Treatment Of Donor Mice Research Articles

Clathrin-mediated Endocytosis of Alpha-1 Antitrypsin is Essential for its Protective Function in Islet Cell Survival.

Cytokine-induced pancreatic β cell death plays a pivotal role in both type 1 and type 2 diabetes. Our previous study showed that alpha-1 antitrypsin (AAT) inhibits β cell death through the suppression of cytokine-induced c-Jun N-terminal kinase (JNK) activation in an islet transplantation model. The aim of this study was to further understand how AAT impacts β cells by studying AAT endocytosis in human islets and a βTC3 murine insulinoma cell line.Methods: In vitro, human islets and βTC3 cells were stimulated with cytokines in the presence or absence of chlorpromazine (CPZ), a drug that disrupts clathrin-mediated endocytosis. Western blot, real-time PCR and cell death ELISA were performed to investigate β cell death. The oxygen consumption rate (OCR) was measured on human islets. In vivo, islets were harvested from C57BL/6 donor mice treated with saline or human AAT and transplanted into the livers of syngeneic mice that had been rendered diabetic by streptozotocin (STZ). Islet graft survival and function were analyzed.Results: AAT was internalized by β cells in a time- and dose-dependent manner. AAT internalization was mediated by clathrin as treatment with CPZ, profoundly decreased AAT internalization, cytokine-induced JNK activation and the downstream upregulation of c-Jun mRNA expression. Similarly, addition of CPZ attenuated cytokine-induced caspase 9 cleavage (c-casp 9) and DNA fragmentation, which was suppressed by AAT. Treatment of donor mice with AAT produced AAT internalization in islets, and resulted in a higher percentage of recipients reaching normoglycemia after syngeneic intraportal islet transplantation.Conclusion: Our results suggest that AAT is internalized by β cells through clathrin-mediated endocytosis that leads to the suppression of caspase 9 activation. This process is required for the protective function of AAT in islets when challenged with proinflammatory cytokines or after islet transplantation.

In vivo engineering of mobilized stem cell grafts with the immunomodulatory drug FTY720 for allogeneic transplantation.

The immunological attributes of stem cell grafts play an important role in the outcome of allogeneic stem cell transplants. Currently, ex vivo manipulation techniques such as bulk T-cell depletion or positive selection of CD34(+) cells are utilized to improve the immunological attributes of grafts and minimize the potential for graft-versus-host disease (GvHD). Here, we demonstrate a novel graft engineering technique, which utilizes the immunomodulatory drug FTY720 for in vivo depletion of naïve T (TN ) cells from donor G-CSF-mobilized grafts without ex vivo manipulation. We show that treatment of donor mice with FTY720 during mobilization depletes grafts of TN cells and prevents lethal GvHD following transplantation in a major mismatch setting. Importantly, both stem cells and NK cells are retained in the FTY720-treated grafts. FTY720 treatment does not negatively affect the engraftment potential of stem cells as demonstrated in our congenic transplants or the functionality of NK cells. In addition, potentially useful memory Tcells may be retained in the graft. These findings suggest that FTY720 may be used to optimize the immunological attributes of G-CSF-mobilized grafts by removing potentially deleterious TN cells which can contribute to GvHD, and by retaining useful cells which can promote immunity in the recipient.

Memory B Cells Are Necessary for the Adoptive Transfer of Murine Peanut Allergy

We hypothesize that memory B cells are necessary and sufficient for the adoptive transfer of murine peanut allergy (PNA). Peanut-allergic donor C3H/HeJ mice (with similar anti-peanut IgE levels, symptom scores (SS) and temperature drops (dT) following intraperitoneal (IP) challenge with crude peanut extract (CPE)) were treated intravenously with either anti-CD20 or isotype control (IC) antibody for 18 weeks. Naïve, lethally irradiated recipients were given bone marrow (BM) and splenocytes (SPL) from either anti-CD20 or IC-treated donors. In a separate experiment, B cells (negatively selected, >96% pure) from SPL of peanut-allergic or naïve donors were injected into naïve, irradiated recipients with or without the addition of naive SPL. Sera were obtained from recipients on days 7 and 17 post-reconstitution, and recipients were challenged IP with CPE on days 8 and 18. Treatment of donor mice with anti-CD20 for 18 weeks significantly reduced B cell numbers in the blood (p<0.0001, >95% reduction) and the spleen (p<0.0001, >98% reduction) but did not affect IgE levels. Recipients (n=15) given BM and SPL from anti-CD20 treated donors were protected from developing anti-peanut IgE (p<0.0001), SS (p<0.0001), and dT (p<0.0001) compared to recipients (n=14) given cells from IC-treated donors. Recipients (n=9) given purified B cells from peanut-allergic donors plus naïve SPL developed significantly elevated anti-peanut IgE (p<0.001), SS (p<0.002), and dT (p<0.003) compared to controls. These results demonstrate that memory B cells are required but not sufficient for the adoptive transfer of murine PNA. Help from a yet-to-be-defined cell population(s) found in naïve SPL is required.

Thrombopoietic effect of VPAC1 inhibition during megakaryopoiesis

Megakaryocytes and platelets express the stimulatory G protein (Gs)-coupled VPAC1 receptor, for which the pituitary adenylyl cyclase-activating peptide (PACAP) and vasoactive intestinal peptide (VIP) are agonists. The neuropeptide PACAP and VPAC1 were previously found to negatively regulate megakaryopoiesis, and inhibition of their physiological pathway was found to have a thrombopoietic effect in conditions where megakaryopoiesis and thrombopoiesis were impaired, such as chemotherapy-induced thrombocytopenia and congenital thrombocytopenia. The present study explored the thrombopoietic effect of VPAC1 inhibition in a murine model of syngeneic bone marrow transplantation (BMT) and in passive immune thrombocytopenia. Treatment of donor mice with a neutralizing anti-VPAC1 antibody stimulated the initial, most critical recovery of the platelets in irradiated mice. In the passive immune thrombocytopenia model, we observed a thrombopoietic effect, resulting in a less severe platelet drop after induction of their removal in the spleen by an anti-platelet antibody. We concluded that inhibition of the physiological PACAP/VPAC1 pathway could stimulate in vivo megakaryopoiesis. This inhibition can be applied to attenuate thrombocytopenia in conditions where platelets are destroyed as the major pathogenetic mechanism, e.g. immune thrombocytopenia purpura, or need to be produced de novo, e.g. after irradiation and BMT.

Effects of fexofenadine on T-cell function in a murine model of allergen-induced airway inflammation and hyperresponsiveness

There is renewed interest in antihistamines for the treatment of allergic asthma. A growing body of literature has shown that the newer compounds can affect inflammatory cell accumulation and cytokine/chemokine production. In a murine model of allergen-induced airway inflammation and hyperresponsiveness, the ability of fexofenadine to affect these outcomes was tested in a primary sensitization and challenge model and after treatment of donor mice before the adoptive transfer of T cells into recipients receiving limited allergen exposure. Mice were sensitized and challenged with allergen (ovalbumin). Airway function after inhaled methacholine was monitored in parallel to the assessment of tissue and airway inflammation and cytokine production. In further experiments, lung T lymphocytes from sensitized/challenged donor mice were transferred into naive recipients before limited airway challenge with the allergen. Administration of fexofenadine before challenge but after sensitization was effective in preventing tissue eosinophilia and airway hyperresponsiveness. Moreover, the treatment of donor mice with fexofenadine before transfer of lung T cells effectively prevented airway hyperresponsiveness and eosinophilia in naive mice exposed to limited airway challenge. These data therefore support the potential for fexofenadine in the treatment of allergen-induced airway hyperresponsiveness and inflammation.

Resident mast cells are important for eotaxin-induced eosinophil accumulation in vivo.

Eotaxin administration intraperitoneally, but not into dorsal air-pouches, of ovalbumin-sensitized mice exhibiting blood eosinophilia induced a threefold increase in eosinophil (E phi s) infiltration. Transfer of 1 x 10(6) mixed peritoneal cavity cells (PCC), containing 3.5 to 4.5 x 10(4) mast cells (MC), from donor mice to air-pouches of sensitized (but not unsensitized) recipient mice, established an E phi infiltration to eotaxin (vehicle, 0.86 +/- 0.27 x 10(6); eotaxin, 1.63 +/- 0.16 x 10(6) E phi s/air-pouch). Neutrophil numbers were also increased. When MC-depleted (-93%) PCC were injected into air-pouches of recipient animals, E phi infiltration was not supported (-52%). Injection of macrophage-depleted (-99%) PCC into air-pouches elicited a full E phi response to eotaxin but not neutrophil infiltration (-81%). Systemic dexamethasone treatment of recipient mice reduced E phi accumulation; treatment of donor mice only reduced neutrophil accumulation. Our study points to a crucial role for MC in E phi recruitment by eotaxin.

Partially Differentiated Ex Vivo Expanded Cells Accelerate Hematologic Recovery in Myeloablated Mice Transplanted With Highly Enriched Long-Term Repopulating Stem Cells

The ability of an infusion of ex vivo expanded hematopoietic cells to ameliorate cytopenia following transplantation of hematopoietic stem cells (HSCs) is controversial. To address this issue, we measured the recovery of circulating leukocytes, erythrocytes, and platelets in lethally irradiated mice transplanted with 10(3) enriched HSCs, with or without their expanded equivalent (EE) generated after 7 days of culture in interleukin-3 (IL-3), IL-6, granulocyte colony-stimulating factor and Steel Factor. Two HSC populations differing in their content of short-term repopulating progenitors were evaluated. Thy-1loLIN-Sca-1+ (TLS) bone marrow (BM) is enriched in colony-forming cells (CFCs), day 8 and day 12 spleen colony-forming units (CFU-S) (435 +/- 19, 170 +/- 30, and 740 +/- 70 per 10(3) cells, respectively), and stem cells with competitive long-term repopulating potential (> or = 1 per 43 cells). Thy-1loSca-1+H-2Khl cells (TSHFU) isolated from BM 1 day after treatment of donor mice with 5-fluorouracil (5-FU) are also highly enriched in competitive repopulating units (CRU, > or = 1 per 55 cells), but are depleted of CFCs, day 8 and day 12 CFU-S (171 +/- 8, 0 and 15 +/- 4 per 10(3) cells, respectively). Recipients of 10(3) TLS cells transiently recovered leukocytes to > or = 2,000/microL in 12 days, but sustained engraftment required 25 days. Platelets recovered to > or = 200,000/microL in 15 days, and erythrocytes never decreased below 50% of normal. Mice transplanted with 10(3) TSHFU cells recovered leukocytes in 15 days, and platelets and erythrocytes in 18 days. Recipients of unseparated normal or 5-FU-treated BM cells (containing 10(3) TLS or TSHFU cells) recovered safe levels of blood cells in 9 to 12 days, suggesting that unseparated marrow contains early engrafting cells that were depleted by sorting. Upon ex vivo expansion, total cells, CFCs and day 12 CFU-S were amplified 2,062-,83- and 13-fold, respectively, from TLS cells; and 1,279-, 259- and 708-fold, respectively, from TSHFU cells. Expanded cells could regenerate the majority of lymphocytes and granulocytes in primary (17 weeks) and secondary (26 weeks) hosts and were only moderately impaired compared to fresh HSCs. The EE of TSHFU cells was more potent than that of TLS cells, suggesting that more highly enriched HSCs are more desirable starting populations for this application. When mice were transplanted with 10(3) TSHFU cells and their EE, the duration of thrombocytopenia was shortened from 18 to 12 days, and anemia was abolished. Leukocytes were also elevated on days 9 to 12, although sustained recovery was not accelerated. Anemia was also abrogated in recipients of 10(3) TLS cells and their EE. Early platelet counts were slightly higher than with TLS cells alone, but leukocyte recovery was not improved. These data confirm that TLS cells contribute to early and sustained hematopoiesis, and demonstrate a benefit of ex vivo expanded cells in accelerating engraftment of more primitive TSHFU stem cells depleted of progenitors.

Bone marrow collected 14 days after in vivo administration of granulocyte colony-stimulating factor and stem cell factor to mice has 10-fold more repopulating ability than untreated bone marrow

Abstract We have examined the repopulating ability of bone marrow and peripheral blood cells collected immediately and at intervals after treatment of donor mice with the combination of granulocyte colony-stimulating factor (G-CSF) and stem cell factor (SCF). Using a competitive repopulation assay we showed that the repopulating ability of peripheral blood cells was highest immediately after cytokine treatment and declined to normal levels within 6 weeks of the termination of treatment with G-CSF and SCF. In contrast the repopulating ability of bone marrow cells was low immediately after cytokine treatment and increased to levels that were 10-fold or more greater than marrow from untreated mice by 14 days after termination of treatment with G-CSF and SCF. This high level of repopulating activity declined to normal levels by 6 weeks after termination of treatment with G-CSF and SCF. The high level of repopulating ability was confirmed by injecting cells from G- CSF- and SCF-treated donors into unconditioned recipients. Peripheral blood cells collected immediately after treatment with G-CSF and SCF engrafted into unconditioned mice sevenfold better than an equivalent number of bone marrow cells from untreated mice. Likewise, bone marrow cells collected 14 days after treatment of the donor animal with G-CSF and SCF engrafted at 10-fold higher levels than an equivalent number of bone marrow cells from untreated mice. We conclude that the treatment of donor mice with G-CSF and SCF causes a transient increase in the repopulating ability of peripheral blood and later of bone marrow. These observations may have applications to clinical hematopoietic stem cell transplantation.

Bone marrow collected 14 days after in vivo administration of granulocyte colony-stimulating factor and stem cell factor to mice has 10-fold more repopulating ability than untreated bone marrow

We have examined the repopulating ability of bone marrow and peripheral blood cells collected immediately and at intervals after treatment of donor mice with the combination of granulocyte colony-stimulating factor (G-CSF) and stem cell factor (SCF). Using a competitive repopulation assay we showed that the repopulating ability of peripheral blood cells was highest immediately after cytokine treatment and declined to normal levels within 6 weeks of the termination of treatment with G-CSF and SCF. In contrast the repopulating ability of bone marrow cells was low immediately after cytokine treatment and increased to levels that were 10-fold or more greater than marrow from untreated mice by 14 days after termination of treatment with G-CSF and SCF. This high level of repopulating activity declined to normal levels by 6 weeks after termination of treatment with G-CSF and SCF. The high level of repopulating ability was confirmed by injecting cells from G-CSF- and SCF-treated donors into unconditioned recipients. Peripheral blood cells collected immediately after treatment with G-CSF and SCF engrafted into unconditioned mice sevenfold better than an equivalent number of bone marrow cells from untreated mice. Likewise, bone marrow cells collected 14 days after treatment of the donor animal with G-CSF and SCF engrafted at 10-fold higher levels than an equivalent number of bone marrow cells from untreated mice. We conclude that the treatment of donor mice with G-CSF and SCF causes a transient increase in the repopulating ability of peripheral blood and later of bone marrow. These observations may have applications to clinical hematopoietic stem cell transplantation.

Treatment of Donor Mice With an αβ T-Cell Receptor Monoclonal Antibody Induces Prolonged T-Cell Nonresponsiveness and Effectively Prevents Lethal Graft-Versus-Host Disease in Murine Recipients of Major Histocompatibility Complex (MHC)-Matched and MHC-Mismatched Donor Marrow Grafts

The purpose of this study was to determine whether the administration of high doses of an anti-T-cell receptor (TCR) monoclonal antibody (H57-597) to donor animals could induce a state of T-cell nonresponsiveness and prevent the development of graft-versus-host disease (GVHD) in murine recipients of major histocompatibility complex (MHC)-matched (B10.BR[H-2k] --> AKR/J[H-2k]) and mismatched (B10.BR[H-2k] --> DBA/2[H-2d]) marrow grafts. Transplantation of H57-597-treated B10.BR T cells into irradiated AKR or DBA mice resulted in protection from GVHD, which was otherwise lethal in transplanted recipients receiving untreated T cells. The administration of H57-597-treated T cells did not compromise alloengraftment in either strain combination and was found to accelerate donor T-cell reconstitution in recipients of MHC-matched marrow grafts. Optimal protection for GVHD was dependent on the duration of antibody exposure in donor mice. T cells from donor exposed to antibody for only 1 day caused lethal GVHD, whereas exposure for at least 4 days was necessary to abrogate graft-versus-host reactivity. The ability of antibody treatment to protect against the development of GVHD could not be ascribed to the antibody-induced production of Th2 cytokines, the induction of a T- or non-T-suppressor cell population, or the preferential depletion of CD4+ T cells by H57-597. Donor T cells exposed to H57-597 antibody were detectable in recipients for up to 5 weeks after transplantation, indicating that these cells were not eliminated in the host immediately after bone marrow transplantation and contributed to enhanced donor T-cell reconstitution. Moreover, in B10.BR --> DBA chimeras that did not have any clinical evidence of GVHD, potentially MIs-reactive donor-derived Vbeta6+ T cells were present in the spleens of recipients at comparable numbers to normal mice but appeared functionally nonresponsive in vivo. These data strongly suggested that protection from GVHD was due to the fact that antibody treatment resulted in a state of prolonged T-cell anergy that persisted despite the presence of potential costimulatory signals in the recipient. This observation is of potential clinical significance in that it shows that the prevention of GVHD can be accomplished without posttransplantation immunosuppression or the need for in vitro or in vivo T-cell depletion.

Treatment ofCandida albicansMannan-Specific Downregulatory Cell Populations with Divergent Concentrations of Monophosphoryl Lipid A and Intact Lipopolysaccharidein VitroAbrogates Their Effect on Delayed Hypersensitivity

We have shown previously that splenocytes from mice injected withCandida albicansmannan (MAN) suppress MAN-specific delayed hypersensitivity (DH) when transferred to immunized recipients and that treatment of donor mice with monophosphoryl lipid A (MLA) derived fromSalmonella typhimuriumorSalmonella minnesotashortly before transfer abrogated the downregulatory activity. We now show that treatment of splenocytesin vitroat 4°C with 5 ng/ml MLA or 0.05 ng/mlS. typhimuriumlipopolysaccharide (LPS) for 30 min before transfer also abrogated downregulatory activity. Higher or lower doses of MLA, 5 μg or 5 pg, appeared to increase the suppressor activity slightly (5 μg) or had no effect (5 pg). LPS induced similar effects but the concentrations of LPS required to show the effects were 100-fold less than those of MLA. The effect of MLA appeared to be on cell(s) in the transfer population involved in MAN-specific DH, in that spleen cells from normal mice treated with MLA prior to transfer had no effect on DH. Finally, the population of MLA-responsive cells mediating downregulation could not be concentrated on MLA-coated plates, suggesting that the MAN-specific downregulatory cell(s) either did not bind to MLA or did not bind to MLA with sufficient avidity to remain attached during the washing procedures. The feasibility of abrogating suppression by treatment of lymphoid cellsin vitrowill allow a more detailed analysis of the mechanism of abrogation.

Antibody to the murine type 3 complement receptor inhibits T lymphocyte-dependent recruitment of myelomonocytic cells in vivo.

We have used the delayed-type hypersensitivity (DTH) response to SRBC or tuberculin to examine the role of the murine type 3 complement receptor in T lymphocyte-dependent inflammatory recruitment. Intravenous injection of 5C6, a CR3-specific rat mAb known to impair myelomonocytic adhesion, divided the DTH to SRBC in actively immunized mice into two phases. The early phase, which lasted 24 h, was characterized by maximal oedema and maximal inflammatory recruitment and was 5C6 inhibitable. The later phase was 5C6 resistant and reached a peak 48 h after antigenic challenge and was superimposable on the declining peak seen in control mice. Passive transfer of reactive T cells mixed with antigen was used to examine the myelomonocytic effector arm of the DTH alone. Both passive transfer of cutaneous DTH to SRBC and passive transfer of the largely monocytic T cell-dependent recruitment to tuberculin in the peritoneal cavity were completely abolished by systemic 5C6 treatment. Injection of 5C6-treated donor leukocytes at the site of passive transfer had no effect. Treatment of donor mice with 5C6 at the time of active immunization did not alter their ability to provide reactive T cells for passive transfer. The myelomonocyte-restricted rat mAb 7/4 and the rapidly cleared F(ab')2 fragment of 5C6 showed no inhibition of the DTH. In all cases, inhibition of footpad swelling correlated with histological evidence of inhibition of myelomonocytic cell recruitment. Peritoneal cell counts after local DTH to tuberculin showed complete inhibition of monocyte recruitment. We conclude that CR3 plays a quantitatively important role in T cell-dependent inflammatory recruitment. This is absolute in passive transfer experiments, but only partial after active immunization. Leukocyte CR3 plays a common role in both immunologically specific and nonspecific inflammatory recruitment and provides a target that could possibly be manipulated to therapeutic advantage.

Role of interleukin 2 (IL 2) and hemopoietin-1 (H-1) in the generation of mouse natural killer (NK) cells from primitive bone marrow precursors.

The development of natural killer (NK) cells from bone marrow (BM) precursors was studied. Recombinant interleukin 2 (IL 2) was able to induce the in vitro development of NK cells when added to cultures of mouse BM cells. Treatment of donor mice with 5-fluorouracil (150 mg/kg i.v.), which eliminates more differentiated cells but spares less differentiated cells, appears to augment NK cell development. The "NK stem cell" was found to be asialo GM1-, Thy-1+, Lyt-2-, and Lyt-1-. The cells generated in vitro had a typical phenotype of NK cells, being asialo GM1+, Lyt-5+, Thy-1+, Lyt-2-, and Lyt-1-. These effector cells also had specificity characteristics of NK cells lysing the NK-susceptible YAC-1 and K562 targets, but not the NK-resistant EL/4 or allogeneic and syngeneic blasts. Hemopoietin-1 (H-1), a factor which acts on very primitive multipotent BM cells, was able to cooperate with IL 2, increasing the development of NK cells. In contrast, other factors such as interleukin 3 or colony-stimulating factor did not cause induction of NK activity when added to cultures of BM cells, indicating that this effect, i.e., induction of NK cell development, is peculiar to IL 2. These results indicate that IL 2 can act as a differentiation as well as growth factor for NK cells, and that H-1 can promote the development of functional activity in a lymphocyte subpopulation as well as affect the differentiation of myelomonocytic and other cell lineages. This experimental system appears quite useful for characterization of BM precursors for NK cells, and should help to better understand the relationship of the NK cell lineage to the T cell or other lineages.

Cellular interactions in the adoptive transfer of contact sensitivity: characterization of an antigen-nonspecific Vicia villosa-adherent T cell needed for adoptive transfer into naive recipients.

The adoptive transfer of delayed-type hypersensitivity (DTH) into naive recipients requires the interaction of two functionally distinct Ly-1+ T cells: and I-J- cell effector cell for DTH which transfers antigen-specific DTH only into animals whose suppressive mechanisms have been compromised, and and I-J+ cell which alone never transfers DTH but allows the transfer of DTH by the I-J- DTH effector cell into naive animals. We investigated the phenotypic and functional characteristics of the cell which "protects" the I-J- DTH effector cell from host suppressive mechanisms and allows the transfer of DTH into naive recipients. This cell was found to express the cell surface phenotype Lyt-1+,2-, L3T4+, and I-J+, and, in contrast to the I-J- DTH effector cell, was found to be adherent to the lectin Vicia villosa (VV). These cells routinely are found in the spleens of both immune or naive animals, and regardless of their origin are antigen-nonspecific in their functional activity in that they complement VV-nonadherent cells to transfer DTH responses of both TNP and oxazolone-primed cells. Treatment of recipient mice with cyclophosphamide (to remove host suppressor mechanisms) or Bordetella pertussis vaccine (which stimulates splenic T cells to circulate) abrogates the need for these cells in the transfer population, whereas treatment of donor mice with B. pertussis functionally depletes these cells from splenic T cell populations. Therefore, it appears that in the adoptive transfer of DTH responses, the antigen-specific I-J- VV-nonadherent cell requires an I-J+ VV-adherent cell in the circulation to overcome host suppressive mechanisms. The importance of these I-J+ cells in DTH responses is discussed.

The effects of polyinosinic: Polycytidylic acid (pl:C) on the GVH reaction: Immunopathological observations

The effects of polyinosinic: polycytidylic acid (pl:C) on the graft-versus-host (GVH) reaction were studied. The drug pl:C rapidly and markedly induces interferon and augments natural killer (NK) cell activity. GVH reactions were induced by injecting parental lymphoid cells intravenously into F 1 hybrid mice. The development of a GVH reaction was monitored by measuring the plaque-forming cell (PFC) response to sheep red blood cells (SRBC) and by histological examination. When 30 × 10 6 B6 lymphoid cells were injected into B6AF 1 mice, the recipients developed profound immunosuppression by 10–12 days post-GVH induction. In addition, pathological changes indicative of GVH reactions were seen in the spleen, lymph nodes, thymus, liver, lung, pancreas, and salivary gland of these mice. However, the treatment of B6AF 1 recipients with pl:C prior to parental cell transfer markedly reduced the degree of suppression of the immune response, as measured by the PFC response to SRBC. Also, such mice failed to demonstrate the histological lesions of GVH disease. Treatment of donor mice with pl:C had no effect in preventing either GVH-induced immunosuppression or pathological changes. This study suggests that a pl:C-induced mechanism, possibly involving NK cells, is capable of regulating the GVH reaction.

Studies of the thymus in mice bearing the Lewis Lung carcinoma. III. Possible mechanisms of tumor-induced thymic atrophy

Adrenalectomy prevents thymic atrophy but not splenomegaly in mice implanted with Lewis Lung carcinoma cells. Surprisingly, the presence of the tumor does not lead to increased levels of corticosterone, which argues against an exclusive role of stress in the tumor-induced involution of the thymus. Interestingly, serum from tumor-bearing hosts in vitro displays strong cytolytical activity against normal syngeneic thymocytes. This thymocytotoxicity depends upon the stage of tumor development, i.e., the size of the local tumor, and is concomitant with the severe thymic atrophy. Treatment of donor mice with zinc chloride or excision of the local tumor, both of which have been shown to prevent this involution of the thymus, also abolishes the cytotoxic effect of the serum. The active component of the serum is a nonimmunoglobulin fraction of molecular weight >25,000 Da. The possible mechanisms of tumor-dependent thymic atrophy as well as the in vivo relevance of this serum-mediated thymocytotoxicity are discussed.

Selective action of alkylating agents against cells participating in suppression of antibody responses

The effect of several alkylating agents on the induction and expression of specific suppressor cell activity induced by supraoptimal immunization (SOI) with (4×10 9) SRBC was studied. Splenocytes taken 8–28 days after SOI and transferred to normal syngeneic recipients together with optimal dose of (3×10 8) SRBC caused 70–90% suppression. By contrast splenocytes harvested 2 days after SOI did not exert a significant suppressive effect. Treatment of donor mice with 30 mg/kg BCNU, CCNU, or MeCCNU, 8 mg/kg Melphalan or as much as 200 mg/kg Cy 2 days before SOI uniformly had no effect on the subsequent development of suppressor cells. By contrast, different drugs had diverse effects when injected after SOI: both BCNU and Cy injected 2 days post SOI alleviated suppression, whereas CCNU, MeCCNU and Melphalan injected 2 days post SOI were without effect. Another diversity between BCNU and Cy was noticed when the administration of drugs was delayed further. While Cy alleviated suppression 12 days post SOI, BCNU was ineffective at this time. Dose response and time course studies revealed that the effect of Cy was most severe when injected 2 days post SOI and gradually diminished with the passage of time after SOI. These results have been discussed in the light of the current concepts of multiple cell participation in the induction and expression of suppressor cell function.

In vivo or in vitro modulating effects of vincristine on the generation of allogeneic cytotoxic lymphocytes in vitro.

Cytotoxic lymphocytes carrying the H-2(i5) haplotype were generated in vitro against alloantigens specified by the IC-S-G-D regions of H-2. Responder lymphocytes were collected from donors B10.A(5R) non-treated or treated with 3 or 0.6 mg/Kg, ip of Vincristinee (VCR) and incubated with irradiated B10 spleen cells. High doses of VCR produced depression of the cytotoxic response (CR), whereas 0.6 mg/Kg of the drug produced substantial increase of the response. Further experiments were conducted adding VCR in vitro to the tissue culture during the generation of cytotoxic lymphocytes. Depression of CR occurred following treatment with high concentrations of VCR. On the other hand, marked increase of CR was found using responder lymphocytes treated in vitro with low concentrations (i.e. 0.016 - 0.0032 microgram/ml) of the drug. The mechanism of VCR-induced enhancement of CR has not been elucidated. However, the present data show the possibility of increasing host immune responses by treatment of donor mice or of responder lymphocytes with VCR at selected doses or concentrations.

Requirement of parental T lymphocytes for the in vitro induction of F1 hybrid anti‐parent cytotoxicity

AbstractPrimary F1 anti‐parent cell‐mediated lympholysis (CML) is a specific response generated in mixed cultures by F1 T lymphocytes with the aid of macrophage‐like cells of responder and stimulator type. A model was described in which the induction of this response required T lymphocytes among stimulators, as judged by the lack of induction following T cell depletion of parental cells by (a) separation on nylon wool columns, (b) treatment with anti‐Thy‐1 antiserum and complement, (c) treatment of donor mice with rabbit anti‐mouse thymocyte serum, or (d) congenital absence of the thymus in nu/nu donors. The parental T lymphocytes required were relatively mature, as indicated by resistance to cortisone, persistence after adult thymectomy, and sensitivity to xenogeneic anti‐thymocyte antiserum. T‐deprived cell populations retained the ability to induce allogeneic CML.Parental lymphoma cells, spleen cells stimulated by mitogens for T or B lymphocytes, normal cells of central and peripheral lymphoid organs, and peritoneal exudate cells, all served to a varying extent as targets for anti‐parent cytotoxic effectors (CTL), but there was no close correlation with their ability to serve as stimulators. Spleen or peritoneal exudate cells of athymic mice were capable of serving as “antigenic” targets for specific F1 anti‐parent CTL, and yet failed to induced the response. Thus, the role of parental T cells in the induction of F1 anti‐parent cytotoxicity was not that of bearing the “antigen”. It was concluded that irradiated parental T lymphocytes provided back stimulation to F1 hybrid responders, either directly or indirectly via the activation of macrophage‐like cells. Under certain circumstances, however, the requirement for parental T cells can be circumvented.

Soluble factors produced by normal or tumor cells, inhibiting the uptake of 5-iodo-2′-deoxy-uridine by cancer cells in vitro

The uptake of 125 I- 5-iodo-2′-deoxy-uridine ( 125 IUdR) by lymphoma cells is inhibited in lethally irradiated hosts incompatible for Hh (hemopoietic-histocompatibility) loci. In the course of attempts to reproduce this phenomenon in vitro, a soluble factor (uptake inhibiting factor, or UIF) was found in the supernatant of cultured ( 16 hr, 37°C) spleen cells. UIF was produced by non-stimulated splenocytes of various strains of conventional and athymic (nude) mice and rats and by various tumor lines as well. UIF was found to be non-strain, non-species and non-tissue specific, since it was capable of inhibiting the 125 IUdR uptake of mouse, hamster and human tumor cells of various tissue origins. Lethal irradiation, silica or IDF (immunodepressive factor) treatment of donor mice, or irradiation or treatment with actinomycin D of spleen cells in vitro, did not prevent UIF production by such cells. On the other hand 5-fluoro- 2′-deoxy-uridine (FUdR) reduced UIF production by 50%. UIF was found to be relatively resistant to heat and proteolytic enzymes in vitro. In addition UIF was capable of cytostatic or cytotoxic effects on tumor cells in tissue culture. UIF-like activity was found also in the plasma of tumor bearing mice. The biological role of UIF seems to be uncertain. Nevertheless it could interfere markedly with in vitro assays involving cell proliferation monitored by DNA-precursor uptake.