Lymphokine-activated killer cells are generated before classical cytotoxic T lymphocytes after bone marrow transplantation in mice

Lymphokine-activated killer (LAK) cells are demonstrable within 2 wk after syngeneic or allogeneic (H-2-compatible) bone marrow transplantation in mice. Classical cytotoxic T lymphocytes (CTL) are not active until at least 4 wk after transplant. Both LAK cells and CTL bear the Thy-1 marker and do not possess the murine natural killer cell marker asialo GM.     

Asialo-GM1+ natural killer cells directly suppress antibody-producing B cells

Natural killer (NK) cells were tested for their ability to suppress antigen-induced antibody responses in vitro. Asialo-GM1+ (ASGM1+) cells were prepared from nylon-wool-nonadherent spleen cells obtained from normal mice. After depletion of Ig+, L3T4+ and Lyt-2+ cells, the ASGM1+-enriched cell population had high NK activity which was abrogated by treatment with anti-ASGM1 and C'. This NK-enriched ASGM1+ cell fraction significantly suppressed the generation of antibody-producing cells when added to in vitro immunization cultures of primed spleen cells. Treatment of the NK-enriched cell population with anti-ASGM1 and C' abrogated the ability of these cells to suppress antibody responses. In vitro antibody production by purified B cells was also suppressed in the presence of the NK-enriched cell population, although the kinetics of the suppression differed from that observed with unfractionated spleen cells. In addition, the NK-enriched cell population suppressed the proliferation of the B cell line WEHI-279.1. Suppression of WEHI-279.1 cells was abrogated when the NK-enriched cell population was treated with anti-ASGM1 and C'. These results suggest that normal NK cells suppress the generation of antibody-producing B cells and that this occurs, at least in part, through a direct regulation of the B cell.     

Repopulation of host lymphohematopoietic systems by donor cells during graft-versus-host reaction in unirradiated adult F1 mice injected with parental lymphocytes.

The graft-vs-host reaction (GVHR) generated by the injection of parental lymphocytes into unirradiated immune-competent F1 hosts is characterized by an acute loss of immune functions, an attack on host tissues, and a gradual recovery of function. Flow cytometric analysis of the donor- and host-derived splenic populations during the course of acute dysfunction and gradual recovery revealed a complex pattern of changes in lymphoid and myeloid populations that resulted in the repopulation of the host with donor-derived cells. Initially, donor-derived T cell populations expanded, particularly CD8+ T cells. Next, host T cell and B cell populations disappeared. Finally, donor-derived cells repopulated the lymphohematopoietic system in the sequence myeloid populations, B cells, and, after a protracted period, T cells. The recovery of immune functions following GVHR-induced immune deficiency was associated with this repopulation of the spleen by donor-derived cells. Donor repopulation of the host lymphohematopoietic system required the presence of both CD4 and CD8 cells in the original donor inoculum. Depletion of donor CD4 populations precluded development of GVHR or any donor engraftment; depletion of CD8 cells resulted in engraftment solely of donor CD4 populations.     

Induction of immune complex glomerulonephritis in F1 hybrid mice: superiority of cortisone-resistant parental thymocytes over spleen cells

Severe immune complex glomerulonephritis developed in (C57B1/10ScSn × DBA/2) F₁ hybrid mice after the injection of various dosages of cortisone-resistant thymus cells from DBA/2 donors. In contrast, significantly lower incidences of glomerulonephritis were induced when the same numbers of DBA/2 spleen cells were injected. The low nephritogenic capacity of spleen cells can be explained by their high content of B cells, assuming that parental B cells are not required for the induction of the disease. As T cells themselves are unable to produce humoral antibodies, the administration of parental cortisone-resistant thymus cells may enable B cells of the F₁ recipients to form the 7S antibodies deposited in the glomerular immune complexes.     

Young F mice spontaneously generate cytotoxic T cells against parental targets.

Spleen cells from young (AKR/J female x BALB/c) or (BALB/c female x AKR/J)F1 mice can spontaneously generate effector cytotoxic T lymphocytes (CTL), in a 5-day primary in vitro culture, which lyse target cells from AKR/J and BALB/c but not allogeneic mice. These spontaneous CTL responses first appear when spleen cells are taken from F1 mice at 3 to 4 weeks of age, are maximum at about 5 weeks, and have declined by week 7. The fact that these spontaneous CTL responses are never detectable in the spleen cell cultures from any ages of parental AKR/J and BALB/c mice makes them unique properties of the F1 mice.     

Graft-vs-host reactions in F1 mice induced by parental lymphoid cells: nature of recruited F1 cells.

Graft versus host (GVH) reactivity of parental lymph node (LN) cells was assayed by measurements of 3H-thymidine incorporation in vivo. Mitomycin (Mit.) treatment of parental cells abolished their proliferative activity but the combination of such Mit.-treated parental cells with F1 LN cells resulted in much higher proliferation than either one population alone. This recruitment into proliferation of F1 cells was prominent on days 3 and 4 after cell injection and amounted to 35 to 51% of the total activity seen after injection of untreated parental cells alone. The F1 cell sensitive to recruitment was resistant to anti-Thy 1.2 treatment, was not removed by carbonyl iron-magnet separation; and was not present in thymus. The parental cell inducing recruitment was, however, sensitive to anti-Thy 1.2. When spleen cells from hapten immune F1 donors were injected together with Mit.-treated parental LN cells and boosted with hapten on another carrier, a typical "allogeneic effect" was observed in the anti-hapten immune response. It was concluded that Mit.-treated parental T cells exerted a mitogenic effect on F1 B cells resulting in extensive recruitment similar to that seen in murine mixed lymphocyte reactions.     

Natural killer T cells promote collagen-induced arthritis in DBA/1 mice

The role of NKT cells in the pathogenesis of collagen-induced arthritis (CIA) remains unclear since most studies have used C57BL/6 (B6) mice, which are less susceptible to CIA than mice with a DBA/1 background. To clarify the immunological functions of NKT cells in CIA, it is necessary to analyze in detail the effects of NKT cell deficiency on CIA development in DBA/1 mice. The incidence and severity of CIA were significantly exacerbated in DBA/1CD1d^+/− mice as compared to DBA/1CD1d^−/− mice. In DBA/1CD1d^+/− mice, antigen-specific responses of B and T cells against CII were remarkably increased and inflammatory cytokine levels were also increased in vivo and in vitro. The number of IL-17-producing NKT cells significantly increased in DBA/1CD1d^+/− mice as the disease progressed. Our results clearly show that NKT cells are involved not only in accelerating the severity and incidence of CIA but also in perpetuating the disease progression.     

Generation of T helper cells in vitro. IV. F1 T helper cells primed with antigen-pulsed parental macrophages are genetically restricted in their antigen-specific helper activity.

A sequential culture technique for the in vitro induction and subsequent assay of T helper cells is employed to examine the histocompatibility requirements for antigen recognition by murine T helper cells. F1 T cells are primed in vitro with antigen-pulsed parental strain macrophages and tested for antigen-specific helper activity in cultures containing anti-Thy 1.2 serum and C treated spleen cells from hapten-primed parental or F1 mice. A semiallogenieic system is used and appropriate controls are included to avoid possible complicating effects of allogeneic interactions. The results indicate that F1 T helper cells preferentially stimulate carrier-specific anti-hapten plaque-forming cell responses in spleen cells which are H-2 identical with the macrophage used initially to prime the T cells. Parental spleen cell cultures do not respond to F1 T helper cells which were primed with the other parental strain macrophage. Supplementing this culture with macrophages which are histocompatible with those used to prime the F1T cells is sufficient to restore T helper cell activity. Thus, the genetic restriction described here is between the primed T cell and the macrophage used to elicit secondary responses and not between the T cell and B cell. The results in this semiallogeneic system, however, do not rule out the possibility of additional allogeneic genetic restrictions in the subsequent interaction of T cells with B cells.     

Fc-receptor-bearing cells in spleen of mice injected with cell-free Ehrlich ascites fluid (EAF)

The kinetics of different cell populations (T and B) and subpopulations (one bearing easily releasable FcR and one bearing stable FcR) was followed in spleens of mice after one single i.p. injection of EAF. The number of FcR bearing cells doubled within 2-7 days after EAF injection. This increase was due to cells bearing temperature sensitive FcR and was accompanied by the doubling of theta positive cells. These results, supported by the demonstration of doubly labeled (theta+FcR+) cells, suggest that EAF injected into normal mice causes the appearance of T-cells expressing easily releasable FcR. These cells, according to Fridman et al. (1977) are suppressor cells. Maximal increase of theta positive cells and of cells with temperature sensitive FcR detected in vitro coincided with the maximum of the suppressive activity of EAF detected in vivo.     

Infiltrating decidual natural killer cells are associated with spontaneous abortion in mice

Immunohistochemistry was used to study a murine model which spontaneously aborts at a frequency of 25 to 30%. Our results show that natural killer (NK) cells are not only the predominant infiltrating cells in aborting feto-placental units, but that they also appear in a similar proportion of feto-placental units before abortion is detectable. The frequency of feto-placental units with significantly elevated NK infiltrates corresponds to the subsequent abortion frequency, indicating a causal relationship. Immunization of the mother with BALB/C splenocytes prevents these NK infiltrates and decreases the abortion frequency to normal levels. These results suggest for the first time that maternal NK cells may have an instrumental role in the etiology of spontaneous abortion.     

T cytotoxic-1 CD8+ T cells are effector cells against pneumocystis in mice

Host defenses are profoundly compromised in HIV-infected hosts due to progressive depletion of CD4+ T lymphocytes. A hallmark of HIV infection is Pneumocystis carinii (PC) pneumonia. Recently, CD8+ T cells, which are recruited to the lung in large numbers in response to PC infection, have been associated with some level of host defense as well as contributing to lung injury in BALB/c mice. In this study, we show that CD8+ T cells that have a T cytotoxic-1 response to PC in BALB/c mice, as determined by secretion of IFN-gamma, have in vitro killing activity against PC and effect clearance of the organism in adoptive transfer studies. Moreover, non-T cytotoxic-1 CD8+ T cells lacked in vitro effector activity and contributed to lung injury upon adoptive transfer. This dichotomous response in CD8+ T cell response may in part explain the clinical heterogeneity in the severity of PC pneumonia.     

Haplotype preference in lymphocyte differentiation. II. F1 hybrid helper T cells generated with antigen-bearing parental macrophages can cooperate with B lymphocytes of either parent.

Carrier-specific helper T cells were generated in F₁ hybrid mice by either conventional immunization procedures or by repeated immunizations with antigen-bearing macrophages derived from either F₁ or parental donors. The F₁ helper T cells generated in these various ways were then analyzed for their capacities to help hapten-primed B lymphocytes derived from each of the two parental strains as well as from F₁ donors in the development of secondary anti-hapten antibody responses. These analyses were conducted using two different types of in vivo assay systems as well as a totally in vitro system. Under all circumstances, helper T cells from F₁ mice, primed either in conventional fashion or with antigen bearing parental or F₁ macrophages, were capable of interacting effectively with B lymphocytes of each parent and of F₁ origin. Moreover, in the case of F₁ helper cells primed with antigen-bearing parental macrophages, there was no evidence of preferential helper activity for parental B lymphocytes corresponding to the type of macrophage used for sensitization; this was true irrespective of whether in vivo or in vitro assay systems were employed. The relevance of these findings and others which are either similar to, or discordant with, them to the general question of genetic restrictions in macrophage-T lymphocyte interactions is discussed.     

Invariant natural killer T cells from rhesus macaque spleen and peripheral blood are phenotypically and functionally distinct populations

BACKGROUND: Natural killer T cells (NKT) possess dual functions of innate and adaptive immune systems, controlling viral infections and regulating autoimmune diseases. Non-human primates (NHP) are penultimate models for advancing therapeutic immunoregulatory strategies for translational application in humans, though, little is known about NHP NKT cells. Here we characterized rhesus macaque NKT cells ex vivo. METHODS: The frequency, phenotype and intracellular cytokine production of V alpha 24+ 6B11+ invariant NKT (iNKT) cells were analyzed by multi-color flow cytometry. V alpha 24J alpha Q mRNA expression was analyzed by real-time RT-PCR. RESULTS: The frequencies of peripheral blood (PB) and spleen V alpha 24+ 6B11+ iNKT cells were not significantly different. The iNKT cell subset in spleen was significantly increased for CD4+ CD8+ and CD3+ CD56+ co-expression as well as intracellular interleukin-4 production, which was rarely observed in circulating PB. CONCLUSION: Spleen iNKT cells in rhesus macaques are Th2 biased and display phenotypically and functionally distinct profiles from their PB counterpart.     

Acute toxoplasma infection of mice induces spleen NK cells that are cytotoxic for T. gondii in vitro

Murine spleen natural killer (NK) cells from normal and Toxoplasma-infected BALB/c mice were examined for their reactivity against RH strain tachyzoites in vitro. First, the effect of suspending medium on survival of extracellular RH tachyzoites was determined. Optimal parasite viability (by ethidium bromide-acridine orange staining) was observed when tachyzoites were incubated in phosphate-buffered saline (PBS) containing 10% horse serum (HS) for as long as 5 hr. In addition, parasite viability in PBS-HS correlated with subsequent infectivity, because freshly harvested and PBS-HS-incubated tachyzoites were equivalent in their ability to cause lethal infections in normal mice and to survive within normal mouse macrophages. Furthermore, viability and tumoricidal capacity of murine spleen NK cells incubated in PBS-HS was comparable to that of NK cells incubated in a standard cytotoxicity medium. Incubation of effector NK cells and target tachyzoites in PBS-HS in vitro revealed that spleen NK cells from 3-day Toxoplasma-infected mice had significantly greater cytotoxicity for extracellular RH tachyzoites than did control cells from uninfected mice. Moreover, Toxoplasma gondii-induced spleen NK cell toxoplasmacidal activity was significant at all effector to target cell ratios tested, and appeared to be mediated by direct contact between the host cell and the parasite. These in vitro results suggest that NK cells may be important in host defense against T. gondii.     

Prostaglandin synthesis in spleen cell cultures of mice injected with Corynebacterium parvum.

Spleen cells of C57BL/6 mice produced high amounts of PGE in vitro when tested 5 to 10 days after injection of heat-killed C. parvum organisms. Little or no PGE was produced by spleen cells from untreated mice or from mice injected with a strain of coryneform bacteria that does not stimulate the lymphoreticular system of mice. Significant release of PGE from spleen cells of C. parvum injected mice could be detected as early as 30 min after initiating the cultures and maximal levels were usually seen after 48 hr. Treatment by indomethacin completely abolished this PGE production. Removal of the adherent population from the spleen cell suspension resulted in markedly decreased levels of PGE, but PGE release of the remaining population was never completely abolished. These data suggest that the cells responsible for most of the PGE synthesis in this system were adherent cells, presumably macrophages. The levels of PGE produced in spleen cells of C. parvum-treated mice were further increased by in vitro addition of C. parvum. This effect could also be observed after addition of zymosan particles indicating that it was not an immunologically specific effect. The reported data suggest that prostaglandins may represent important mediator molecules of the described immunostimulatory and immunosuppressive effects of C. parvum.