Regulatory T Cells in γ Irradiation-Induced Immune Suppression

Sublethal total body γ irradiation (TBI) of mammals causes generalized immunosuppression, in part by induction of lymphocyte apoptosis. Here, we provide evidence that a part of this immune suppression may be attributable to dysfunction of immune regulation. We investigated the effects of sublethal TBI on T cell memory responses to gain insight into the potential for loss of vaccine immunity following such exposure. We show that in mice primed to an MHC class I alloantigen, the accelerated graft rejection T memory response is specifically lost several weeks following TBI, whereas identically treated naïve mice at the same time point had completely recovered normal rejection kinetics. Depletion in vivo with anti-CD4 or anti-CD25 showed that the mechanism involved cells consistent with a regulatory T cell (T reg) phenotype. The loss of the T memory response following TBI was associated with a relative increase of CD4+CD25+ Foxp3+ expressing T regs, as compared to the CD8+ T effector cells requisite for skin graft rejection. The radiation-induced T memory suppression was shown to be antigen-specific in that a third party ipsilateral graft rejected with normal kinetics. Remarkably, following the eventual rejection of the first MHC class I disparate skin graft, the suppressive environment was maintained, with markedly prolonged survival of a second identical allograft. These findings have potential importance as regards the immunologic status of T memory responses in victims of ionizing radiation exposure and apoptosis-inducing therapies

Effective Antigen-Specific Immunotherapy in the Marmoset Model of Multiple Sclerosis

Mature T cells initially respond to Ag by activation and expansion, but high and repeated doses of Ag cause programmed cell death and can suppress T cell-mediated diseases in rodents. We evaluated repeated systemic Ag administration in a marmoset model of experimental allergic encephalomyelitis that closely resembles the human disease multiple sclerosis. We found that treatment with MP4, a chimeric, recombinant polypeptide containing human myelin basic protein and human proteolipid protein epitopes, prevented clinical symptoms and did not exacerbate disease. CNS lesions were also reduced as assessed in vivo by magnetic resonance imaging. Thus, specific Ag-directed therapy can be effective and nontoxic in primates. The Journal of Immunology, 2001, 166: 2116 -2121. M ultiple sclerosis (MS) 4 is a paralytic disease involving destruction of myelin sheaths surrounding axons in the CNS (1, 2). MS affects young adults, most often women residing in northern latitudes. The disease exhibits relapsing and remitting symptoms including disturbances in vision, speech, coordination, and cognition as well as weakness, spasticity, and paralysis (1, 2). Lymphocytic infiltration in the CNS white matter and immune reactions against myelin Ags indicate an autoimmune etiology for MS (1-8). Allergic encephalomyelitis was first observed as a side effect of the rabies vaccine prepared from rabbit brains by Pasteur in the 1880s (see Ref. 3). Rivers and others showed that the CNS inflammation was caused not by the rabies virus but by immune sensitization to the combination of adjuvant and brain tissue contaminating the vaccine (3, 4). Experimental allergic encephalomyelitis (EAE) models in various animal species, typically rodents, were later developed by immunization with myelin proteins in adjuvant or by the adoptive transfer of myelinreactive T cells, causing inflammatory damage to the white matter (1-6). Rodent EAE is the most widely used disease model despite important differences from MS (2). Encephalitogenic CD4 ϩ T cells are believed to initiate and perpetuate EAE and MS and thus constitute a therapeutic target (1-8). Abundant myelin protein Ags, including myelin basic protein (MBP) and proteolipid protein (PLP) as well as the less abundant Ags, myelin oligodendrocyte glycoprotein (MOG) and myelin-associated glycoprotein (MAG), are recognized by T cells in MS patients (9 -11). T cell responses against MBP and PLP may occur at an increased frequency in MS patients compared with controls (1, 2, 11, 12). Ag-specific immunotherapies directed at T cells could avoid the harmful side effects of general immunosuppressive treatments. We have investigated a potential immunotherapy for MS based on our observation that T cells undergo apoptosis both in vitro and in vivo when exposed to high or repeated doses of their cognate Ag (13, To present a broad array of potential epitopes to reactive T cells, we constructed MP4, a protein chimera of the 21.5-kDa isoform of human MBP, and a modified form of human PLP, termed PLP4, that lacks the hydrophobic domains of the protein but includes all of the known T cell epitopes (19 -21). MP4 is processed into multiple determinants and can eliminate rodent EAE by promoting tolerance to different epitopes In a few instances, EAE and Ag treatments have been studied in nonhuman primates. EAE was originally induced in rhesus macaques using CNS homogenates or purified MBP (3, 4, 30 -32). It was also found that repeated injections of MBP could arrest EAE The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact

AGM1+ Spleen Cells Contain Gamma Interferon (IFN-γ) Gene Transcripts in the Early, Sex-Dependent Production of IFN-γ After Picornavirus Infection

Encephalomyocarditis D variant (EMCV-D)-infected spleen cell cultures prepared from diabetes-resistant ICR Swiss female mice produce more gamma interferon (IFN-gamma) activity over a 24-h period than do spleen cell cultures from diabetes-susceptible male mice of this strain. Pretreatment of mice with anti-asialo GM1 eliminates early in vitro IFN-gamma production from 4 to 16 h postinfection (p.i.) and reduces IFN-gamma production from 16 to 24 h p.i. In this study, depletion of spleen cells with anti-Thy-1 by panning greatly reduced IFN-gamma activity in EMCV-D-infected spleen cell cultures throughout a 24-h period. Populations of asialo GM1 (AGM1), L3T4, and Lyt-2-positive cells were isolated from cells harvested at 9 h p.i. from EMCV-D-infected spleen cell cultures by a modified panning technique on polystyrene microscope slides. By in situ hybridization with a [35S]dATP-labeled IFN-gamma cDNA probe, only the AGM1-bearing cells were found to contain detectable IFN-gamma gene transcripts. An AGM1+, Thy-1+ natural killer-like cell is the probable producer of the early, sex-dependent IFN-gamma activity in this system

AGM1+ Spleen Cells Contain Gamma Interferon (IFN-γ) Gene Transcripts in the Early, Sex-Dependent Production of IFN-γ After Picornavirus Infection

Encephalomyocarditis D variant (EMCV-D)-infected spleen cell cultures prepared from diabetes-resistant ICR Swiss female mice produce more gamma interferon (IFN-gamma) activity over a 24-h period than do spleen cell cultures from diabetes-susceptible male mice of this strain. Pretreatment of mice with anti-asialo GM1 eliminates early in vitro IFN-gamma production from 4 to 16 h postinfection (p.i.) and reduces IFN-gamma production from 16 to 24 h p.i. In this study, depletion of spleen cells with anti-Thy-1 by panning greatly reduced IFN-gamma activity in EMCV-D-infected spleen cell cultures throughout a 24-h period. Populations of asialo GM1 (AGM1), L3T4, and Lyt-2-positive cells were isolated from cells harvested at 9 h p.i. from EMCV-D-infected spleen cell cultures by a modified panning technique on polystyrene microscope slides. By in situ hybridization with a [35S]dATP-labeled IFN-gamma cDNA probe, only the AGM1-bearing cells were found to contain detectable IFN-gamma gene transcripts. An AGM1+, Thy-1+ natural killer-like cell is the probable producer of the early, sex-dependent IFN-gamma activity in this system

In vivo state of antiviral CTL precursors. Characterization of a cycling cell population containing CTL precursors in immune mice

The in vivo state of CD8+ mouse memory CTL specific to lymphocytic choriomeningitis virus (LCMV) was characterized. During acute LCMV infection, the majority of the LCMV-specific CTL activity tested immediately ex vivo was mediated by CD8+ L-selectin- Mac-1+ CTL. The L-selectin- population of CD8+ cells elicited during acute infection also carried \u3e 99% of the restimulatable CD8+ CTL precursors (CTLp) to LCMV, and these required added IL-2 for development into effectors in vitro. In contrast with the acute infection, most of the virus-specific CTLp in immune mice were L-selectin+. Examination of CD8+ T cells in LCMV-immune mice revealed that a L-selectin+ blast-size population of cycling CD8+ cells contained CTLp, which developed into effector CTL in the absence of added IL-2. These cells also expressed Mac-1 and IL-2R. Flow cytometric sorting for IL-2R+ and IL-2R- CD8+ cells in the immune animal revealed, by limiting dilution analysis, similar frequencies of CTLp in both populations. In bulk restimulation assays, the CD25+ CTLp did not require added IL-2 for their in vitro development into effectors, whereas the CD25- CTLp did. Hence, the different requirements for CTLp to effector development in vitro reflect qualitative differences in the in vivo state of the CTLp in the various subpopulations. LCMV-specific memory CTLp that did not require added IL-2 for differentiation were also found in the small-size, noncycling, CD8+L-selectin- cells. In contrast, the small-size, noncycling, CD8+L-selectin+, and CD8+IL-2R- populations also carried CTLp, but these required added IL-2 for development into effector CTL. Hence, T cell memory to LCMV is distributed among various lymphocyte subpopulations in immune animals, and the presence of an activated cycling cell component may account for the long-term perpetuation of antiviral immunologic memory

Enhanced Capacity of Females for Early Interferon-γ Production by Natural Killer-Like Cells Following Stimulation by Staphylococcal Enterotoxin A

Staphylococcal enterotoxin A (SEA) induced the production of interferon-γ (IFN-γ) by spleen cells from ICR Swiss mice during the first 24 h of culture. Splenocytes from females produced higher levels of IFN-γ than did those from males at 8,12, and 16 h. By 20 h after SEA stimulation, IFN-γ production by spleen cells from males was similar to that of females. The cell types involved in IFN-γ production in this SEA/spleen cell system were analyzed by depletion studies. Removal of Thy-1+ cells by panning prevented production of IFN-γ in the 24 h after SEA stimulation. In vivo depletion of asialo GM1+ (AGM1+) cells prevented production of IFN-γ through 16 h of culture with SEA, but permitted a modest IFN-γ response at 20 h that was similar in magnitude in both sexes. Following removal of L3T4+ and Lyt-2+ cells by panning, IFN-γ production was detected at 12 h after SEA stimulation and maintained through 24 h of culture with cells from females producing higher levels of IFN-γ. These data suggest that male ICR Swiss mice are deficient in the activity of Thy-1+, AGM1+, L3T4−, and Lyt-2− cells in the early (8–16 h) production of IFN-γ following SEA stimulation of spleen cells

CD11b (Mac-1): a marker for CD8+ cytotoxic T cell activation and memory in virus infection

We have found that CD11b, a cell surface integrin of macrophages, granulocytes, and NK cells, is expressed by a subset of CD8+ T cells that include both the active virus-specific CTL and the virus-specific memory CTL populations. CD8+CD11b+ cells comprise less than 3% of naive mouse splenocytes, but after lymphocytic choriomeningitis virus (LCMV) infection increase by 9- to 12-fold by the peak (day 8) of the virus-specific CTL response. Depletion of day-8 splenocytes with anti-Mac-1 and C\u27 or enrichment by sorting for CD11b+ or CD8+CD11b+ spleen cells demonstrated that LCMV-specific CTL are CD11b+. The CD11b+ subpopulation also contained the bulk of the IL-2-responsive CD8+ cells. MEL-14, a homing marker down-regulated on activated T cells, was down-regulated on the majority of CD8+ cells that became CD11b+. Less than 1% of LCMV-immune splenic lymphocytes expressed CD11b. Antibody and C\u27 depletion of this population severely impaired the ability of immune splenocytes to respond to in vitro secondary stimulation with LCMV-infected peritoneal macrophages, but did not affect the generation of a primary allospecific CTL response in MLC. Mixing of CD8-depleted and CD11b-depleted LCMV-immune splenocytes failed to restore the ability of these cells to mount a virus-specific memory CTL response, indicating that a cell coexpressing CD8 and CD11b is essential for this response. As determined by limiting dilution analysis, the precursors for the LCMV-specific memory CTL response were enriched in the CD11b+ population of LCMV-immune splenocytes. CD11b stained far fewer CD8+ splenocytes from naive mice than did CD44 (Pgp-1), and among immune splenocytes it identified a small subpopulation of CD44hi cells, indicating that CD11b may be the best single marker available for discriminating between naive and memory CD8+ T cells

Enhanced Capacity of Females for Early Interferon-γ Production by Natural Killer-Like Cells Following Stimulation by Staphylococcal Enterotoxin A

Staphylococcal enterotoxin A (SEA) induced the production of interferon-γ (IFN-γ) by spleen cells from ICR Swiss mice during the first 24 h of culture. Splenocytes from females produced higher levels of IFN-γ than did those from males at 8,12, and 16 h. By 20 h after SEA stimulation, IFN-γ production by spleen cells from males was similar to that of females. The cell types involved in IFN-γ production in this SEA/spleen cell system were analyzed by depletion studies. Removal of Thy-1+ cells by panning prevented production of IFN-γ in the 24 h after SEA stimulation. In vivo depletion of asialo GM1+ (AGM1+) cells prevented production of IFN-γ through 16 h of culture with SEA, but permitted a modest IFN-γ response at 20 h that was similar in magnitude in both sexes. Following removal of L3T4+ and Lyt-2+ cells by panning, IFN-γ production was detected at 12 h after SEA stimulation and maintained through 24 h of culture with cells from females producing higher levels of IFN-γ. These data suggest that male ICR Swiss mice are deficient in the activity of Thy-1+, AGM1+, L3T4−, and Lyt-2− cells in the early (8–16 h) production of IFN-γ following SEA stimulation of spleen cells