Loci controlling lymphocyte production of interferon c after alloantigen stimulation in vitro and their co-localization with genes controlling lymphocyte infiltration of tumors and tumor susceptibility

Abstract

Low infiltration of lymphocytes into cancers is associated with poor prognosis, but the reasons why some patients exhibit a low and others a high infiltration of tumors are unknown. Previously we mapped four loci (Lynf1–Lynf4) controlling lymphocyte infiltration of mouse lung tumors. These loci do not encode any of the molecules that are involved in traffic of lymphocytes. Here we report a genetic relationship between these loci and the control of production of IFNγ in allogeneic mixed lymphocyte cultures (MLC). We found that IFNγ production by lymphocytes of O20/A mice is lower than by lymphocytes of OcB-9/Dem mice (both H2pz) stimulated in MLC by irradiated splenocytes of C57BL/10SnPh (H2b) or BALB/ cHeA (H2d) mice, or by ConA. IFNγ production in MLCs of individual (O20 9 OcB-9)F2mice stimulated by irradiated C57BL/10 splenocytes and genotyped for microsatellite markers revealed four IFNγ-controlling loci (Cypr4-Cypr7), each of which is closely linked with one of the four Lynf loci and with a cluster of susceptibility genes for different tumors. This suggests that inherited differences in certain lymphocyte responses may modify their propensity to infiltrate tumors and their capacity to affect tumor growth.

Fig. 1

The MLC reactivity of O20, B10.O20, and OcB-9 strains. Proliferative response of spleen cells in RPMI medium only and stimulated by C57BL/10Sn (H2 ^b) (B10), BALB/c (H2 ^d), and CBA/Ph (H2 ^k) alloantigens. Spleen cells (1.5 × 10⁵ cells per well) were mixed with 2 × 10⁵ irradiated (3000 R) B10, BALB/c or CBA/Ph stimulator cells in 200 μl in 96-well tissue culture plates. [3H]-thymidine (0.5 μCi/well) was added into the cultures for the last 6 h of the 96-h incubation period as described previously [17]. The data show the mean ± SE from three independent experiments. Reproduced with the kind permission from Ref. [17]

Fig. 2

a Comparison of concentration of IFNγ in supernatants of the spleen cells of the strains OcB-9 and O20 after alloantigen stimulation. Spleen cells (6 × 10⁵ cells per well) were mixed with 8 × 10⁵ irradiated (3000 R) C57BL/10 stimulator cells in 800 μl in 24-well tissue culture plates. Cell supernatants were analyzed 48, 72, and 96 h after stimulation. Data summarize the result of 13 independent experiments. Both female and male mice were used in our analysis, but no influence of sex on strain difference was observed. The columns show the means ± SE of IFNγ concentration in ng/ml. Filled square: OcB-9, unfilled square: O20. b Comparison of concentration of IFNγ in supernatants of the spleen cells of the strains OcB-9 and O20 after stimulation by C57BL/10Sn (H2^b), BALB/c (H2^d), CBA (H2^k), and DBA/1 (H2^q) alloantigens. Spleen cells (6 × 10⁵ cells per well) were mixed with 8 × 10⁵ irradiated (3000 R) C57BL/10Sn, BALB/c, CBA, or DBA/1 stimulator cells in 800 μl in 24-well tissue culture plates. Cell supernatants were analysed 72 and 96 h after stimulation. Data summarize the result of 13 independent experiments

Fig. 3

Comparison of concentration of IFNγ in supernatants of the spleen cells of the strains OcB-9 and O20 after stimulation with 2.5 μg/ml ConA. To test IFNγ concentration, 800 μl of spleen cells (4 × 10⁵ cells per well) were incubated in 24-well tissue culture plates in complete RPMI 1640 medium with 2.5 μg/ml ConA. After 24, 48, or 72 h of incubation supernatants were collected and assayed for IFNγ presence. The columns show the means ± SE of IFNγ concentration in ng/ml. Data summarize the result of four independent experiments. Square: OcB-9, unfilled square: O20

Fig. 4

a Comparison of concentration of IL-2 in supernatants of the spleen cells of the strains OcB-9 and O20 after alloantigen stimulation. Spleen cells (6 × 10⁵ cells per well) were mixed with 8 × 10⁵ irradiated (3000 R) C57BL/10 stimulator cells in 800 μl in 24-well tissue culture plates. Cell supernatants were analysed 48, 72, and 96 h after stimulation. Data summarize the result of 13 independent experiments. Both female and male mice were used in our analysis, but no influence of sex on strain difference was observed. The columns show the means ± SE of IL-2 concentration in ng/ml. Filled square: OcB-9, unfilled square: O20. b Comparison of concentration of IL-4 in supernatants of the spleen cells of the strains OcB-9 and O20 after alloantigen stimulation. Spleen cells (6 × 10⁵ cells per well) were mixed with 8 × 10⁵ irradiated (3000 R) C57BL/10 stimulator cells in 800 μl in 24-well tissue culture plates. Cell supernatants were analysed 48, 72, and 96 h after stimulation. Data summarize the result of 13 independent experiments. Both female and male mice were used in our analysis, but no influence of sex on strain difference was observed. The columns show the means ± SE of IL-4 concentration in ng/ml. Filled square: OcB-9, unfilled square: O20