The localization of aggregated human γ-globulin in the spleens of normal mice

The localization of aggregated human γ-globulin in the germinal centres of the white pulp of the spleens of normal mice was found to be dependent on the ability of the spleen to concentrate this material from the blood and of lymphoid cells in the mantle layer of the Malpighian bodies to take it up on their surface membranes. The fact that lymphoid cells resident in the spleen perform this function was shown by the inability of lymphoid cells transferred to recipient animals to transport the aggregated material to the spleens of the latter.

Ionizing radiation in relatively high doses prevented the localization of aggregated human γ-globulin and was effective only when given before or less than 6 hours after administration of the aggregated material. Spleens shielded during whole body irradiation were capable of localizing the material in germinal centres, but local irradiation of the spleens with protection of the rest of the body prevented splenic localization.

These results support the conclusion that localization of altered heterologous antibody is a function of lymphoid cells already present in the spleens at the time of injection of this material and is not dependent on its transport into the white pulp by migrating cells which pick it up in the blood or elsewhere.

Antilymphocytic serum also prevented localization of aggregated human γ-globulin, suggesting a direct effect of this antiserum on non-circulating lymphoid cells. However, the manner of its action requires further study.

     

Tolerance of turkey γ-globulin in chickens

The effect of injection of turkey γ-globulin (TGG) at hatching upon the subsequent response to immunization with TGG has been studied in chickens, using, for measurement, elimination of [¹³¹I]TGG. Tolerance was obtained, which depended in duration on the quantity of TGG initially injected, over the dose range of 10–100 mg. The duration of tolerance could be extended by subsequent injection of TGG up to at least 29–31 weeks. The period of susceptibility to a tolerance-prolonging dose of TGG is itself of limited duration.     

Binding of γ-globulin fragments to muscle tissue

Serum γ-globulins from normal persons and from twenty-four patients with myasthenia gravis were tested in the antiglobulin consumption test before and after pepsin digestion.

F(ab')₂ fragments prepared from normal sera did not bind to muscle tissue, while Fc fragments did. Accordingly, muscle binding γ-globulins from normal sera bind to the tissue only by the Fc portion of the molecule.

F(ab')₂ preparations from sera from patients with myasthenia gravis combined with the tissue. In twelve of these sera, the amount of muscle binding γ-globulin was shown to be quantitatively within normal limits. The difference in binding capacity registered between the different patient sera is quantitative, while the data indicate a qualitative difference between normal and patient sera.

     

Immunogenic potency of human γ-globulin in mice

Heat-treated, but not untreated, human γ-globulin (HGG) is a potent antigen for the initiation of primary antibody response in mice. Thus a dose as low as 10^-7 g of heat-treated HGG was sufficient to induce an excellent primary response in intact mice. However, the primary response was meagre and negative in the cell-transfer and diffusion-chamber culture systems of dispersed spleen cells, respectively. In contrast, no significant difference could be demonstrated between heat-treated and untreated HGG preparations in their capacity to induce a secondary response in intact mice. Furthermore, good secondary responses were obtained in both the cell-transfer and diffusion-chamber culture systems. This suggests differences between primed and non-primed spleen cells in their requirements of cellular organization for the initiation of antibody response. Both the primary and the secondary responses were found to be antigen—dose dependent, although the optimum antigen dose range was broad. Moreover, doses of heat-treated HGG (≤10^-12 g) lower than that reported for the excellent immunogen, flagella, were sufficient to induce secondary responses, thereby emphasizing the utility of HGG for studying the role of antigen in the initiation of primary and secondary antibody responses.     

Studies on passive cutaneous anaphylaxis in the rat with disodium cromoglycate: I. Cutaneous reactions induced by an anti-DNP 7Sγ2 antibody

The 4-hour passive cutaneous anaphylaxis reaction (PCA) in rats induced by rat anti-DNP 7Sγ₂ antibody has been investigated and compared with the rat reagin induced PCA reaction. Time course studies revealed that the PCA reaction was made up of at least two parts, an early immediate reaction which involved mast cell degranulation, was inhibited by disodium cromoglycate and by cyproheptadine and a late reaction unaffected by disodium cromoglycate, by cyproheptadine and by an anti-SRS-A agent, diethyl-carbamazine.

The early part of the 7Sγ₂ reaction was found to be comparable to the rat reagin PCA reaction whereas the later part of the 7Sγ₂ reaction does not appear to involve similar pathways or mediators.

     

In vitro absorption of γ-globulin by neonatal intestinal epithelium of the pig

1. An in vitro method, using fluorescent gamma-globulin and everted neonatal pig's intestinal slices, for the study of the active transport of large molecules is described.2. Uptake of gamma-globulin occurred within 15 min and required no exogenous substrates.3. In vitro absorption of gamma-globulin by intestinal epithelium was limited to the neonatal pig and 5-day-old mouse. No uptake was seen in intestines from a mature mouse, a pig with diarrhoea, a normal pig, a mature rabbit, a guinea-pig, a chick, and a chick embryo. Chick embryo yolk sac readily took up gamma-globulin.4. Rings of everted intestinal epithelium remained active (still absorbed gamma-globulin) after incubating for 4-6 hr in balanced salt solution (BSS).5. Uptake of gamma-globulin required oxygen and sodium and was reversibly inhibited by metabolic antagonists such as iodoacetate, arsenate, fluoride, 4,6-dinitro-varphi-cresol, phlorrhizin, anaerobiosis and cold. Under the conditions of the test, large colloidal molecules did not inhibit uptake of gamma-globulin.6. Similar results (although not as clear-cut) with metabolic inhibitors were obtained with preparations of chick embryo yolk sacs.7. Injuring mature pig's intestinal epithelium with surface-active agents did not produce non-specific absorption artifacts that resembled the specific absorption found in immature pig's intestinal epithelium.     

Effects of different drugs on passive cutaneous anaphylaxis elicited in the mouse ear at 1.5 hours

IgG1, antibody-mediated homologous passive cutaneous anaphylaxis at 1.5 h (1.5-hour PCA) was elicited in the ears of mice and the effects of different drugs were studied. The reaction, as measured by the amount of extravasated dye, was inhibited by antihistamines, antiserotonins, cyclic AMP-elevating agents, tranilast and ketotifen, but not by an SRS-A antagonist (FLP 55712), lipoxygenase inhibitors, cyclooxygenase inhibitors and disodium cromoglycate. These results suggest that the pharmacological profile of 1.5-hour PCA resembles that of 48-hour PCA mediated by IgE antibody in the mouse ear.     

Studies on passive cutaneous anaphylaxis in the rat with disodium cromoglycate: II. A comparison of the rat anti-DNP 7Sγ2 and rat reagin induced cutaneous reactions

A study of the passive cutaneous anaphylactic reaction induced by anti-DNP 7Sγ₂ antibodies was undertaken in rats to compare this anaphylactic reaction with that induced by rat reagin. This study revealed that disodium cromoglycate was capable of inhibiting the 7Sγ₂ induced PCA reaction immediately following antigen challenge but not the slow developing late reaction. Using various doses of disodium cromoglycate on a series of rat reagin PCA reactions and on a series of early 7Sγ₂ induced PCA reactions, similarities in dose dependence were demonstrated suggesting that certain pathways might be common to both reactions. Studies using 48/80 treatment after sensitization revealed inhibition of both early and late 7Sγ₂ reactions. Concomitant sensitization with rat reagin and 7Sγ₂ at various concentrations demonstrated that either antibody was capable of inhibiting the reaction induced by the other antibody, suggesting similar sensitization sites might be involved. In actively sensitized rats with a high reagin titre we were unable to induce a 7Sγ₂ or rat reagin PCA reaction whereas a cutaneous reaction of the `Arthus' type was readily induced. It is concluded that passive sensitization by anti-DNP 7Sγ₂ antibodies has certain factors in common with sensitization by rat reagin and that the sensitized mast cell plays a vital role in the elicitation of both early and late 7Sγ₂ PCA reactions.     

Presence of aggregated γG-globulin in certain rheumatoid synovial effusions

A precipitin reaction occurred between rheumatoid arthritis sera with high titre rheumatoid factor (RF) and certain rheumatoid synovial effusions. The precipitating factor in the sera was shown to be γM-type RF, reacting mainly with human γG-globulin. The precipitating component of synovial effusions was structurally altered, γG-globulin sedimenting much faster than native γG-globulin by density gradient ultracentrifugation. Following exposure to increasing hydrogen ion concentrations, an increasing amount of the RF-precipitating γG-globulin attained a slower sedimentation rate. Evidence for the in vivo presence of this altered γG-globulin was given, indicating that it may represent a stimulus for RF production.     

Passive haemagglutination for estimation of early and late anti-human γ-globulin antibodies

The influence of various concentrations of human γ-globulin (HGG) employed to sensitize tanned sheep erythrocytes on haemagglutination titres with various early and late mouse and rabbit antisera to HGG has been studied. The concentration of HGG employed to sensitize tanned erythrocytes which gives the highest haemagglutination titres with early, mercaptoethanol-sensitive antibodies was not optimal for obtaining highest haemagglutination titres with late, mercaptoethanol-insensitive antibodies. Similar results were obtained with heavy and light sucrose gradient ultracentrifugation fractions of a late and early rabbit antiserum. These findings may account for past discrepancies and should be considered when employing the haemagglutination test to detect early and late antibodies.     

Transmission of human γ-globulin to rabbit foetus and its inhibition by conjugates with ferritin

The transmission of a ferritin—human γ-globulin conjugate across the rabbit yolk sac splanchnopleur has been investigated by means of the fluorescent antibody technique, and a comparison made with the transmission of the separate components. Ferritin alone was not transmitted to the foetal circulation and neither was the conjugate, whereas human γ-globulin alone was readily transmitted. However, both the conjugate and the separate components were readily transmitted to the exocoelomic and amniotic fluids by traversing the paraplacental chorion. These results are discussed in the light of a proposed hypothesis to explain selective transfer of proteins.     

Immune injury of mouse peritoneal mast cells by antibodies to mouse serum and mouse γ-globulin

In vitro degranulation of mouse peritoneal mast cells and release of histamine were induced by the addition at 37° of anti-mouse serum or antimouse γ-globulin rabbit antibody. The release of histamine does not depend upon complement, and seems to be due to the reaction of the antibody with the homologous γ-globulin present on the surface of the mast cells.     

Proteolytic activity during the absorption of [131I]γ-globulin in the new-born calf

1. Proteolytic activity within the small intestine of unsuckled calves less than 20 hr of age, anaesthetized with sodium pentobarbitone, has been assessed from the break-down of [(131)I]bovine serum gamma-globulin infused into the duodenum.2. Absorption of [(131)I]gamma-globulin was measured by analysis of venous blood, the levels of radioactivity attained in which were comparable with those when [(131)I]PVP K.60 (mean mol.wt. 160,000) was administered. When lymph collected from the thoracic duct during the absorption of [(131)I]gamma-globulin was injected into the femoral vein, the levels of radioactivity in the blood were close to those expected if the labelled material in the lymph had been retained within the plasma. These observations suggested that [(131)I]gamma-globulin was absorbed into the circulation of the anaesthetized young calf without significant break-down.3. Gel-filtration of lymph and plasma from calves fed [(131)I]gamma-globulin has confirmed that proteolysis before and during absorption was slight, since little (131)I labelled material of low mol.wt. was found.4. Gel-filtration of the contents of the alimentary tract from calves fed [(131)I]gamma-globulin showed that some hydrolysis occurred in the abomasum and duodenum and that this was reduced by barbiturate anaesthesia. Protein break-down in the terminal ileum was slight both in the conscious animal and in animals anaesthetized with sodium pentobarbitone.     

Processing of Mycobacterium tuberculosis Bacilli by Human Monocytes for CD4+ αβ and γδ T Cells: Role of Particulate Antigen

Mycobacterium tuberculosis readily activates both CD4⁺ and Vδ2⁺ γδ T cells. Despite similarity in function, these T-cell subsets differ in the antigens they recognize and the manners in which these antigens are presented by M. tuberculosis-infected monocytes. We investigated mechanisms of antigen processing of M. tuberculosis antigens to human CD4 and γδ T cells by monocytes. Initial uptake of M. tuberculosis bacilli and subsequent processing were required for efficient presentation not only to CD4 T cells but also to Vδ2⁺ γδ T cells. For γδ T cells, recognition of M. tuberculosis-infected monocytes was dependent on Vδ2⁺ T-cell-receptor expression. Recognition of M. tuberculosis antigens by CD4⁺ T cells was restricted by the class II major histocompatibility complex molecule HLA-DR. Processing of M. tuberculosis bacilli for Vδ2⁺ γδ T cells was inhibitable by Brefeldin A, whereas processing of soluble mycobacterial antigens for γδ T cells was not sensitive to Brefeldin A. Processing of M. tuberculosis bacilli for CD4⁺ T cells was unaffected by Brefeldin A. Lysosomotropic agents such as chloroquine and ammonium chloride did not affect the processing of M. tuberculosis bacilli for CD4⁺ and γδ T cells. In contrast, both inhibitors blocked processing of soluble mycobacterial antigens for CD4⁺ T cells. Chloroquine and ammonium chloride insensitivity of processing of M. tuberculosis bacilli was not dependent on the viability of the bacteria, since processing of both formaldehyde-fixed dead bacteria and mycobacterial antigens covalently coupled to latex beads was chloroquine insensitive. Thus, the manner in which mycobacterial antigens were taken up by monocytes (particulate versus soluble) influenced the antigen processing pathway for CD4⁺ and γδ T cells.

Mycobacterium tuberculosis, the etiologic agent of human tuberculosis, is spread readily from person to person by inhalation of aerosolized mycobacteria (8). A hallmark of M. tuberculosis infection is the ability of most healthy individuals to control the infection by mounting an acquired immune response, in which antigen-specific T cells and mononuclear phagocytes arrest the growth of M. tuberculosis bacilli and maintain control over dormant bacilli within granulomas (reviewed in reference 25). This protective cellular immune response results in conversion of the tuberculin skin test from negative to positive and probably in increased resistance to reinfection with tubercle bacilli.CD4⁺ αβ-T-cell-receptor (αβ TCR)-bearing T cells (CD4⁺ T cells) are readily activated by mycobacterial antigens and have a dominant role in the protective immune response to M. tuberculosis in humans (2, 34). These CD4⁺ T cells not only secrete cytokines but also serve directly as cytotoxic effector cells against M. tuberculosis-infected macrophages (6). In addition to CD4⁺ T cells, M. tuberculosis antigens activate other human T-cell subsets such as γδ TCR⁺ T cells (γδ T cells) (15, 16, 18). Vδ2⁺ and Vγ9⁺ γδ T cells are particularly responsive to live M. tuberculosis (15). A role for both γδ and CD4⁺ T cells in protective immunity to acute M. tuberculosis infection has been demonstrated in murine models (20, 21, 26, 27). A recent study of humans suggests that Vγ9⁺ and Vδ2⁺ γδ T-cell numbers and function are reduced in tuberculosis patients (23).Functional comparisons of human CD4⁺ and γδ T-cell responses of healthy tuberculin-positive persons demonstrate that both T-cell subsets have similar cytotoxic effector functions for M. tuberculosis-infected monocytes and produce large amounts of gamma interferon (IFN-γ), with γδ T cells being slightly more efficient producers of IFN-γ than CD4⁺ T cells (37). Despite similarities in function, these two T-cell subsets differ in the mycobacterial antigens recognized by their TCRs and the manners in which antigens are presented to them by M. tuberculosis-infected mononuclear phagocytes. CD4⁺ T cells recognize a wide diversity of mycobacterial peptides in the context of class II major histocompatibility complex (MHC) molecules, which include secreted as well as somatic antigens (6, 13, 33, 37). In contrast, Vγ9⁺ and Vδ2⁺ γδ T cells, the dominant γδ TCR subsets activated by M. tuberculosis, recognize mycobacterial antigens in a non-MHC-restricted manner and the repertoire of antigens includes small phosphate-containing antigens such as TUBag’s (5, 9, 19, 22, 29, 36).Both blood monocytes and alveolar macrophages infected with M. tuberculosis are efficient antigen-presenting cells for mycobacterial antigen-specific CD4⁺ and γδ T cells (1, 5). However, little is known about how M. tuberculosis-infected mononuclear phagocytes process antigens for these two T-cell subsets. M. tuberculosis bacilli are taken up by mononuclear phagocytes through a variety of surface receptors, including complement receptor 4, mannose receptor, and complement receptor 3 (17, 31, 32). Within mononuclear phagocytes, the mycobacteria reside within phagosomes and modulate the phagosome by preventing fusion with acidic lysosomal compartments (7). Although the vacuolar membranes surrounding the phagosome acquire endosomal markers, the vesicular proton ATPase is actively excluded, resulting in an elevated pH of 6.3 to 6.5 compared to the normal lysosomal pH of 4.5 (7, 35). The elevated pH in the phagosome does not appear to inhibit the ability of mycobacterial antigens to be processed and presented to CD4⁺ and Vδ2⁺ γδ T cells. This study was undertaken to gain insight into the mechanisms used by monocytes infected with live M. tuberculosis bacilli to process mycobacterial antigens for presentation to both CD4⁺ and γδ T cells.     

The break-down of [131I]γ-globulin in the digestive tract of the new-born pig

1. The intestinal absorption of [(131)I]porcine and bovine serum gamma-globulin after oral administration has been investigated in conscious pigs less than 20 hr old. Absorption was measured by the concentration of (131)I in venous blood during the 6 hr after feeding and also by the distribution of (131)I between homogenates of the alimentary tract and the rest of the animal at the end of the experiment.2. The concentration of (131)I in the blood was always low after feeding [(131)I]gamma-globulin, although a large proportion of the isotope fed was found to have left the alimentary tract. This indicated that much of the [(131)I]-gamma-globulin had been hydrolysed into fragments of low mol.wt. which were not retained in the plasma. There were no significant differences between results obtained with homologous and heterologous gamma-globulin.3. Examination by gel-filtration confirmed that, after feeding [(131)I]-serum gamma-globulin, much of the (131)I in the plasma was associated with material of mol.wt. less than 12,400 and demonstrated that the break-down of bovine gamma-globulin was comparable with that of homologous gamma-globulin.4. Comparison of the absorption of [(131)I]serum gamma-globulin from colostrum with that from a chloride solution with a similar Na(+) and K(+) concentration showed that, although the blood concentration remained low, colostrum reduced the hydrolysis of the labelled protein.5. This effect of colostrum could be simulated by the addition to the chloride solution of either the synthetic trypsin inhibitor Trasylol or a higher concentration of unlabelled protein.6. Gel-filtration of samples of the contents of the stomach, duodenum and terminal ileum after feeding [(131)I]serum gamma-globulin showed that proteolysis occurred at all these sites.     

Single-cell analysis reveals the origins and intrahepatic development of liver-resident IFN-γ-producing γδ T cells

γδ T cells are heterogeneous lymphocytes located in various tissues. However, a systematic and comprehensive understanding of the origins of γδ T cell heterogeneity and the extrathymic developmental pathway associated with liver γδ T cells remain largely unsolved. In this study, we performed single-cell RNA sequencing (scRNA-seq) to comprehensively catalog the heterogeneity of γδ T cells derived from murine liver and thymus samples. We revealed the developmental trajectory of γδ T cells and found that the liver contains γδ T cell precursors (pre-γδ T cells). The developmental potential of hepatic γδ T precursor cells was confirmed through in vitro coculture experiments and in vivo adoptive transfer experiments. The adoptive transfer of hematopoietic progenitor Lin⁻Sca-1⁺Mac-1⁺ (LSM) cells from fetal or adult liver samples to sublethally irradiated recipients resulted in the differentiation of liver LSM cells into pre-γδ T cells and interferon-gamma⁺ (IFN-γ⁺) but not interleukin-17a⁺ (IL-17a⁺) γδ T cells in the liver. Importantly, thymectomized mouse models showed that IFN-γ-producing γδ T cells could originate from liver LSM cells in a thymus-independent manner. These results suggested that liver hematopoietic progenitor LSM cells were able to differentiate into pre-γδ T cells and functionally mature γδ T cells, which implied that these cells are involved in a distinct developmental pathway independent of thymus-derived γδ T cells.     

CFTR is a negative regulator of γδ T cell IFN-γ production and antitumor immunity

CFTR, a chloride channel and ion channel regulator studied mostly in epithelial cells, has been reported to participate in immune regulation and likely affect the risk of cancer development. However, little is known about the effects of CFTR on the differentiation and function of γδ T cells. In this study, we observed that CFTR was functionally expressed on the cell surface of γδ T cells. Genetic deletion and pharmacological inhibition of CFTR both increased IFN-γ release by peripheral γδ T cells and potentiated the cytolytic activity of these cells against tumor cells both in vitro and in vivo. Interestingly, the molecular mechanisms underlying the regulation of γδ T cell IFN-γ production by CFTR were either TCR dependent or related to Ca²⁺ influx. CFTR was recruited to TCR immunological synapses and attenuated Lck-P38 MAPK-c-Jun signaling. In addition, CFTR was found to modulate TCR-induced Ca²⁺ influx and membrane potential (V_m)-induced Ca²⁺ influx and subsequently regulate the calcineurin-NFATc1 signaling pathway in γδ T cells. Thus, CFTR serves as a negative regulator of IFN-γ production in γδ T cells and the function of these cells in antitumor immunity. Our investigation suggests that modification of the CFTR activity of γδ T cells may be a potential immunotherapeutic strategy for cancer.     

Integrins αvβ3 and α5β1 Mediate Attachment of Lyme Disease Spirochetes to Human Cells

Borrelia burgdorferi (sensu lato), the agent of Lyme disease, is able to cause chronic, multisystemic infections in human and animal hosts. Attachment of the spirochete to host cells is likely to be important for the colonization of diverse tissues. The platelet-specific integrin α_IIbβ₃ was previously identified as a receptor for all three species of Lyme disease spirochetes (B. burgdorferi sensu stricto, B. garinii, and B. afzelii). Here we show that B. burgdorferi also recognizes the widely expressed integrins α_vβ₃ and α₅β₁, known as the vitronectin and fibronectin receptors, respectively. Three representatives of each species of Lyme disease spirochete were tested for the ability to bind to purified α_vβ₃ and α₅β₁. All of the strains tested bound to at least one integrin. Binding to one integrin was not always predictive of binding to other integrins, and several different integrin preference profiles were identified. Attachment of the infectious B. burgdorferi strain N40 to purified α_vβ₃ and α₅β₁ was inhibited by RGD peptides and the appropriate receptor-specific antibodies. Binding to α_vβ₃ was also shown by using a transfected cell line that expresses this receptor but not α_IIbβ₃. Attachment of B. burgdorferi N40 to human erythroleukemia cells and to human saphenous vein endothelial cells was mediated by both α₅β₁ and α_vβ₃. Our results show that multiple integrins mediate attachment of Lyme disease spirochetes to host cells.     

Passive Protection of Mice Against Intraperitoneally Injected Vibrio cholerae by γG and γM Antibody

Fractions of rabbit anti-Vibrio cholerae serum containing gammaG antibodies were compared with fractions containing gammaM antibodies for their ability to protect mice against lethal infection resulting from the intraperitoneal injection of organisms suspended in mucin. About twice as much gammaG as gammaM (estimated by quantitative precipitation) was required to protect against approximately 1,000 50% lethal doses when the antibody was given intraperitoneally 4 hr before challenge. When protective serum fractions were given subcutaneously, however, the amount of gammaM required to protect was increased about 40-fold, whereas gammaG was about equally effective subcutaneously and intraperitoneally.