Gustavo Cudkowicz

Do natural killer cells engage in regulated reactions against self to ensure homeostasis

Host reactivities not requiring immunization in the mouse, especially natural resistance of irradiated animals to accept grafts of normal or malignant hemopoietic cells, were compared with NK activity against the YAC-1 lymphoma. The effects of several independent variables known to influence natural resistance in vivo had a similar effect on the NK system. Figure 12 lists an impressive array of shared properties and positive correlations. In contrast, the distinctions were few and minor. Many of the positive correlations were of particular significance since the experimental variables either have opposing or no effects on conventional induced immunity. The multiplicity and pervasiveness of these correlations suggest that the cellular mechanisms underlying natural reactivities are similar or common. Cytotoxic effectors mediating natural resistance to normal cells, tumors, and cells infected with intracellular pathogens may be distinct in terms of target selectivity, yet belong to a single cell lineage subject to common regulatory influences for differentiation and function. Regulation of reactivity via suppressor cells was studied in the NK system only. The spleens of mice selected for low levels of NK activity (resulting from young age, irradiation, and treatment with the macrophage-active agents l-carrageenan or hydrocortisone acetate) contained cells capable of inhibiting the lytic function of NK effectors taken from untreated adult donors. All the suppressor cells studied were thymus-independent, as judged by their occurrence in spleens of genetically athymic mice; the suppressive function was resistant to 2000 rads of gamma-rays administered in vitro and was not restricted by the major histocompatibility complex, without exception. However, two major classes of suppressors were identified: (a) macrophagelike cells inducible by l-carrageenan or hydrocortisone acetate, and (b) nonadherent cells found in spleens of untreated infants and of irradiated adult mice. It is proposed that the suppression of NK cytolysis demonstrated in vitro was a manifestation of regulatory mechanisms modulating the level of NK activity in vivo. Macrophagelike cells that are induced, activated, or inactivated by bacteria, viruses, hormones, and other agents may act as regulators of differentiation, maturation, and function of cells belonging to the NK lineage. Nonadherent cells could be either a distinct class of suppressors or immature NK cells capable of binding but not lysing target cells. In the latter case, regulation would be achieved via competitive binding of targets by pre-NK cells presumably in dynamic equilibrium with functional (i.e. matured) NK effectors.

Carrageenan-induced decline of natural killer activity: I. In vitro activation of adherent non-T-suppressor cells

Abstract Short-term in vitro exposure of spleen cells of (C57BL/6 × C3H)F 1 or BALB/c mice to ι-carrageenan (100 μg/ml) induces the expression of suppressor activity. Suppressor function is assessed in vitro as inhibition of natural cytotoxicity against YAC-1 lymphoma targets. The responsible cells are not post-thymic T lymphocytes as they can be found among treated splenocytes of athymic BALB/c nu/nu mice; suppressor activity is neither restricted by the major histocompatibility complex (MHC) nor impaired by 2000 rad of γ irradiation. The ι-carrageenan-induced suppressor cells are adherent to Sephadex G-10 columns, but are neither adherent to glass wool columns nor phagocytic for, or adherent to, carbonyl iron particles. The lytic function of natural killer cells is also inhibited by cell-free supernatants in which suppressor splenocytes had been incubated. The short time required for induction of suppressor activity (24–48 hr), the lack of MHC restriction, and the effectiveness of supernatants suggest that ι-carrageenan simply “activates” precursor spleen cells to become nonspecific suppressors. The paradox of suppressor cell removal by Sephadex G-10 filtration but not by other adherence techniques complicates the assignment of such cells to a particular cell lineage or monocyte-macrophage subtype.

Density Gradient Separation of Marrow Cells Restricted for Antibody Class

Primitive cells of (C3H x C57BL/10)F1 mouse bone marrow, participating with thymocytes in immune responses to sheep erythrocytes, are already committed to the immunoglobulin M or immunoglobulin G antibody class. By equilibrium centrifugation in discontinuous gradients of bovine serum albumin, cells responsible for production of IgM immunocytes migrate to the denser regions, whereas those responsible for IgG immunocytes remain in the lower density regions.

Natural resistance of irradiated 129-strain mice to bone marrow allografts: Genetic control by theH-2K region

A major genetic determinant of natural resistance to bone marrow allografts, designated asHh-3, was mapped to theH-2K region. This gene may code for or regulate the expression of cell surface structures selectively expressed on donor hemopoietic cells and recognized by naturally occurring cytotoxic effectors. Resistance was observed as failure of donor cell growth in the spleen of irradiated 129-strain (H-2bc) recipients of H-2k bone marrow cells. The mapping was accomplished by substituting donor cells bearingk alleles throughout theH-2 complex with cells of recombinant mouse lines bearingk alleles at definedH-2 regions. The host antigraft reaction underlying resistance was abrogated by pretreating 129-strain mice with either rabbit antimouse lymphocyte serum or the antimacrophage agent silica. Grafting of H-2Kk cells into mice ancestrally unrelated to 129 but sharing theH-2bc or the similarH-2b haplotype, and intoH-2b/k,H-2k/bc, andH-2k/d F1 hybrids revealed that resistance was unique to 129 mice, since mice of the other strains, including F1 hybrids, were susceptible to the grafts. Thus,Hh-3 incompatibility was a necessary but insufficient condition for the manifestation of allogeneic resistance; other genetic factors not associated withH-2 conferred responder status to 129-strain mice and nonresponder status to D1.LP, B10.129(6M), B10, B6, and possibly to F1 hybrid mice. The possible relationships between allogeneic resistance to H-2k marrow grafts, hybrid resistance to H-2k lymphomas, and F1 hybrid antiparental H-2k cytotoxicity induced in vitro are discussed.

Cellular suppression of murine ADCC and NK activities induced by Corynebacterium parvum

SummaryAdministration of a single dose of C. parvum (CP) induces depression of splenic NK activity in mice after a lag period of 3–5 days and this depression lasts about 2 weeks. The depressed levels of NK activity noted in this study depended on time of CP administration and were associated with the induction of suppressor cell activity. Neonatally thymectomized or sublethally irradiated mice had unimpaired ability to generate suppressor cells following CP treatment. Depletion of adherent/phagocytic cells by carbonyl iron plus magnetism, Sephadex G-10 filtration, or both neither enriched NK activity nor removed suppressor activity from the spleens of CP-treated mice. Antibody-dependent cellular cytotoxicity (ADCC) against lymphoma targets was also depressed in CP-treated mice, accompanied by a concomitant appearance of suppressor cells that interfere with ADCC at the effector level.

Density gradient separation of marrow precursor cells restricted for antibody specificity.

Potentially immunocompetent cells of (C57BL/6 x DBA/2) F1 mouse bone marrow are committed to antigenic determinants of sheep or burro erythrocytes prior to interaction with thymus-derived cells and participation in immune responses to administered antigens. At this stage of differentiation marrow cells of this particular mouse strain are not yet restricted for the immunoglobulin M or immunoglobulin G antibody class. By equilibrium centrifugation in discontinuous gradients of bovine serum albumin, precursors of cells that produce antibody to sheep erythrocytes migrate to denser regions, whereas the precursors of immunocytes that produce antibody to burro erythrocytes remain in the lower density regions. cursors for all specificities.

Sensitivity to radiation and insensitivity to vinblastine of the inducer function of thymus-derived cells.

Abstract Thymic antigen-reactive cells stimulated by sheep erythrocytes generate specific inducer cells facilitating differentiation and maturation of marrow precursors into immunocytes (direct and indirect plaque-forming cells). This function is abolished by in vitro exposure of inducer cells to 1000 rads of gamma rays, but not by their in vivo exposure to the drug vinblastine which is cytocidal for cells entering mitosis. Thus, the cooperative function of inducer cells in anti-sheep immune responses is radiosensitive even when the cells are not dividing. Other helper functions of thymus-derived cells are radioresistant, e.g., the function of carrier-specific cells and of cells reconstituting spleens of thymectomized mice. Differences in radiosensitivity of helper functions suggest that more than one cell type and/or mechanism may be utilized for triggering marrow-derived precursors of immunocytes.

Thymic antigen-reactive cells do not specify serological properties of antibody.

Abstract Irradiated mice of the (C3H × C57BL/10)F 1 and (C57BL/6 × DBA/2)F 1 strains were reconstituted with an excess of syngeneic bone marrow cells containing precursors of immunocytes, and with graded limiting numbers of thymocytes containing antigen-reactive cells (ARC), and then injected with sheep erythrocytes. The number of ARC and their possible specialization for serological properties of antibody were investigated by determining the titer of 2-mercaptoethanol-sensitive serum hemagglutinins and hemolysins 11 days after grafting. The limiting dilution assays indicated that the number of detectable ARC/10 6 thymocytes was of the same order of magnitude for both antibody responses. Agglutinins and lysins were associated in most recipient mice receiving an average of 1 ARC. Hence, serological properties of antibodies were not dictated by ARC, but by other cells participating in the immune responses, presumably of nonthymic origin.

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

Abstract The role of macrophage-like cells in the in vitro generation of specific B6D2F 1 hybrid anti-parental B6 cytotoxic T lymphocytes (CTL) was investigated by means of silica particles (SIL). Depression of this cell-mediated response resulted from the addition of 12.5 or 25 μg of SIL to mixed F 1 /parent spleen cell cultuers, and full abrogation resulted from the addition of 125 or 250 μg of SIL. The treatment was effective if applied during the first 48 hr of culture. When treatment was delayed, responsiveness did not decline nor did the lytic function of mature CTL exposed to SIL. Moreover, no depression of the anti-allogeneic cell mediated response resulted from the addition of 250 or 500 μg of SIL to mixed F 1 /allogeneic instead of F 1 /parent spleen cell cultures. Abrogation of the F 1 hybrid anti-parent response was attributed to SIL-induced impairment of an accessory function presumably exerted by macrophage-like cells during the early phases of responder T cell activation. If so, the F 1 anti-parent response was considerably more dependent than the allogeneic response on the integrity of accessory cells. Injection of 5 mg of SIL to donors of responder cells likewise resulted in loss of F 1 anti-parent and occasionally of anti-allogeneic in vitro responsiveness. This in vivo effect of SIL was prevented by pretreating mice with the lysosomal stabilizer poly-2-vinylpyridine N -oxide. Because unresponsiveness induced in vivo was not selective for F 1 anti-parent responses and lasted for up to 10 days, it may be attributable not only to depletion of accessory macrophages by SIL but also to the induction of suppressor macrophages.

Bone marrow precursor cells specify serological properties of antibody

Abstract Irradiated mice of the (C3H × C57BL 10 )F 1 and C57BL 6 × DBA 2 )F 1 strains were reconstituted with an excess of syngeneic thymocytes containing antigen-reactive cells, and with graded limiting numbers of bone marrow cells containing precursors of antibody-forming cells (P-AFC), and then injected with sheep erythrocytes. The number of P-AFC and their possible specialization for serological properties of antibody were investigated by determining the titer of 2-mercaptoethanol-sensitive serum hemagglutinins and hemolysins 11 days after grafting. The limiting dilution assays gave different results in the two mouse strains, but clearly indicated that the number of detectable P-AFC per unit number of bone marrow cells was of the same order of magnitude for both antibody responses. However, agglutinins and lysins were not associated in a significant number of recipient mice receiving an average of 1 P-AFC. Hence, marrow precursor cells were restricted for serological properties of antibody reflecting structural features not equivalent to those underlying molecular class or subclass. Circumstantial evidence suggested that a subpopulation of marrow P-AFC characterized by larger burst size was detected by serum titrations, whereas more numerous P-AFC with smaller burst size were previously detected by enumeration of splenic plaque-forming cells.