Distinct subsets of accessory cells activate Thy-1+ triple negative (CD3-, CD4-, CD8-) cells and Th-1 delayed-type hypersensitivity effector T cells.

The SJL strain of mice possess a unique developmental delay in the ability to exhibit delayed-type hypersensitivity (DTH) responses after immunization with a wide variety of Ag. Similar to other models of DTH, the adoptive transfer of syngeneic Ag-pulsed macrophages from DTH-responsive mice into these DTH-unresponsive mice results in the activation of Ag-specific, CD4+ DTH effector Th1 T cells. The absence of other defects in APC-dependent immune responses indicate that the macrophages is the sole APC required for the induction of DTH effector T cells in SJL mice. The defect occurs during the sensitization phase of the DTH response; however, it has not been determined whether a Th cell, which is required for the induction of CD4+ DTH effector T cells, was present in the DTH unresponsive SJL mice. In this study, we have determined that the Thy-1+ helper cell is induced upon Ag stimulation of nonresponder mice and present evidence for the existence of an accessory cell distinct from the macrophage that induces CD4+ DTH effector T cells. Our data indicate that CD4+ DTH effector T cells are induced in an Ag-specific and MHC-restricted manner by an adherent macrophage that expresses the Mac-1+, Mac-2-, Mac-3+, I-A+ phenotype. Adoptive transfer of as few as 100 of the Mac-1+, Mac-2-, or Mac-3+ subsets from DTH responsive donors to DTH unresponsive recipients is able to overcome the DTH deficit. The activation of CD4+ DTH effector T cells in the SJL mouse cells also requires a Thy-1+, Lyt-1+, CD3-, CD4-, CD8-, helper cell. In contrast to the Mac-1+, Mac-3+, I-A+ accessory cell, this helper cell requires an adherent, irradiation resistant, accessory cell that expresses the Mac-1+, Mac-2-, Mac-3-, I-A- surface phenotype for activation. Further, the interaction between this accessory cell and the Thy-1+ helper cell is neither Ag-specific nor MHC restricted. This is the first demonstration of an accessory cell requirement for the Thy-1+, Lyt-1+, B220-, CD4-, CD8-, CD3- DTH Th cell. These data indicate that the activation of the triple negative helper cells and subsequent activation of the CD4+ effector T cells are regulated by two distinct macrophage subpopulations.     

Antigen presentation by Langerhans cells in vivo: donor-derived Ia+ Langerhans cells are required for induction of delayed-type hypersensitivity but not for cytotoxic T lymphocyte responses to alloantigens

T cell activation in response to allogeneic stimulation and hapten-specific delayed-contact hypersensitivity responses in vivo can be initiated by Ia-bearing epidermal Langerhans cells (LC). By using a murine heterotopic corneal allograft model, we have investigated the requirement for allogeneic LC as antigen-presenting cells (APC) in the in vivo induction of delayed-type hypersensitivity (DTH) and cytolytic T lymphocyte (CTL) responses to alloantigens in fully allogeneic and H-2 I region-disparate strain combinations. LC-deficient, avascular central corneal allografts from BALB/c donors failed to induce DTH responsiveness when grafted to a subdermal bed on C57BL/6 recipients (p greater than 0.05), yet antigen-specific primary CTL reactivity developed within 7 days after grafting. LC-containing corneal-limbus allografts or central corneal allografts containing a latex bead-induced infiltrate of LC resulted in intense DTH as well as CTL responsiveness when grafted in this same strain combination. Similarly, LC-containing but not LC-deficient corneal allografts from A.TL donors induced DTH responsiveness in I region-disparate A.TH hosts despite the fact that these grafts survived for prolonged duration (less than 28 days). By contrast, CTL induction in I region-disparate hosts was independent of the presence of allogeneic LC. Corneal epithelial cells of grafts removed from I region-disparate hosts 7 days posttransplantation were shown by immunohistology to express the Iak antigens of donor origin. The possibility that bone marrow-derived allogeneic LC were a sufficient requirement for DTH induction was confirmed in experiments performed with CB6F1----B6 bone marrow chimeras used as corneal allograft donors. Corneal-limbus grafts obtained from mice 90 days after chimerization were shown by immunohistology to contain Iad-bearing CB6F1 LC as a sole source of class II alloantigens. When grafted to C57BL/6 recipients, LC-containing chimeric corneas induced DTH responsiveness that was similar in magnitude to that observed in C57BL/6 mice grafted with chimeric skin, yet no DTH response to LC-deficient chimeric central corneal grafts was observed. Moreover, in all cases, the chimeric corneal and skin allografts survived for prolonged duration (greater than 28 days). These results demonstrate that donor-derived LC act as APC in the induction of DTH responsiveness to allogeneic tissue; however, there was no apparent requirement for allogeneic LC in the induction of CTL responses to class I or class II MHC alloantigens.(ABSTRACT TRUNCATED AT 400 WORDS)     

Direct in vitro evidence for different susceptibilities to 4-hydroperoxycyclophosphamide of antigen-primed T cells regulating humoral and cell-mediated immune responses to sheep erythrocytes: a possible explanation for the inverse action of cyclophosphamide on humoral and cell-mediated immune responses

Suppressor T cells of humoral immune responses, effector T cells mediating DTH, suppressor T cells of DTH, and helper T cells of humoral immune responses, all with specificity to SRBC, were produced in mice. The biologic activity was tested in adoptive transfer experiments. In vitro treatment with different doses of 4-hydroperoxycyclophosphamide (4-HPCy) yielded the result that the various activities tested were not uniformly sensitive to the action of this drug: Suppressor T cells of humoral immune responses and effector T cells mediating DTH were resistant to doses of 4-HPCy that eliminated the activities of suppressor T cells of DTH and helper cells of the humoral immune response. These findings help to explain the various effects cyclophosphamide has on the in vivo immune response and may help to form a basis for the rational manipulation of the immune response by drugs that selectively affect different subgroups of immune cells.     

The selection of effector T cell phenotype by contrasuppression modulates susceptibility to autoimmune injury

The genetic susceptibility to murine alpha TBM disease is a dominant trait that maps to H-2K. In previous studies we have shown that the critical difference between susceptible (SJL) and nonsusceptible (B10.S(8R] mice is the phenotype of the tubular Ag-specific effector T cells (TDTH). In SJL mice, these TDTH are Lyt-2+, whereas in B10.S(8R) mice the TDTH are L3T4+. These phenotypic differences have an important functional correlate: Lyt-2+ TDTH are nephritogenic, whereas L3T4+ TDTH are typically not nephritogenic. Both mouse strains have the potential to differentiate both L3T4+ and Lyt-2+ TDTH. The preferential selection of a single TDTH phenotype in each is the result of differential T cell regulation. In the present studies, we have examined the contribution of suppressor and contrasuppressor T cells in the regulation of TDTH phenotype selection. Our studies show that in both SJL and B10.(8R) mice, after exposure to Ag, a suppressor T cell subpopulation functions to inhibit the nephritogenic Lyt-2+ TDTH. In SJL, but not B10.S(8R) mice, this suppression is counterbalanced by Lyt-2+, Vicia Villosa lectin-adherent T cells. Such contrasuppressor function is mediated through a T cell-derived soluble protein (TcsF), which is Ag-binding and recognized by alpha I-JS antisera. This functional TcsF activity maps, as does susceptibility to disease, to H-2K. In the presence of genetically compatible TcsF, the TDTH phenotype in nonsusceptible mice switches to that of susceptible mice. These Lyt-2+ TDTH from nonsusceptible mice are fully capable of inducing tubulointerstitial nephritis following adoptive transfer. Our studies describe a new role for Tcs cells and augment our understanding of their etiopathogenetic role in autoimmunity.     

The in vitro suppression of spontaneous erythrocyte autoimmune responses with lymphocytes activated with concanavalin A.

When normal mouse spleen cells are cultured in vitro, large numbers of cells develop that produce antibody toward antigens found on bromelain-treated mouse erythrocytes (BrMRBC). The in vitro culture also generates T cells that mediate DTH toward these antigens. We have suggested that under in vivo conditions, suppressor T cells maintain these immune responses at a low level but that this suppression wanes when the cells are cultured in vitro. The present study examines the effect of concanavalin A (Con A) on the in vitro development of humoral and cell-mediated immunity to Br-MRBC. Mitogenic concentrations of Con A prevented the development of both the PFC and TDTH responses toward BrMRBC. The Con A-induced suppression was due to the induction of suppressor T cells; thus the addition of Con A-activated cells to fresh spleen cell cultures prevented the development of both the PFC and TDTH response against BrMRBC.     

Influence of 2'-deoxyguanosine upon the development of DTH effector T cells and suppressor T cells in vivo

Subcutaneous (s.c.) immunization of mice with allogeneic spleen cells can induce delayed-type hypersensitivity (DTH) to both major and minor histocompatibility antigens. Intravenous immunization with allogeneic spleen cells, however, induces a poor state of DTH. Furthermore, i.v. immunization with allogeneic spleen cells, especially if they have been irradiated, induces suppressor T lymphocytes. These suppressor T cells are capable of suppressing the host-vs-graft (HvG) DTH reactivity that normally arises after s.c. immunization. Moreover, they can suppress the development of anti-host DTH effector T cells during graft-vs-host (GvH) reactions. These models for HvG and GvH DTH reactivity were used to study the influence of 2'-deoxyguanosine (dGuo) and guanosine (Guo) on the generation of DTH-reactive T cells and suppressor T cells in vivo. It was found that daily i.p. administration of 0.01 mg dGuo to mice immunized i.v. partially prevented the generation of suppressor T cell activity, whereas daily administration of 0.1 or 1 mg dGuo resulted in a complete abolition. Administration of dGuo has no effect on the anti-host DTH reactivity by spleen cells from nonsuppressed donors except for when a daily dose of 10 mg is administered. This dose proved to be toxic for precursors of DTH effector T cells. Daily i.p. injection of Guo had no effect on the generation of suppressor T cells nor on the generation of DTH effector T cells. The effect of dGuo was found to be due to a direct effect on suppressor T cells and not to the induction of contrasuppressor cells. These data suggest a differential sensitivity of DTH-reactive T cells and suppressor T cells for dGuo. Because suppressor T cells and DTH-reactive T cells require proliferation for expressing maximal functional activity in the systems used, both cell types probably have different enzyme activities involved in the purine metabolism and similar deoxycytidine kinase activities, but have different nucleotidase (5'NT) activities, those in suppressor T cells being the lowest. If so, suppressor T cells will accumulate deoxyguanosine triphosphate, which causes an inhibition of the ribonucleotide reductase activity and thus of the DNA synthesis by these cells.     

Tumor progression despite efficient tumor antigen cross-presentation and effective "arming" of tumor antigen-specific CTL

To determine whether APC function or "arming" of CTL for lytic function are the points at which Ags from a nonimmunogenic tumor fail to induce an effective immune response, we established a murine tumor model that expressed intracellular OVA and selected a clone (cOVA-9) that remained susceptible to lysis by specific CD8(+) T cells throughout tumor growth. Viable cOVA-9 tumor cells grew in normal mice at a rate similar to the parental tumor, and vaccination with irradiated cOVA-9 cells did not induce protection against itself or the parental line, confirming its nonimmunogenic status. In vivo evaluation during tumor growth demonstrated persisting tumor Ag cross-presentation accompanied by the generation of potent, specific CTL which were detectable when tumors were barely palpable. Despite the presence of highly active CTL in the tumor-draining lymph nodes, there was no apparent lysis of tumor-associated APC. These data show that tumor-draining APC are not dysfunctional with regard to two crucial processes, in vivo tumor Ag cross-presentation and specific CTL arming, and that failure to prevent tumor growth is not in the induction phase, but in the effector phase and occurs within the tumor itself before the tumor matrix is established.     

Ongoing hapten-specific delayed-type hypersensitivity prevents generation of cytolytic T lymphocytes to the same hapten

Cytolytic T lymphocytes (CTL) specific for trinitrophenyl (TNP)-altered self antigens can be generated in vivo through the simultaneous injection of TNP-modified syngeneic spleen cells and H-2-compatible, minor histocompatibility locus (Mls)-disparate auxiliary spleen cells into the footpads of mice. The latter stimulates host helper cells to produce differentiative and proliferative signals required for the generation of CTL. Advent of this protocol allowed investigation of the initiation of two different cell-mediated immune responses, delayed-type hypersensitivity (DTH) and the generation of CTL, in the same experimental animal. Mice presensitized for CTL were able to develop DTH as well as normal controls. However, when mice were first sensitized for DTH, they were thereafter incapable of generating CTL. This effect was hapten specific, relatively long lasting, and preventable by treating mice with cyclophosphamide before sensitizing for DTH. Adoptive transfer of lymphoid cells from DTH-immune mice conferred DTH reactivity upon naive recipients but not a suppressed CTL response. Therefore, cells mediating DTH were not responsible for suppression of CTL. The mechanism for suppression has been discussed from the viewpoint of the suppressor-T-cell circuits that are known to be generated when animals are sensitized for DTH and which are susceptible to treatment with cyclophosphamide.     

Characterization of Theiler's murine encephalomyelitis virus (TMEV)-specific delayed-type hypersensitivity responses in TMEV-induced demyelinating disease: correlation with clinical signs

After intracerebral inoculation of Theiler's murine encephalomyelitis virus (TMEV), certain mouse strains develop a persistent central nervous system (CNS) infection and inflammatory demyelinating lesions containing infiltrates of mononuclear cells and macrophages. Previous findings demonstrating a strong correlation between disease incidence, the presence of particular H-2 region genotypes, and development of high levels of TMEV-specific delayed-type hypersensitivity (DTH) supported an immune-mediated basis for myelin breakdown. These findings led us to examine whether a possible causal relationship would be supported by a temporal analysis comparing the onset of clinical disease and the development of TMEV-specific cellular or humoral immune responses in susceptible and resistant strains. In susceptible SJL/J mice, TMEV-specific DTH and T cell proliferative (Tprlf) responses developed within 10 to 14 days postinfection, preceded the onset of clinical signs, and remained elevated for 6 mo. In contrast, resistant BALB/c mice developed low levels of TMEV-specific Tprlf and no measurable DTH. However, both strains attained comparable levels of TMEV-specific serum antibody responses with parallel kinetics. Both DTH and Tprlf responses in susceptible SJL/J mice were shown to be specific for TMEV and mediated by L3T4+, Lyt-1+2-, class II-restricted T cells. A model is proposed implicating an effector role for TMEV-specific DTH, wherein lymphokine release by virus-specific DTH T cells leads to the recruitment, accumulation, and activation of macrophages in CNS tissue, which cause bystander myelin destruction and provide a permissive population of host cells for TMEV persistence.     

The mechanism of antigen presentation by dendritic cells and splenic adherent cells in the induction of an allogeneic cytotoxic T-lymphocyte response to H-2Kk liposomes

Induction of an allogeneic cytotoxic T-lymphocyte (CTL) response to purified alloantigen is partially dependent on uptake and processing of the class I alloantigen by antigen-presenting cells (APC) followed by recognition of the alloantigen and self Ia by helper T cells (TH). The activated TH provides the helper signal(s) to the alloantigen-specific CTL for proliferation and differentiation into an active effector CTL. The role of antigen processing and presentation of major histocompatibility complex alloantigens was examined and the ability of different types of APC to present purified H-2Kk liposomes was investigated. Splenic adherent cells (SAC), splenic dendritic cells (DC), and B-cell lymphoblastoid lines were all shown to be effective in the presentation of H-2Kk liposomes. The relative ability of these cells to serve as APC was determined to be DC greater than B-cell tumors greater than SAC. The role of processing of H-2Kk liposomes by SAC and DC was examined by investigating the effect of weak bases on pulsing of the APC. These experiments suggest that presentation of alloantigen by both SAC and DC involves a step which is sensitive to inhibition by weak bases. We examined whether the TH were activated by similar mechanisms when stimulated by the various APC. The functional involvement of the T-cell surface marker L3T4 was demonstrated in the induction of TH. In contrast, L3T4 was not involved in the subsequent generation of CTL since monoclonal antibody (MAb) specific for L3T4 was not effective in blocking CTL function in the presence of nonspecific T helper factor (THF). Similarly, Ia on the APC was shown to be involved in the stimulation of the TH pathway but not directly in the differentiation of the CTL. Thus, DC and B cells in addition to SAC can present H-2Kk to TH. The presentation of alloantigen by both cell types may involve an intracellular route as demonstrated by the blocking of the TH response by weak bases. Both Ia and L3T4 are required on the APC for induction of the TH response. The minimal requirements for activation of the CTL were H-2Kk liposomes and a source of THF.     

T-cell responses induced by the parenteral injection of antigen-modified syngeneic cells. III. Dissociation of primed cytolytic T-cell and efferent suppressor-T-cell activity following intravenous injection of trinitrophenol-modified syngeneic spleen cells

The parenteral injection of ligand-coupled syngeneic spleen cells has profound effects on immune responsiveness. In this regard, it was examined whether the primed in vitro trinitrophenol (TNP)-specific cytotoxic T-lymphocyte (CTL) responses observed in splenic T-cell populations from mice injected intravenously (iv) with syngeneic TNP-modified spleen cells (TNP-SC) are related to the efferent-acting suppressor-T-cell (Ts) activity observed in splenocytes from iv primed mice. Treatment of mice with cyclophosphamide, adult thymectomy, or monoclonal anti-I-J antiserum prior to the iv injection of TNP-SC was found to eliminate the ability of splenic Ts from these mice to suppress the passive transfer of delayed-type hypersensitivity (DTH) mediated by trinitrochlorobenzene-immune T cells. In contrast, spleen cells from these pretreated mice showed no impairment in the development of augmented TNP-specific CTL responses upon in vitro restimulation with TNP-SC. Separation of the two activities was also achieved in a kinetic analysis. It is concluded that specific enhancement of CTL responsiveness induced by the iv injection of TNP-SC is related to the expansion of a population prelytic Lyt 2+ CTL effector cells which does not appear to contain efferent-acting Lyt 2+ Ts active in suppressing DTH expression.     

Generation of alloreactive cytotoxic T lymphocytes: production of T cell and macrophage helper factors in addition to IL 1 and IL 2 by peritoneal cells from mice immunized to Listeria monocytogenes

We investigate the production and biological activity of soluble helper factors produced by peritoneal T cells and macrophage derived from mice primed in vivo with Listeria monocytogenes. Supernatant fluids from co-cultures of these immune T cells and activated macrophages contained Interleukin 1 (IL 1) and Interleukin 2 (IL 2), and had the ability to assist the generation of cytotoxic T lymphocytes (CTL) from a population of nylon wool nonadherent spleen cells sensitized to allogeneic heat-treated thymocytes. The ability to assist CTL development involved T cell and macrophage factors in addition to IL 1 and IL 2. Immune T cells cultured alone produced a factor, devoid of significant IL 2 activity, that assisted CTL development only if adherent cells were present in the responding population. Activated macrophage produced a 38,000 dalton component, distinct from IL 1 on the basis of m.w., that assisted the development of CTL from nylon wool nonadherent splenic cells. Supernatants fluids from co-cultures of immune T cells and allogeneic, nonactivated macrophage contained a CTL helper factor but did not contain IL 1 or IL 2 activities. In contrast, supernatant fluids from co-cultures of immune T cells and syngeneic, nonactivated macrophage contained all 3 activities. This suggests a genetic restriction for the production of IL 1 and IL 2 that does not restrict the production of a CTL helper factor. These results demonstrate that T cell- and macrophage-derived helper factors distinct from IL 1 and IL 2 participate in the development of CTL.     

The effect of T cell growth factor on the generation of cytolytic T cells.

The development of cytotoxic effector cells through primary allogeneic mixed tumor-lymphocyte culture (MTLC) was found to be accompanied by the production of T cell growth factor (TCGF). Addition of supplemental TCGF to MTLC resulted in the generation of significantly greater quantities of effector cells, and these effector cells displayed augmented cytotoxic activity. The TCGF-induced effect could not by duplicated by the addition of fresh medium or a mitogenic concentration of concananvalin A. Although TCGF augmented the proliferation of antigen-nonreactive cells, antigen-reactive cells appeared to be preferentially stimulated by TCGF. Finally, it was shown that depletion of TCGF from MTLC resulted in an impairment of proliferation and differentiation of cytotoxic effector cells. These findings demonstrate that soluble factors are involved in the regulation of in vitro cell-mediated immune responses in an analogous manner to similar factors that have been shown to regulate humoral immune responses. Therefore, the forces affecting TCGF production may modulate the amplitude of a T cell-mediated cytolytic response.     

IL-12 signaling drives CD8+ T cell IFN-gamma production and differentiation of KLRG1+ effector subpopulations during Toxoplasma gondii Infection

IFN-gamma-producing CD8(+) T lymphocytes are essential effector cells that mediate protective immunity during murine toxoplasmosis, and yet their effector development remains poorly characterized. Vaccination with the carbamoyl phosphate synthase (CPS) mutant strain of Toxoplasma gondii was used to examine the CD8(+) T cell response in the peritoneal effector site. Four CTL subpopulations with varying effector potentials were defined based on the expression of effector molecules and the cell surface activation markers CD62L and killer cell lectin-like receptor G1 (KLRG1). Further phenotypic analysis revealed that the acquisition of KLRG1 among effector subpopulations correlated with the down-regulation of both IL-7R and CD27, suggesting that KLRG1 marks dominant, end-stage effector cells. Using gene-targeted mice, we tested the in vivo requirements of key IL-12 signaling components for effector CTL differentiation. Contrary to established models of viral and bacterial infection, CD8(+) T cell-intrinsic IL-12 signaling was required for the generation of IFN-gamma-producing CTLs in response to T. gondii. Importantly, the development of the KLRG1(+) effector subpopulations, but not the memory precursor-containing KLRG1(-) effector subset, was critically reliant on IL-12. Furthermore, IL-12 signaling-dependent T-bet expression was also found to be important for differentiation of KLRG1(+) effectors. Our results underscore a vital role for IL-12 in not only the induction of IFN-gamma expression but also in the development of heterogeneous subpopulations of effector CD8(+) T cells generated in response to the intracellular parasite T. gondii.     

Non-H-2-linked genetic regulation of cytotoxic responses to hapten-modified syngeneic cells. I. Non-H-2-linked Ir gene defect expressed on T cells is not predetermined at the stage of bone marrow cells

Spleen cells from C3H/He or BALB.K mice immunized to the newly synthesized amino-reactive hapten 5-sulfo-1-naphthoxy acetic acid N-hydroxysuccinimide ester (AED-NH2) were stimulated in vitro with AED-NH2-modified syngeneic cells. After 5 days of culture, effector cells were assayed for their cytotoxic activity against AED-NH2-modified target blast cells. C3H/He and BALB.K mice exhibited the respective high and low anti-AED-NH2 cytotoxic T lymphocyte (CTL) responses. This contrasted with the observation that both of these H-2k strains generated potent CTL responses against aminoreactive haptens, e.g., trinitrophenyl (TNP). Because C3H.SW and BALB.B strains, which are the H-2b counterpart of the above two strains, also represented the respective high and low responders to AED-NH2 hapten, this hapten model enabled us to investigate cellular mechanisms underlying the above non-H-2-associated genetic regulation of CTL responses (C3H vs BALB non-H-2 backgrounds). The results demonstrated that there was no detectable difference between C3H/He and BALB.K strains in the lysability of target cells and the ability of stimulating cells to activate primed spleen cells. Anti-AED-NH2 CTL responses were only marginal when antigen-presenting cells (APC) were eliminated from the primed spleen cells of high responder C3H/He or (C3H/He X BALB.K)F1 mice. The addition of APC to cultures free of APC regained an appreciable CTL response in C3H/He or (C3H/He X BALB.K)F1 mice, irrespective of whether APC were derived from high (C3H/He) or low (BALB.K) responders. We have also demonstrated that allogeneic radiation bone marrow chimera (BALB.K----C3H/He) exhibited a CTL response comparable to that induced by C3H/He mice, whereas the reverse direction of allogeneic chimera (C3H/He----BALB.K) induced a marginal CTL response. These results indicate that this non-H-2-associated Ir gene defect is expressed on T cells (CTL precursors and/or helper T cells) rather than APC, and that this T cell defect is not predetermined at the level of bone marrow cells. The results are discussed in the light of the genetic and cellular mechanisms underlying non-H-2-linked Ir gene control.     

Induction of cytotoxic T lymphocytes in vivo with protein antigen entrapped in membranous vehicles.

Intravenous administration of APC such as splenocytes loaded with a soluble protein Ag has been shown to prime for an Ag-specific CTL response. It is thought that the APC directly presents loaded Ag in a MHC-restricted manner. However, it is demonstrated in this study that allogeneic splenocytes, MHC-free RBC, and even synthetic lipid vesicles (liposomes) after loading with OVA can elicit an OVA-specific and MHC-restricted CTL response. Biodistribution studies of these Ag-associated vehicles showed that the liver, spleen, and lung were the major organs responsible to scavenge these carriers, suggesting that the monocyte-macrophage system was involved in the Ag presentation for CTL. Depletion of macrophages by a specific macrophage killer, Cl2MDP, containing liposomes, abolished the CTL induction by immunization with OVA Ag carried by these vehicles except the induction by syngeneic splenocytes. Thus, the syngeneic splenocytes present Ag directly to the T cells, but other membranous vehicles carry the Ag to the host APC including macrophages, which then present it to the T cells. These results indicate that formulation of an Ag in membranous/colloidal vehicles may be a way to prime for a CTL response.     

Induction of cytotoxic T lymphocytes against herpes simplex virus type i: role of accessory cells and amplifying factor

Spleen cells from mice primed with herpes simplex virus type 1 (HSV-1) could be induced to differentiate into cytotoxic T lymphocytes (CTL) by in vitro culture with infectious HSV-1 but not by heat-inactivated virus. Induction of CTL failed to occur if the spleen cells were depleted of adherent cells by passage over columns of nylon wool before culture with virus. The CTL response could be restored by adding normal syngeneic peritoneal cells (PC) or L cell fibroblasts but not by allogeneic PC or BALB/c 3T3 fibroblasts. Thus, the induction of HSV-1-specific CTL was H-2 restricted. The response of HSV-1-stimulated nylon wool-depleted spleen cells could also be restored by adding amplifying factor (AF) produced in 24 hr mixed lymphocyte cultures. The addition of AF to nondepleted spleen cells also permitted the generation of CTL with heat-inactivated HSV-1 as a viral stimulant. Our results indicated that induction of a HSV-1 CTL response requires two signals, one provided by virus and a second, presumably nonspecific, by helper T cells. It was suggested that only the helper cells require H-2 restriction and need to be presented virus in the context of a macrophage.     

Protective immunity and delayed type hypersensitivity reaction are uncoupled in experimental Leishmania major infection of CCR6-negative mice

The chemokine receptor CCR6 is expressed on naïve B cells, dendritic cell and T-cell subpopulations and is involved in cell navigation during organogenesis and recruitment in response to inflammatory stimuli. Gene-deficient C57BL/6 CCR6^−/− mice infected with the protozoan parasite Leishmania (L.) major were able to mount a protective immune response and survived the infection. Whereas macrophage production of nitric oxide (NO), the key leishmanicidal effector molecule during the immune response to L. major, did not require CCR6, the migration of CD4⁺ T cells to the site of infection was reduced in CCR6^−/− mice. Furthermore, the induction of a T-cell-dependent delayed-type-hypersensitivity (DTH) reaction was defective in CCR6^−/− mice, whereas resistance to re-infection was maintained in the absence of CCR6. We conclude that CCR6 contributes to the recruitment of T cells to the site of infection, but is largely dispensable for the control of L. major parasites during primary or secondary infection.     

Influence of the H-2u haplotype on immune function in F1 hybrid mice. I. Antigen presentation

Previously, we reported that myelin basic protein (MBP) peptide 1-37 contains an encephalitogenic epitope for PL/J mice, and MBP peptide 89-169 is encephalitogenic for SJL/J mice. (SJPL)F1 hybrid mice do not respond to immunization with these peptides in a co-dominant manner because the encephalitogenic response to peptide 1-37 dominates. To examine this phenomenon more closely, we tested the ability of MBP-primed parental or F1 T cells to respond to MBP or MBP peptides in the context of PL, SJL, or F1 antigen-presenting cells (APC). It was found that the F1 T cells responded to either the protein or the peptides when these were presented in the context of F1 or PL APC. However, F1 T cells would not respond to MBP in the context of SJL APC, although the latter cells were functionally intact. This effect was not antigen-specific because SJL APC would not present ovalbumin or PPD to primed F1 T cells. F1 T cells from mice immune to the strongly antigenic bacterium Listeria monocytogenes responded to bacterial antigens presented by SJL APC, although at a significantly lower level compared to the results obtained when these antigens were presented by F1 or PL APC. This finding implied that unbalanced antigen presentation was a quantitative rather than a qualitative phenomenon. When F1 hybrid mice from other strain combinations were tested, a similar effect was observed whenever one of the parental strains was PL/J. This effect was mapped to the MHC in MHC-congenic B10 mice.     

Synergism of T lymphocyte subsets in the response to Mls-locus coded antigens during graft-versus-host reaction

After transplantation of lymphoid cells into lethally irradiated (semi)allogeneic mice specific anti-host directed effector T cells are generated. This can be demonstrated using a delayed type hypersensitivity (DTH) assay. In H-2 compatible combinations, Mls-locus antigens, but no other minor histocompatibility antigens, can induce the generation of such effector T cells. This paper shows that maximal anti-host DTH responses are obtained when the lymphoid cells transplanted constitute of a mixture of long-lived, recirculating T2 cells and short-lived, sessile T1 cells. It was demonstrated that anti-Mls locus-directed DTH effector T cells are the progeny of T2 cells, and that T1 cells amplify this response. The latter, however, are by themselves incapable of displaying anti-Mls DTH reactivity. The T1 cells were found to be of the Lyt-1+2+ phenotype, and the T2 cells of the Lyt-1+2- phenotype. The same Lyt phenotypes were found for T1 and T2 cells synergizing in the GvH reaction against H-2 alloantigens.