Natural cytotoxicity against mouse hepatitis virus-infected target cells. I. Correlation of cytotoxicity with virus binding to leukocytes

Spleen cells from uninfected control mice selectively lysed BALB/c 3T3 fibroblasts infected with mouse hepatitis virus (MHV), a murine coronavirus. Lysis of infected cells occurred within 3 hr, and histocompatibility between effector and target cells was not required. This natural, cell-mediated, virus-associated cytotoxicity differed from NK cell- and T cell-mediated lysis. Spleen cells from animals infected with MHV were enriched in NK activity and were more cytotoxic to YAC-1 target cells, but did not show enhanced cytotoxicity for MHV-infected target cells. Spleen cells from beige mice, which are deficient in NK cell activity, were able to lyse MHV-infected target cells, as were spleen cells from nude mice, which are deficient in T cell activity. Lysis of MHV-infected target cells could be mediated by cells from the spleen and, to a lesser extent, by cells from the bone marrow, but not by resident peritoneal cells or thymocytes. We suggest the term "virus killer (VK) activity" for this phenomenon. VK activity of splenocytes from different mouse strains correlated with the ability of the splenocytes to bind purified radiolabeled MHV virions. MHV virions caused agglutination of spleen leukocytes from susceptible mouse strains, indicating that leukocyte agglutination or adsorption may provide a useful assay for coronaviruses such as MHV which lack hemagglutinating activity. SJL mouse splenocytes did not bind MHV and did not lyse infected targets. MHV bound relatively well to splenocytes of other mouse strains, but poorly to thymocytes and erythrocytes. Binding of MHV to leukocytes was not influenced by 6 mM EDTA or EGTA, indicating a lack of requirement for Mg++ or Ca++. VK activity was also resistant to EDTA and EGTA, in contrast to NK activity, which was sensitive to those chelating agents. VK activity was also unaffected by actinomycin D, cycloheximide, or puromycin, indicating that new protein synthesis was not required for lysis. Antibody to interferon-alpha/beta did not block lysis, nor was there substantially enhanced lysis mediated by leukocytes from mice infected with virus and thus exposed to high levels of interferon. VK activity was blocked by antibody directed against the peplomeric glycoprotein E2 of MHV. VK activity required infected target cells, because cells with adsorbed MHV virions were not lysed by splenocytes.(ABSTRACT TRUNCATED AT 400 WORDS)     

Genetically determined resistance to mouse hepatitis virus 3 is expressed in hematopoietic donor cells in radiation chimeras

Differences in mouse hepatitis virus 3 (MHV3) sensitivity among mouse strains are mainly determined by H-2-related and -nonrelated genetic factors. Reciprocal chimerism was therefore established between two H-2a compatible pairs of strains that differ widely in their susceptibility to MHV3: a) A/J and B10.A, respectively resistant and highly susceptible; b) A/J and A/Sn, respectively resistant and semisusceptible. Chimeric mice were challenged with 100 LD50 of MHV3, 30 or 90 days after X-irradiation (900 R) and bone marrow reconstitution. Results showed that sensitivity of recipients was similar either to that of the recipient strain or to that of the donor strain when chimeric mice were tested 30 or 90 days, respectively, after reconstitution. In addition, no paralysis occurred in surviving animals. These data indicate, therefore, that resistance or susceptibility to MHV3 is expressed intrinsically in some population(s) of hematopoietic-derived cells, which is radioresistant and has a life span of more than 30 days and less than 90 days. Additional experiments showed that X-irradiated A/J recipients reconstituted with A/J bone-marrow cells were protected against MHV3 challenge with spleen cells, with a mixture of spleen cell populations or of adherent spleen cells and thymocytes originating from A/J donors. Transfer of protection to recipients by using similar cell populations provided by semisusceptible A/Sn donors required the administration of five times more cells. Results suggest that two complementary mechanisms are required to confer resistance to MHV3: a) a gene(s) for resistance that may operate at the level of macrophages, and b) cells capable of mounting an efficient immune response. The reduced efficiency of A/Sn spleen cells suggests that semisusceptibility to MHV3 may be related to partial quantitative or functional immune defect.     

Neonatal susceptibility to MHV3 infection in mice. I. Transfer of resistance.

Up to 3 weeks of age, mice of the resistant A/J strain are fully susceptible to mouse hepatitis virus type 3 infection (MHV3). Immune deficiency, however, resulting from neonatal thymectomy or long term ALS administration led A/J animals to remain susceptible when tested at adult age. Whole spleen cells transferred from normal adult A/J donor mice protected suckling syngeneic recipients from i.p. infection with MHV3. Such a protective capacity of spleen cells was abolished after treatment with anti-theta serum and complement. Spleen cell separation by means of adherence to plastic also showed that neither the nonadherent nor the adherent populations injected separately were able to confer resistance to young mice challenged with the virus. Protection was not achieved with peritoneal cells originating from adult syngeneic animals. Transfer of resistance to MHV3 was obtained, however, when peritoneal cells were associated with adherent spleen cells. This study indicated that two types of mature cells, at least, were required for transferring MHV3 resistance into newborn mice of the A/J strain: T lymphocytes and an adherent spleen cell population.     

Efficient natural defense mechanisms against Listeria monocytogenes in T and B cell-deficient allogeneic bone marrow radiation chimeras. Preactivated macrophages are the main effector cells in an early phase after bone marrow transfer

Radiation chimeras in the early phase after bone marrow transplantation are a good model to study the efficiency of the body's nonspecific defense system represented by macrophages (M phi), polymorphonuclear cells (PMN), and NK cells. These cell types are present in large numbers in spleen and liver at that time, whereas the specific immune system represented by T and B cells is functionally deficient. We previously reported enhanced activities in vitro of M phi (and PMN) from recipient animals in an early phase after allogeneic bone marrow transfer. We here demonstrate that these activities result in enhanced spontaneous resistance against Listeria monocytogenes in vivo: CFU of L. monocytogenes in spleen and liver 48 h after infection were about 1 or 2 to 4 log steps less than in untreated control mice of donor or host haplotype. This enhanced resistance decreased over the 4-mo period after marrow transfer. Preactivated M phi were identified as the most important effector cells. Isolated from spleen and peritoneal cavity, they performed enhanced killing of phagocytosed Listeria. Such preactivated M phi occurred in recipient animals after transfer of allogeneic but not of syngeneic bone marrow. The precise mechanism of M phi activation in the allogeneic radiation chimera in the complete absence of any detectable T cell function is not clear at present. However, these preactivated M phi display an important protective effect against L. monocytogenes: chimeras could eliminate Listeria without acquisition of positive delayed-type sensitivity when infected with 10(3) bacteria. An inoculum of 5 . 10(3) L. monocytogenes resulted either in prolonged survival compared with normal mice of the recipient haplotype or in definitive survival accompanied by a positive delayed-type sensitivity. We concluded that enhanced nonspecific immune functions can in part compensate for the defective specific immune system after bone marrow transfer.     

Genetically determined resistance to foreign bone marrow transplantation in mice: characterization of the effector cells

Mice of most strains show a genetically determined ability to reject a variety of foreign marrow grafts even after lethal irradiation. The phenomenon is both host strain and donor marrow graft-dependent. To characterize the effector cell responsible for graft rejection, attempts were made to 1) determine to what morphologic subclass it belongs; 2) determine its life span; and 3) establish whether genetically different host environments influence the functioning of the effector cell. Mice of the 129/J strain (normally nonresistant), C57BL/6 strain (made non-resistant), and the homozygous mutants of C57BL/6, i.e., C57BL/6 (bg/bg), were recipients of C57BL/6 marrow or spleen cells. After lethal irradiation, hosts were given marrow or spleen cells from normal, strongly resistant C57BL/6 donors pretreated with a) 950 R whole body irradiation or b) twice daily injections for 4 days of the cell cycle toxic drug hydroxyurea followed by 950 R. In other cases, hosts were recipients of the lymphoid cell-rich fraction of marrow from irradiated C57BL/6 donors or adherent cells taken from cultures of marrow cells of unirradiated C57BL/6 donors. Three hours after receiving C57BL/6 marrow or spleen cells, irradiated hosts were given allogeneic DBA/2 marrow (always strongly rejected by C57BL/6 mice and always accepted by 129/J strain mice). Seven days later, host spleens were removed and the numbers of microscopic colonies were counted from subserial sections. The results demonstrate that 1) mice either normally or rendered nonresistant to a marrow allograft can be made to develop resistance by the administration of either whole spleen cells or marrow lymphoid cells from lethally irradiated strongly resistant donors; 2) adherent cells from cultures of marrow from strongly resistant mice are ineffective in conferring resistance; 3) the cell effective in conferring resistance has a life span greater than 4 but less than 7 days; and 4) the effector cell can function in genetically different environments of nonresistant strains.     

Specific inhibition of hybrid resistance in F1 hybrid mice pretreated with parent strain spleen cells. I. Induction of a nylon-adherent, Thy-1+Lyt-1+2- suppressor cell

Hybrid resistance, which is observed in certain strain combinations when parent-strain bone marrow cells are grafted into lethally irradiated F1 hybrids, can be specifically overcome by the i.v. injection, 1 wk before the graft, of spleen cells syngeneic with the bone marrow graft. This phenomenon is due to a suppressor mechanism, induced in the spleen of the F1 hybrid by the injection of parent-strain spleen cells and mediated by a nylon-adherent Thy-1+Lyt-1+2- cell population of hybrid origin, because hybrid resistance can be inhibited by the transfer into a normal B6D2F1 of nylon-adherent Thy-1+Lyt-1+2- spleen cells from B6D2F1 mice pretreated with B6 spleen cells 1 wk earlier (B6-pretreated B6D2F1); spleen cells from B6-pretreated B6D2F1 mice not depleted of their nylon-adherent subpopulation cannot restore hybrid resistance when they are injected into a B6D2F1 rendered nonresistant by split-dose irradiation; and spleen cells from normal B6D2F1 mice cannot restore hybrid resistance when they are injected into B6-pretreated B6D2F1 hybrids. The suppressor cells specifically inhibit resistance against bone marrow cells syngeneic with the spleen cells used for pretreatment, because transfer of nylon-adherent B6-pretreated B6D2F1 spleen cells into a normal B6D2F1 does not enhance syngeneic B6D2F1 or parent-strain D2 bone marrow growth, and when injected into normal B6D2F1 hybrids, nylon-adherent spleen cells from B6D2F1 mice pretreated with D2 spleen cells 1 wk earlier (D2-pretreated B6D2F1) are not able to transfer the inhibition of hybrid resistance against B6 bone marrow cells. Moreover, the activity of the suppressor cells depends on the genetic environment of the hybrid host mice, because nylon-adherent B6-pretreated B6D2F1 spleen cells injected into normal B6C3F1 hybrids do not transfer an inhibition of hybrid resistance, and when injected into B6C3F1 hosts previously rendered nonresistant by split-dose irradiation, spleen cells from B6-pretreated B6D2F1 mice can, in contrast, transfer hybrid resistance.     

The effects of polyinosinic:polycytidylic acid (pI:C) on the graft-vs-host (GVH) reaction. II. Increased NK-mediated rejection on C57BL/6 lymphocytes by (C57BL/6 X A)F1 mice

We previously demonstrated that treatment of (C57BL/6 X A)F1 (F1) recipient mice with polyinosinic:polycytidylic acid (pI:C) before injection with 30 X 10(6) C57BL/6 (B6) lymphocytes prevents both the immunosuppression and pathologic lesions typical of graft-vs-host (GVH) reactions. We now report the further characterization of this phenomenon. Donor spleen and lymph node cells were labeled with fluorescein in vitro and injected into pI:C-treated or untreated mice. Two days later, recipient splenocytes were analyzed for the presence of fluorescein-labeled donor cells by flow microfluorometry. Treatment of F1 mice with pI:C resulted in a sharp reduction in the recovery of labeled B6 but not A strain parental cells. Treatment with pI:C had no effect when syngeneic recipients were used, or when F1 cells were injected into A, B6, or F1 recipients. These results suggest that pI:C treatment induces rejection of B6 but not A or F1 lymphocytes by F1 hybrid mice at least as early as 2 days after donor cell transfer. As F1 cells are not rejected by either parent, rejection does not seem to be directed against classical alloantigens. These observations are compatible with the previously described model of hybrid resistance (HR) against bone marrow grafts. The rapidity of rejection strongly suggested that natural cytotoxic mechanisms were involved, thus, natural killer (NK) cell and macrophage (M phi) cytotoxic activities were tested throughout the time when the parental cell graft was being rejected. Over this period, pI:C treatment increased cytotoxic activity against the NK-sensitive target cell line YAC-1 but had no effect on spontaneous M phi tumoricidal activity against the L5178Y and MDAY-D2 cell lines. The results suggest that NK cells, but not M phi, may be involved in the elimination of B6 parental cells by the pI:C-treated F1 mice. NK cells have been demonstrated to be radioresistant; thus, as a test of our hypothesis, we examined the effects of irradiation on the capacity of pI:C treated F1 mice to reject B6 lymphocytes. The results show that this capacity was not blocked by 750 cGy, a dose of radiation that abrogates most T and B cell functions. Furthermore, rejection of parental cells could be prevented by treatment of recipient F1 mice with antibodies to asialo GM1, a treatment that suppresses NK activity. These data demonstrate that pI:C-mediated protection from GVH-induced changes is due to increased rejection of grafted B6 parental cells by F1 NK cells, a phenomenon very similar, if not identical, to HR to bone marrow grafts.     

In vitro splenic T cell responses of diverse mouse genotypes after oronasal exposure to mouse hepatitis virus, strain JHM.

Mortality rates among BALB/cByJ, A/JCr, C3H/HeSnJ, and C57BL/6NCr mice inoculated oronasally with mouse hepatitis virus (MHV) strain JHM, ranged from 25 to 67%. Spleen cells harvested from the first three genotypes at 5 days postinoculation proliferated poorly in response to concanavalin A stimulation and produced significantly less interleukin (IL) 2 than cells from uninfected control mice. The function of spleen cells harvested at 14 days postinoculation varied and was host genotype-dependent. Despite clinical signs among some infected C57BL/6NCr mice, spleen cell function was relatively unaffected. C57BL/10ScNCr, B10.A, and SJL/JCr mice remained clinically normal after MHV inoculation. Proliferation and IL2 production by cells from inoculated C57BL/10ScNCr and B10.A mice were similar to responses of their respective controls. In contrast, cells from inoculated SJL/JCr mice were hyper-responsive and produced peak levels of IL2 earlier than control cells. Among the seven genotypes tested, only BALB/cByJ and C3H/HeSnJ spleen cells produced detectable IL4 after primary stimulation with concanavalin A or after priming and restimulation. Primary IL4 production by cells from these two genotypes was significantly reduced if donors were inoculated with MHV 5 days prior to spleen harvest. IL4 production by cells from acutely infected BALB/cByJ mice was considerably enhanced by priming and restimulation.     

Hybrid resistance to parental mast cell precursors in the skin of (WB X C57BL/6)F1-W/Wv mice

When bone marrow cells of (WB X C57BL/6)F1-+/+ (WBB6F1-+/+) and WB-+/+ (WB) mice were directly injected into the skin of genetically mast cell-deficient WBB6F1-W/Wv mice, mast cell clusters appeared at the injection sites. However, the number of WB bone marrow cells necessary for appearance of mast cell clusters was significantly larger than when bone marrow cells of WBB6F1-+/+ mice were used. When WB bone marrow cells were mixed either with WB thymus cells or with silica particles, the proportion of injection sites at which mast cell clusters appeared increased to the level that was observed after the injection of the same number of WBB6F1-+/+ bone marrow cells. When suckling WBB6F1-W/Wv mice of less than or equal to 18 days of age were used as recipients, bone marrow cells of WBB6F1-+/+ and WB mice produced mast cell clusters with a comparable efficiency. Both syngeneic thymus cells and silica particles are known to abrogate the hybrid resistance that is observed in the spleen against parental hematopoietic stem cells. The hybrid resistance in the spleen is not detectable in suckling mice, either. Thus, the poor growth of mast cell precursors in the skin and the poor growth of hematopoietic stem cells in the spleen seem to be regulated by the same mechanism.     

20alpha-hydroxysteroid dehydrogenase: a T lymphocyte-associated enzyme.

20alpha-Hydroxysteroid dehydrogenase (20alpha-SDH), an enzyme which reduces progesterone to 20alpha-dihydroprogesterone, was found to be associated with T lymphocytes. 20alphaSDH activity was present in spleen cells bearing theta antigen, spleen cells nonadherent to nylon wool (T lymphocyte-enriched population), and in thymocytes. Almost no enzymatic activity was found in bone marrow cells from normal mice and in spleen cells from neonatally thymectomized or athymic nude mice. T cell mitogens (PHA and Con A), but not the B cell mitogen LPS, induced high levels of enzymatic activity 48 hr after addition to spleen cell cultures. The level of 20alphaSDH activity in lymphocytes was age dependent. At the age of 4 weeks 20alphaSDH activity in thymocytes, spleen cells, and lymph node lymphocytes was 3 to 5 times higher than at 8 and 16 weeks. Progesterone (5.0 X 10(-7) M) was found to inhibit thymocyte proliferation after exposure to mitogens, but not 20alpha-dihydroprogesterone (10(-6) M). 20alpha SDH may protect the embryonic thymocytes against high concentrations of progesterone.     

Heterogeneity within the hematopoietic stem cell compartment: evidence for a marrow-seeding stem cell distinct from CFU-s

Using a chromosome marker within a syngeneic system, we investigated the seeding characteristics of murine hematopoietic stem cells after transplantation to irradiated hosts. The chromosome-marked test cells were allowed to compete with normal marrow cells in repopulating the spleen and marrow of irradiated mice. Although the seeding behavior of normal marrow could be predicted from the number of colony-forming units-spleen (CFU-s) transplanted, the marrow seeding of melphalan-treated marrow was 7-fold greater than expected. Repopulation of marrow by spleen cells was less effective than expected from the CFU-s content, while the reverse was true after repopulation by fetal liver cells. These differences were emphasized after treatment of cell donors with melphalan. The results were due primarily to differences in the lodging properties of the transplanted cells, those seeding in the marrow were less sensitive to melphalan than CFU-s. In some instances marrow-repopulating ability could be separated from peak CFU-s activity on a density gradient, suggesting a marrow-repopulating cell exists that is distinct from CFU-s.     

Evidence for extrathymic development of TNK cells. NK1+ CD3+ cells responsible for acute marrow graft rejection are present in thymus-deficient mice.

The predominant mechanism responsible for acute specific rejection of allogeneic and parental bone marrow by irradiated mice is due to a cell (TNK) that expresses the NK cell surface markers NK1 and ASGM1 as well as TCR. Here we analyze the question as to whether TNK cells require a functional thymus for their development. Using adoptive cell transfer assays, evidence is presented that, as is the case in normal mice, NK1+ CD3+ effector cells are responsible for rejection in thymus-deficient nude mice and that the specificity of rejection is indistinguishable from that of normal mice. To reveal the presence of TNK cells in the spleen of nude mice, double staining for NK1 and CD3 followed by FACS analysis was done. It is shown that NK1+ CD3+ cells are present in the spleens of nude but not euthymic mice, suggesting that the lack of a functional thymus stimulates either Ag expression or the number of TNK cells. In support of this finding, the treatment of irradiated marrow reconstituted mice with cyclosporin A leads to the appearance of TNK cells in the spleen. The relative efficiency of spleen cells from nude and cyclosporin A-treated mice to transfer resistance in adoptive cell transfers was assessed and found to be higher than that of normal spleen, consistent with the higher frequency of these cells in thymus-defective mice. The fate of NK1+ CD3+ cells subsequent to stimulation with an allogeneic marrow graft indicates that these cells proliferate in nude mice without gaining cytolytic activity. In euthymic mice, however, NK1+ CD3+ cells appear transiently but disappear in favor of CD4+ and CD8+ cells that proliferate in response to an allogeneic marrow graft. The CD8+ cells express cytolytic activity with specificity similar to that of the acute rejection mechanism, consistent with the suggestion that TNK cells differentiate into CD8+ killer cells. The reason why TNK cells in nude mice fail to differentiate into CD8+ CTL is explained by the lack of Th cells.     

The induction of hapten-specific immunological tolerance and immunity in B lymphocytes. II. Temporal and dose response relationships of tolerance induction in B lymphocytes of various differentiation states.

The induction of TNP-specific B lymphocyte tolerance by TNBS in sources representing various differentiation states was studied in an adoptive cell transfer system. An adoptive assay was utilized in which the delay of immunization with the T-independent antigen TNP-LPS resulted in an enhanced PFC response. TNBS induced tolerance in spleen cells which was independent of T cell activity, was dose dependent, and could be adoptively transferred. While bone marrow and spleen cells were susceptible to tolerogenesis after cell transfer, TNBS treatment of the donor induced unresponsiveness in splenocytes but not marrow cells. The tolerance dose response relationship and the effect of the temporal relationship between cell transfer and tolerogenesis were studied in B lymphocytes from various sources. Adult spleen cells were resistant to tolerance induction late in the adoptive response, and the tolerance induced by TNBS administration 1 hr after cell transfer was dose dependent. Athymic nude spleen cells and adult bone marrow cells displayed similar characteristics while fetal liver cells were somewhat more susceptible to the induction of unresponsiveness. Neonatal spleen cells were rendered tolerant at much lower doses and at any stage of the adoptive response. The hierarchy obtained in these studies in the order of decreasing resistance to tolerance induction is: adult normal and athymic nude spleen and adult bone marrow, fetal liver, and neonatal spleen. This variation in tolerogenesis appears to be due to the maturity of the cell types which may reflect differences in B lymphocyte sub-populations.     

Studies of the T-lymphocytes resident in the spleens of thymectomized, irradiated, bone marrow-reconstituted (TXB) mice.

The T-lymphocytes resident in the spleens of thymectomized, lethally irradiated mice that had been reconstituted with syngeneic bone marrow (TXB) were characterized. Both recently reconstituted N-TXB, (approximately 3 weeks after bone marrow injection) and aged (>6 months after reconstitution) A-TXB animals were studied. The T-lymphocytes from spleens of recently reconstituted N-TXB donors did not respond to PHA but did react significantly to Concanavalin A (Con A). The lack of PHA sensitivity was not due to dilution of reactive cells by other cell types. Removal of adherent cells, likewise, did not restore N-TXB spleen cell PHA responsiveness. N-TXB splenic T-cells were cortisone resistant. N-TXB spleen cells by themselves did not cause a graft vs host response. However, N-TXB spleen cells amplified the graft vs host response of normal lymph node cells but not N-TXB lymph node cells. Addition of cyclic GMP enhanced [³H]thymidine uptake of N-TXB spleen cells caused by Con A. N-TXB spleen cells were exclusively spleen seeking. The Con A reactive cell within N-TXB spleens was demonstrated to be of donor origin. Fetal liver as well as syngeneic bone marrow contained cells capable of reconstituting the Con A response. Spleen cells from aged. (>6 months) A-TXB were found to be PHA sensitive. Competitive inhibition assays measuring θ expression in A-TXB spleen cells indicate a significant increase in the θ positive lymphocyte population occurred with time. The data indicate that considerable reconstitution of θ positive cells had occurred in A-TXB donors. The results also suggest that the T-lymphocyte population of the TXB spleen may be a unique subpopulation of T-lymphocytes that resides exclusively in spleen and bone marrow.     

Dual regulation of resistance against Toxoplasma gondii infection by Lyt-2+ and Lyt-1+, L3T4+ T cells in mice

Studies were performed to attempt to define the T cell subset responsible for resistance to Toxoplasma gondii. A temperature-sensitive mutant (ts-4) strain of T. gondii was used for immunization because it causes infection but does not persist in the host. Immunization with this strain induced marked resistance against lethal challenge infection with virulent strains of T. gondii in mice. The resistance could be transferred to normal recipient mice by i.v. injection of spleen cells from ts-4-immunized mice. Marked inhibition of cyst formation in the recipient mice was also noted. The protective activity of immune spleen cells was removed by pretreatment of the spleen cells with anti-Thy-1.2 and C, indicating that T cells are responsible for the observed protection. Pretreatment of immune spleen cells with anti-Lyt-2.2 and C completely ablated their protective effect; pretreatment with anti-Lyt-1.2 or anti-L3T4 and C had lesser effects on their ability to transfer resistance. The effect of anti-Lyt-1.2 was the same as that obtained with anti-L3T4. This suggested that one T cell subset that is partially responsible for protection has both Lyt-1.2 and L3T4 markers on the cell surface. These results indicate that there are substantial roles for both the Lyt-2+ and Lyt-1+, L3T4 T cell subsets in dual regulation of resistance against toxoplasma infection and that Lyt-2+ T cells are the principal mediator of the resistance.     

Immunity to transplantable carcinogen induced fibrosarcomas in B2/B2 chickens. II. Macrophage dependency of adoptive T cell mediated tumor immunity.

The nature of immunity to transplantable chemically induced fibrosarcomas in SC (B2/B2) chicken was examined using Winn tests performed in the wing web. Immunity in spleen cell donors was induced by pretreatment with C. parvum or BCG followed by tumor cells + bacterial adjuvant in one and tumor cells alone in the other wing web. The T cells mediating the adoptive immunity were sensitive to anti-T + C, nylon wool nonadherent, mitomycin resistant and radiation (1000 R) sensitive. The adoptive immunity could not be expressed in heavily irradiated recipients or in hosts pretreated with trypan blue or silica. The host contribution could be reconstituted by iv injection of spleen or bone marrow cells from agamma-globulinemic (Aγ) unimmunized donors 2 days prior to Winn tests or by the local injection into wing webs of spleen cells or purified peripheral blood monocytes from Aγ donors. It was concluded that cooperation between immune donor T cells and normal monocytes of host origin mediated the inhibition of SCFS growth in Winn tests.     

Transfer of agammaglobulinemia in the chicken. I. Generation of suppressor activity by injection of bursa cells

Spleen cells from adult agammaglobulinemic (bursectomized) chickens taken 1 to 3 weeks after an injection of histocompatible bursa cells can inhibit the adoptive antibody response to B. abortus of normal spleen or bursa cells in irradiated recipients. Spleen cells from Aγ chickens not injected with bursa cells generally do not. Moreover, bursectomized chickens which have been reconstituted with spleen cells within the first week after hatching do not respond with suppressor cell formation upon bursa cell injection. This apparent “autoimmunization” with bursa cells induces suppressor T cells which are only minimally sensitive to treatment with mitomycin C or to 5000 R γ irradiation. The suppressor activity is neither induced nor potentiated by concanavalin A in vivo. It is much stronger in spleen than in thymus cells and appears to be macrophage independent and to require intact cells. The cell component which stimulates the suppressor activity is more pronounced on bursa than on spleen cells, and is at most present to a very limited extent on bone marrow, thymus, or peritoneal exudate cells. It is better represented in comparable cell numbers of Day 17 than of Day 14 embryonic bursa. The inducing cell component is present in the membrane fraction of disrupted bursa cells. Immunization with bursa cells from B locus histoincompatible chickens leads to suppressor activity against histocompatible bursa cells. Although the removal of Ig-bearing cells from bursa greatly diminishes its immunizing capacity, injection of serum IgM and IgG does not induce suppressor cells. It is suggested that tolerance to a B-cell antigen is lacking in adult Aγ chickens, resulting in an autoimmune response upon exposure to B cells. The B-cell antigen may be a cell surface-specific form of Ig, a complex of Ig and a membrane component, or a differentiation antigen which appears simultaneously with Ig during ontogeny.     

Use of lymphokine-activated killer cells to prevent bone marrow graft rejection and lethal graft-vs-host disease

Prompted by our recent finding that lymphokine-activated killer (LAK) cells mediate both veto and natural suppression, we tested the ability of adoptively transferred LAK cells to block two in vivo alloreactions which complicate bone marrow transplantation: resistance to transplanted allogeneic bone marrow cells, and lethal graft-vs-host disease. Adoptive transfer of either donor type B6D2 or recipient-type B6 lymphokine-activated bone marrow cells, cells found to have strong LAK activity, abrogated or inhibited the resistance of irradiated B6 mice to both B6D2 marrow and third party-unrelated C3H marrow as measured by CFU in spleen on day 7. The ability of lymphokine-activated bone marrow cells to abrogate allogeneic resistance was eliminated by C lysis depletion of cells expressing asialo-GM1, NK1.1, and, to a variable degree, Thy-1, but not by depletion of cells expressing Lyt-2, indicating that the responsible cells had a LAK cell phenotype. Similar findings were obtained by using splenic LAK cells generated by 3 to 7 days of culture with rIL-2. Demonstration that allogeneic resistance could be blocked by a cloned LAK cell line provided direct evidence that LAK cells inhibit allogeneic resistance. In addition to inhibiting allogeneic resistance, adoptively transferred recipient-type LAK cells prevented lethal graft-vs-host disease, and permitted long term engraftment of allogeneic marrow. Irradiation prevented LAK cell inhibition of both allogeneic resistance and lethal graft-vs-host disease. These findings suggest that adoptive immunotherapy with LAK cells may prove useful in preventing graft rejection and graft-versus-host disease in human bone marrow transplant recipients.     

Thymus-dependent and thymus-independent effector functions of mouse lymphoid cells. Comparison of cytotoxicity and primary antibody formation in vitro

We have compared two effector functions, antibody formation and cytotoxic capacity in vitro, of mouse cells of various origin with special reference to the T lymphocyte dependence of these processes. We have used addition of PHA and coating of target chicken erythrocytes (CRBC) with antibody as the two means of inducing cytotoxicity. Antibody formation in vitro has been studied both against thymus-dependent sheep erythrocytes (SRBC) and thymus-independent (E. coli) antigens. Spleen cells from thymectomized, lethally irradiated bone marrow-, or fetal liver-repopulated mice were deprived of phagocytic cells by uptake of colloidal iron. They did perform better than normal spleen cells in the antibody-induced cytotoxicity and were also induced to cytotoxicity by PHA. PHA did not induce increased DNA synthesis in these T cell-deprived spleen cell preparations, which could not make primary antibodies to SRBC but were able to do so against E. coli antigens. Fresh bone marrow and fetal liver cells, deprived of phagocytic cells, were also induced into a highly efficient cytotoxicity by anti-CRBC as well as by PHA. Pretreatment of spleen cells with an alloantiserum (θ) against T lymphocytes reduced but did not abolish the PHA-induced cytotoxicity. In contrast, it did not affect the antibody-induced cytotoxicity. Such treated cells could not make antibodies to SRBC but could do so against E. coli. Pretreatment of spleen cells with a heteroantiserum (MBLA) against mouse B lymphocytes completely abolished all cytotoxic- and antibody-forming abilities of the cells, although experiments with combinations of θ-treated and MBLA-treated cells suggested that the MBLA treatment had left behind a significant portion of helper T cells needed for the in vitro antibody response. From these data we conclude, as have others, that the antibody-induced cytotoxicity is independent of T lymphocytes. It can be induced in immature precursor cells from fetal liver or bone marrow, and these cells may also become cytotoxic on interaction with PHA. However, in normal spleen cells, at least part of the PHA-induced cytotoxicity is T cell dependent. Some preliminary data suggest that this PHA-induced cytotoxicity of normal spleen cells may be a joint process between T lymphocytes and other cells.     

Evidence for the functional binding in vivo of tumor rejection antigens to antigen-presenting cells in tumor-bearing hosts

Spleen cells of BALB/c mice bearing a syngeneic CSA1M fibrosarcoma were treated with anti-Thy-1.2 antibody plus C, yielding a T cell-depleted, APC-containing fraction. The APC-containing fraction was first tested for its capacity to present exogenous modified-self or another tumor (Meth A) Ag after in vitro pulsing. The results showed comparable Ag-presenting capacities to those obtained by APC-containing fraction from normal spleen cells, indicating that APC function is not affected in tumor-bearing mice. We next examined whether APC from CSA1M-bearing mice bind endogenously generated CSA1M tumor Ag onto its surfaces to stimulate tumor-specific T cells. Five rounds of inoculation of APC-containing fraction from CSA1M-bearing mice without further in vitro pulsing resulted in the induction of potent anti-CSA1M immune resistance. The involvement of anti-CSA1M T cells in the induction of anti-CSA1M immunity was excluded by the fact that the in vivo immunity was excluded by the fact that the in vivo immunity was delivered by Thy-1+ cell-depleted, but not by Thy-1+ cell-enriched fractions of spleen cells from CSA1M-bearing mice. Moreover, the failure of Sephadex G10-passed spleen cells to deliver anti-CSA1M resistance demonstrated the absolute requirement of APC for inducing the in vivo immunity. Finally, this in vivo resistance was found to be tumor specific, because APC fractions from CSA1M-bearing and Meth A-bearing BALB/c mice induced immune resistance selective against the corresponding tumor cell challenge. These results indicate that APC from tumor-bearing hosts can not only exert unaffected APC function against exogenous Ag, but also function to present tumor Ag generated endogenously in the tumor-bearing state and to produce tumor-specific immunity in vivo.