In vitro primary induction of T cells mediating delayed footpad reaction and acquired cellular resistance to Listeria monocytogenes

We established an in vitro system generating L. monocytogenes-specific T cells primarily from unprimed spleen cells of mice. Normal spleen cells were cultured for 5 days in the presence of L. monocytogenes in vitro. Viable cells were harvested and assessed for their capacity to confer acquired cellular resistance (ACR) and delayed footpad reaction (DFR) upon local passive transfer to naive syngeneic recipient mice. When normal spleen cells were stimulated with viable L. monocytogenes, the viable cells that were recovered after 5 days of culture conferred a high level of ACR and DFR. Negative selection revealed that the effector cells obtained in primary in vitro culture were Thy 1+, L3T4+, Lyt2- cells. T cells mediating ACR could not be generated in the culture of normal spleen cells with heat-killed bacteria; however, cells mediating only DFR were generated in the presence of a large number of killed L. monocytogenes. The expression of DFR and ACR by T cells generated in this primary culture system was Listeria-specific; reactions were not observed against unrelated bacterial antigens including S. typhimurium, S. aureus, E. coli and PPD. FACS analysis of the cells in culture showed that L3T4+ and Lyt2- T cells were being enriched during culture. The primary generation of antigen-specific T cells in vitro was also possible with spleen cells from NTx mice but not with cells from nude mice, suggesting the presence of Listeria-specific precursors in NTx mice.     

Listeria monocytogenes reactive T lymphocytes in healthy individuals

Peripheral blood mononuclear cells of healthy individuals were enriched for T lymphocytes and stimulated with killed organisms of Listeria sp. Different strains of Listeria sp. induced comparable T cell responses independent from their pathogenicity and virulence. Evidence is presented that killed L. monocytogenes organisms lacked mitogenic activity for human B and T lymphocytes. It is concluded that Listeria reactive T lymphocytes are commonly present in healthy individuals and hence may contribute to the rare occurrence of listeriosis in normal adults.     

rIL-1 alpha enhances adoptive transfer of resistance to Listeria monocytogenes infection.

We have previously demonstrated that administration of recombinant rIL-1 alpha enhances resistance against Listeria monocytogenes infection in mice. In this study we considered the possibility that this cytokine might also augment adoptive immunity conferred by the transfer of listeria-immune spleen cells. Concomitant administration of rIL-1 alpha with large numbers (2 x 10(7) or 10(8)) of listeria-immune spleen cells reduced the protection mediated by the transferred cells. Conversely, rIL-1 alpha co-administered with suboptimal numbers (1-5 x 10(6)) of immune splenocytes augmented anti-listeria resistance in an additive fashion. Although transfer of 10(6) listeria-immune spleen cells alone did not result in significant protection, when 10(6) immune cells were incubated with rIL-1 alpha prior to transfer they conferred significant protection to naive recipients. Time course experiments indicated that the greatest protection was achieved when listeria-immune spleen cells were pretreated with rIL-1 alpha for 2 h prior to adoptive transfer. The protection transferred by 10(6) rIL-1 alpha-pretreated immune spleen cells was not inhibited by TGF beta. This study is the first to use rIL-1 alpha to potentiate the adoptive transfer of resistance to an infectious agent by immune cells.     

Prolongation of acquired cellular resistance to Listeria monocytogenes

Intracutaneous immunization of mice with 10⁵ or 10⁶ viable listeria resulted in acquired cellular resistance (ACR) of short duration (7 days) and in delayed-type hypersensitivity (DH) lasting at least 27 days. The ACR was partially non-specific, as 50% of the mice were also protected against a lethal challenge with Salmonella enteritidis. The specific element of the ACR could be transferred by non-adherent spleen cells from immune mice to normal recipient mice. Such transfer was not possible with adherent spleen cells from immune mice or with spleen cells from normal mice.

Two systems of multiple immunizations to extend the period during which mice were protected against a challenge with 50 LD₅₀ listeria were used. In the first system, mice were immunized with 10⁶ viable listeria and subsequently challenged with 50 LD₅₀ (= 10⁷) viable listeria. Mice surviving the challenge were actually boosted at the challenge injection for ACR. In the second system mice were immunized and boosted with 10⁸ killed listeria mixed with the adjuvant dimethyl dioctadecyl ammonium bromide (DDA). In the former system after each booster injection with viable listeria the interval during which the mice were protected doubled and reached a maximum of 31 days. In the latter system all intervals between two booster injections were equally long and never exceeded 28 days.

In both systems the existence of immunological memory was suggested. The difference in results obtained after immunization with viable listeria and killed listeria mixed with DDA are discussed.

     

The fate of adoptively transferred antigen-specific T cells in vivo

In this study, anti-major histocompatibility complex class I L^d T cell receptor (TCR)-transgenic cells were adoptively transferred into severe-combined immunodeficient (Scid) mice which express the L^d Ag on all nucleated cells, and the fate of transferred antigen-specific T cells (ASTC) was followed in vivo. It was found that, after encountering antigen (Ag) in vivo, the number of ASTC increased 10–15-fold followed by a decline in number to a value that was still above the starting value. The expansion of ASTC could be abrogated by blocking CD28 on the T cells prior to Ag stimulation. Using the technique which simultaneously stains ASTC surface markers and apoptotic cells, we have not only demonstrated directly that the ASTC that disappeared from the periphery died by activation-induced apoptosis but also studied their kinetics in vivo. The remaining ASTC moderately down-regulated both TCR and CD8 on their cell surface and were fully unresponsive when cultured with L^d+ cells even in the presence of exogenous interleukin (IL)-2 and IL-4. However, they were still susceptible to apoptosis when transfereed into a secondary host that provided a new source of Ag and antigen-presenting cells. These studies indicate that peripheral T cell tolerance can be induced by multiple mechanisms in which activation-induced ASTC apoptosis plays an important role.     

Use of recombinant interleukin-2 to enhance adoptive transfer of resistance to Listeria monocytogenes infection.

In vitro incubation of Listeria-immune spleen cells (LISC) with recombinant interleukin-2 (rIL-2) for at least 3 days increased their ability to transfer antilisteria resistance to recipient mice. This effect was blocked by the in vitro addition of transforming growth factor beta 1. The level of protection afforded by the transfer of rIL-2-incubated LISC was further elevated by the in vivo administration of rIL-2 at a dose that by itself did not significantly increase antilisteria resistance. The antilisteria resistance of recipient mice remained elevated for approximately 7 days and then rapidly declined to undetectable levels by 10 days. After cell transfer, recipient mice were protected against challenge with Listeria monocytogenes but not Salmonella typhimurium, Yersinia enterocolitica, or Streptococcus pyogenes. Flow cytometric analyses revealed an increase in the percentages of CD8+, NK+, and gamma delta T cell receptor+ cells but no change in the percentage of CD4+ cells as a result of LISC coculturing with rIL-2. In vitro depletion of CD4+ cells just prior to transfer had no significant effect on the adoptive transfer of resistance; depletion of CD8+ cells reduced the level of resistance by approximately 25%. Combined depletion of Thy-1.2+, CD4+, and CD8+ cells just prior to adoptive transfer diminished the level of protection in the spleens but not the livers of recipient mice. These data suggest that rIL-2 can be used to augment adoptive immunotherapy for bacterial infection in a manner similar to adoptive immunotherapy of human cancer patients. Although the protective cell population was not definitively identified, it appeared to be independent of CD4+ cells and only partly dependent on CD8+ cells.     

Targeting cytotoxic T cells to antigen-specific B lymphocytes

A recent development in immunomanipulation involves the targeting of cytotoxic T lymphocytes (CTL) to cell-bound antigens using bispecific antibodies. These antibodies have been engineered such that specificity is directed against the T cell receptor (TCR) or TCR-associated T3 molecules, as well as against the chosen antigen. The present study was aimed to force interactions between T and B cells by bridging their receptors. F23.1 antibodies, which are specific for gene products of the TCR V beta 8 gene family, were conjugated with TNP (2,4,6-trinitrophenyl) and this construct was used to bridge the receptors of V beta 8+ T cells with the receptors of TNP-specific B cells. The bridging was demonstrated by direct killing of both a TNP-specific B hybridoma and of blast cells from mice transgenic for mu, kappa of the TNP-specific antibody Sp6. Further, F23.1-TNP constructs in conjunction with V beta 8+ CTL were shown to specifically deplete Ig-secreting B cells from Sp6 transgenic mice. Conjugates of TCR-specific antibodies and antigen are theoretically useful in vivo to either deplete or expand B cells of a given specificity by coupling their receptors to the TCR of CTL or T helper cells, respectively.     

Enhanced resistance to Listeria monocytogenes in splenectomized mice.

Mice infected with live Listeria monocytogenes intravenously from 1 week to 3 months following splenectomy exhibit greatly enhanced antibacterial resistance to this micro-organism as compared to normal or sham-splenectomized mice. They survive a dose of Listeria 100 times higher than is the LD50 of this parasite for normal mice. Initially, the same number of viable micro-organisms lodge in the livers of splenectomized and normal hosts. However, within 24 h after infection, the number of viable Listeria which can be recovered from the livers of splenectomized animals is significantly reduced in comparison with control mice. This effect of splenectomy is transient and gradually disappears spontaneously within 3 months following splenectomy. Enhancement of anti-listerial resistance in splenectomized mice can be abrogated by the transfer of normal spleen cells. The presence of a normal splenic cell population that controls macrophage activation is postulated.     

Control of natural killer cell-mediated innate resistance against the intracellular pathogen Listeria monocytogenes by gamma/delta T lymphocytes.

Listeria monocytogenes is an intracellular bacterium which causes an acute infectious disease in mice. Initial host resistance depends on innate immunity mediated primarily by natural killer (NK) cells followed by specific alpha/beta T cells, which are central to acquired specific immunity. Gamma/delta T lymphocytes seem to provide a link between the innate and the specific immune response. All these lymphocyte populations produce gamma interferon (IFN-gamma), which, because of its macrophage-activating potential, is central to antibacterial protection. IFN-gamma from NK cells not only contributes to early host resistance but also promotes development of protective T-cell responses of helper T type 1 (Th1) type. Here, we show that innate resistance and early IFN-gamma production in listeriosis are markedly impaired in T-cell receptor (TCR)-delta-/- but not TCR-beta-/- gene disruption mutant mice. By two-color cytofluorimetry, we demonstrate that NK cells rather than gamma/delta T lymphocytes are the major cellular source of IFN-gamma in immunocompetent mice and that IFN-gamma production by NK cells is impaired in the TCR-delta-/- mutants. Probably, reduced tumor necrosis factor production in listeria-infected TCR-delta-/- mutants contributed to impaired NK cell activation. Our data reveal a novel function of gamma/delta T cells as regulators of innate resistance against sublethal infection with an intracellular pathogen.     

Administration of interleukin-10 abolishes innate resistance to Listeria monocytogenes

We have used severe-combined immunodeficient (SCID) mice to examine the immunoregulatory effects of interleukin (IL)-10 on innate resistance to infection with Listeria monocytogenes. Addition of heat killed Listeria to spleen cells from naive SCID mice resulted in secretion of interferon (IFN)-γ from natural killer cells in vitro. This response was enhanced up to 15-fold in the presence of exogenous IL-2, but was completely ablated by addition of IL-10 with an IC₅₀ of < 0.5 U/ml. Infection of SCID mice with viable Listeria in vivo resulted in a prolonged course of infection eventually causing death by 12–14 days, whereas daily administration of IL-10 increased bacterial replication in the liver and spleen by up to 1000-fold resulting in death by day 4 post-infection. The immunosuppressive actions of IL-10 in vivo were also observed in immunocompetent BALB/c mice, where doses as low as 100 U/day converted a sublethal infection to 100% mortality. To study the events controlling expression of endogenous IL-10, peritoneal macrophage monolayers were challenged with Listeria after pre-incubation with a panel of recombinant cytokines. IFN-γ primed macrophages for enhanced tumor necrosis factor (TNF) secretion, but inhibited IL-10 production, whereas granulocyte/macrophage colony-stimulating factor (CSF), macrophage CSF and also IL-4 enhanced macrophage IL-10 responses after ingestion of Listeria in vitro. Finally, monoclonal antibody neutralization of IFN-γ during infection of SCID mice with Listeria inhibited TNF-α mRNA, but augmented expression of IL-10 mRNA in infected tissues. These results demonstrate that exogenous IL-10 is a potent immunosuppressive cytokine in the context of infection with an intracellular bacterium and that expression of endogenous IL-10 versus TNF is differentially regulated by the cytokine environment of the macrophage.     

Stimulation of activated rat T cells in vitro by Listeria monocytogenes antigens.

A soluble extract of Listeria monocytogenes bound firmly and in similar amounts to a variety of rat cells. Cells that bound this material differed in their capacity to stimulate the in vitro proliferation of lymphocytes obtained from the thoracic duct of Listeria-immune donors. The capacity of cells to serve as antigen-presenting cells in this system coincided or closely overlapped the expression on these cells of an Ia antigen-like structure. Three lines of evidence indicate that T cells respond to L. monocytogenes antigen: the responder cells are members of a nylon-wool nonadherent population that lacks readily detectable surface immunoglobulin; they express determinants recognized by the W3/25 monoclonal antibody (a surface marker of rat peripheral T cells); and they are stimulated optimally by L. monocytogenes antigen when the latter is displayed on cells that share a haplotype with the responder lymphocytes.     

Passive transfer of acquired resistance to Listeria monocytogenes infection is independent of mononuclear cell granuloma formation.

This study documents the formation of leukocyte foci in the livers of mice infused with either normal or immune T cells and then challenged intravenously with Listeria monocytogenes. The results show that the transfer of antilisterial resistance occurred before mononuclear cell granuloma formation and was associated instead with the appearance of foci of infiltrating lymphocytes and neutrophils. Numbers of these foci remained low in mice which received immune cells but increased progressively until death in mice which received normal cells. These findings do not support the previous hypothesis that a major component of acquired resistance against Listeria infection involves the rapid generation of mononuclear cell granuloma formation under the control of immune T cells.     

In vivo and in vitro activation and expansion of gammadelta T cells during Listeria monocytogenes infection in humans.

Serial flow cytometry analyses of peripheral blood mononuclear cells obtained from 8 patients infected with Listeria monocytogenes showed a higher percentage (P < 0.01) of gammadelta T cells (median, 11.7; range, 3.7 to 35.3) than did 16 age-matched uninfected controls (1.7, 0.4 to 13). Most in vivo-expanded gammadelta T cells expressed the Vgamma9 and Vdelta2 gene products and displayed a memory phenotype (CD45RO[high]), and patients' gammadelta T cells expressed significantly more (P < 0.01) activation marker HLA-DR than did controls (19.8% [median] and 0.9 to 87.6% [range] versus 2.3% and 0 to 4.7%, respectively). When peripheral blood mononuclear cells from normal donors were cultured in vitro with heat-killed Listeria cells, analysis of CD25 and HLA-DR expression on gammadelta and alphabeta T cells indicated that a high percentage of gammadelta T cells was activated early compared to alphabeta T cells. In addition, depletion of gammadelta T cells before culture abrogated the early lymphocyte proliferative response induced by the pathogen. Taken together, these results argue for the involvement of gammadelta T cells during L. monocytogenes infection in humans.     

Use of the CD107 mobilization assay reveals that cytotoxic T lymphocytes with novel MHC-Ib restriction are activated during Listeria monocytogenes infection

Detection of cytotoxic activity by pathogen-specific T cells of unknown antigenic specificity is difficult due to the limitations of using infected cells, instead of peptide-pulsed cells, as targets. We report here that the recently described CD107 mobilization assay readily allowed for the ex vivo detection of cytotoxic T lymphocytes (CTL) with a novel MHC-Ib restriction that specifically recognized Listeria monocytogenes-infected macrophages. The CD107 mobilization assay is likely to be a useful tool for detection of CD8(+) T cells that recognize a wide variety of intracellular pathogens.     

Mechanism of depletion of T lymphocytes from the spleen of mice infected with Listeria monocytogenes.

Marked changes in the splenic lymphocyte populations during murine infection with Listeria monocytogenes were observed histologically and quantitated by the immunofluorescence of Thy-1+ immunoglobulin (Ig-) (T) and Ig+ (B) cells. Cells were depleted from the T-dependent areas of the spleen, and the number of T cells in suspensions prepared from spleens of mice 1 to 3 days after primary or secondary infection were less than 1/10 of normal. High numbers of alcohol-killed Listeria sp. did not cause any depletion. Depletion was not prevented by adrenalectomy. Although injected radiolabeled T cells distributed normally between spleen, liver, lymph node, and gut in infected mice, there appeared to be a barrier to their entry into depleted T-dependent areas of the spleen. Evidence for the destruction of T cells, but not of B cells, in the infected mouse spleen was obtained.     

Recombinant interleukin-12 enhances resistance of mice to Listeria monocytogenes infection

The effect of recombinant murine IL-12 (rIL-12) or anti-IL-12 antibody administration on resistance to murine listeriosis was investigated. Mice given a single 0.5 μg dose of rIL-12 had 1.5 log₁₀ fewer listeriae in their spleens and livers as compared with control infected mice 3 days after L. monocytogenes challenge. Conversely, administration of anti-IL-12 IgG caused an equivalent increase in the cfu of L. monocytogenes recovered from the spleens and livers as compared to control mice. This is the first report of such a protective effect from a single dose of rIL-12. Treatment of uninfected mice with rIL-12 induced IFN-γ mRNA production in their livers. Infection of mice with L. monocytogenes caused a similar increase in IFN-γ mRNA levels that was not increased further by concurrent treatment with rIL-12. Treatment of mice with an anti-IFN-γ MAb eliminated the protective effect of IL-12 on Listeria infection. Expression of TNF-γ, IL-10 and IL-12p40 mRNA in L. monocytogenes-infected mice were not significantly altered by administration of either anti-IL-12 IgG or rIL-12. rIL-12 administration was associated with increased serum AST levels, a measure of liver damage, 1 day after treatment in L. monocytogenes-infected mice. In addition, rIL-12 administration was associated with the increased presence of small inflammatory foci and necrotic hepatocytes in both infected and uninfected mice, suggesting a proinflammatory role for IL-12 in the liver.     

Phenotypic expression of genetically controlled host resistance to Listeria monocytogenes.

Several inbred mouse strains, all of them derived from the C57BL background, have genetically determined increased resistance to infection with Listeria monocytogenes, whereas a variety of other strains are relatively sensitive to this infection. Comparison of the host response to L. monocytogenes in the sensitive A strain and the resistant C57BL/6 (B6) strain revealed that the B6 mice were superior to A mice both in the T-cell-independent and in the T-cell-dependent phase of the response. Although animals of both strains had equal ability to clear their circulation of intravenously administered Listeria and to take up comparable amounts of bacteria in their livers and spleens, already 24 to 48 h after infection the genetic advantage of B6 strain mice to suppress bacterial proliferation was apparent. Both the primary (early and late) and the secondary responses as well as the ability to inactivate the bacterial load after adoptive protection by syngeneic immune lymphocytes were more efficient in the B6 animals, suggesting that the common effector macrophage arm of the antilisterial resistance rather than the lymphocyte arm (mediating the T-cell-dependent phase of response) is genetically controlled.     

Induction of protective CD8+ T lymphocytes by an attenuated Listeria monocytogenes actA mutant.

We tested the ability of an attenuated actA mutant of Listeria monocytogenes to induce protective immunity in mice. This mutant can enter and multiply in the cytosol of the infected host cell, but is deficient in actin-dependent cell-to-cell spread. It was found to be of attenuated virulence for inbred C3H mice: the LD50 after i.v. injection was 1000-fold higher than that of the wild-type strain. Mutant bacteria multiplied up to the fourth day in the liver, but only for 1 day in the spleen. A single infection with the maximum sublethal dose of the actA mutant induced long-lasting immunity; the LD50 of virulent wild-type L. monocytogenes increased 100-fold and growth of wild-type L. monocytogenes was controlled in liver and spleen of these mice. The presence of Listeria-reactive T cells in spleen of C3H mice infected 7 days previously with the actA mutant was monitored, through their ability to protect naive syngeneic recipients against wild-type L. monocytogenes. Protection was mainly conferred by Thy-1+ CD8+ T lymphocytes; depletion of CD4+ T cells had no significant effect on the level of transferred protection. Such attenuated mutants may be used to develop live vector vaccines for delivery of heterologous proteins into the cytosol, thereby favoring the induction of a CD8+ T cell response.     

Impaired resistance to Listeria monocytogenes in mice chronically exposed to cadmium.

It is shown in this work that resistance to Listeria monocytogenes is greatly impaired in C57BL/6 mice chronically exposed to cadmium (Cd) chloride. Animals received 0.5 mg/kg Cd by an intraperitoneal route three times a week during a 4-week period and were then infected with L. monocytogenes. Susceptibility to this pathogenic bacteria was not due to a defect of the specific immune response, since mice developed normal levels of anti-Listeria T cell-mediated immunity and did not show any impairment of macrophage activation. In fact, bacterial growth in organs was rapid in Cd-exposed mice during the early phase of infection, suggesting an impairment of non-specific defence mechanisms. Experimental data indicate that the susceptibility to L. monocytogenes might be due to a defect of macrophage recruitment in sites of infection during the early phase of the host response.