μg poly(IC) intravenously 1 d (open circles), or 1 and 3 d (closed circles) before testing splenic cytotoxic activity on NK-sensitive YAC-1 target cells (d), MC57G target cells pulsed with LCMV gp33 (e), or unlabeled (  f   ). Spontaneous release was <23%.