Subpopulations of human T lymphocytes. II. Effect of thymopoietin, corticosteroids, and irradiation.

Lymphoid cells from normal SJL/J mice gave high proliferative responses but failed to develop cytotoxic activity to γ-irradiated cells from syngeneic transplantable reticulum cell sarcomas (X-RCS). In spite of a vigorous in vivo proliferative response to X-RCS, cytotoxic activity was never generated to detectable levels in vivo. After repeated injections of X-RCS, spleen and, to a lesser degree, lymph node cells acquired the ability to give moderate secondary cytotoxic responses in vitro upon co-culture with X-RCS. This immunity was T-cell mediated and specific for RCS although it did not distinguish between different transplantable RCS lines. SJL/J mice also developed resistance to RCS growth after injection of X-RCS, which showed a transient RCS-line-specific component. (SJL/J × C₅₇B1/6)F₁ mice showed 60% less RCS growth than did SJL/J mice, and their lymphoid cells gave slightly lower proliferative responses than did cells from SJL/J mice, whereas (SJL/J × BALB/c)F₁ mice showed little tumor growth, and their spleen cells proliferated only minimally to X-RCS. B10.S mice allowed moderate RCS growth. Cytotoxic activity was generated in co-cultures with X-RCS of immunized F₁ spleen cells even after a single immunization in vivo but not in cultures of normal F₁ cells with X-RCS.     

Adjuvant-induced nonspecific supressor cells: in vitro and in vivo studies

The in vitro mitogen responses of spleen cells from mice injected ip with the nonantigenic adjuvant, Al(OH)₃, are markedly depressed. This depressed reactivity was found to be mediated by a population of nylon wool adherent, Fc-receptor-bearing suppressor cells. Suppressor cells were detected only in the spleens of the adjuvant-treated mice, as the response of lymph node cells to mitogenic stimulation in vitro was found comparable to that of normal controls. Moreover, elevated levels of suppressive activity could be detected in sera of Al(OH)₃-treated mice during the first week after adjuvant administration, which, however, did not correlate with either the long-lasting presence of suppressor cells or the in vivo normal immune response of the adjuvant-treated animals. Studies designed to test the effect of suppressor cells on the generation of splenic PFC in vivo revealed that both the direct and indirect PFC responses against SRBC inoculated iv were enhanced rather than suppressed, as compared to those of the normal controls. Furthermore, the level of cytotoxic lymphocytes generated in spleens of Al(OH)₃-treated mice immunized with allogeneic tumor cells was equal to or higher than that of the normal controls. In view of the present results, we feel that the concept that splenic, nonspecific suppressor cells (macrophages) are immunosuppressive in vivo as well as the in vivo relevance of in vitro findings should be carefully reevaluated.     

Enhancement by interferon of natural killer cell activity in mice.

Injection of mice with several interferon inducers, Newcastle Disease virus, polyinosinic-polycytidylic acid and tilorone resulted in an increase in spleen cell cytotoxicity for ⁵¹chromium-labeled mouse YAC tumor target cells in 4-hr in vitro assays. This increase in spleen cell cytotoxicity was abrogated by injection of mice with potent anti-mouse interferon globulin. Inoculation of mice with mouse interferon (but not human leucocyte or mock interferon preparations) also resulted in a marked enhancement of spleen cell cytotoxicity. The extent of enhancement of spleen cell cytotoxicity was directly proportional to the amount of interferon injected and a significant increase was observed after inoculation of as little as 10³ to 10⁴ units of interferon. An effect could be detected as soon as 1 hr after injection of interferon. The increase of spleen cell cytotoxicity after inoculation of an interferon inducer was not due to a localization and accumulation of cytotoxic cells in the spleen but reflected a general increase in cytotoxic cell activity in various lymphoid tissues (except the thymus). The splenic cytotoxic cells from interferon or interferon-inducer-injected mice had the characteristics of natural killer (NK) cells since (i) interferon enhanced spleen cell cytotoxicity in athymic (nu/nu) nude mice, (ii) classical spleen cell fractionation procedures by nylon wool columns, anti-Thy 1.2 serum plus complement, anti-Ig columns, and depletion of FcR+ rosette-forming cells, failed to remove the effector cells generated in vivo or in vitro. Therefore like NK cells, interferon-induced cytotoxic cells lack the surface markers of mature T and B lymphocytes, are not adherent, and are devoid of avid Fc receptors. Furthermore like NK cells, the spleen cells from interferon-treated mice lysed various target cells (known for their sensitivity to NK cells) without H-2 or species restriction. Incubation in vitro of normal spleen cells with interferon also resulted in an increase in cytotoxicity for YAC tumor cells. We conclude that interferon acts directly on NK cells and enhances the inherent cytotoxic activity of these cells.     

Suppression of murine NK activity induced by Corynebacterium parvum

Summary Formalin-killed Corynebacterium parvum (CP), given at a dose of 0.4–0.7 mg/mouse IV or IP, induced suppressor cells for NK activity in B6C3F₁ mice. The suppressor cells belong to at least two different populations, plastic adherent and nonadherent, and were not depleted by antibodies specific for Thy-1.2, Ia^k, or NK-1.2 surface markers. Administration of p-I:C, an interferon-inducer, to animals 18 h before the assay did not affect the suppressor activity. Hypotonic shock treatment of splenocytes abrogated the in vitro suppressive activity, and subsequent reconstitution of the shock-treated cells with RBC failed to restore the suppressive activity. SJL/J mice, which have low NK activity, exhibited suppressor activity comparable to B6C3F₁ mice following CP treatment, whereas CP-treated BALB/c athymic and euthymic mice showed a lower ability to generate suppressors for NK as compared to B6C3F₁ mice.     

Mechanisms of killing of measles virus-infected cells by human lymphocytes: interferon associated and unassociated cell-mediated cytotoxicity

The recent interest in natural killer (NK) cells in immunosurveillance and the ability of infection with certain organisms to modulate NK activity led us to examine the influence of Toxoplasma gondii infection on mouse NK cells. Infection of BALB/c mice with 5 × 10³ virulent Toxoplasma intraperitoneally (ip) resulted in significantly enhanced NK activity in peritoneal exudate cells (PC) and in spleen cells (SC). Intravenous (iv) and subcutaneous (sc) challenge of BALB/c mice with Toxoplasma also resulted in enhanced natural killer (NK) activity in PC and SC. In BALB/c mice, as well as in other strains (A/J, C57BL/6, C3H/HeJ, CeH/HeN, [A/J × C3H]F₁), peak augmentation of PC and SC NK activity was observed 3 days following ip Toxoplasma challenge. Administration of silica to mice abolished Toxoplasma-induced NK cytotoxicity. BALB/c mice chronically infected with Toxoplasma had significantly higher endogenous NK activity than did controls in PC but not in SC. Chronically infected BALB/c mice boosted with virulent Toxoplasma ip exhibited significantly enhanced NK activity in PC but not in SC. Thus, acute and chronic infection with Toxoplasma modulates NK activity in addition to macrophage activation and thereby provides a system that should facilitate study of the relative contribution of NK cells and activated macrophages in resistance to tumor growth and spread.     

The in vitro radiosensitivity of hemopoietic stem cells from control and preirradiated infant mice

Summary The radiosensitivity of hemopoietic stem cells isolated from infant mice (6 or 9 days of life), of infant preirradiated mice (exposed to 126 rad on day 6 and assayed at day 9 of life) and of adult C57/B1 mice was assayed on the basis of their capacity to form spleen colonies and to incorporate iododeoxyuridine after transplantation into heavily irradiated hosts. Stem cells of infant non-irradiated mice have a D₀ of 115 rad compared to 72 rad for adult mice whereas the D₀ of preirradiated infant mice has diminished to 80 rad. No significant difference in D₀ was seen between spleen and bone marrow cells or between total cells and cells not sensitive to³H-thymidine. It is postulated that this sensitization of stem cells caused by a preirradiation is responsible for the greater mortality of infant mice after fractionated exposure compared to a single one.     

Alteration of allospecific t-cell receptors after differentiation from prethymic precursor cells in semiallogeneic environments

Murine bone marrow cells (strain A) have been allowed to differentiate in vivo in syngeneic (A) or semiallogeneic hosts (A × B) to produce mature splenic T lymphocytes. After stimulation of these cells with irradiated allogeneic (C) spleen cells in tissue cultures, the cytotoxic T-cell blasts (CTL) were purified by velocity sedimentation and used to immunize (A × C) F₁ hybrid mice, to produce antisera recognizing the receptor structure (for C) on the relevant A cytotoxic cells (and their precursors). Using these sera we have been able to show that the T-cell receptor for alloantigen C on strain A cytotoxic precursor lymphocytes (CTLp) seems to differ according to the host environment in which those T cells differentiate from immature bone marrow precursors.     

The role of Ia antigens and Fc receptor-bearing T-cells in the inhibition of macrophage receptors for cytophilic antibody induced by soluble immune complexes

Soluble antigen-antibody complexes composed of 3 M KCl-extracted L1210 antigens and alloantibody to L1210 given to C3H mice caused immunosuppression in the mice. This was reflected in part by the inhibition of cytophilic antibody receptors on macrophages which could be used as a measure of the suppression. Thymocytes or splenic T cells from mice treated with immune complexes could adoptively transfer the suppression to normal syngeneic mice. These cells, which we have termed suppressor inducers, were found to be Ia positive: specifically, I-A⁺, I-J^?. Thus, treatment of the inducers with anti-la or anti-I-A antibodies and complement in vitro abrogated their ability to transfer the suppression to normal mice. In contrast treatment with anti-I-J serum and complement had no effect. Through a similar approach, the cooperating (acceptor) T cells were found to be I-A⁺, I-J^?. Pretreatment of mice with anti-Ia or anti-I-A serum before the administration of antigen-antibody complexes prevented the inhibition of macrophages. This was due at least in part to steric hindrance of adjacent Fc receptors on the FcR⁺ T cells with which the complexes interacted. Early interaction of immune complexes with FcR⁺ T cells was in fact demonstrated directly by the inability of the complexes to induce suppression when FcR⁺ T cells were depleted. The thymocytes or splenic T cells from anti-Ia-pretreated mice failed to transfer the suppression to recipient mice. In contrast, treatment with either anti-Ia or anti-I-A after the immune complexes did not abrogate the generation of suppressor inducers. Treatment of normal recipient mice with anti-Ia serum in vivo before they received the suppressor inducer cells did not prevent cooperation between the two types of cells. By the same token, blocking of Ia antigens of the inducers in vitro with anti-Ia serum (without complement) also did not impair the cooperative interaction. These results indicate that antigen-antibody complexes generate I-A-positive, I-J-negative T-suppressor inducer cells from FcR⁺ naive T cells. These in turn interact with Ia-positive (I-A⁺ and I-J^?) normal thymocytes or spleen T cells. This interaction most likely generates the ultimate suppressor T cells that suppress cytophilic antibody receptors on macrophages in vivo. However, the I-region determined antigens did not appear to be directly involved in the T-T interaction of suppressor inducer and acceptor cells.     

Differential effect of dietary vitamin D supplementation on natural killer cell activity in lean and obese mice

Vitamin D has an immunoregulatory effect on both innate and adaptive immunity. Contradictory results regarding vitamin D and natural killer (NK) cell functions have been reported with in vitro studies, but little is known about this in vivo. We investigated whether vitamin D levels (50, 1000 or 10,000 IU/kg of diet: DD, DC or DS) affect NK cell functions in mice fed a control or high-fat diet (10% or 45% kcal fat: CD or HFD) for 12 weeks. The splenic NK cell activity was significantly higher in the CD-DS group than the HFD-DS group, and the CD-DS group showed significantly higher NK cell activity compared with the CD-DD and CD-DC groups. However, no difference in NK cell activity was observed among the HFD groups fed different levels of vitamin D. The splenic population of NK cells was significantly higher in the CD-DS group than the HFD-DS group. There was no difference in the intracellular expression of IFN-γ and the surface expression of NKG2D and CD107a in NK cells by both dietary fat and vitamin D content. The splenic mRNA expression of Ifng and Ccl5 was significantly lower in the HFD groups compared with the CD groups, but there was no difference in the mRNA levels of Vdup1 and Vdr among the groups. Taken together, these results suggest that dietary vitamin D supplementation can modulate innate immunity by increasing NK activity in control mice but not in obese mice. This effect might be mediated through alternation of the splenic NK cell population.     

Analysis of subpopulations of glass-adherent mouse skin cells controlling resistance/susceptibility to infection with Leishmania tropica,and correlation with the development of independent proliferative signals to Lyt-1+/Lyt-2+ T lymphocytes

A comparison has been made between the course of Leishmania tropica infection of BALB/ c, CBA, and (BALB/c × CBA)F₁ mice in vivo and the growth of the parasite in isolated adherent skin cells in vitro. The susceptible phenotype of the BALB/c mouse was reflected in an innate susceptibility of a discrete subpopulation of adherent skin cells to permit extensive and prolonged growth and replication of the parasite in tissue culture. When cells infected in culture were used to stimulate proliferation of immune lymphocytes from “cured” mice, the skin cells of susceptible BALB/c mice were deficient in their ability to induce proliferation of lymphocytes of BALB/c, CBA, or BCF₁ origin (all immunized in the appropriate bone marrow reconstituted irradiated BCF₁ hosts). In contrast, these skin cells were able to induce proliferation of immune lymphocytes if the L. tropica antigen source used was a soluble excreted extract (EF), rather than that produced by a live parasite infection. Stimulation of naive lymphocytes using an infected adherent skin cell population from BALB/c mice was found to produce a cell population(s) (Thy-1.2⁺, Lyt-2⁺ and including some Lyt-1⁺ cells) able to inhibit subsequent sensitization of normal BCF₁ lymph node cells by L. tropica antigens. The susceptibility of the BALB/c mouse in vivo thus may be attributable to the early contact of T-lymphocyte subsets in BALB/c mice with the high-antigen load maintained in this discrete skin cell population. These particular skin cells were also found to express low levels of Ia antigens.     

Depressive effect of silica particles on F1 hybrid anti-parent cell-mediated lympholysis induced in vitro.

The role of macrophage-like cells in the in vitro generation of specific B6D2F₁ hybrid anti-parental B6 cytotoxic T lymphocytes (CTL) was investigated by means of silica particles (SIL). Depression of this cell-mediated response resulted from the addition of 12.5 or 25 μg of SIL to mixed F₁/parent spleen cell cultuers, and full abrogation resulted from the addition of 125 or 250 μg of SIL. The treatment was effective if applied during the first 48 hr of culture. When treatment was delayed, responsiveness did not decline nor did the lytic function of mature CTL exposed to SIL. Moreover, no depression of the anti-allogeneic cell mediated response resulted from the addition of 250 or 500 μg of SIL to mixed F₁/allogeneic instead of F₁/parent spleen cell cultures. Abrogation of the F₁ hybrid anti-parent response was attributed to SIL-induced impairment of an accessory function presumably exerted by macrophage-like cells during the early phases of responder T cell activation. If so, the F₁ anti-parent response was considerably more dependent than the allogeneic response on the integrity of accessory cells. Injection of 5 mg of SIL to donors of responder cells likewise resulted in loss of F₁ anti-parent and occasionally of anti-allogeneic in vitro responsiveness. This in vivo effect of SIL was prevented by pretreating mice with the lysosomal stabilizer poly-2-vinylpyridine N-oxide. Because unresponsiveness induced in vivo was not selective for F₁ anti-parent responses and lasted for up to 10 days, it may be attributable not only to depletion of accessory macrophages by SIL but also to the induction of suppressor macrophages.     

Structurally characterized arabinogalactan from Anoectochilus formosanus as an immuno-modulator against CT26 colon cancer in BALB/c mice

In this study, the innate immuno-modulatory effects and anti-cancer action of arabinogalactan (AG), a derivative of a well-known orchid, Anoectochilus formosanus, were investigated. The innate immuno-modulatory effects of AG were determined in vitro using RAW 264.7 cells for microarray analysis, and in vivo using BALB/c mice administrated with AG at 5 and 15 mg/kg intra-peritoneally for 3 weeks. The anti-cancer activity of AG was evaluated by CT26 colon cancer-bearing BALB/c mice. The microarray analysis was performed to evaluate the innate immunity and demonstrated that AG significantly induced the expression of cytokines, chemokines, and co-stimulatory receptors, such as IL-1α, CXCL2, and CD69. An intraperitoneal injection of AG in mice increased the spleen weight, but not the body weight. The treatment of mitogen, LPS significantly stimulated splenocyte proliferation in AG treated groups. The AG treatment also promoted splenocyte cytotoxicity against YAC-1 cells and increased the percentage of CD3⁺CD8⁺ cytotoxic T cells in innate immunity test. Our experiments revealed that AG significantly decreased both tumour size and tumour weight. Besides, AG increased the percentage of DC, CD3⁺CD8⁺ T cells, CD49b⁺CD3⁻ NK cells among splenocytes, and cytotoxicity activity in tumour-bearing mice. In addition, the immunohistochemistry of the tumour demonstrated that the AG treatments increased the tumour-filtrating NK and cytotoxic T-cell. These results demonstrated that AG, a polysaccharide derived from a plant source, has potent innate immuno-modulatory and anti-cancer activity. AG may therefore be used for cancer immunotherapy.     

Irradiated chimeric antigen receptor engineered NK-92MI cells show effective cytotoxicity against CD19+ malignancy in a mouse model

Background aimsAnti-CD19 chimeric antigen receptor (CAR)-modified T cells have shown dramatic cytotoxicity against B-cell malignancies. Currently, autologous T cells are conventionally used to manufacture CAR T cells. Low quality or insufficient quantity of autologous T cells may lead to failure of CAR T preparations. Moreover, CAR T preparation usually takes 1–2 weeks, which is too long for patients with rapid disease progression to successfully infuse CAR T cells. Thus, the development of a ready-to-use CAR immunotherapy strategy is needed. NK-92, a natural killer (NK) cell line derived from an NK lymphoma patient, has been gradually applied as a CAR-modified effector cell. To avoid the potential development of secondary NK lymphoma in patients, large doses of radiation are used to treat NK-92 cells before clinical application, which ensures the safety but reduces the cytotoxicity of NK-92 cells. Therefore, it is crucial to explore a suitable radiation dose that ensures short life span and good cytotoxicity of CAR NK-92 cells.MethodsNK-92MI, a modified IL-2-independent NK-92 cell line, was used to establish an anti-CD19 CAR NK. The suitable radiation dose of CAR NK was then explored in vitro and validated in vivo, and the specific cytotoxicity of irradiated and unirradiated CAR NK against CD19⁺ malignant cells was assessed.ResultsCAR NK exhibited specific cytotoxicity against CD19⁺ malignant cells. Irradiation ensured a short life span of CAR NK in vitro and in vivo. Encouragingly, irradiated CAR NK displayed an anti-CD19⁺ malignancy capacity similar to that of unirradiated CAR NK.ConclusionsFive Gy is a suitable radiation dose to ensure the safety and effectiveness of CD19 CAR NK-92MI cells.     

Regulation of cell-mediated cytotoxicity. II. Synergism of two types of thymus dependent cells in vivo.

Sublethal (500 rads) doses of radiation given to mice before the intravenous injection of allogeneic spleen cells induced the development of an increased cell-mediated cytotoxicity (CMC) of the recipients' spleen cells. The effector cells from the irradiated animals were shown to carry the θ alloantigenic marker and to be capable of transferring adoptive immunity in vivo. On the other hand, irradiation of mice with the same dose before the administration of skin or tumor allografts induced a suppression of CMC. The response of irradiated mice treated with tumor allografts was restored with small numbers of spleen or lymph node cells from syngeneic or semi-allogeneic F₁ hybrid donors. With the use of the appropriate cytotoxic alloantisera, it was demonstrated that the majority of the effector cells generated in the spleens of mice restored with semiallogeneic cells were of host origin. These results demonstrate that the precursors of the cytotoxic lymphocytes are radioresistant and indicate that for their stimulation some radiosensitive T cells are necessary to amplify their reaction to nonlymphoid allografts. Allogeneic lymphoid cells, on the other hand, supply a stimulus which does not require the intervention of such amplifier cells. In this case, irradiation induces a stronger CMC response probably by inactivating radiosensitive cells with suppressor activity.     

Partially restorative role of T cells for low interleukin 2 dependent growth of NK cell progenitors from nude mice

The frequency of cells in the spleens of nude mice which could be grown in conditioned medium containing interleukin 2 and of those which developed natural killer (NK)-like activity was evaluated. Although BALB/c nu/nu spleen cells have higher spontaneous NK activity than euthymic mice, they showed a substantially lower frequency of proliferating and cytotoxic cells as compared to BALB/c nu/+ littermates. This defect in cells of nu/nu mice was reversed in part by culturing nu/nu responder cells in the presence of irradiated (3,000 R) splenic or thymic feeder cells that included T cells. In contrast to the dissociation of NK activity and progenitor frequencies in nude mice, the results of parallel studies with spleen cells from euthymic mice indicated that the limiting dilution assay correlated well with previously described features of NK activity. High-NK-reactive CBA/J mice were found to have a considerably higher frequency of interleukin 2 dependent NK cell progenitors than low-NK-reactive strains of mice when assessed against NK-susceptible YAC-1 targets. The frequency of progenitors of cells cytotoxic against YAC-1 was higher in spleens of high-NK-reactive mice than that of cells reactive against the NK-insensitive target P-815. Furthermore, the phenotype of the progenitor cells and of the cultured effector cells was consistent with that of NK cells rather than cytotoxic T cells in that the cells expressed asialo GM1, some Thy-1, but no detectable Lyt-1 or Lyt-2 antigens. Thus, the present observations suggest that the subpopulation of NK cell progenitors in nude mice which can grow and develop cytotoxic reactivity in vitro in the presence of interleukin 2 is small, that it can be increased appreciably in the presence of T cells, but that this does not represent the major pathway for development of NK cells in athymic individuals.     

Concomitant induction of cytotoxic and suppressor cells: modulation by theophylline

Previous studies in man have shown that T cells with suppressor activity were mainly found among a subset bearing Fc receptors for IgG (T_γ). Recently, we found that virus-induced cytotoxic effector cells were also found predominantly among T_γ cells. In the present studies, we present evidence that similar, possibly overlapping T-cell populations can mediate both suppressor and cytotoxic activities when sensitized in vitro with virus-infected cells. In fact, both activities are found within the positively selected T_γ subset, but not in the T_γ-depleted population; both activities are abolished by irradiation but not by treatment with mitomycin C; a 1-hr exposition to theophylline at the onset of sensitization enhances both cytotoxic and suppressor activities. The data suggest that development of antiviral cell-mediated immune responses in vivo may also be accompanied by a concurrent induction of nonspecific suppressor cells. Such suppressor activity may play a role in the depressed cellular immune responsiveness which is associated with several systemic virus infections.     

Development of mesothelin-specific CAR NK-92 cells for the treatment of gastric cancer

Background: The application of chimeric antigen receptor (CAR) NK cells in solid tumors is hindered by lack of tumor-specific targets and inefficient CAR NK cell efficacy. It has been reported that mesothelin (MSLN) may be an ideal immunotherapy target for gastric cancer. However, the feasibility of using anti-MSLN CAR NK cells to treat gastric cancer remains to be studied.Methods: MSLN expression in primary human gastric cancer, normal tissues and cell lines were detected. MSLN and CD19 targeted CAR NK-92 (MSLN- and CD19-CAR NK) cells were constructed, purified and verified. N87, MKN-28, AGS and Huh-7 cells expressing the GFP and luciferase genes were transduced. Cell- and patient-derived xenograft (PDX) were established via NSG mice. The ability of MSLN-CAR NK cells to kill MSLN-positive gastric cancer cells were evaluated in vitro and in vivo.Results: MSLN-CAR NK cells can specifically kill MSLN-positive gastric cancer cells (N87, MKN-28 and AGS), rather than MSLN negative cell (Huh-7), in vitro. Moreover, compared with parental NK-92 cells and CD19-CAR NK cells, stronger cytokine secretions were secreted in MSLN-CAR NK cells cocultured with N87, MKN-28 and AGS. Furthermore, MSLN-CAR NK cells can effectively eliminate gastric cancer cells in both subcutaneous and intraperitoneal tumor models. They could also significantly prolong the survival of intraperitoneally tumor-bearing mice. More importantly, the potent antitumor effect and considerable NK cell infiltration were observed in the patient-derived xenograft treated with MSLN-CAR NK cells, which further warranted the therapeutic effects of MSLN-CAR NK cells to treat gastric cancer.Conclusion: These results demonstrate that MSLN-CAR NK cells possess strong antitumor activity and represent a promising therapeutic approach to gastric cancer.     

Evidence for histamine-induced auto-anti-idiotypic antibody immunoregulation in vivo

The in vivo effects of histamine injection in LAF₁ male mice on the immune reactivity to trinitrophenylated bovine γ-globulin was studied using plaque-forming cell (PFC) responses and their avidity distributions. Splenic anti-trinitrophenyl (anti-TNP) PFC responses of mice treated with histamine (5 × 10^?6 mol or 1 mg, intravenously) were significantly reduced in number and restricted in heterogeneity and characterized by a preferential loss of high-avidity IgG PFCs. The reduced PFC response in histamine-treated mice was dose and time dependent. No evidence of suppressor cell activity in the spleens from histamine-treated mice was demonstrable. Only histamine-treated mice produced a significantly high percentage of anti-idiotype-blocked, hapten-augmentable IgG PFCs, suggesting the presence of auto-anti-idiotypic activity. Immune sera taken from histamine-treated mice caused an inhibition of anti-TNP PFC in vitro. This PFC-inhibiting factor in immune sera of histamine-treated mice was an antibody of the IgG₁ and IgG_2a class, lacked anti-TNP antibody activity, but reacted with anti-TNP antibody of LAF₁ origin. Passive hemagglutination study of this sera showed anti-(anti-TNP F(ab′)₂-IgG) titer. Thus, the results of this study suggest that histamine in combination with antigen induces auto-anti-idiotypic antibody which, in turn, is involved in the normal regulation of the immune response to trinitrophenylated bovine γ-globulin in vivo.     

Phloroglucinol (1,3,5-trihydroxybenzene) protects against ionizing radiation-induced cell damage through inhibition of oxidative stress in vitro and in vivo

Exposure of cells to γ-rays induces the production of reactive oxygen species (ROS) that play a main role in ionizing radiation damage. We have investigated the radioprotective effect of phloroglucinol (1,3,5-trihydroxybenzene), phlorotannin compound isolated from Ecklonia cava, against γ-ray radiation-induced oxidative damage in vitro and in vivo. Phloroglucinol significantly decreased the level of radiation-induced intracellular ROS and damage to cellular components such as the lipid, DNA and protein. Phloroglucinol enhanced cell viability that decreased after exposure to γ-rays and reduced radiation-induced apoptosis via inhibition of mitochondria mediated caspases pathway. Phloroglucinol reduced radiation-induced loss of the mitochondrial membrane action potential, reduced the levels of the active forms of caspase 9 and 3 and elevated the expression of bcl-2. Furthermore, the anti-apoptotic effect of phloroglucinol was exerted via inhibition of mitogen-activated protein kinase kinase-4 (MKK4/SEK1), c-Jun NH₂-terminal kinase (JNK) and activator protein-1 (AP-1) cascades induced by radiation exposure. Phloroglucinol restored the level of reduced glutathione (GSH) and protein expression of a catalytically active subunit of glutamate-cysteine ligase (GCL), which is a rate-limiting enzyme in GSH biosynthesis. In in vivo study, phloroglucinol administration in mice provided substantial protection against death and oxidative damage following whole-body irradiation. We examined survival with exposure to various radiation doses using the intestinal crypt assay and determined a dose reduction factor (DRF) of 1.24. Based on our findings, phloroglucinol may be possibly useful as a radioprotective compound.     

Suppressor T cells and suppressor macrophages induced by Corynebacterium parvum

Corynebacterium parvum, injected intravenously into C57B1/6 mice (H-2^b) previously alloimmunized with P815 (H-2^d) mastocytoma cells, generated splenic suppressor cells that inhibited the development of primary cytotoxic lymphocytes in vitro. These suppressor cells differed from those generated by intravenous C. parvum injection of naive C57B1/6 mice. The former suppressor cells were effectively induced by administration of 700 μg of C. parvum whereas the latter suppressor cells were dependent upon higher doses (1400 μg) of adjuvant for their activation. Furthermore, suppressor cells generated in alloimmunized mice could only suppress C57B1/6 anti-P815 in vitro cytotoxic responses whereas suppressor cells generated in naive mice could suppress C57B1/6 anti-CBA (H-2^k) responses as well. Suppressor cells were not H-2 restricted in their action. Fractionation of spleens from alloimmunized, C. parvum-treated mice revealed the presence of suppressor T cells and suppressor macrophages. We were unable, however, to determine which cell was responsible for “antigen specificity” of suppression since the fractionation procedures seemed to trigger both suppressor cell types prior to adding them to the primary culture.