Generation of Listeria monocytogenes-specific T cells mediating delayed footpad reaction and protection in neonatally thymectomized mice but not in nude mice

Neonatally thymectomized (NTx) mice, sham-operated control mice and congenitally athymic nude mice were immunized with viable Listeria monocytogenes and their spleen cells examined for the capacity to transfer both delayed footbad reaction and protection against challenge at the site of local transfer. Cells from immune NTx mice conferred significant degrees of delayed footpad reaction and protection comparable to sham mice, while cells from immune nude (nu/nu) mice did not. This abilty was completely eliminated by the treatment of cells with anti-Thy1, anti-Lytl or anti-L3T4 antibody plus complement but not with anti-Lyt2 antibody plus complement. These results indicated that NTx mice can normally mount the immunity to L. monocytogenes by generating Lyt1⁺2^–, L3T4⁺ T cells. Immune competence of NTx mice and thymus dependency of various immune responses are discussed.     

Failure of FK 506 to suppress the T cell-mediated immunity of mice to Listeria monocytogenes.

Listeria monocytogenes belongs to the group of intracellular bacteria, which means that they reside and multiply within host cells. The protective immunity against such an infection is mediated by cellular immune mechanisms. Whereas the CD8+ T cell population plays a major role therein, the CD4+ T cells are held to be of minor importance in this defence system. Consequently, one can understand that immune suppression with FK 506 which acts primarily on this latter T cell subset, does not interfere with protective immunity of mice infected with L. monocytogenes. We have demonstrated that the drug blocks neither curing of primary infection, nor formation of granulomas, nor induction of cell populations capable of mediating adoptive transfer of immunity, nor expression of pre-existing immunity.     

Active and memory immunity to Listeria monocytogenes infection in mice is mediated by phenotypically distinct T-cell populations.

The results of this study show that acquired protective immunity to Listeria monocytogenes, measured as the capacity of splenic T cells to passively transfer acquired resistance to normal syngeneic recipients, was mediated by a population of cyclophosphamide-sensitive CD4- CD8+ T cells. The results further show, in contrast, that memory immunity to listeriosis, which was defined as resistance transferred by T cells harvested from infected animals that had received ampicillin chemotherapy, was shown to be mediated by cyclophosphamide-resistant CD4+ CD8- T cells.     

Self-reactive T cells are activated by the 65-kDa mycobacterial heat-shock protein in neonatally thymectomized mice

To elucidate the mechanism of autoimmune disease in neonatally thymectomized (NTX) mice, we have investigated the responsiveness of the self-reactive T cells which have not undergone clonal deletion in such animals. Consistent with a recent report (Yuuki et al., Eur. J. Immunol. 1990. 20: 1475), T cells bearing V beta 11-gene products capable of recognizing I-E-encoded molecules were readily detected in the mature T cell pool of NTX BALB/c (I-Ed, Mls-2a) mice. The V beta 11-bearing T cells in NTX mice expressed interleukin 2 receptors and responded normally to signals delivered through the T cell receptor. Notably, these T cells in NTX mice proliferated significantly after culture with the 65-kDa mycobacterial heat-shock protein, whose amino acid sequence is highly homologous to that in eukaryotes. These results suggest that self-reactive T cells in NTX mice may be activated by heat-shock proteins derived from various pathogens and/or stressed autologous cells, resulting in the development of autoimmune diseases in such animals.     

Immune-mediated cholangiohepatitis in neonatally thymectomized mice: the role of T cells and analysis of T-cell receptor Vbeta usage

We have previously reported the induction of immune-mediated cholangiohepatitis following injection of a hybrid recombinant proteins containing the E2 of the pyruvate dehydrogenase (PDC-E2) and the branched-chain keto-acid dehydrogenase (BCOADC-E2) to neonatally thymectomized (Tx) A/J mice. Further, we demonstrated that intrahepatic infiltrating mononuclear cells could transfer pathology to other Tx mice. To further our observations, we examined intrahepatic infiltrating mononuclear cells by flow cytometry and used cell transfer experiments to identify the phenotype involved. Interestingly, following immunization of neonatally Tx A/J mice and immunization with the bihybrid molecule, the number of CD3+infiltrating mononuclear cells were significantly higher (77.8%) compared with the control group. There was a small although not significant increase among intrahepatic infiltrating mononuclear cells and splenic cells of Vbeta 5.1,5.2+, Vbeta7+and Vbeta17+. In addition, Vbeta14+cells accounted for 20.4% of the infiltrating T-cells (P<0.01 vs. the control group). In further experiments, CD3+, CD4+or CD8+cells were isolated and removed from intrahepatic infiltrating mononuclear cells and subpopulations of mononuclear cells transferred to Tx mice. Both CD3+CD4+cells and CD3+CD8+cells are required for development of the lesion, and the damage is mediated by CD3+Vbeta14+cells.     

Restricted appearance of self-reactive clones into T cell receptor intermediate cells in neonatally thymectomized mice with autoimmune disease

Neonatally thymectomized (NTx) mice fall victim to such autoimmune diseases as gastritis and pancreatitis with aging. Self-reactive T cell clones are known to be consistently generated through TCR intermediate (i.e. TCR^int) cell differentiation in normal mice (i.e. via the extrathymic pathways and an alternative intrathymic pathway). It was investigated whether the generation of such clones in NTx mice follows this rule or whether they are generated by default via mainstream T cell differentiation in the thymus. The majority of T cells generated in NTx mice were TCR^int cells in all organs tested. In contrast to athymic mice, which carry only TCR^int cells with aging, a leaky appearance of high TCR (i.e. TCR^hi) cells emerged in the lymph nodes and other organs of NTx mice. Self-reactive clones estimated by anti-Vβ monoclonal antibodies in conjunction with the Mls system were confined to TCR^int cells in all tested organs, including a target organ, the stomach, in NTx mice. A leaky population of TCR^hi cells did not contain a significant number of self-reactive clones. Moreover, such self-reactive clones among TCR^int cells in NTx mice with autoimmune disease were shown to be nonanergic in the proliferation assay. The present results suggest that the generation of self-reactive clones is totally due to TCR^int cell differentiation, although it is still undetermined whether the expanding TCR^int cell population is generated via the extrathymic pathway or an alternative intrathymic pathway. It is shown here not to be due to a failure of the TCR^hi cell-differentiation pathway in NTx mice.     

Administration of superantigens protects mice from lethal Listeria monocytogenes infection by enhancing cytotoxic T cells

Superantigens stimulate T-cell-receptor Vbeta-selective T-cell proliferation accompanying the release of cytokines, which may eventually protect the host from microbial infections. We investigated here whether superantigens can rescue the host from lethal bacterial infection. Mice were pretreated with Staphylococcus aureus enterotoxin B (SEB) 1 and 2 days before bacterial infection, and the mortality of infected mice was assessed. SEB pretreatment protected mice from lethal infection with Listeria monocytogenes but not from lethal infection with Streptococcus pyogenes. This enhanced protection was also observed upon pretreatment with recombinant streptococcal pyrogenic exotoxin A. Furthermore, L. monocytogenes-specific delayed-type hypersensitivity (DTH) due to type 1 helper T (Th1) cells and the cytotoxicity of CD8(+) T cells were significantly enhanced after SEB administration and bacterial infection. Depletion of either CD4(+) T cells or CD8(+) T cells in SEB-pretreated mice completely abolished this protection. This phenomenon was ascribed to the elimination of L. monocytogenes-specific CD8(+) cytotoxic T lymphocytes (CTL). It was found that CD4(+) T cells contributed to the induction of the CTL populations. Furthermore, SEB pretreatment of heat-killed L. monocytogenes-immunized mice enhanced the protection from challenge of L. monocytogenes. Taken together, these results indicated that administrations of superantigens protected mice from infection with L. monocytogenes, which was dependent on the enhanced L. monocytogenes-specific CTL activity in the presence of CD4(+) T cells, and superantigens exhibited adjuvant activity in the immunization against intracellular pathogens.     

Effect of cyclosporin A on immunity to Listeria monocytogenes.

The effect of the immunosuppressive drug cyclosporin A (CS-A) on immunity to the facultative intracellular bacterium Listeria monocytogenes was investigated in unprimed and primed mice. Different treatment protocols were followed to evaluate the time dependence of CS-A-mediated immune suppression and the effect of CS-A on immunological memory to L. monocytogenes. The effect of CS-A was observed only during and after activation of T cell-mediated immunity, whereas early resistance exerted by macrophages assessed 6 and 70 min after challenge remained unaffected. CS-A suppressed efficient elimination of L. monocytogenes even when given after day 3 of a primary infection. This contrasts with findings in other models, including viral infections, where CS-A must be administered very early in an immune response to suppress it. CS-A suppressed antibacterial resistance in mice primed at various times before challenge; suppression of protection was time dependent and was virtually complete in livers, whereas CS-A-resistant memory persisted in spleens for up to 10 months.     

Alteration of cell-mediated immunity to Listeria monocytogenes in protein-malnourished mice treated with thymosin fraction V.

Cell-mediated immune reactivity, measured by lymphocyte responsiveness to phytohemagglutinin, was higher in both young or aged mice fed a 4% casein diet compared with age-matched controls. Treatment in vivo with bovine thymosin fraction V decreased the responsiveness to phytohemagglutinin of lymphocytes from mice fed either the control or moderately protein-deficient diets when compared with mice treated in vivo with saline. Resistance against Listeria monocytogenes, known to be a cell-mediated immune function, was impaired in young and aged mice which were fed the low-protein diet. Treatment with thymosin was able to significantly improve the cell-mediated immune resistance to L. monocytogenes of moderately protein-malnourished mice. Thymosin treatment impaired the resistance to L. monocytogenes of young or aged mice fed the control diet. The splenic natural killer cell cytotoxicity of protein-malnourished mice was impaired compared with that of mice fed the control diet. Treatment with thymosin did not restore the natural killer cell cytotoxic activity in protein-malnourished mice, but did enhance that activity in control mice.     

The mechanism of reduction of cell-mediated cytotoxicity in neonatally thymectomized mice.

The effect of neonatal thymectomy at various times after birth (Tx-1, Tx-7) on effector and suppressor T cells responsible for cell-mediated cytotoxicity (CMC) for allogenic antigens was determined. Following in-vitro primary mixed lymphocyte cultures, in the absence of T-cell growth factor (TCGF), alloreactive CMC was not detected in spleen cells of Tx-1 mice, but was detected in spleen cells of Tx-7 mice at as high levels as in those of sham-operated mice. However, in the presence of TCGF, as much alloreactive CMC was detected in spleen cells of Tx-1 mice as in those of Tx-7 mice. Furthermore, TCGF production was not detected in spleen cells of Tx-1 mice but was detected in those of Tx-7 mice. In in-vivo experiments, inhibition of allogeneic tumour growth and CMC in spleen cells showed the same pattern as in in-vitro experiments. These results support the concept that the reduction of CMC in Tx-1 mice might be due to a defect in helper function (TCGF-producing capacity) rather than to a defect in cytotoxic T lymphocytes and/or cytotoxic T lymphocyte precursors. Alloreactive suppressor T cells could not be induced in spleen cells of Tx-1 mice but were induced in spleen cells of Tx-7 mice. Therefore, it was suggested that alloreactive suppressor T cells require the presence of the thymus for 7 days after birth in their development.     

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.     

Evidence for a significant role of CD4+ T cells in adoptive immunity to Listeria monocytogenes in the liver.

Although the ability of CD8+ T cells to adoptively immunize mice against Listeria monocytogenes in the spleen is well established, the role of different T-cell subsets in anti-bacterial protection in the liver, a major target of Listeria infection, remains unclear. Therefore, the ability of sorted CD4+ and CD8+ T cells to adoptively immunize mice against a L. monocytogenes infection in the liver was studied. The results show that positively sorted CD4+ T cells from day 7 Listeria-immune mice were as effective as sorted CD8+ cells in transferring significant anti-Listeria protection in the liver. Similar findings were obtained when CD4+ and CD8+ T cells, negatively selected by antibody-induced complement-mediated depletion in vitro, were used for adoptive transfer. CD8+ T cells, however, were more efficient than CD4+ T cells in transferring protection in the spleen. Taken together, the results show that CD4+ T cells are at least as protective as CD8+ T cells against a L. monocytogenes infection in the liver, thereby arguing against the view that CD4+ T cells are of limited importance in adoptive immunity against listeriosis.     

Listeria pneumonitis: induction of immunity after airborne infection with Listeria monocytogenes.

After implantation of approximately 10(3) Listeria monocytogenes organisms into the lungs, mice develop an acute pneumonitis with dissemination of infection to a mediastinal lymph node (MedLN), liver, and spleen. The infections in a MedLN and spleen resolve in approximately 7 days, but the lung infection persists for a few days longer. Pneumonitis is accompanied by a lymphoproliferative response in a MedLN and spleen, and immunity to Listeria is conferred adoptively with MedLN and spleen cells but not with mesenteric lymph node cells. Although the spleen appears to be the major repository of sensitized lymphocytes, splenectomized mice combat Listeria pneumonitis as effectively as normal mice. It is concluded that the induction of immunity to lung infection with L. monocytogenes is efficient and that the cause for the rather protracted pneumonitis is due to a defect in the expression of the cell-mediated immunity effector mechanism.     

Differentiation of distinct long-lived memory CD4 T cells in intestinal tissues after oral Listeria monocytogenes infection

Mucosal antigen-specific CD4 T-cell responses to intestinal pathogens remain incompletely understood. Here we examined the CD4 T-cell response after oral infection with an internalin A ‘murinized’ Listeria monocytogenes (Lm). Oral Lm infection induced a robust endogenous listeriolysin O (LLO)-specific CD4 T-cell response with distinct phenotypic and functional characteristics in the intestine. Circulating LLO-specific CD4 T cells transiently expressed the ‘gut-homing’ integrin α₄β₇ and accumulated in the intestinal lamina propria and epithelium where they were maintained independent of interleukin (IL)-15. The majority of intestinal LLO-specific CD4 T cells were CD27⁻ Ly6C⁻ and CD69⁺ CD103⁻ while the lymphoid LLO-specific CD4 T cells were heterogeneous based on CD27 and Ly6C expression and predominately CD69⁻. LLO-specific effector CD4 T cells transitioned into a long-lived memory population that phenotypically resembled their parent effectors and displayed hallmarks of residency. In addition, intestinal effector and memory CD4 T cells showed a predominant polyfunctional Th1 profile producing IFNγ, TNFα, and IL-2 at high levels with minimal but detectable levels of IL-17A. Depletion of CD4 T cells in immunized mice led to elevated bacterial burden after challenge infection highlighting a critical role for memory CD4 T cells in controlling intestinal intracellular pathogens.     

Pattern of distribution of syngeneic spleen cells in the lymphoid organs of neonatally thymectomized mice

Newborn mice were thymectomized and intraperitoneally inoculated with syngeneic spleen cells on days 0, 3, 7 and 20 after birth. Donor cells were chromosomally distinguishable from host cells (T6). Cytological analysis performed between days 2 and 118 post-natal revealed greatly increased proportions of donor cells in host lymph nodes if comparison were made with similarly injected non-thymectomized hosts. The effect is highly specific, since the proportions of donor cells in the host spleen and bone marrow remained similar to those in control, non-thymectomized mice (syngeneic standard). The mechanism behind this phenomenon is unclear.     

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.     

Induction by killed Listeria monocytogenes of effector T cells mediating delayed-type hypersensitivity but not protection in mice.

Using a local passive transfer system, we found that effector T cells mediating delayed-type hypersensitivity (DTH) but not acquired cellular resistance (ACR) to Listeria monocytogenes (strain EGD) were generated in mice immunized with killed Listeria, although immunized mice did not express DTH or ACR. When non-adherent cells of peritoneal, lymph node, or spleen cells from mice immunized with killed Listeria were transferred into the footpad of naive recipient mice along with eliciting antigen, positive delayed footpad reaction (DFR) was elicited. However, there was no evident protection against challenge at the site of the local transfer. Cells from mice immunized with viable Listeria conferred significant degrees of DFR and ACR on the recipients. DFR transferred by cells immunized with killed Listeria was mediated by L3T4+ T cells in an antigen-specific manner. The antigen-specific proliferative response of T cells from mice immunized with killed Listeria was much lower than that of T cells from mice immunized with viable Listeria. The production of macrophage chemotactic factor (MCF) by cells from killed Listeria-immune mice was much the same as that by cells from viable Listeria-immune mice. In contrast, the production of interleukin-2 (IL-2) and macrophage activating factor (MAF) was much lower in cells from killed Listeria-immune mice. The elimination of L. monocytogenes (strain L461), a strain of low virulence, was enhanced at the site of DFR transferred with cells from killed Listeria-immune mice. These results suggest that stimulation with killed bacteria is effective for the generation of DTH-mediating effector T cells, and that different effector T cells mediating DTH or ACR are involved in cell-mediated immunity to L. monocytogenes.