Passive enhancement of mouse tumor allografts by alloantibodies is Fc-dependent.

Enhancement of growth of a B10.D2 fibrosarcoma in B6AF1 recipients could be induced by administration of B6AF1 anti-B10.D2 lymphocyte serum. The role of the Fc part in this phenomenon was studied by treatment of the B6AF1 recipients with F(ab')2 fragments of enhancing alloantibodies. A highly purified F(ab')2 preparation was used to exclude any effects of undigested IgG. Administration of F(ab')2 did not lead to enhancement of the tumor allografts, not even when given in a dose that was 22 times the molar amount of the lowest enhancing dose of undigested IgG. We therefore conclude that passive enhancement of mouse tumor allografts by alloantibodies is Fc dependent.     

Immune regulation by monoclonal antibodies: failure to enhance mouse skin allografts

Monoclonal anti-H-2k alloantibodies were analyzed for their capacity to enhance the survival of B10.A skin grafted onto B10.D2 recipients. Included were five anti-class I and four anti-class II antibodies. In contrast to conventional B10.D2 anti-B10.A serum, none of the individual anti-class I or anti-class II monoclonal antibodies induced enhancement. The same negative results were obtained with various mixtures of anti-class I, anti-class II, or anti-class I + anti-class II antibodies. The failure to induce enhancement was not due to inefficient antigen binding in vivo, because monoclonal antibodies were as effective as conventional B10.D2 anti-B10.A serum in the induction of acute antibody-mediated graft rejection, and in the opsonization of 51Cr-labeled B10.A leukocytes injected into B10.D2 recipients pretreated with antibody. These results demonstrate that monoclonal antibodies cannot always substitute for conventional sera, at least not in immune regulation. They also show that although opsonization may be a prerequisite for the induction of enhancement, it does not guarantee that enhancement will invariably occur.     

Modulation of natural cytotoxicity by alloantibodies. I. Alloantisera enhancement of cytotoxicity of mouse spleen cells toward a human myeloid cell line.

Natural killer activity of spleen cells obtained from different strains of mice against the human myeloid leukemia cell line, K562, and two mouse cell lines P815 and L1210 was measured by using the 4-hr chromium release assay. The level of cytotoxic activity of spleen cells against the K562 target was usually less than 4% lysis. However, treatment of the spleen cells with a specific anti-H-2 antiserum resulted in a dose-dependent augmentation of the degree of lysis of K562 cells. The augmentation of cytotoxic activity could be obtained by pretreatment of the spleen cells with antisera or by directly adding the antisera to the cytotox-incubation medium. Anti-thy-1 and anti-immunoglobulin antisera had no enhancing effect under similar conditions. The specific alloantisera-treated spleen cells did not show any increase in cytotoxicity against P815 and L1210 target cells. Spleen cells responsible for the alloantiserum-mediated augmentation of cytotoxicity against K562 cells appear to be different from T or B cells as indicated by their resistance to anti-thy-1 and complement treatment and lack of adherence to nylon wool columns.     

Acid-induced aggregation propensity of nivolumab is dependent on the Fc

Nivolumab, an anti-programmed death (PD)1 IgG₄ antibody, has shown notable success as a cancer treatment. Here, we report that nivolumab was susceptible to aggregation during manufacturing, particularly in routine purification steps. Our experimental results showed that exposure to low pH caused aggregation of nivolumab, and the Fc was primarily responsible for an acid-induced unfolding phenomenon. To compare the intrinsic propensity of acid-induced aggregation for other IgGs subclasses, tocilizumab (IgG₁), panitumumab (IgG₂) and atezolizumab (aglyco-IgG₁) were also investigated. The accurate pH threshold of acid-induced aggregation for individual IgG Fc subclasses was identified and ranked as: IgG₁?1?2?4. This result was cross-validated by thermostability and conformation analysis. We also assessed the effect of several protein stabilizers on nivolumab, and found mannitol ameliorated the acid-induced aggregation of the molecule. Our results provide valuable insight into downstream manufacturing process development, especially for immune checkpoint modulating molecules with a human IgG₄ backbone.     

Receptor modulation by Fc gamma RI-specific fusion proteins is dependent on receptor number and modified by IgG.

The high-affinity IgG receptor, FcgammaRI (CD64), is constitutively expressed exclusively on professional APCs. Human FcgammaRI binds monomeric IgG with high affinity and is, therefore, saturated in vivo. The binding of IgG to FcgammaRI causes receptor recycling, while Abs that cross-link FcgammaRI cause rapid down-modulation of surface FcgammaRI. Because studies performed in the absence of ligand may not be representative of FcgammaRI modulation in vivo, we investigated the ability of FcgammaRI-cross-linking Abs and non-cross-linking derivatives to modulate FcgammaRI in the presence and absence of ligand. In the absence of ligand mAb H22 and wH22xeGFP, an enhanced green fluorescent protein (eGFP)-labeled fusion protein of H22, cross-linked and rapidly down-modulated surface FcgammaRI on the human myeloid cell line, U937, and its high FcgammaRI-expressing subclone, 10.6. This effect was dependent on the concentration of fusion protein and the level of FcgammaRI expression and correlated with internalization of both wH22xeGFP and FcgammaRI, itself, as assessed by confocal microscopy. A single-chain Fv version, sFv22xeGFP, which does not cross-link FcgammaRI, was unable to modulate FcgammaRI in the absence of IgG. However, if ligand was present, treatment with either monovalent or cross-linking fusion protein led to intracellular receptor accumulation. These findings suggest at least two alternate mechanisms of internalization that are influenced by ligand and demonstrate the physiologic potential of FcgammaRI to transport a large antigenic load into APCs for processing. These studies may lead to the development of better FcgammaRI-targeted vaccines, as well as therapies to down-modulate FcR involved in autoimmune diseases.     

Effects of monoclonal anti-H-2Ld and anti-H-2Dd alloantibodies in the immunological enhancement of skin grafts and neonatal heart grafts in the mouse

Three anti-H-2Ld and two anti-H-2Dd monoclonal alloantibodies were analyzed for their capacity to enhance skin graft and neonatal heart graft survival. Of two anti-H-2Ld antibodies with the same specificity but with different isotypes, IgG2a antibody 30-5-7S prolonged graft survival in a skin graft combination with an Ld difference, whereas IgM antibodies did not. A second IgG2a antibody, but with a specificity different from 30-5-7S, was ineffective on its own. However, when mixed with 30-5-7S, skin graft survival was augmented as compared with the prolongation by 30-5-7S alone. Enhancement by anti-H-2Ld antibodies was dependent on the extent of the H-2 graft barrier. It was abrogated on extension of the graft barrier to a D-end H-2 difference by using the B10.A----B10.BR combination. Also, anti-Dd antibodies, either alone or in combination with anti-Ld, were ineffective in this skin graft combination. By using the same graft combination but the less immunogenic neonatal heart graft model, anti-Ld antibodies were still ineffective, but both anti-Dd antibodies were able to enhance graft survival from 15 to 22 days. When mixed with anti-Ld antibody 30-5-7S, graft survival was augmented further to 30 days. These results indicate that two kinds of enhancing alloantibodies may be distinguished. One category interacts with immunodominant epitopes on H-2 molecules, but their effectiveness may be limited to a particular H-2 difference, because immunodominance may vary from one graft barrier to another. In the second category, antibodies are ineffective on their own but they are able to potentiate the effects of antibodies of the first kind. These allocations are relative, however, because they are dependent on the type of graft examined.     

The neonatal Fc receptor is not required for mucosal infection by mouse mammary tumor virus.

The milk-borne mouse mammary tumor virus (MMTV) infects newborn mice via the intestine. Infection is initially restricted to Peyer's patches and later spreads to the epithelial cells of the mammary gland. The receptor that mediates uptake and transport of MMTV across the intestinal barrier has not yet been identified, The neonatal Fc receptor (nFcR), which is expressed by enterocytes during the first two weeks of life, is downregulated at weaning, and its disappearance correlates with the onset of intestinal resistance to MMTV. To test whether the nFcR mediates transport and allows infection, we foster nursed on infected MMTV mothers beta2 microglobulin-deficient (beta2m-deficient) newborn mice that are unable to express the nFcR at the surface of their enterocytes. Exposure of beta2m-deficient mice to milk-borne virus resulted in the deletion of peripheral blood T cells reactive to the superantigen encoded by MMTV. Since beta2m-deficient newborn mice are susceptible to MMTV infection despite the lack of the nFcR, we conclude that the nFcR is not required for MMTV transport.     

IgG-mediated enhancement of antibody responses is low in Fc receptor gamma chain-deficient mice and increased in Fc gamma RII-deficient mice.

Immunization with IgG/Ag or IgE/Ag complexes leads to a higher production of specific Abs than immunization with Ag alone. The enhancing effect of IgE is exclusively dependent upon the low-affinity receptor for IgE, Fc epsilon RII, whereas the mechanism behind IgG-mediated enhancement is unknown. We have investigated whether receptors for the Fc part of IgG are required for responses to IgG/Ag. Mice lacking the gamma subunit of Fc receptors (FcRs) (FcR gamma-/-), Fc gamma RII (Fc gamma RII-/-), or Fc gamma RIII (Fc gamma RIII-/-) were immunized with BSA-2,4,6-trinitrophenyl (TNP) alone or BSA-TNP complexed to monoclonal TNP-specific IgG1, IgG2a, or IgG2b. As expected, all subclasses enhanced the Ab-response to BSA in wild-type mice. Enhancement was in the same order of magnitude in Fc gamma RIII-/- mice (相似文献   

Allergen-induced airway disease is mouse strain dependent

We investigated the development of airway hyperreactivity (AHR) and inflammation in the lungs of nine genetically diverse inbred strains of mice [129/SvIm, A/J, BALB/cJ, BTBR+(T)/tf/tf, CAST/Ei, C3H/HeJ, C57BL/6J, DBA/2J, and FVB/NJ] after sensitization and challenge with ovalbumin (OVA). At 24, 48, and 72 h post-OVA exposure, the severity of AHR and eosinophilic inflammation of the mouse strains ranged from relatively unresponsive to responsive. The severity of the airway eosinophilia of some strains did not clearly correlate with the development of AHR. The temporal presence of T helper type 2 cytokines in lung lavage fluid also varied markedly among the strains. The levels of IL-4 and IL-13 were generally increased in the strains with the highest airway eosinophilia at 24 and 72 h postexposure, respectively; the levels of IL-5 were significantly increased in most of the strains with airway inflammation over the 72-h time period. The differences of physiological and biological responses among the inbred mouse strains after OVA sensitization and challenge support the hypothesis that genetic factors contribute, in part, to the development of allergen-induced airway disease.     

Glucose enhancement of LDL oxidation is strictly metal ion dependent

Recent evidence suggests that lipoprotein oxidation is increased in diabetes, however, the mechanism(s) for such observations are not clear. We examined the effect of glucose on low-density lipoprotein (LDL) oxidation using metal ion-dependent and -independent oxidation systems. Pathophysiological concentrations of glucose (25 mM) enhanced copper-induced LDL oxidation as determined by conjugated diene formation or relative electrophoretic mobility (REM) on agarose gels. Similarly, iron-induced LDL oxidation was stimulated by glucose resulting in 4- to 6-fold greater REM than control incubations without glucose. In contrast, glucose had no effect on metal ion-independent LDL oxidation by aqueous peroxyl radicals. The effect of glucose on metal ion-dependent LDL oxidation was associated with enhanced reduction of metal ions, and in the case of iron-induced LDL oxidation, was completely inhibited by superoxide dismutase. The effect of glucose was mimicked by other reducing sugars, such as fructose and mannose, and the extent to which each sugar enhanced LDL oxidation was closely linked to its metal ion-reducing activity. Thus, promotion of LDL oxidation by glucose is specific for metal ion-dependent oxidation and involves increased metal ion reduction. These results provide one potential mechanism for enhanced LDL oxidation in diabetes.     

Tolerance to antigen-presenting cell-depleted islet allografts is CD4 T cell dependent

Pretreatment of pancreatic islets in 95% oxygen culture depletes graft-associated APCs and leads to indefinite allograft acceptance in immunocompetent recipients. As such, the APC-depleted allograft represents a model of peripheral alloantigen presentation in the absence of donor-derived costimulation. Over time, a state of donor-specific tolerance develops in which recipients are resistant to donor APC-induced graft rejection. Thus, persistence of the graft is sufficient to induce tolerance independent of other immune interventions. Donor-specific tolerance could be adoptively transferred to immune-deficient SCID recipient mice transplanted with fresh immunogenic islet allografts, indicating that the original recipient was not simply "ignorant" of donor antigens. Interestingly, despite the fact that the original islet allograft presented only MHC class I alloantigens, CD8+ T cells obtained from tolerant animals readily collaborated with naive CD4+ T cells to reject donor-type islet grafts. Conversely, tolerant CD4+ T cells failed to collaborate effectively with naive CD8+ T cells for the rejection of donor-type grafts. In conclusion, the MHC class I+, II- islet allograft paradoxically leads to a change in the donor-reactive CD4 T cell subset and not in the CD8 subset. We hypothesize that the tolerant state is not due to direct class I alloantigen presentation to CD8 T cells but, rather, occurs via the indirect pathway of donor Ag presentation to CD4 T cells in the context of host MHC class II molecules.     

Lowering of interstitial fluid pressure after neurogenic inflammation in mouse skin is partly dependent on mast cells

Neurogenic inflammation is known to induce lowering of interstitial fluid pressure (P(if)) in mouse skin. This study examined the possible role of mast cell activation secondary to neuropeptide release in lowering of P(if) by using Kit(W)/Kit(W-v) mice, which are devoid of mast cells, including connective tissue mast cells (CTMCs). P(if) was measured in paw skin of anesthetized (fentanyl-fluanison and midazolam, 1:1) mice with glass capillaries connected to a servo-controlled counterpressure system. In contrast to wild-type mice, intravenous administration of mast cell-activating compound 48/80 induced no lowering of P(if) in Kit(W)/Kit(W-v) mice. Intravenous challenge with substance P (SP), calcitonin gene-related peptide (CGRP), or capsaicin induced a significant (P < 0.05) lowering of P(if) in wild-type mice to -2.16 +/- 0.28, -1.96 +/- 0.11, and -2.22 +/- 0.19 mmHg, respectively, compared with vehicle (-0.49 +/- 0.11 mmHg). In Kit(W)/Kit(W-v) mice the P(if) response to SP was completely abolished (-0.53 +/- 0.32 mmHg) while the response to CGRP and capsaicin was attenuated (-1.33 +/- 0.13 and -1.42 +/- 0.13 mmHg, respectively) although significantly (P < 0.05) lowered compared with vehicle. Immunohistochemical analysis revealed no difference in distribution or density of SP- and CGRP-immunoreactive fibers in paws of Kit(W)/Kit(W-v) compared with wild-type mice. We conclude that lowering of P(if) normally depends on mast cells. However, the sensory nerves can also elicit a lowering of P(if) that is independent of mast cells.     

Variable effect of anH-21 barrier on response to skin allografts in the mouse

(AQR×B10)F₁ mice were grafted with skin from donors differing in theK, I, KI, andISD regions of theH-2 complex. A dichotomy was observed in the fate of theH-2I-disparate grafts: either they were rejected acutely within the second week or were accepted indefinitely. Acceptances were much more common among male than female hosts. Acceptor status was limited to the I group, was unpredictable in occurrence, was not well-correlated with positive serum anti-Ia titers, and did not confer protection of grafts that were alike atH-2I but different atH-2K orH-2D. Since theH-2I barrier studied here elicited such divergent responses in genetically identical hosts, it is unlikely that any histocompatibility typing test could predict graft fate.Abbreviations used in this paper are MST median survival time - MHC major histocompatibility complex - CTL cytotoxic T lymphocyte - B10 C57 BL/10 - 6R BIO.T(6R) - B10.A BIO. ASn - H-2-Ia serologically detected antigens coded in theI region ofH-2 This term is used in preference toIa, since it has recently been shown that Ia-like alloantigens may be coded outside the MHC (Dickleret al. 1975).     

Long-term survival of corneal allografts is dependent on intact CD1d-reactive NKT cells

BALB/c mice that tolerate the allogeneic grafts develop allogeneic-specific anterior chamber-associated immune deviation. Because CD1d-reactive NKT cells are essential for anterior chamber-associated immune deviation, we postulated that the survival of C57BL/6 (B6) cornea graft in BALB/c mice was also dependent on CD1d-reactive NKT cells. The B6 corneal graft rejection rate in BALB/c vs Jalpha281 knockout (KO) mice, which lack NKT cells, was measured. While there were no difference in the early phase of rejection, the survival rates at 12 wk after grafting for BALB/c and Jalpha281 KO mice were 50 and 0%, respectively. Because anti-CD1d mAb abrogated the corneal graft survival in the wild-type mice we concluded that CD1d-reactive NKT cells were essential for graft survival. Moreover, allospecific T regulatory (Tr) cells correlated with acceptance of B6 grafts in BALB/c mice, and the adoptive transfer of these allospecific Tr cells to Jalpha281 KO mice allowed a 50% survival rate of B6 cornea grafts. In conclusion, CD1d-reactive NKT cells are required for induction of allospecific Tr cells and are essential for survival of corneal allografts. Mechanisms that contribute to cornea graft acceptance may lead to new therapies for improvement in graft survival in high-risk corneas and other transplanted tissues and grafts.     

Denitrification by Alcaligenes eutrophus is plasmid dependent.

Curing of the hydrogenase-specifying megaplasmid pHG indigenous to strains of the facultative lithoautotrophic bacterium Alcaligenes eutrophus was correlated with a loss of denitrifying ability (Nitd). The retransfer of plasmid pHG1 reconstituted the Nitd phenotype. Plasmid-free mutants were still capable of converting some nitrate to nitrite, but they did not metabolize nitrite under anaerobic conditions.