Inhibition of the T suppressor circuit of delayed-type hypersensitivity by interferon

The effects of electrophoretically pure murine interferon (Mu-IFN-alpha beta) on the T suppressor pathway and on the T effector cell of delayed hypersensitivity (TDH) were investigated in BALB/c mice, in a 2,4-dinitrofluorobenzene (DNFB) contact-sensitivity model. Various T cell subpopulations, suppressor T cells of the afferent (Ts-aff) and efferent (Ts-eff) types, an auxiliary Ts (Ts-aux), as well as TDH were induced, and their function was assessed in transfer experiments. The results were as follows. At a dose of 5 X 10(3) U, IFN was shown to inhibit the Ts-aff response, when given to the donor animal shortly after induction of the Ts-aff subpopulation or when injected into the recipient 2 hr after spleen cell transfer. Pretreatment in vitro with IFN of the splenic cells to be transferred also abolished the Ts-aff response. Similar amounts of IFN were able to inhibit the generation of Ts-eff in the donor animals, whereas 10-fold-higher amounts were needed in vivo or in vitro to block the functional expression of Ts-eff in the recipient animal. Intravenous injection of IFN into recipients of Ts-eff on day 0 and 1 after sensitization inhibited the expression of the Ts-eff transferred 1 day before ear challenge. This suggests that the Ts-aux response required for the TDH suppression by Ts-eff is blocked by IFN. Secretion of a suppressor factor by Ts in vitro was not blocked by IFN. Treatment of the donor of suppressor factor-secreting Ts with IFN, however, blocked the induction of this Ts. The TDH were not sensitive to IFN even at amounts approximately 100 times higher than those used for the Ts inhibition in vivo as well as in vitro. These results demonstrate that low amounts of IFN may selectively block the suppressor pathway, because induction of these regulatory T cell subsets appears to be particularly sensitive to IFN. The exact mechanism of the IFN-mediated inhibition of Ts is not yet clear. The data suggest an important regulatory function of IFN in delayed-type hypersensitivity (DTH) reactions.     

Regulatory responses in contact sensitivity: afferent suppressor T cells inhibit the activation of efferent suppressor T cells.

Two types of suppressor cells regulate the contact sensitivity (CS) response to picryl chloride (PCL). Afferent suppressor T cells (Ts-aff) inhibit the generation of CS responses to PCL, while efferent suppressor T cells (Ts-eff) inhibit the activity of Th 1 cells that mediate CS reaction. Intravenous injection of mice with TNP-substituted peritoneal exudate cells (TNP-PEC) induces Ts-eff cells that block the adoptive transfer of contact sensitivity. The induction of Ts-eff cells is prevented by the presence of Ts-aff cells, which in turn are induced by the injection of TNP-PEC coupled with antibodies of the IgG2a and IgG2b isotype (TNP-PEC-Ab). If an animal is injected with TNP-PEC prior to or simultaneously with TNP-PEC-Ab, it generates only Ts-aff cells, while if it is injected with TNP-PEC alone or TNP-PEC prior to TNP-PEC-Ab, it generates Ts-eff cells. Ts-aff cells effect only the generation of Ts-eff cells, as the addition of Ts-eff cells to assays for Ts-eff cells has no inhibitory effect on the suppressive effects of Ts-eff cells in adoptive transfer. Our experiments show that Ts-aff cells induced by TNP-PEC-Ab are phenotypically either Lyt 1+2- or Lyt 1-2+, but only the latter inhibit the generation of Ts-eff cells in vivo. The Ts-aff cells that inhibit Ts-eff activity adhere to the lectin Vicia villosa (VV), while Ts-eff cells are VV nonadherent. In addition, Ts-aff cells can prevent the generation of Ts-eff to linked haptens presented on the same PEC. It appears that a cascade of Ts cell interactions are involved in the regulation of CS responses.     

Regulation of contact sensitivity reaction: contrasuppressor T cells and contrasuppressor factor downregulate efferent T suppressor cells.

Contact sensitivity (CS) reaction mediated by CD 4+8- Th 1 cells is under the control of several antigen-specific regulatory lymphocytes. Reaction is downregulated at the induction stage by T afferent suppressor T cells (Ts-aff) that prevent immunization and at the effector stage by efferent T suppressor cells (Ts-eff) that made immune Th 1 cells inoperative. Both suppressor cells are CD 4-8+ Th 1 effector cells and are protected against the suppressive action of Ts-eff cells by CD 4+8- contrasuppressor T cells (Tcs). As has been already shown there are also regulatory interactions between regulatory cells themselves and Ts-aff cells in addition to their effect on precursors of Th 1 cells, also preventing the induction of Ts-eff cells. The present experiments extend these findings and demonstrate that Ts-eff cells are also under negative control of Tcs lymphocytes. Likewise, antigen-specific factor produced by contrasuppressor T-T cell hybridoma, used in lieu of Tcs cells, impedes the activation of Ts-eff cells. In both cases regulation is aimed at the precursors of Ts-eff cells. Our experiments demonstrate that the outcome of immunization is dependent not only on the balance between immune cells and regulatory cells, but also on interactions between regulatory cells themselves.     

Murine antibody-dependent cellular cytotoxicity to herpes simplex virus-infected target cells.

Freshly collected peritoneal cells (PC) and cultured spleen cells (SC) (but not fresh SC) from nonimmune mice could mediate antibody-dependent cellular cytotoxicity (ADCC) against herpes simplex virus (HSV)-infected cells in the presence of mouse or human sera containing antibody to HSV. PC also demonstrated variable natural killer cell cytotoxicity to infected cells. Both PC and cultured SC required high concentrations of antibody and high effector to target cell ratios for optimal ADCC. The time kinetics of the reaction appeared to depend on the state of activation of the effector cells. In both PC and SC populations, ADCC activity was limited to adherent cells, and was profoundly inhibited by particulate latex or silica. The murine effector cell found in PC and SC able to mediate ADCC to HSV-infected cells appears to be a macrophage.     

The use of Staphylococcus aureus rich in protein A in the detection of herpes simplex virus antigens

Herpes simplex virus (HSV) type 1 antigens were detected in infected human embryonic lung cells with the aid of specific antiserum and Staphylococcus aureus rich in protein A. When such staphylococci carrying specific anti-HSV IgG on their surface were interacted with various suspension of virus, a reduction in the initial virus titre of about 65% was obtained. However, no direct coagglutination was observed between cell-free supernatants of HSV or HSV-infected cells and sensitized staphylococci. When monolayers or suspended cells infected with the virus were treated with dilutions of specific anti-HSV antiserum followed by non-sensitized staphylococci (indirect method), an "aureola" of the bacteria was detected around the cells expressing the viral antigens. A similar picture was observed when infected cells were interacted directly with sensitized staphylococci. Viral antigens were detected already 12 hours post infection, well before the appearance of cytopathic effect. The sensitivity of the indirect method was found to be higher than that of the direct one and dependent on the multiplicity of infection and the serum dilution used. The method is proposed as a rapid means of identifying viral antigens in diagnostic and experimental virology.     

Production of soluble suppressor factors by herpes simplex virus-stimulated splenocytes from herpes simplex virus-immune mice.

Indirect evidence indicates that herpes simplex virus (HSV)-specific cytotoxic-T-lymphocyte induction is regulated by suppressor cells. To search for such suppressor effects, supernatant fluids from splenocyte cultures from normal and HSV-immune mice cultured either with or without viral stimulation were tested for their ability to inhibit HSV-specific cytotoxic-T-lymphocyte induction. Only the supernatant fluid from the HSV-stimulated, HSV-immune cultures contained a suppressor activity (HSV-SF). HSV-SF was produced by nylon-wool-purified Thy 1+ cells. HSV-SF was detectable after 3 days of culture and would only suppress cytotoxic-T-lymphocyte induction if HSV-SF was added within 24 h of initiation of the test cultures. HSV-SF was neither dialyzable nor heat stable. Molecular sieve chromatography of HSV-SF yielded multiple peaks of suppressor activity. Although most of these peaks exhibited nonspecific suppressor activity, the suppression mediated by the 90,000 to 150,000-molecular-weight fractions was antigen specific and genetically restricted. These results provide direct evidence for the regulation of HSV-cytotoxic-T-lymphocyte induction by a novel suppressor factor.     

Crossed immunoelectrophoretic studies of the solubility immunogenicity of herpes simplex virus antigens.

The nonionic detergent Triton X-100 was capable of solubilizing 90% of the protein content in herpes simplex virus (HSV)-infected rabbit cornea cells. The solubilized HSV antigens formed well-characterized precipitates by crossed immunoelectrophoresis in Triton X-100-containing agarose gel, allowing both identification and relative quantitation. Water-soluble and detergent-requiring HSV antigens were identified by different solubilization procedures in buffer with and without detergent. Five glycoprotein antigens were solubilized only in the presence of detergent, indicating their membrane-bound state. One non-glycosylated antigen was present in both a water-soluble and a membrane-bound form. Based upon the crossed immunoelectrophoretic precipitating patterns of Triton X-100-solubilized HSV antigens, it has been estimated that infected cells yield an amount of virus-specific protein equivalent to 2,000 enveloped virions per cell. Rabbits inoculated intracutaneously with Triton X-100-solubilized HSV antigens developed neutralizing antibodies against HSV almost as effectively as rabbits with an active HSV infection. Precipitins against individual HSV antigens in sera from rabbits infected with HSV and immunized with the Triton X-100-solubilized HSV antigens were assayed by the crossed immunoelectrophoretic technique. Sera from infected rabbits reacted more strongly and with a higher number of HSV antigens than sera from immunized rabbits.     

The immune response and the eye. I. The effects of monoclonal antibodies to T suppressor factors in anterior chamber-associated immune deviation (ACAID)

We report the effects of two monoclonal antibodies (mab) specific for murine T suppressor (Ts) factors (TsF) in anterior chamber (AC)-associated immune deviation (ACAID), as induced by AC inoculation of TNP-coupled syngeneic spleen cells (TNP-Spl). One mab (14-12) is specific for Ts effector factor and can block the induction of Ts cells in ACAID if given before or after AC injection of TNP-Spl. The other mab (14-30) is specific for Ts inducer factors and blocks suppression only after given after TNP-Spl. We also studied the surface phenotype of the Ts cells induced by AC injection of TNP-Spl. We show that at least two cells are required for the adoptive transfer of suppression in TNP-ACAID. One is Lyt-2+ and 14-12+, the other is I-J+. These Ts cells have the surface phenotype of Ts effector cells as seen in other systems. These results indicate that mab which bind TsF in other systems affect Ts cells in TNP-ACAID, and that the Ts cells induced in TNP-ACAID are only of the Ts effector type.     

Tumor bearer T cells suppress Bacillus Calmette-Guérin-potentiated antitumor responses. III. Identification of an auxiliary efferent suppressor-T-cell population

T cells (Ts-eff) induced in BALB/c mice by subcutaneous (sc) growth of syngeneic Meth A tumors can adoptively suppress the effector phase of delayed-type hypersensitivity (DTH) in Bacillus Calmette-Guérin (BCG)-primed and unprimed recipients which have been sensitized with irradiated Meth A cells but they do not inhibit the augmented DTH response in recipients inoculated with cyclophosphamide (CY) 2 days prior to sensitization. By reconstituting CY-treated immunized recipients with selected spleen cell populations, it has been demonstrated that Ts-eff suppress DTH by interacting with a second or auxiliary suppressor cell population present in immune but not normal spleens. These auxiliary suppressor cells (Ts-aux) are Thy+, Lyt 1-2+ and I-J+, phenotypically similar to Ts-eff. Their activity is not influenced by B-cell depletion. Unlike Ts-eff, Ts-aux do not bear receptors specific for Meth A cells. Ts-aux and Ts-eff share similar sensitivity to irradiation and high dose (100 mg/kg) CY but unlike Ts-eff, Ts-aux are cortisone sensitive, nondividing, nonadherent cells which are absent from the thymus. The phenotype and mechanism of action of Ts-aux resemble those of the auxiliary or Ts3 cells defined in models of contact sensitivity, DTH to simple haptens, and in vitro antibody responses.     

An in vitro system for the generation of suppressor cells and the requirement for B cells in their induction

An in vitro method for the generation of effector suppressor cells (Ts3) was developed. By utilizing this protocol, it was possible to investigate both the cellular and genetic requirements for suppressor cell induction. It was determined that populations containing Ts3 cells can be induced after a 4-day culture of spleen cells and antigen. These Ts3 cells are similar to Ts3 cells generated by in vivo immunization. Both populations are I-J+, bind NP hapten, bind NP hapten, bear receptors which share NPb idiotypic determinants with anti-NP antibodies, function during the effector phase of the immune response, and require activation with Ts2 cells. Generation of Ts3-containing populations required both nylon wool-nonadherent T cells and a nylon-adherent, B cell-enriched population from an Igh-identical donor. T cells cultured with antigen alone or with syngeneic macrophages and antigen did not develop suppressive activity. Lytic treatment of the nylon-adherent population with a B cell-specific monoclonal antibody (J11d) removed the ability to generate suppressor cells. These results imply that the induction of suppressor T cells requires B lymphocytes, and that this induction process is dependent on Igh-linked gene products.     

Suppressor T cell recognition of major and minor histocompatibility alloantigens: selected suppression of MHC-directed responses by minor alloantigen Ts

The induction of suppression by i.v. administered alloantigens in the murine host was analyzed as a model of the possible effects of blood transfusion on transplant survival. The results indicated that suppressor T cells (Ts) specific for minor histocompatibility alloantigens could be readily induced by the i.v. presentation of minor alloantigen-disparate spleen cells. In contrast, similar priming with cells differing solely at the H-2 major histocompatibility complex stimulated only positive T cell immunity, with no evidence of suppression. The induction of H-2 directed Ts activity could be accomplished only by i.v. priming with major plus minor incompatible donor cells, suggesting that suppressor cell recognition of minor alloantigens may have facilitated the generation of Ts against H-2-encoded major transplantation antigens. A role for minor histocompatibility antigens in the regulation of H-2-specific immunity at the effector level was also indicated. Ts induced by i.v. pretreatment with minor antigen-disparate donor cells not only suppressed the delayed-type hypersensitivity (DTH) response to the relevant minor alloantigens, but also inhibited DTH against unrelated H-2 alloantigens introduced during subsequent intradermal immunization. Suppression of H-2-directed T cell reactivity was specific in that the presence of the Ts-inducing minor alloantigens was also required and occurred only when the minor and unrelated major alloantigens were presented within the same inoculum, if not on the same cell surface. The capacity of Lyt-2+Ts or Ts-derived suppressive factors specific for one set of cell surface molecules to modulate responses to an unrelated group of surface antigens does not appear to represent a general phenomenon, because similar suppression of immunity to unrelated tumor-specific transplantation antigens by minor-specific Ts was not observed. These results are discussed with respect to the possible mechanism of H-2-directed suppression and the role of the I region in Ts recognition of antigen.     

Antibody-dependent cell-mediated cytotoxicity to target cells infected with type 1 and type 2 herpes simplex virus.

The phenomenon of antibody-dependent cell-mediated cytoxicity (ADCC) has been extended to include target cells acutely infected with herpes simplex type 1 virus (HSV-1) or herpes simplex type 2 virus (HSV-2) in an in vitro system that employs immune human serum and human blood mononuclear cells. The cytotoxic reaction was detectable after 1 hr of incubation and was complete between 4 and 8 hr. The amount of ADCC noted was directly proportional to the logarithm(10) of the effector: target cell ratio (E:T), and ADCC was noted at E:T as low as 1:1. The mononuclear effector cell was present in the blood of both HSV immune and non-immune individuals. The immune serum factor was demonstrated to be an antibody with specificity for HSV membrane antigen(s) and was reactive with target cells infected with either of the two HSV types. The antibody rendered the mononuclear cell cytotoxic by sensitization of the target cell rather than by direct attachment to or "arming" of the mononuclear cell. The physiochemical properties of the antibody as well as its presence in cord blood demonstrated that it is an immunoglobulin on the IgG class.     

Delayed hypersensitivity and immune protection against herpes simplex virus: suppressor T cells that regulate the induction of delayed hypersensitivity effector T cells also regulate the induction of protective T cells

We have been studying delayed hypersensitivity (DH) to herpes simplex virus (HSV) in order to examine the role of this response in host defense against acute and recurrent HSV infections. In previous reports the basic parameters of DH to HSV have been characterized by using a murine ear swelling model, and also the regulation of DH to HSV induced by i.v. injection of the virus. In this paper, we describe a murine protection system and our use of the ability to specifically regulate DH to HSV to examine the correlation between T cells that transfer DH (TDH) and cells that transfer protection from acute HSV infection. Both DH and protection can be transferred with lymph node cells from mice immunized subcutaneously 4 days previously. The effector cell appears to be a T cell, because serum from these donors confers no protection and treatment of immune cells with anti-Thy-1.2 plus complement reduced their ability to protect. Tolerance of DH to HSV was induced by i.v. injection 7 days before subcutaneous immunization. Tolerized mice were unable to generate protective cells. Furthermore, tolerized mice contained suppressor T cells that suppressed not only DH but also the development of protective cells. Regulation of protective cells was shown to be virus specific, because mice tolerized with vesicular stomatitis virus (VSV) were not impaired in their ability to generate T cells that protected from HSV infection. The correlation between the TDH cell and cells that transfer protection from acute HSV infection is discussed.     

Herpes simplex virus-specific antibodies present in tears during herpes keratitis

We examined the specificity and levels of antibodies present in rabbit tears after induced infection of the rabbit cornea. Two strains of herpes simplex virus-1 (HSV) with different patterns of ocular disease were used: RE which produces stromal disease, and F which produces epithelial disease. We found that (i) IgG, IgA, and IgM antibodies were produced, (ii) the number of specific HSV antigens recognized by these antibodies was no significantly different, and (iii) postinfection (PI) timing and concentration of antibodies varied according to the disease pattern of the virus strain. The animals infected with strain F produced high levels of IgG antibodies early PI which remained constant, while IgA and IgM antibodies also increased early PI but declined after Day 16 PI. Animals infected with strain RE showed low levels of IgA and IgM antibodies which remained low. IgG antibodies increased early PI but declined at Day 16 PI. These differences in times of appearance and in amounts of antibodies in tears may be related to the clinical course of the disease. It has been shown that stromal disease has an immunopathologic basis. Inflammation, cellular infiltration of lymphocytes, and plasma cells are seen in the stroma of RE-infected animals, but these are not present in the stroma of F-infected animals. Infectious virus was not isolated from corneal explants taken from animals during the quiescent stage of the disease. The difference in pathogenicity cannot be explained in terms of specificity of tear antibodies. Even though the disease patterns were different, the number and types of HSV polypeptides recognized by both sets of tears was similar. Consequently, we believe that the immunopathology seen in the stromal disease may be due to the anatomical site of HSV antigens, rather than to differences in specificity of tear antibodies.     

Herpes simplex virus glycoprotein D mediates interference with herpes simplex virus infection.

We showed that the expression of a single protein, glycoprotein D (gD-1), specified by herpes simplex virus type 1 (HSV-1) renders cells resistant to infection by HSV but not to infection by other viruses. Mouse (LMtk-) and human (HEp-2) cell lines containing the gene for gD-1 under control of the human metallothionein promoter II expressed various levels of gD-1 constitutively and could be induced to express higher levels with heavy metal ions. Radiolabeled viruses bound equally well to gD-1-expressing and control cell lines. Adsorbed viruses were unable to penetrate cells expressing sufficient levels of gD-1, based on lack of any cytopathic effects of the challenge virus and on failure to detect either the induction of viral protein synthesis or the shutoff of host protein synthesis normally mediated by a virion-associated factor. The resistance to HSV infection conferred by gD-1 expression was not absolute and depended on several variables, including the amount of gD-1 expressed, the dosage of the challenge virus, the serotype of the challenge virus, and the properties of the cells themselves. The interference activity of gD-1 is discussed in relation to the role of gD-1 in virion infectivity and its possible role in permitting escape of progeny HSV from infected cells.     

Membrane phenotype of murine effector and suppressor T cells involved in delayed hypersensitivity and protective immunity to herpes simplex virus

The membrane phenotype of T cells involved in delayed hypersensitivity (DH), protective immunity, and suppression of delayed hypersensitivity to herpes simplex virus (HSV) has been determined. T cells from immune lymph nodes transferring DH and antiviral immunity to normal recipients were characterized as Lyt 1+2-. There appeared to be no detectable antiviral role for Lyt 1-2+ cells in the transferred cell suspension. Splenic T cells suppressing the induction of DH to HSV were characterized as being both Lyt 1+2- and Lyt 1-2+ 4 weeks after their induction. At earlier times, i.e., after 7 days, the suppression was mediated solely by the Lyt 1+2- population. Thereafter, a progressive increase in the contribution of the Lyt 1-2+ suppressor was observed. Both the early and later phases of suppression were due to I-J positive cells. The nature of the two suppressor cell types is discussed in relation to suppressor cell "cascades" and to the pathogenesis of herpes simplex virus infection.     

Interactome analysis of herpes simplex virus 1 envelope glycoprotein H

Herpes simplex virus 1 (HSV‐1) envelope glycoprotein H (gH) is important for viral entry into cells and nuclear egress of nucleocapsids. To clarify additional novel roles of gH during HSV‐1 replication, host cell proteins that interact with gH were screened for by tandem affinity purification coupled with mass spectrometry‐based proteomics in 293T cells transiently expressing gH. This screen identified 123 host cell proteins as potential gH interactors. Of these proteins, general control nonderepressive‐1 (GCN1), a trans‐acting positive effector of GCN2 kinase that regulates phosphorylation of the α subunit of translation initiation factor 2 (eIF2α), was subsequently confirmed to interact with gH in HSV‐1‐infected cells. eIF2α phosphorylation is known to downregulate protein synthesis, and various viruses have evolved mechanisms to prevent the accumulation of phosphorylated eIF2α in infected cells. Here, it was shown that GCN1 knockdown reduces phosphorylation of eIF2α in HSV‐1‐infected cells and that the gH‐null mutation increases eIF2α in HSV‐1‐infected cells, whereas gH overexpression in the absence of other HSV‐1 proteins reduces eIF2α phosphorylation. These findings suggest that GCN1 can regulate eIF2α phosphorylation in HSV‐1‐infected cells and that the GCN1‐binding viral partner gH is necessary and sufficient to prevent the accumulation of phosphorylated eIF2α. Our database of 123 host cell proteins potentially interacting with gH will be useful for future studies aimed at unveiling further novel functions of gH and the roles of cellular proteins in HSV‐1‐infected cells.     

Preparation of stable target cells for anti-herpes simplex virus cytotoxic T lymphocytes

Using an avirulent strain of herpes simplex virus (HSV), SKa, and a methylcholanthrene induced sarcoma cell line, Meth A cells, we have developed a reliable target cell system for detection of cell-mediated cytotoxicity directed against HSV-infected cells. SKa-infection in Meth A produced no progeny virus but induced HSV-specific surface antigens as revealed by radioimmunoassay using 125I-labeled HSV antibody. Spontaneous release of 51Cr from the SKa-infected Meth A cells was no more than that from uninfected control cells but a strong spontaneous 51Cr release was produced in Meth A cells infected with KOS, a virulent strain which produced a progeny virus in Meth A and was lytic for the cells. When used as a target, SKa-infected Meth A cells could detect HSV-specific cytotoxicity by spleen and lymph node lymphocytes of mice immunized with SKa and KOS. This system also detected effector cytotoxic lymphocytes stimulated in vitro by mixed cultures of immune spleen cells and KOS-infected Meth A cells. Thus, the system should be valuable in studies of cell-mediated cytotoxicity directed against HSV-infected cells.     

Persistence of herpes simplex virus type 1 in rat neurotumor cells.

Herpes simplex virus type 1 (HSV-1) infection of a rat central nervous system tumor cell line led to almost complete destruction of the cells. Cells that survived the infection could be isolated and shown to produce infectious HSV particles for variable lengths of time in culture ranging from 20 to 57 passages. Even though infectious virus production eventually ceased, the cell lines continued to produce herpes-specified proteins as measured by immunological techniques. These cells also showed herpesvirus-like structures in the electron microscope. The persistently infected cells that produced HSV antigens and bore HSV sequences were resistant to superinfection by HSV-1. The resistance was not due to failure of adsorption of the virus or to the production of interferon by the cells. The nature of the block in HSV replication in these neurotumor cells, which contain and partially express the HSV genome, is unknown, but may offer an interesting parallel to the known latency of HSV in neural tissues.