Ko-ichi Ando

Dynamics of cytotoxic T lymphocyte precursors in vivo assessed by change in the radiation sensitivity: evidence for development of radiation-sensitive memory cells without clonal expansion

The dynamics of cytotoxic T lymphocyte precursors (CTL‐p) in mice injected with allogeneic spleen cells (SC) was studied with special reference to changes in their radiation sensitivity. Whole‐body 400 rad X‐ray irradiation of allo‐SC‐primed and unprimed mice virtually abolished the capacity of their SC to proliferate and to generate CTL in primary or secondary mixed leucocyte culture (MLC). However, the impaired ability of SC to generate CTL in the primary MLC was restored by interleukin 2 (IL‐2). This showed that helper cells whose activity was replaceable with IL‐2 (IL‐2‐producing cells) were functionally more radiation‐sensitive than CTL‐p in unprimed mice. In contrast, the radiation‐impaired activity in secondary MLC was not restored by IL‐2, suggesting that memory CTL‐p in allo‐SC‐primed mice were unexpectedly sensitive to radiation. The D37 values determined from the percentage of residual CTL‐p activity of SC in bulk cultures 1 day after irradiation were 525 rad for virgin CTL‐p and 75 rad for memory CTL‐p. Further studies demonstrated that the radiation‐sensitive memory CTL‐p were generated from relatively radiation‐resistant precursors, largely independent of radiation‐sensitive IL‐2‐producing cells and of cellular proliferation. The mean frequency of CTL‐p in SC measured by limiting dilution assay was not significantly increased by the priming. This supports our conclusion that the development of the memory CTL‐p activity in allo‐SC‐primed mice did not depend on clonal expansion. Whole‐body 400 rad‐irradiation reduced the frequency of CTL‐p in SC from unprimed‐mice 1/2–1/3 and that in SC from allo‐SC‐primed mice to 1/8–1/15. This supports the view that the majority of radiation‐resistant virgin CTL‐p functionally mature to radiation‐sensitive memory CTL‐p without cellular proliferation in allo‐SC‐primed mice.

Aberrancy in immunogenicity and cell-surface expression of H-2 antigens on erythrocytes

Immunogenicity for T cell-independent B-cell response assessed by splenic plaque-forming cell (PFC) response and cell-surface expression measured by laser flow cytometry of various class I H-2 antigens on mouse red blood cells (RBC) were compared. It was found that the order of magnitude of both immunogenicity and cell-surface expression on RBC is H-2Dd ≫ H-2Db > H-2Kd, H-2Kb. Furthermore, H-2d public antigens and H-2Ld antigens were neither immunogenic nor easily demonstrable on RBC. These findings contrasted with poor immunogenicity for PFC response (Nakashima et al. 1982, 1983) and proportionally strong expression of H-2 antigens on lymphoid cells. Immunogenicity and cell-surface expression of H-2Dd antigen on RBC were not shown to be controlled by the action of genes outside H-2D. It was therefore suggested that a number of H-2 antigens, including H-2Kd private, H-2Kb private, and H-2d public specificities are at least functionally defective on RBC. This is possibly due to the structural characteristics of the antigens. Since immunogenicity and cell-surface expression were in parallel, the expression of H-2 antigens on RBC must be dictated by a subset of B cells whose activity was assessed by PFC response. This finding supports the view that the H-2 molecules display a new category of activity which is different from their ability to activate T cells and depends on their expression on RBC.

Genetic Controls of T Cell-Independent Thy-1 Alloantibody Responses

Early and late primary IgM antibody responses of mice to Thy-1.1 antigens showed different antigenic and cellular requirements. We studied genetic controls of the early primary responses, which could be induced by subcellular thymocyte antigens independently of host T-cell activity. All Thy-1.2 mouse strains of Igha(BALB/c and BC8), Igh-VaCb(BAB14), Ighd(AKR/Cum), Ighj(CBA/J, C3H/HeN, C3H.SW, and C3H.JK), and Ighn(NZB) definitely responded early to Thy-1.1 antigens from AKR/J (Ighd), A.Thy-1.1 (Ighe), or B10.Thy-1.1 (Ighb) mice or SD rats, whereas all strains of Ighb(C57BL/6, C57BL/10, B10.D2, B10.BR, B10.A, CB20 and CWB), Ighc(DBA/2), Ighe(A/J), and Igho(C.AL20) responded poorly to the same antigens. This contrasts with the observation that both strains of Ighj(C3H/HeN) and Ighb(B10.BR) responded well at later times. As was the case for late responses, the matching of H-2 between donor and recipient resulted in early responses of exceptional quality in high-responder strains. It was concluded that under the influence of H-2, whose incompatibility between donor and recipient partially interferes with responses, early but not late primary Thy-1.1-specific antibody responses are selectively controlled by Igh-V or closely linked Ir gene(s) as a new VHmarker.

Ontogeny of the transplantation immunity of mice for rejecting ascitic allogeneic tumors.

Ontogeny of the murine transplantation immunity for rejecting ascitic allogeneic tumors (chemically-induced RG lymphoma and L1210 leukemia) as a model of in vivo cytotoxic T cell immunity was studied. Challenge by 10(6) to 10(7) allogeneic tumor cells per 20 g body weight (b.w.) of the mouse was fatal to 1-3 day-old mice, whereas 7-30 day-old mice rejected the tumor. In newborn mice however some yet undetermined mechanism worked to temporally depress the initial tumor growth. Injection of low (10(6) cells per 20 g b.w.) to moderate (10(7)) doses of semiallogeneic spleen cells into newborn mice prepared for second set rejection of the tumor carrying the same alloantigens as the spleen cells, although injection of high dose (3 X 10(8)) cells reduced the tumor rejecting immunity. This second set rejection occurred even against the allogeneic tumor inoculated as early as 3 days old, if the mice had been primed with the alloantigens at birth. It appears therefore that newborn and early suckling mice are protected from tumor invasion by cytotoxic immunity more powerfully than expected from earlier in vitro works.

Biphasic radiation-sensitivity of the transplantation immunity for rejection of an allogeneic ascites tumor.

Transplantation immunity for second-set rejection of an allogeneic ascites tumor was induced by sensitizing mice with H-2-identical allogeneic spleen cells, and radiation-sensitivity of this immunity was studied. The immunity was not severely affected by 400 rads whole-body X-ray irradiation given at one day before the initial antigenic stimulation. In contrast, it was totally inactivated by 300-400 rads irradiation that was given one day before tumor challenge. An adoptive cell transfer experiment showed that alloreactive memory cells responsible for the immunity were unexpectedly highly radiosensitive. The immunity (memory), however, became resistant to 400 rads irradiation soon (one day) after challenge with the allogeneic tumor, and was resistant to 1500 rads for rejection of the tumor that was challenged at the second time when the initially challenged tumor was rejected. Corresponding to these observations, cells for allospecific CTL responses in peritoneal cavity of mice showed corresponding biphasic radiation-sensitivity.

Direct activation of Sendai virus (HVJ)-specific cell-mediated immunity of mice for second set rejection of the virus-infected syngeneic tumor by noninfectious virus-sensitized spleen cells

Syngeneic spleen cells (SPC) sensitized in vitro with noninfectious NVJ were shown to effectively stimulate mice to generate the HVJ-specific cell-mediated immunity for second set rejection (SSR) of virus-infected syngeneic leukemia cells. As few as 10(4) live but not disrupted SPC either infected with a temperature-sensitive mutant of HVJ (HVJts) or sensitized passively with ultraviolet (UV)-inactivated HVJts were active as immunogen. Syngeneic SPC as the carrier of virus could be replaced by allogeneic SPC or L cells, a fibroblast cell line, without reduction of the immunogenicity. Further study demonstrated that a special density of antigen on the surface of HVJts-sensitized SPC is required for high immunogenicity. It was suggested that live cells appropriately sensitized with noninfectious virus would serve as an excellent vaccine for virus-specific cell-mediated immunity.

Depression of afferent arc of the in vivo cytotoxic T-Cell immunity by bacterial lipopolysaccharides

The afferent arc of the in vivo cytotoxic T-cell immunity assessed by second set rejection of ascitic allogeneic tumors was shown to be depressed by bacterial lipopolysaccharide (LPS) that was administered simultaneously with or 1 day before injection of allogeneic spleen cells as stimulators. Two different LPSs from Escherichia coli O55 and Klebsiella O3 displayed similar activities whereas dextran sulfate, concanavalin A, or poly A:U was not effective. Stimulator activities of allogeneic cells was not directly modified by LPS. Any definite suppressor activity on afferent or efferent arc of the T-cell response was not demonstrable in mice receiving LPS and allogeneic cells. Further, the LPS effect for immune depression was not diminished by whole body X-ray irradiation to the recipient at 300 R, which ablated the B-cell reactivity to LPS for polyclonal activation, or by treatment of the recipient with carrageenan, a known toxic agent to macrophages. It was suggested from these results that LPS suppresses the cytotoxic T-cell immunity by modulating responder T cells to be temporarily refractory to the allogeneic stimulus rather than by activating suppressor cells such as radiation-sensitive lymphocytes and carrageenan-sensitive macrophages.

Control of T-cell-dependent antibody responses of mice to rat antigens by donor cell types

Administration of rat red blood cells (RBC) into mice induced a rat antigen-specific T-cell-dependent primary antibody response that was detected by a plaque assay using rat RBC as a target. This response was not induced by rat thymocytes or rat spleen cells that should share rat-specific antigens with rat RBC. We then demonstrated that rat spleen cells but not rat thymocytes, which were administered with rat RBC, partially inhibit the action of rat RBC for induction of the anti-rat response. This inhibition required live donor FcR+ cells, and seemed to be specific to rat antigens common to RBC and spleen cells. The findings supported the idea that the control by donor cell types, which was originally shown to work for T-cell-independent antibody response [3,5], should be effective for T-cell-dependent response beyond the species barrier.