Suppression of primary immune response by heterologous anti-lymphocyte sera.

The effect of anti-lymphocyte serum (ALS) on the primary immune response to sheep erythrocytes (SRBC) in rats was studied by the Jerne Plaque technique. ALS against normal rat lymph node cells, ALS(N) suppressed the immune response to SRBC when administered before the antigen and had no effect on the immune response when administered either with or after the antigen. ALS(I), which was produced against lymph node cells from SRBC immunized rats, produced significant immunosuppression when administered either before or after the antigen.     

Effects of cyclosporin A, antilymphocyte serum and donor-specific transfusions on murine delayed-type hypersensitivity and skin graft survival

The effect of immunosuppressive reagents cyclosporin A (CsA) and rabbit anti-mouse antilymphocyte serum (ALS) on the response to alloantigens was studied in inbred mouse strains. Alloantigen was given either as a cell suspension which induced a delayed-type hypersensitivity reaction (DTH), or as a full-thickness skin graft. Dose-response studies showed that DTH reactions in CBA mice sensitised to BALB/c cells were reduced to background levels when recipient mice were treated with 100 mg/kg CsA on days 0, 4 and 6 after primary alloantigenic challenge. The response to a second challenge was significantly decreased by CsA treatment during primary or secondary exposure to alloantigen and CsA was as effective as ALS in abrogating both primary and secondary DTH reactions. Survival of full-thickness grafts of BALB/c skin on CBA mice was increased from 9 to 23 days by ALS treatment on days -1 and +2, with grafts given on day 0. Long-term treatment with CsA, from day -14 to +12, also prolonged graft survival from 9 to 18 days but donor-specific transfusions, with or without concomitant ALS or CsA treatment, decreased graft survival and often sensitised the recipients. This occurred with transfusions administered from -63 to -7 days and on the day of grafting. Thus, in H-2 mismatched mice, both CsA and ALS treatments produced a state of tolerance when administered during short-term exposure to alloantigen.(ABSTRACT TRUNCATED AT 250 WORDS)     

Specific suppression of delayed hypersensitivity response to sheep erythrocytes by heterologous anti-lymphocyte serum.

The development of a heterologous anti-lymphocyte serum (ALS) capable of specifically suppressing the delayed hypersensitivity (DH) response is reported. This ALS, termed ALS(CMI), was prepared against lymph node cells from rats which had been immunized against sheep erythrocytes (SRBC) following treatment with cyclophosphamide which is known to enhance the DH response and suppress the humoral immune response. The effect of ALS(CMI) on the primary DH response to SRBC using the footpad swelling test was studied. Its effect on the primary humoral immune response to SRBC was also studied using the Jerne plaque assay technique. ALS(CMI) suppressed the humoral antibody response to SRBC and the DH response to a third party antigen only when administered before the antigen, having no effect when administered post-antigenically. On the other hand, ALS(CMI) significantly suppressed the primary DH response to SRBC when administered either before or after the antigen.     

The requirement of intrathymic mixed chimerism and clonal deletion for a long-lasting skin allograft tolerance in cyclophosphamide-induced tolerance

Mechanisms of cyclophosphamide (CY)-induced tolerance were studied. When C3H/He Slc (C3H; H-2k, Mls-1b) mice were primed i.v. with 1 x 10(8) viable spleen cells from H-2-identical AKR/J Sea (AKR; H-2k, Mls-1a) mice and treated with 200 mg/kg of CY 2 days later, a long-lasting skin allograft tolerance to AKR was established. When [C57BL/6 Sea (B6; H-2b, Mls-1b) x AKR]F1 (B6AKF1) cells were used as the tolerogen, however, only a moderate, but not long-lasting, skin tolerance to AKR was observed. In the C3H mice treated with AKR cells and CY, the intrathymic clonal deletion of V beta 6+ T cells, which are strongly correlated with reactivity to Mls-1a antigens, was observed in the chimeric thymus on day 35, although neither the clonal deletion of V beta 6-bearing T cells nor the mixed chimerism was observed in the thymus on day 14. In the C3H mice treated with B6AFKF1 cells followed by CY, however, neither the clonal deletion of V beta 6+ T cells nor the mixed chimerism was observed in the thymus throughout the test period. In the lymph nodes of the C3H mice treated with AKR cells and CY, only CD4+ V beta 6+ T cells, bur not CD8+V beta 6+ T cells, had selectively decreased by day 14, and they were hardly detectable on day 35. The selective decrease of CD4+V beta 6+ T cells in the lymph nodes was also observed by day 14 when B6AKF1 cells were used as the tolerogen, although CD4+V beta 6+ T cells gradually increased on day 35, at which time almost all skin grafts from AKR had already been rejected. These results strongly support the necessity of the intrathymic mixed chimerism and clonal deletion of donor-reactive T cells for a long-lasting skin allograft tolerance in CY-induced tolerance.     

Suppression of secondary immune response by antilymphocyte serum: time relationship between immunization and administration of antilymphocyte serum.

The effect of antilymphocyte serum (ALS) on the secondary humoral immune response to sheep erythrocytes (SRBC) in rats was studied by the Jerne plaque assay technique. Its effect was also studied on the delayed hypersensitivity (DH) response to SRBC by the foot pad swelling test. ALS(N), which was prepared against lymphocytes from normal rats, had no effect on the secondary humoral and cellular response or on the primary cellular response, when administered postantigenically. ALS(I), which was raised against lymph node cells from SRBC immunized rats produced significant immunosuppression of the secondary response to SRBC when administered either before or after the antigenic injections. In the case of DH, ALS(I) behaved just like ALS(N) having no effect on the secondary response and suppressing the primary only when administered prior to the antigen.     

MULTIPLE FOREIGN NON-H-2 DETERMINANTS ON THE SURFACE OF A CHEMICALLY-INDUCED MURINE SARCOMA*

By immunizing BALB/c (H-2^d mice against normal tissues from C57BL/6J (H-2^b), C3Hf (H-2^k) and DBA/2 (H-2^d), but not from AKR (H-2^k) strains, resistance was induced to the subsequent challenge of the ‘syngeneic’ methyl-cholanthrene-induced BALB/c sarcoma ST5; lymph node cells from allo-immune BALB/c mice were also able to exert a parallel cytotoxic effect against in vitro cultured ST5 cells. The involvement of foreign H-2 specificities in the observed cross-reactions was ruled out by absorptions of H-2 monospecific sera and by the interallelic combinations used, thus suggesting that non-H-2 histocompatibility antigens were responsible for the above findings. By using the indirect isotopic antiglobulin assay, BALB/c anti-C57BL/6J and anti-C3Hf polyspecific sera were found to bind specifically to cultured ST5 cells. C57BL/6J and C3Hf, but not DBA/2, lymph node cells were able to absorb the anti-ST5 activity of the anti-C57BL/6J serum. These results indicated that ST5 cells expressed on their surface at least two different sets of foreign non-H-2 antigens: one shared by C57BL/6J and C3Hf tissues, and detected by both cell-mediated and serological techniques; the other one belonging to DBA/2 tissues, and revealed mainly at the cell-mediated level.     

Cytotoxicity in vitro of preleukaemic lymphoid cells on syngeneic monolayers of embryo or thymus cells

Lymphoid cells from preleukaemic AKR mice were cytotoxic for monolayers of syngeneic embryo and thymus target cells in tissue culture. This reactivity was detectable with cells from mice aged 3–36 weeks but was not present with cells from younger mice. Cytotoxic reactions to syngeneic embryo tissues were also seen with lymphoid cells from high leukaemia strain C3H mice carrying Moloney virus but not with lymphoid cells from normal low leukaemic strain C3H/Bi or DBA/2 mice. Lymphoid or lymphoma cells from leukaemic AKR mice showed reduced reactivity. Phytohaemagglutinin was not necessary for the reaction of preleukaemic AKR cells against AKR monolayers and cytotoxicity was inhibited by preincubation of target cells with an antiserum directed against AKR G+ cells.

The reactivity of preleukaemic AKR and C3H lymphoid cells against syngeneic monolayers may represent some type of allogeneic inhibition due to acquired antigenic differences between aggressor and target cell but the data fit best an interpretation that some lymphoid cells in preleukaemic AKR and C3H mice acquire immunological reactivity to virus-induced G+ or M+ antigens exhibited by the target cells.

     

Induction of tolerance to skin homografts with a methylhydrazine derivative. Synergism with antilymphocytic serum and morphologic responses of the lymphoid system

CBA mice were treated before skin grafting with a course of either the methylhydrazine derivative Ro 4-6824 or cyclophosphamide, combined with donor type lymphoid cells. No further immunosuppressive treatment was applied after the grafting. Marked prolongation of graft survival and specific tolerance to (CBAxA)F₁ and A skin homografts could be induced with Ro 4-6824, but not with cyclophosphamide. Tumors occurred in two tolerant mice after 10 months. Besides by administration of donor lymphoid cells, the effect of Ro 4-6824 pretreatment is also potentiated by heterologous antilymphocytic serum (ALS) and by thymectomy. The mice treated with Ro 4-6824 showed more profoud depletion of lymphoid tissue in the lymph nodes than after ALS. In addition, marked atrophy of the thymus occurred. In the spleen, Ro 4-6824 prevented the plasma cell response to ALS.     

In Vitro Studies on the Effect of Anti-Lymphocyte Serum on the Humoral Immune Response

The effect of ALS (I), a heterologous anti-lymphocyte serum prepared against lymph node cells from rats pre-immunised with sheep erythrocytes (SRBC), on plaque forming cells (PFC) to SRBC was studied in vitro. ALS (I) reduced the number of both IgM and IgG PFC when complement was included in the reaction. This ability of ALS (I) to inhibit FFCs in vitro was absorbed out by the IgG fraction of anti-SRBC serum. Thus ALS (I) was thought to possess an anti-idiotypic antibody directed against B-cells at a later stage of differentiation.     

Properties of antisera against lymphocytes of nude mice.

The activity of rabbit antisera against nu/nu BALB/c lymphocytes was estimated in vivo and in vitro. It was found that anti-lymphocyte serum (ALS) against nu/nu lymph node cells suppressed the alloantigen reaction and the spontaneous rosette-forming cell (sRFC) or plaque-forming cell (PFC) formation for T-dependent (sheep red blood cells) and T-independent (lipopolysaccharide) antigens. ALS against nu/nu spleen cells affected only the sRFC and PFC for T-independent antigen. The former serum exhibited a high cytotoxicity for the suspensions enriched or depleted in B cells, while the latter was more cytotoxic for the suspension enriched in B cells. This may indicate that ALS anti-nu/nu spleen cells is specific for B lymphocytes, and ALS anti nu/nu lymph node cells is directed not only to B cells but also to a subpopulation of T lymphocytes. It may suggest the existence of a subpopulation of T lymphocytes in nu/nu lymph node cells.     

Properties of antisera against lymphocytes of nude mice.

This paper reports results of a study on the activity of rabbit antisera against nu/nu Balb/c lymphocytes in vivo and in vitro. It was found that ALS against nu/nu lymph node cells suppressed the alloantigen reaction and the sRFC or PFC formation for T-dependent (SRBC) and T-independent (LPS) antigens. ALS against nu/nu spleen cells affected only the sRFC and PFC for T-independent antigen. The former serum exhibited a high cytotoxicity for the suspensions enriched or depleted in B cells while the latter one was more cytotoxic for the suspension enriched in B cells. It indicates that ALS anti nu/nu spleen cells is specific for B lymphocytes and ALS anti nu/nu lymph node cells is directed not only to B cells but also to a subpopulation of T lymphocytes. It suggests the existence of a subpopulation of T lymphocytes in nu/nu lymph node cells.     

Effect of antilymphocyte serum (ALS) on shock in rats.

Effects of treatment with rabbit antirat anti-lymphocyte serum and globulin (ALS and ALG) on shock survival were studied in Sprague-Dawley derived male rats. Because of their known cytotoxic capability, it was postulated that lymphocytes might play a role in the pathogenesis of shock and that suppression of lymphocyte function by ALS/ALG treatment should then protect against shock. Shock models used were tourniquet, endotoxin, and hemorrhagic shock. Protection against tourniquet shock was found for ALS made against thymocytes but not for ALS against spleen cells or lymph node cells. The shock-protective factor was found in the ALG-containing serum fraction but not in the primarily albumin fraction. No significant protection was found for ALS treatment against either endotoxin or hemorrhagic shock. ALS effects on blood cell counts, reticulo endothelial system clearance, and inflammation were studied to help identify effects of ALS on shock survival. It was concluded from these studies that thymic or thymus-processed lymphocytes could play a role in the pathogenesis of shock but that multiple effects of ALS/ALG treatment necessitate further studies to elucidate any role for lymphocytes in shock.     

Quantitative studies of the growth and rejection of allogeneic tumour cells in mouse cerebrospinal fluid. Elimination in the absence of H-2 differences.

Following intracerebral (i.c.) inoculation DBA/2 (H-2d) mastocytoma cells (P815) grow to concentrations of greater than 10,000/mul in cerebrospinal fluid (CSF) of H-2 compatible BALB/c mice, but are completely eliminated from 90% of such animals within 12 days. A similar pattern occurs in allogeneic C57BL (H-2b) and random-bred WEHI mice, whereas syngeneic DBA/2 mice die within 7 days from unrestricted tumour growth. Rejection in BALB/c mice is enhanced by prior exposure to the tumour, but is severely depressed by pretreatment with cyclophosphamide. Leucocytes responsible for eliminating the mastocytoma are apparently not active against sarcoma 180 cells injected simultaneously. Subsequent to intraperitoneal inoculation with mastocytoma cells both BALB/c and C57BL mice generate significant cytotoxic activity in spleen, as measured by an in vitro 51Cr release assay. Cytolysis is abrogated by prior incubation of spleen cells with AKR anti-O ascitic fluid and complement, but not by normal AKR ascitic fluid and complement. Following i.c. exposure, however, much lower levels of cytotoxic activity are found in spleen, though specifically sensitized lymphocytes are also present in lymph nodes of the cervical chain.     

Oral Administration of Chlorella Vulgaris Augments Concomitant Antitumor Immunity

Chlorella vulgaris, an unicellular green algae, or its acetone-extract (Ac-Ex) were administered orally to Meth A tumor bearing BALB/c or (BALB/c DBA/2)F1 (CDF1) mice. When CDF1 mice were fed daily with 10% dried powder of Chlorella vulgaris (CVP) containing diet before and after Meth A tumor inoculation, the growth of rechallenged Meth A tumor was significantly suppressed in an antigen-specific manner. Augmentation of antitumor resistance was exhibited also by Winn assay using lymph node cells of tumor-bearing mice orally administered with CVP or Ac-Ex. Antigen-specific concomitant immunity in these mice were mediated by cytostatic T cells but not by cytotoxic T cells. Natural killer cells seemed not to contribute in antitumor resistance in this system.     

Approach to withdrawal from tacrolimus in a fully allogeneic murine skin graft model

With few exceptions, transplant patients must take immunosuppressants throughout their lives. In this study, we used anti-T-cell receptor (TCR/CD3) monoclonal antibodies (mAbs) to induce immunological tolerance to alloantigens after withdrawal from tacrolimus in a fully allogeneic murine skin graft model. Skin grafts from AKR donor mice were maintained in C57BL/6 recipients by administering tacrolimus for one month. Anti-T-cell receptor (TCR) alphabeta mAb was administered to recipient mice on the day of withdrawal from tacrolimus administration. Seven days after mAb administration, the recipient mice were treated with various combinations of the following treatments: low-dose whole body irradiation, AKR bone marrow transfer (BMT), and anti-CD3 mAb administration. The control recipient mice did not receive treatment with either mAb, nor any other treatment. All the control recipient mice showed rejection of AKR skin grafts 42 days after tacrolimus withdrawal (mean skin graft survival: 77 days). Mice treated with a combination of anti-TCR alphabeta antibody, low-dose irradiation and AKR BMT showed stable chimerism in their peripheral blood lymphocytes and significantly prolonged skin graft survival (mean skin graft survival: >151.2+/-15.3 days). Mice given the combination of anti-TCR alphabeta mAb, anti-CD3 mAb, low-dose irradiation, and AKR BMT exhibited more stable chimerism but had earlier skin graft rejection (mean skin graft survival: 116.7+/-17.6 days) than the mice that did not receive anti-CD3 mAb. These results suggest that anti-TCR alphabeta mAb, but not anti-CD3 mAb, in combination with low-dose irradiation and BMT, is useful for long-lasting allograft survival after withdrawal from tacrolimus in mice with fully allogeneic skin grafts.     

Spontaneous in vivo retrovirus-infected T and B cells, but not dendritic cells, mediate antigen-specific Fas ligand/Fas-dependent apoptosis of anti-retroviral CTL

C57BL/6 (H-2b), but not spontaneous virus-expressing AKR.H-2b congenic, mice generate retrovirus-specific CD8+ CTL responses to the immunodominant Kb-restricted epitope, KSPWFTTL. AKR.H-2b non-responsiveness is mediated by a peripheral tolerance mechanism. When co-cultured with primed B6 antiviral pCTL, AKR.H-2b splenocytes are recognized by the antiviral TcR as "veto" cells, which inhibit by an exquisitely virus-specific, MHC-restricted, veto cell FasL/responder T cell Fas, mediated apoptotic mechanism. Here, AKR.H-2b thymus, lymph node, and bone marrow cells are also shown to inhibit antiviral CTL generation. Purified AKR.H-2b CD4+ and CD8+ T cells, and B cells, served effectively as FasL-dependent veto cells. In contrast, AKR.H-2b dendritic cells (DC) did not efficiently veto antiviral CTL responses, despite expressing sufficient MHC class I/viral peptide complexes for TcR recognition. AKR.H-2b DC also expressed FasL mRNA and cell surface protein, albeit at a lower level than AKR.H-2b T and B cells. These findings suggest a fail-safe escape mechanism by virus-infected cells for escape from CTL-mediated immunity.     

CD4+CD8+ thymocytes develop into CD4 or CD8 single-positive cells in athymic nude mice

A differentiation pathway from CD4+CD8+ cells to CD4+CD8- or CD4-CD8+ cells was investigated in athymic nude mice. Using fluorescence-activated cell sorter, CD4+CD8+ cells were sorted out from AKR thymocytes (H-2k, Thy-1.1) stained with two monoclonal antibodies against CD4 and CD8 (anti-L3T4 and anti-Ly-2). These CD4+CD8+ AKR thymocytes were injected i.v. into CBA or C3H nude mice (H-2k, Thy-1.2) which had received 650 rads and had been reconstituted with syngeneic nude bone marrow cells. The lymph node cells of the nude recipients at 4 wks post-thymocyte transfer were shown to contain 50% AKR-derived Thy-1.1+ cells. The majority of the Thy-1.1+ cells were found to express either CD4 or CD8 alone but not to express both CD4 and CD8. These findings indicate that CD4+CD8+ thymocytes can develop into CD4+CD8- and CD4-CD8+ single-positive cells in extrathymic tissues.     

Adherence of Candida albicans to tissues from mice with genetic immunodeficiencies.

Ex vivo adherence comparisons were made between immunocompetent and immunocompromised mouse tissues, and the roles of serum immunoglobulin and macrophages in the adherence of Candida albicans were investigated. Spleen, lymph node, and kidney tissues were harvested from congenitally immunodeficient mice, including AKR/scid, C.B-17, C3Hscid, and N:NIH nu/bg/xid mice, and their normal counterparts into which the defects were bred (AKR/J, C3H/HeSnJ, and BALB/c-ByJ). Tissues were compared for the ability to bind C. albicans 219 in an ex vivo assay. In general, immunodeficiencies significantly decreased binding of C. albicans to spleen but not to lymph node or kidney tissue compared with immunocompetent mice. In C3Hscid and AKRscid mice, spleen tissues from "nonleaky" mice bound significantly fewer yeast cells (P = 0.0005 and 0.0009, respectively) than did those from C3H/HeSnJ or AKR/J mice. Numbers of adherent yeast cells were similar in "leaky" AKRscid and AKR/J mice. Yeast adherence to spleen tissue from N:NIH nu/bg/xid mice correlated with mouse age (P = 0.01). Measurements of total serum immunoglobulin indicated that the scid defect was most complete in C3Hscid mice and that yeast adherence in spleen tissue correlated with immunoglobulin titers. Results of adherence assays and macrophage-specific immunostains suggested that factors determining adherence differ among reticuloendothelial organs.