A predominant role for antibody in acquired immunity to chlamydial genital tract reinfection

Acquired immunity to murine Chlamydia trachomatis genital tract reinfection has long been assumed to be solely dependent on cell-mediated immunity. However, in this study, we identify a previously unrecognized protective role for Ab. Immunity develops in Ab-deficient mice following the resolution of primary chlamydial genital infection. Subsequent depletion of CD4+ T cells, but not CD8+ T cells, in those immune Ab-deficient mice before secondary infectious challenge, resulted in an infection that did not resolve. Passive immunization with immune (convalescent) serum conferred a marked level of protective immunity to reinfection, which was characterized by a striking decrease in bacterial shedding, from >100,000 inclusion forming units to fewer than 10 inclusion forming units, and a shortened duration of infection. Furthermore, mAbs to the chlamydial major outer membrane protein and LPS conferred significant levels of immunity to reinfection and reduced chlamydial shedding by >100-fold. Anti-heat shock protein 60 mAb had no protective effect. In contrast to the marked protective efficacy of immune serum on reinfection, the course of primary infection was essentially unaltered by the passive transfer of immune serum. Our results convincingly demonstrate that Abs contribute importantly to immunity to chlamydial genital tract reinfection, and that Ab-mediated protection is highly dependent on CD4+ T cell-mediated adaptive changes that occur in the local genital tract tissues during primary infection. These results impact our understanding of immunity to chlamydial genital infection and may provide important insight into vaccine development.     

Interdependence of CD4+ T cells and malarial spleen in immunity to Plasmodium vinckei vinckei. Relevance to vaccine development

We studied immunity to the blood stage of the rodent malaria, Plasmodium vinckei vinckei, which is uniformly lethal to mice. BALB/c mice develop solid immunity after two infections and drug cure. The following experiments define the basis of this immunity. Transfer of pooled serum from such immune mice renders very limited protection to BALB/c mice and no protection to athymic nu/nu mice. Moreover, B cell-deficient C3H/HeN mice develop immunity to P. vinckei reinfection in the same manner as immunologically intact mice, an observation made earlier. In vivo depletion of CD4+ T cells in immune mice abrogates their immunity. This loss of immunity could be reversed through reconstitution of in vivo CD4-depleted mice with fractionated B-, CD8-, CD4+ immune spleen cells; however, adoptive transfer of fractionated CD4+ T cells from immune spleen into naive BALB/c or histocompatible BALB/c nude mice does not render recipients immune. In vivo depletion of CD8+ T cells did not influence the parasitemia in nonimmune or immune mice. Splenectomy of immune mice completely reverses their immunity. Repletion of splenectomized mice with their own spleen cells does not reconstitute their immunity. We conclude that some feature of the malaria-modified spleen acts in concert with the effector/inducer function of CD4+ T cells to provide protection from P. vinckei. To be consistent with this finding, a malaria vaccine may require a combination of malaria Ag to induce immune CD4+ T cells and an adjuvant or other vaccine vehicle to alter the spleen.     

Studies on the transfer of protective immunity with lymphoid cells from mice immune to malaria sporozoites.

In an effort to understand the mechanisms involved in the protective immunity to malarial sporozoites, an A/J mouse/Plasmodium berghei model was studied. Protective immunity could consistently be adoptively transferred only by using sublethal irradiation of recipients (500 R); a spleen equivalent (100 X 10(6))of donor cells from immune syngeneic mice; and a small booster immunization (1 X 10(4)) of recipients with irradiation-attenuated sporozoites. Recipient animals treated in this manner were protected from lethal challenge with 1 X 10(4) nonattenuated sporozoites. Immune and nonimmune serum and spleen cells from nonimmune animals did not protect recipient mice. Fewer immune spleen cells (50 X 10(6)) protected some recipients. In vitro treatment of immune spleen cells with anti-theta sera and complement abolished their ability to transfer protection. This preliminary study suggests that protective sporozoite immunity can be transferred with cells, and that it is T cell dependent.     

Role of IL-5 in innate and adaptive immunity to larval Strongyloides stercoralis in mice

Protective immunity to Strongyloides stercoralis infective larvae in mice has been shown to be dependent on IL-5 based on mAb depletion studies. The goal of this study was to determine the functional role of IL-5 during the innate and adaptive immune response to larval S. stercoralis in mice. In these studies, three strains of mice were used: wild-type C57BL/6J (WT), IL-5 knockout (KO), and IL-5 transgenic (TG). Innate responses to the larvae indicated that there was enhanced survival in the KO animals and decreased survival in the TG animals compared with WT. Furthermore, killing of larvae in TG mice was associated with eosinophil infiltration and degranulation. In studying the adaptive immune response, it was observed that immunization of KO mice did not lead to the development of protective immunity. Experiments were then performed to determine whether KO mice reconstituted with Abs or cells could then develop protective immunity. KO mice displayed protective immunity via a granulocyte-dependent mechanism following injection of purified IgM from immune wild-type animals. Immunity in KO mice could also be reconstituted by the injection of eosinophils at the time of immunization. These eosinophils did not participate in actively killing the challenge infection, but rather were responsible for the induction of a protective Ab response. We conclude that IL-5 is required in the protective immune response for the production of eosinophils, and that eosinophils were involved in larval killing during innate immunity and in the induction of protective Abs in the adaptive immune response.     

Transfer of Protection to Murine Typhoid Conferred by L-Form Salmonella typhimurium in Dependence of Cooperation between L Form-Adopted Macrophages and L Form-Induced Lyt-2+ T Cells

The effector cells responsible for protection to Salmonella typhimurium in C3H/HeJ mice, conferred by L-form S. typhimurium, were determined by cell transfer test. Nonfractionated spleen cells from 6-week immune mice but not from 24-week immune animals transferred anti-S. typhimurium immunity. Treatment with anti-macrophage antiserum and complement most effectively abolished protective capacity in 6-week immune cells, while anti-T cell monoclonal antibody plus complement reduced it to a lesser extent. However, adoptive protection was achieved only by transfer of immune macrophages along with Lyt-2⁺ T cells selected from 6-week immune spleen cells. These Lyt-2⁺ T cells were cytotoxic to Kupffer cells from C3H/HeJ mice which had been infected 48 hr previously and from the mice which had been immunized 1 week previously, but not to the cells from 6-week immune mice and from normal animals. Moreover, protective capacity in immune macrophages seemed to be correlated to the degree of colonization by the L forms, and the inability to transfer immunity of 24-week immune spleen cells may be due to the decrease in the L form-colonization. These results suggest that cooperation between the L form-colonized macrophages and L form-induced cytotoxic Lyt-2⁺ T cells contributes to anti-S. typhimurium immunity, and might imply the immunological difference between the 6-week immune phagocytes and the cells at an early stage of infection or immunization.     

Cellular subsets involved in cell-mediated immunity to murine Plasmodium yoelii 17X malaria

Cell mediated immunity to nonlethal Plasmodium yoelli 17X (PY17X-NL) was examined in the CBA/CaJ mouse by adoptive transfer of sensitized T lymphocyte subsets. In intact mice, PY17X-NL causes a self-limiting infection with parasitemia levels ranging from 10 to 25% of total red blood cells. Upon recovery, mice are refractory to subsequent challenge with the homologous parasite. In T cell-depleted mice, PY17X-NL infections are extremely virulent and result in death of the host after parasitemia levels reach 50% or higher. The transfer of either Lyt-1 T cells or Lyt-2 T cells from immune animals into normal, naive animals produced accelerated recovery to subsequent infection. However, this adoptive transfer of immunity by either subset was dependent upon the presence of an I-J+, Lyt-null cell in the immune population. T cell deprivation precluded the ability of animals to control blood-stage infections. When T cell-depleted mice were reconstituted with naive, Ig-negative (T cell-enriched) spleen cells, parasitemia levels were controlled and the parasites were eliminated. When T cell-deprived animals were reconstituted with naive Lyt-1+2-, Ig-negative spleen cells, they experienced twofold higher parasitemias of longer duration than mice receiving unfractionated cells. Two of six of these Lyt-1 mice died of fulminant infections, suggesting that the presence of naive Lyt-2 cells enhances the degree of protection. Immune Lyt-2 T cells were highly protective in T cell-depleted animals. Protection by sensitized Lyt-1 T cells correlated with the induction of a monocytosis. On the other hand, protection by Lyt-2T cells occurred in the absence of monocytosis. The possibility that the immunity induced by each T cell subset is mediated by a different effector mechanism is discussed.     

Studies of delayed hypersensitivity to L. Monocytogenes in mice: nature of cells involved in passive transfers

C3Hf/Umc mice were immunized by an intravenous injection of a sublethal dose of live Listeria monocytogenes. The animals developed delayed-type hypersensitivity (DH) concomitant with infectious immunity to this organism. Delayed hypersensitivity could be transferred to normal lethally irradiated mice with spleen cells from immune animals. The immune cells cells responsible for transfer of adoptive immunity were susceptible to in vitro cytolytic action of anti-theta iso-antibody and complement, since such treatment rendered these cells incapable of further passive transfer of specific immunity to Listeria. The acquired DH to Listeria persisted in mice after 900 R lethal irradiation, provided normal syngeneic bone marrow cells were also administered, thus indicating the persistance of a cell population in the immune irradiated mice, resistant to effects of radiation. The radio resistant nature of this immune cell population was further demonstrated by passive transfer with spleen cells, derived from preimmunized lethally irradiated mice to normal syngeneic mice or to lethally irradiated nonimmunized hosts reconstituted with normal bone marrow which then responded to antigenic challenge with DH.Treatment of the immune radio resistant spleen cells in vitro with anti-theta and complement eliminated passive transfers of DH by these cells; however, this effect was less obvious than similar treatment of the immune, nonirradiated, spleen cells.     

Tumor-specific immunity induced by somatic hybrids: III. Persistence of adoptively transferred immunity specific for TEPC-15 plasmacytoma in normal BALB/c mice

Immunity against TEPC-15 tumor cells was induced in BALB/c mice by injecting semi-allogeneic hybrid cells derived from fusion of TEPC-15 tumor cells with LM(TK^?) cells of the C3H origin. Adoptive transfer of spleen cells from the immune mice into normal BALB/ c recipients rendered them free from tumors following tumor challenge; the recipients were most significantly protected from the tumor when tumor cells were injected 7–14 days after the adoptive transfer of immune cells. Such immunity following adoptive transfer appeared to persist in the recipients for at least 60 days. Moreover, the tumor-specific immunity was consecutively transferable (more than nine passages) into normal BALB/c recipients by serially passing spleen cells from the recipients every 14 days, without further stimulation with the hybrid cells or inactivated TEPC-15 tumor cells. Such consecutive transfer of the immune spleen cells induced splenomegaly in the recipients: a two- to five-fold increase over normal spleen cell recipients. The ability of spleen cells to transfer immunity, but not splenomegaly, was abrogated by treatment with mitomycin C. These results suggest that proliferation of donor cells is necessary to transfer immunity, and that splenomegaly alone does not manifest such immunity in the recipients.     

Hymenolepis nana: passive transfer of mouse immunity by T-cell subset of phenotype Lyt-1

Mesenteric lymph node cells obtained from donor mice (BALB/c strain) actively immunized by oral inoculation with Hymenolepis nana eggs were syngeneically transferred by intravenous injection into athymic nude mice previously uninfected. The adoptively immunized recipients were then challenged with 1000 H. nana eggs 2 days after cell transfer. The degree of immunity transferred was assessed by examining cysticercoids developed in the intestinal villi of the recipients on Day 4 of challenge infection. The criterion for success in cell transfer of immunity was the complete rejection of cysticercoids as was generally expected in mice infected previously. The transfer of 1.5 X 10(8) immune mesenteric lymph node cells obtained from donors immunized 4 days before cell collection resulted invariably in the complete rejection of cysticercoids, though not less than this cell dosage. The immunity was passively transferable to recipients by T cells, especially by T-cell subset of phenotype Lyt-1 but not those of phenotype Lyt-2.3 and Lyt-1.2.3. However, 1.5 X 10(8) immune mesenteric lymph node cells obtained from donors immunized 21 days before cell transfer and 1.5 X 10(8) immune spleen cells obtained from donors immunized 4 days before cell transfer had little or no effect on the rejection of cysticercoids.     

Tumor-specific immunity induced by somatic hybrids. II. Elicitation of enhanced immunity against the parent plasmacytoma.

Hybrid cells derived from fusion of a BALB/c plasmacytoma (TEPC-15) and L cells (C3H origin) were used to stimulate tumor-specific immunity against the parental plasmacytoma cells. Live hybrid cells induced tumor-specific immunity against TEPC-15 more effectively than mitomycin-treated hybrid or TEPC-15 tumor cells. Adoptive transfer of immunity with spleen cells of mice immunized with hybrid cells was also more effective than that with mitomycin-treated tumor cells. The immunity induced by the hybrid cells was specific to the TEPC-15 tumor because the mice that received immune spleen cells were not protected against challenge with either HOPC-8 or McPC-603 plasmacytomas. T cell populations were primarily responsible for the transfer of specific immunity based on the sensitivity of immune cells to anti-Thy 1.2 and complement. Mice that had established solid tumors were treated with 5 x 10(7) spleen cells to evaluate the therapeutic value of the hybrid-induced immune cells. Tumors in the mice that received immune cells gradually regressed over a 40-day period, whereas tumors on the control mice continued to grow. These results suggest that a rearrangement of tumor-specific antigens on allogeneic hybrid cells can enhance their immunogenicity.     

Immunity to Trichinella spiralis in irradiated mice

Irradiation prevented the accelerated expulsion of Trichinella spiralis from mice immunized by transfer of immune mesenteric lymph node cells (IMLNC) or by prior infection. Nevertheless, worms in irradiated immune mice were smaller and less fecund than those in controls. In adoptively immunized and irradiated mice expulsion could not be achieved by increasing the numbers of IMLNC transferred, although the effect upon worm length was more severe. Thus IMLNC express a direct, anti-worm immunity which is independent of their role in worm expulsion. IMLNC cause expulsion in irradiated mice only when adequate levels of bone marrow-derived cells are available. The results are discussed in terms of a possible antibody-mediated basis for direct anti-worm immunity.     

Immunologic responses to Candida albicans. III. Effects of passive transfer of lymphoid cells or serum on murine candidiasis

Passive transfer of immune serum gave a significant degree of protection against deep seated candidiasis in mice. Repeated attempts to transfer resistance by the transfer of sensitized lymphoid cells gave negative results, even though cutaneous delayed hypersensitivity was transferred by the cells. The results suggest that cell-mediated immunity is not of primary importance in this model of murine candidiasis, and that humoral immunity contributes to protection.     

Role of thymus-derived lymphocytes in acquired immunity to salmonellosis in mice

The relative role of thymus-derived (T-) lymphocytes and bone marrow-derived (B-) cells in acquired immunity to salmonellosis was examined in mice. The results demonstrate that the protective capacity of the donor immunized mice could be passively transferred to the recipient mice by spleen cells but not with peritoneal exudate cells or sera. A high cell number of spleen cells (2 X 10(8)/mouse) were required before passive transfer of immunity could be obtained. Of the T-lymphocytes and B-cell populations of spleen cells, T-cells from immune mice were effective in conferring protection to the recipient mice.     

Absolute requirement for an active immune response involving B cells and Th cells in immunity to Plasmodium yoelii passively acquired with antibodies to the 19-kDa carboxyl-terminal fragment of merozoite surface protein-1.

Vaccination of mice with the leading malaria vaccine candidate homologue, the 19-kDa carboxyl terminus of merozoite surface protein-1 (MSP119), results in sterile immunity to Plasmodium yoelii, with no parasites detected in blood. Although such immunity depends upon high titer Abs at challenge, high doses of immune sera transferred into naive mice reduce parasitemia (and protect from death) but do not result in a similar degree of protection (with most mice experiencing high peak parasitemias); this finding suggests that ongoing parasite-specific immune responses postchallenge are essential. We analyzed this postchallenge response by transferring Abs into manipulated but malaria-naive mice and observed that Abs cannot protect SCID, nude, CD4+ T cell-depleted, or B cell knockout mice, with all mice dying. Thus, in addition to the Abs that develop following MSP119 vaccination, a continuing active immune response postchallenge is required for protection. MSP119-specific Abs can adoptively transfer protection to strains of mice that are not protected following vaccination with MSP119, suggesting that the Ags targeted by the immune response postchallenge include Ags apart from MSP119. These data have important implications for the development of a human malaria vaccine.     

Determinants of immunity to murine salmonellosis: studies involving immunization with lipopolysaccharide-lipid A-associated protein complexes in C3H/HeJ mice

We have earlier demonstrated that the C3H/HeJ Salmonella hypersusceptible mouse can be protected against infection with this organism by prior immunization with lipopolysaccharide (LPS)-lipid A-associated protein (LAP) complexes, but not with LPS alone. In the current studies, protection has been shown to correlate with the induction of LPS-specific antibody in immunized mice. LPS was demonstrated to be a relevant target antigen for Salmonella immunity since C3H/HeJ mice were afforded higher survival rates when they were challenged with Salmonella that shared the same LPS O-antigen as the vaccine. Although low levels of LPS-specific antibody can be detected 14 days after immunization with LAP-LPS, significant antibody is present only after 21-28 days. In addition, anti-LAP specific antibodies can be detected after 14 days of immunization with LAP-LPS. Adoptive transfer of either day 28 anti-LAP-LPS immune serum or day 28 LAP-LPS immune splenocytes alone to naive recipients affords mice minimal, if any, survival against lethal S. typhimurium LT2 challenge. In contrast, transfer of day 28 anti-LAP-LPS immune serum and day 28 LAP-LPS immune splenocytes together is able to transfer Salmonella immunity to naive C3H/HeJ mice. Further, equivalent transfer of only day 28 anti-LAP-LPS immune serum to C3H/HeJ mice immunized 7 days previously with LAP-LPS provides protection similar to that found in mice adoptively transferred with immune cells and serum. These results suggest that a host cellular factor or factors responsive to LAP-LPS, in addition to day 28 anti-LAP-LPS immune serum, may contribute to the protection afforded C3H/HeJ mice following immunization with LAP-LPS.     

Cutting Edge: IFN-gamma-producing CD4 T lymphocytes mediate spore-induced immunity to capsulated Bacillus anthracis

Virulent strains of Bacillus anthracis produce immunomodulating toxins and an antiphagocytic capsule. The toxin component-protective Ag is a key target of the antianthrax immune response that induces production of toxin-neutralizing Abs. Coimmunization with spores enhances the antitoxin vaccine, and inactivated spores alone confer measurable protection. We aimed to identify the mechanisms of protection induced in inactivated-spore immunized mice that function independently of the toxin/antitoxin vaccine system. This goal was addressed with humoral and CD4 T lymphocyte transfer, in vivo depletion of CD4 T lymphocytes and IFN-gamma, and Ab-deficient (muMT(-/-)) or IFN-gamma-insensitive (IFN-gammaR(-/-)) mice. We found that humoral immunity did not protect from nontoxinogenic capsulated bacteria, whereas a cellular immune response by IFN-gamma-producing CD4 T lymphocytes protected mice. These results are the first evidence of protective cellular immunity against capsulated B. anthracis and suggest that future antianthrax vaccines should strive to augment cellular adaptive immunity.     

Transplacental transfer and subsequent neonate utilization of herpes simplex virus-specific immunity are resilient to acute maternal stress

Neonates are severely compromised in the ability to generate an immune response to pathogens and thus rely heavily on maternally derived immunity that is acquired by transplacental and transmammary means. The passive transfer of maternal herpes simplex virus (HSV)-specific antibody is critical in determining the outcome of neonatal HSV infection. In adults, psychological stress alters immune responsiveness via the increased level of corticosterone that is produced as a result of hypothalamic-pituitary-adrenal axis activation. Although the behavioral and neuroendocrine effects of pre- and postnatal stress-induced increases in corticosterone are well documented, the effects of maternal stress on the efficacy of prenatally transferred and neonatally developed viral immunity has yet to be addressed. By using a well-established prenatal restraint-and-light stress mouse model, we investigated the effects of increased maternal corticosterone on the passive transfer of total and HSV-specific immunoglobulin G (IgG) antibody and subsequent neonatal susceptibility to HSV infection. Serum corticosterone levels in pregnant mice were significantly increased in response to restraint-and-light stress, and fetuses derived from these stressed mice had significantly elevated levels of corticosterone. Despite the increases in corticosterone, the passive transfer of total and HSV-specific IgG antibody persisted and, in turn, protected the neonate from systemic viral spread. Therefore, prenatal stress did not increase the susceptibility of neonates to HSV type 2-associated mortality. These findings demonstrate the resiliency of the passive transfer of protective HSV-specific immunity under conditions of acute psychological stress.     

Pathogenesis of age dependent paralysis by a temperature sensitive mutant (tsl) of Moloney murine leukemia virus-TB.

The tsl mutant of Moloney murine leukemia virus-TB produces neurological disease leading to fatal hind limb paralysis when inoculated in newborn BALB/c mice. The present study was under taken to assess the role of T and B lymphocytes in age dependent resistance to tsl induced paralysis in BALB/c mice. The adoptive transfer of non-immune splenic unseparated lymphoid cells, T cells and B cells and tsl immune B cells and T cells to newborn BALB/c mice infected with tsl did not prevent the development of paralysis. However, adoptive transfer of immune splenic unseparated lymphoid cells and immune T cells delayed the onset of paralysis by 5 to 10 days as compared to the mice which did not receive the immune lymphocytes. Athymic BALB/c nude mice inoculated with tsl at days 1 and 10 after birth failed to develop the paralytic disease. Transfer of tsl neutralising antibody also delayed the onset of paralysis. Mice (10 days old) treated with cyclophosphamide, cyclosporine A, cortisone acetate and anti-T cell serum when inoculated with tsl also did not develop neurological disease. The results suggest that age related resistance to neurological disease may not be associated with B cell mediated immunity.     

SAP is required for Th cell function and for immunity to influenza

Ab is a crucial component of protective immunity to infection, but Ab responses do not proceed normally when defects occur in a protein called signaling lymphocytic activation molecule-associated protein (SAP). To explain this Ab defect, we analyzed B cell and plasma cell responses under conditions of SAP deficiency. Our results demonstrate that SAP-deficient (SAP knockout (KO)) mice have a profound CD4 T cell-intrinsic defect in generating Ag-specific plasma cells following challenge with model Ags or influenza virus, resulting in low Ag-specific Ab titers. We also show that SAP is required in CD4 T cells for normal division and expansion of B cells. These B cell and plasma cell defects were observed during the expansion phase of the primary immune response, indicating early defects in Th cell activity. In fact, additional experiments revealed a nearly complete lack of T cell help for B cells in SAP KO mice. Our work suggests that the ability of SAP to promote T-dependent humoral immune responses is important for antiviral immunity because mice lacking SAP are unable to prevent high dose secondary influenza infection, and because passive transfer of IgG in immune serum from wild-type, but not SAP KO mice can protect mice from an otherwise lethal influenza infection. Overall, our results demonstrate that SAP is required in CD4 T cells for their ability to help B cell responses and promote influenza-specific immunity.     

Attempts to transfer immunity against Clonorchis sinensis in nude and DS mice.

The effects of peritoneal exudate cells (PEC) and sera of athymic nude and DS mice infected with Clonorchis sinensis metacercariae or sensitized by injection of metabolic products into footpad on transfer of immunity against the fluke to the syngeneic mice were studied. There was no significant difference in eggs per gram pattern between the sensitized and control groups, and between nude and DS mice. However, the worm burdens were slightly greater in nude mice than in DS mice. Also, a few plaque forming cells were found in only DS mice given PEC and serum from Group II DS mice. In the light of these results, it is likely that PEC and sera of nude or DS mice which are deficient, at least partially, in the cellular immune system are unable to transfer immunity against C. sinensis to syngeneic recipients.