The induction of peripheral T cell unresponsiveness in adult mice by monomeric human gamma-globulin

Monomeric human gamma-globulin (HGG), when injected into adult mice, induces a state of specific immunologic unresponsiveness to further challenge with immunogenic forms of HGG. In this report we have directly determined the role of the thymus in the induction of HGG tolerance and the proliferative responsiveness of T cells from normal and HGG-tolerant mice. Draining lymph node T cells were isolated from HGG-tolerized and -challenged mice, and tested for their proliferative response to HGG in vitro. T cells from untreated but challenged adult CBA/CaJ and A/J mice proliferate in response to HGG, whereas such mice given monomeric HGG before challenge fail to show an HGG-specific proliferative response. APC from tolerant or nontolerant mice were equally effective in the support of Ag-specific proliferation of primed T cells. The influence of the thymus gland on HGG-induced T cell unresponsiveness was assessed by determining whether thymectomized mice could be tolerized to HGG. The results suggest that the generation of T cell tolerance to HGG is independent of thymic function as assayed by both antibody production in vivo and T cell proliferation in vitro. Unresponsiveness of T cells from tolerant mice was not a result of the presence of CD8+ cells since removal of CD8+ cells from lymph node T cells did not alter unresponsiveness to HGG in vitro. Further, mixing tolerant T cells with normal HGG-primed T lymphocytes did not inhibit proliferation of the HGG-primed cells. The results of this investigation suggest that this mouse model of tolerance to HGG represents a thymus-independent unresponsiveness of mature peripheral T cells to a nonself-Ag. Understanding the regulation of tolerance to HGG may give additional insight into the mechanisms required for the maintenance and possibly the induction of tolerance to certain self-Ag in peripheral lymphoid organs.     

Immunologic suppression after oral administration of antigen. III. Activation of suppressor-inducer cells in the Peyer's patches

Mice were orally administered sheep erythrocytes (SRBC) in a regimen previously known to produce systemic tolerance to SRBC. Cellular interactions and movement from the gut-associated lymphoid tissue (GALT) to the spleen were found to occur using both in vivo and in vitro transfer systems. The cell in the GALT which initiates the suppression circuit migrates from the GALT to the spleen shortly after contacting antigen. This cell is a T suppressor-inducer (Tsi) cell which interacts with splenic lymphocytes to induce the formation of an effector T suppressor cell (Ts). The Tsi and Ts can be separated from each other by their differential sensitivities to cyclophosphamide. In addition, the Tsi can be separated from other GALT T cells by its inability to bind the lectin, peanut agglutinin. Thus, cell migration and cellular interaction among T cells must occur to result in orally induced tolerance.     

Immunologic suppression after oral administration of antigen. I. Specific suppressor cells formed in rat Peyer's patches after oral administration of sheep erythrocytes and their systemic migration.

Rats given 10(10) sheep erythrocytes (SRBC) orally were found to contain specific suppressor cells to SRBC in their Peyer's patches (PP) and mesenteric lymph nodes (MLN) after 2 days of feeding. After 4 days of feeding, similar suppressor cells were found in the thymus and spleen, but they were missing in the PP or MLN. These suppressor cells effectively blocked IgM and IgG plaque-forming cell responses to SRBC in Mishell-Dutton cultures and delayed-type-hypersensitivity responses to SRBC when transferred to syngeneic recipients, but they did not affect responses to horse erythrocytes. The orally induced specific suppressor cells appeared to be T2 cells since their activity was eliminated by in vivo treatment of SRBC-fed rats with anti-rat lymphocyte serum but not by adult thymectomy. Because carrageenan partially relieved the suppression observed in culture, the actual suppressive mechanism may also involve a macrophage.     

Molecular and cellular requirements for enhanced antigen cross-presentation to CD8 cytotoxic T lymphocytes

MHC class I-mediated cross-priming of CD8 T cells by APCs is critical for CTL-based immunity to viral infections and tumors. We have shown previously that tumor-secreted heat shock protein gp96-chaperoned peptides cross prime CD8 CTL that are specific for genuine tumor Ags and for the surrogate Ag OVA. We now show that tumor-secreted heat shock protein gp96-chaperoned peptides enhance the efficiency of Ag cross-priming of CD8 CTL by several million-fold over the cross-priming activity of unchaperoned protein alone. Gp96 also acts as adjuvant for cross-priming by unchaperoned proteins, but in this capacity gp96 is 1000-fold less active than as a peptide chaperone. Mechanistically, the in situ secretion of gp96-Ig by transfected tumor cells recruits and activates dendritic cells and NK cells to the site of gp96 release and promotes CD8 CTL expansion locally. Gp96-mediated cross-priming of CD8 T cells requires B7.1/2 costimulation but proceeds unimpeded in lymph node-deficient mice, in the absence of NKT and CD4 cells and without CD40L. Gp96-driven MHC I cross-priming of CD8 CTL in the absence of lymph nodes provides a novel mechanism for local, tissue-based CTL generation at the site of gp96 release. This pathway may constitute a critically important, early detection, and rapid response mechanism that is operative in parenchymal tissues for effective defense against tissue damaging antigenic agents.     

Generation of human plaque-forming cells in culture: tissue distribution, antigenic and cellular requirements.

A system for the induction of specific, hemolytic plaque-forming cells from normal human lymphocytes in vitro (HcPFC) has been established and cells from various normal lymphoid tissues have been investigated. Normal values for anti-SRBC HcPFC responses in cultures of 107 Ficoll-Hypaque separated lymphocytes range from 2000 (bone marrow) to 7000 (spleen) and 15,000 (tonsillar and peripheral blood lymphocytes). HcPFC responses to ovalbumin were lower by factor of 2 to 4. Anti-SRBC as well as anti-ovalbumin responses required the cooperation of T lymphocytes and IgM-bearing B lymphocytes and the magnitude of the response was antigen dose dependent. Addition of adherent cells as well as of 2-mercaptoethanol enhanced the response. On the basis of the data obtained in experiments examining the role of B and T lymphocytes, a tentative model of cellular interaction has been postulated, suggesting a major role for antigen concentration in the modulation of the response via reactive T lymphocytes.     

IgE-selective and antigen-specific unresponsiveness in mice. I. Induction of the unresponsiveness by administration of ovalbumin-pullulan conjugate.

An experimental model was demonstrated in mice for the induction of IgE-selective unresponsiveness to ovalbumin, a protein antigen. An administration of ovalbumin, conjugated with pullulan, a linear polymer of glucose, (OA-pullulan) into mice resulted in the induction of a long lasting, IgE-selective unresponsiveness to the subsequent immunization with native OA in the form optimal to elicit IgE antibody response. The IgE-selective unresponsiveness is antigen specific and is infectious to normal mice by transferring the spleen cells from mice receiving OA-pullulan conjugate at least 2 weeks before. In contrast to other modified antigens, OA-pullulan was found to elicit good IgM and IgG antibody responses, but not an IgE response, without the aid of an adjuvant.     

Molecular, cellular, and antigen requirements for development of age-associated T cell clonal expansions in vivo

T cell aging manifests itself both at the cellular (cell-autonomous defects in signaling) and at the population (age-related dysregulation of T cell homeostasis) levels. A prominent contributor to the latter is the appearance of T cell clonal expansions (TCE), with a potential to impair immune defense. In this study, we investigated molecular, cellular, and Ag requirements for TCE development. Of the mutant mice tested, old animals lacking MHC class I exhibited 7-fold fewer TCE than controls, with a 7-fold reduction in TCE. By contrast, animals lacking only one of the MHC class I molecules (Kb or Db), or IL-7R, or devoid of T cell renewal via adult thymectomy, all exhibited significant increases in TCE incidence. This increase directly correlated to lymphopenia, increased CD8 T cell turnover and an accumulation of memory-phenotype T cells. These data suggested that homeostatic cell division in the CD8 compartment enhances the formation of TCE. Repeated immunization with peptide/adjuvant did not result in an increase in Ag-specific TCE; however, adjuvant alone increased TCE incidence. In these experiments, therefore, nonspecific and/or homeostatic proliferation was more efficient in generating TCE in mice than repeated Ag-driven stimulation, suggesting that many, if not most, TCE in specific pathogen-free laboratory mice may be Ag-independent.     

Cocaine administration induces human splenic constriction and altered hematologic parameters

Cocaine is a potent vasoconstrictor that hasbeen shown to alter hemoglobin, hematocrit, and red blood cell countsin both animals and humans. The present study evaluated whether cocaine administration induces splenic constriction in men and whether spleen-volume changes temporally correlate with altered hematologic parameters. Spleen volume was assessed at baseline and after cocaine administration (0.4 mg/kg) by using magnetic resonance imaging. A groupof five healthy men, aged 31 ± 2 (SE) yr and reporting occasionalcocaine use (13 ± 5 lifetime exposures), participated. Cocainereduced spleen volume by 20 ± 4%(P < 0.03) 10 min after drugadministration. Spleen volume returned to normal (101 ± 3% baseline) within 35 min after cocaine administration, indicating thatthe reduction is a transient phenomenon. In subjectsadministered cocaine from whom blood samples were obtained(n = 3), cocaine increased hemoglobinlevels, hematocrit, and red blood cell count to 104.5 ± 0.9, 105.6 ± 1.2, and 106.5 ± 1.0% of baseline levels, respectively(P < 0.03), but it did not alterwhite blood cell and platelet counts. Placebo administration(n = 5) did not alter hematologicparameters. These results suggest that cocaine induces splenicconstriction in humans, and this may contribute to temporally concordant hematologic parameter changes. These events may help topreserve or increase tissue oxygenation in periods of high oxygendemand and/or increased vascular resistance.

     

Dose-dependent induction of immunologic enhancement and suppression after oral administration of antigen

The effects of feeding various quantities of a particulate antigen, sheep red blood cells (SRBC), on plaque-forming cells (PFC) in the spleen were determined. Mice were given various numbers of SRBC orally daily for 14 days, then injected with SRBC intravenously. Splenic IgA PFC responses to SRBC were enhanced in the mice fed 5 X 10(8) SRBC and splenic IgG PFC responses to SRBC were depressed in the mice fed 5 X 10(9) SRBC. Adoptive transfer experiments showed that enhancement of splenic IgA PFC responses and suppression of splenic IgG PFC responses were induced by the T-cell rich fraction from Peyer's patches (PP) and the spleen in 5 X 10(8) SRBC- and 5 X 10(9) SRBC-fed mice, respectively. Kinetic studies revealed that IgA helper cells or IgG suppressor cells appeared in PP 2 days after oral administration and 4 days after it in the spleen.     

In vitro anamnestic antibody response: differential cellular and calcium requirements for induction by antigen and regulation by cyclic AMP.

Lymph node cells from rabbits, immunized 6 clays previously with keyhole limpet hemocyanin (KLH) were fractionated on columns containing nylon fibers. The non-retained population (effluent cells) and the retained population (adherent cells) were subsequently characterized by various criteria. The addition of dibutyryl cAMP (DbcAMP) or cholera enterotoxin (CT) during induction by 1 and 100 μg KLH resulted in a >100% increase in antibody synthesis over the control (KLH only) responses in the unfractionated and adherent cell populations. In the effluent population CT and DbcAMP failed to enhance the 1 μg response, but did increase the 100 μg response. Antibody forming cells, as judged by ongoing antibody synthesis during the first 24 hr of culture, were deficient in the effluent population. Both the effluent and adherent cells responded to the mitogens concanavalin A, phytohemagglutinin, and goat anti-rabbit Fab'. The control, effluent, and adherent populations each contained approximately 45% surface Ig positive cells as judged by direct immunofluorescence. The removal of calcium from the medium during induction (0–24 hr) also demonstrated that induction of the antibody response by KLH was separable from the cAMP mediated enhancement of antibody synthesis.     

An in vitro model for induction of immunological unresponsiveness to turkey gamma-globulin in primed mouse spleen cells. I. The secondary in vitro response and induction of unresponsiveness.

Spleen cells from mice previously immunized with turkey γ-globulin (TGG) were shown to give a vigorous secondary response in vitro when challenged in Mishell-Dutton cultures with TGG covalently coupled to pig erthrocytes (TGG-PRBC). However, 90–100% of the response could be abrogated by the incorporation of soluble TGG (sTGG) into the culture medium at concentrations greater than 1 mg/ml. Unresponsiveness, as measured by the absence of plaque-forming cells (PFC) in cultures receiving sTGG, was found to be antigen specific in that these cultures were still able to give normal PFC responses to sheep or burro erythrocytes. Spleen cells incubated with sTGG for short periods of time were shown to remain unresponsive after removal of sTGG from the culture and addition of TGG-PRBC. A 1-hr exposure period resulted in greater than 70% Unresponsiveness and a complete unresponsive state required only 8 hr of exposure. In contrast to the continued Unresponsiveness of sTGG-treated cells in vitro, spleen cells incubated with sTGG for 24 hr were fully responsive to an immunogenic challenge with alum-precipitated TGG when they were transferred into irradiated syngeneic mice. These data suggest that the readily induced unresponsive state in cultures of TGG primed cells may involve either a reversible antigen blockade of antigen-sensitive lymphocytes or a peripheral inhibition of reactive cells by suppressor lymphocytes.