Lung T cells in hypersensitivity pneumonitis: phenotypic and functional analyses

Cells recovered from bronchoalveolar lavage (BAL) and tissue sections from transbronchial lung biopsies were studied in 16 patients with symptomatic hypersensitivity pneumonitis (HP) and in six subjects with a similar history of exposure but without features of disease by using a series of monoclonal antibodies (MoAb) detecting different lymphocyte subpopulations, including T and T subsets, B lymphocytes, and natural killer (NK) cells. Their functional activities in cytotoxic and suppressor assays and the microenvironment in the lung by using immunohistological techniques were also evaluated. It has been demonstrated that the majority of cells recovered from BAL of HP patients are represented by T8 lymphocytes, with a relevant imbalance of the T4/T8 ratio (p less than 0.001). HNK-1+ cells were markedly increased (p less than 0.001), whereas the frequency of cells bearing other NK-related markers (NK-15, VEP 13, Ab8.28, T10, M1, and Fc gamma R) were not significantly increased with respect to controls. Immunohistological study confirmed that the majority of cells infiltrating lung parenchyma are T8+ lymphocytes. The number of HNK-1+ cells detected on lung biopsies was very low in all cases, even in patients with the highest values on BAL suspensions. The evidence of cells bearing the proliferation-associated markers (Tac and T9 antigens) seems to support the hypothesis of a local proliferation in the lung. In terms of phenotypic analysis, the results observed in the group of asymptomatic individuals are qualitatively superimposable on those observed in the HP group, but the magnitude of the phenomenon is less prominent and therefore the data are not as statistically significant as that produced by the comparison between HP patients and the same controls. Functional analysis of BAL T cells from both HP patients and asymptomatic individuals showed suppressor activity in vitro, as determined by the ability to influence a pokeweed mitogen (PWM)-driven B cell differentiation assay. BAL cells from HP patients were also able to display a definite cytotoxic function in vitro, whereas BAL lymphocytes from asymptomatic subjects did not. Taken together, these data demonstrated that cells responsible for the alveolitis in patients with HP are characterized by the expansion of T cells with the phenotype and functions of both suppressor and/or cytotoxic lymphocytes. This expansion is likely to be related to a local immunologic response to the antigenic stimulus and may provide new insights into the pathogenetic mechanisms of this disease, its pathological pattern, and its management.     

Effect of concanavalin A on lymphocyte interactions involved in the antibody response to type III pneumococcal polysaccharide I. Comparison of the suppression induced by con A and low dose paralysis.

Concanavalin A (Con A) administered at the time of immunization induces suppression of the in vivo splenic plaque-forming cell (PFC) response to type III pneumococcal polysaccharide (SSS-III). As with low dose paralysis of the PFC response to SSS-III, Con A-induced suppression could not be demonstrated in congenitally athymic (nu/nu) mice and could be eliminated partially by treatment with anti-lymphocyte serum (ALS). The kinetics for Con A-induced suppression paralleled those for low dose paralysis of the antibody response to SSS-III. These findings support the view that Con A-induced suppression is produced in vivo by suppressor T cells and that this form of suppression shares with low dose paralysis a common pathway through which suppression is mediated.     

Longitudinal cellular immune responses in asymptomatic and symptomatic Brugia malayi-infected Indian leaf monkey Presbytis entellus

To investigate the cell-mediated immune (CMI) responses of the host during the development of acute filarial disease manifestations, we studied the sequential changes in CD4+ and CD8+ T-cell subsets, leukocyte migration inhibition (LMI) response to Brugia malayi adult worm antigen, and concanavalin-A (ConA) and filarial antigen-induced lymphocyte transformation (LT) in the Indian leaf monkey (Presbytis entellus)-B. malayi model. Filarial infection was established in monkeys by subcutaneous inoculations of infective larvae (L3) (700-1,250 L3/monkey) in multiple doses, and the infected monkeys were categorized as symptomatic (Sym) and asymptomatic (Asym) depending on whether or not acute clinical manifestations were shown by them. In Sym monkeys, LMI response to homologous adult parasite antigen was significantly suppressed as compared to Asym monkeys. In Asym monkeys, LMI response varied among the animals; 2 showed an increase throughout the study period and 2 showed suppression at different time points. When compared with Asym monkeys, CD8+ T cells in Sym monkeys showed a trend of significant increase after day 180 postinoculation (PI). CD4+ T cells remained within the normal range till day 300 (PI), after which they showed a marginal increase. ConA-stimulated LT was suppressed in Asym monkeys from day 60 PI. Antigen-stimulated LT was unresponsive in both Asym and Sym animals. Thus, the host's LT response to ConA is suppressed in Asym animals, and alteration in CD8+ T-cell number and LMI response in Sym monkeys may be involved in the development of the acute disease manifestations in this model.     

Dopamine turnover is upregulated in the caudate/putamen of asymptomatic MPTP-treated rhesus monkeys

In Parkinson's disease (PD) and experimental parkinsonism, losses of up to 60% and 80%, respectively, of dopaminergic neurons in substantia nigra, and dopamine (DA) in striatum remain asymptomatic. Several mechanisms have been suggested for this functional compensation, the DA-mediated being the most established one. Since this mechanism was recently challenged by striatal DA analysis in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated monkeys, we present data on several DAergic parameters in three groups of rhesus monkeys: MPTP-treated asymptomatic animals; symptomatic MPTP-treated animals with stable parkinsonism; and untreated sex and age matched controls. We determined ratios of striatal and nigral 3,4-dihydroxyphenyl acetic acid (DOPAC) to DA levels and tyrosine hydroxylase (TH) enzyme activity to DA levels, in addition to the commonly used homovanillic acid (HVA)/DA ratios which, as such, might be less reliable under the conditions of partial denervation. We found that in the asymptomatic MPTP monkeys the DOPAC/DA ratios in putamen and caudate nucleus were shifted with high statistical significance 1.9-5.8-fold, as compared to controls, the shifting of the ratios being in the same range as the 2.6-5.4-fold shifts in the symptomatic animals. Also TH/DA ratios were significantly increased in both, the asymptomatic and the symptomatic MPTP-treated monkeys, with shifts in the putamen and caudate nucleus of 3- and 2.7-7.0-fold, respectively. In the substantia nigra, DOPAC levels and TH activity were strongly decreased after MPTP (-77 to -97%), but the ratios DOPAC/DA and TH/DA were not changed in this brain region. Collectively, our findings support the concept of DAergic compensation of the progressive striatal DA loss in the presymptomatic stages of the parkinsonian disease process.     

体内CD8^＋T细胞剔除对无症状期SHIV感染猴的影响

目的 CD8＋T细胞在一些病毒感染疾病的免疫反应中起着重要的作用,但CD8＋T细胞在HIV无症状期的作用尚不明确,本研究通过体内CD8＋T细胞剔除,研究CD8＋T细胞对SHIV感染猴的影响,进一步了解艾滋病的发病机制。方法选择8只SHIV病毒感染的恒河猴,均处于无症状期,随机分成两组,实验组4只恒河猴在0、3、7 d注射抗CD8＋T抗体cM-T807,不同的时间取外周血、腹股沟淋巴结。流式细胞术测定恒河猴外周血和淋巴结中CD8＋T细胞数目,Real-time RT-PCR法测定实验猴血浆病毒载量,并使用IFN-γElispot方法测定其对猴细胞免疫的影响。结果 CD8＋T细胞敲除后,4只猴的病毒载量都转阳,但反应性不一,HIV-1的靶细胞CD4＋T细胞有轻微下降,后反弹,与病毒载量无相关性;CD8敲除猴的感染情况（血浆病毒载量和CD4细胞）比SHIV病毒急性感染轻,这与ELIPOT结果一致。结论 CD8＋T细胞在HIV无症状期发挥重要的作用,但其作用具有个体差别。     

Mlsa generated suppressor cells. I. Suppression is mediated by double-negative (CD3+CD5+CD4-CD8-) alpha/beta T cell receptor-bearing cells.

Grafting of cells from B10.D2 (H-2d) donors into H-2 compatible lethally irradiated (DBA/2 x B10.D2)F1 hosts results in a severe graft-vs-host reaction (GVHR), developed against DBA/2 non-H-2 Ag, with only 0 to 10% of animals surviving. This GVHR mortality rate is dramatically reduced (90 to 100% of animals survive) by donor preimmunization against Mlsa determinants. The protection against GVHR correlates with a decreased B10.D2 anti-DBA/2 proliferative response in vitro. Both in vivo and in vitro phenomena are associated with activation of CD5+ suppressor T cells in the spleens of immunized mice. The present work was designed to study the origin of these suppressor cells and to further characterize their phenotype. The results show that significant suppression is not inducible in "B" mice. In contrast, in mice that were only thymectomized or else pretreated in vivo with anti-CD4 or anti-CD8 mAb, the suppressor cells are activated as efficiently as in normal mice. The suppression of GVHR mortality and proliferative responses in vitro is lost after depletion from preimmunized splenocytes of CD5+ T cells and remains unaltered after depletion of CD4+ or CD8+ T cells or both. Depletion of asialo GM1+ cells removes all NK activity, whereas the suppression is decreased only slightly. FACS analysis showed that double-negative (DN) cells from normal and immunized mice contain both CD3+ and CD3- cells; the vast majority of the CD3+ DN T cells express the alpha/beta T cell receptor. Suppression of GVHR and of proliferative responses in vitro are abrogated after elimination of CD3+ cells. These results suggest that Mlsa generated suppressor cells: 1) are derived from post-thymic long-lived T cell precursors; 2) are low asialo GM-1+ but do not exhibit NK activity; 3) belong to a subset of peripheral CD5+ DN T cells bearing a CD3-associated alpha/beta-heterodimer.     

Live attenuated lentivirus infection elicits polyfunctional simian immunodeficiency virus Gag-specific CD8+ T cells with reduced apoptotic susceptibility in rhesus macaques that control virus replication after challenge with pathogenic SIVmac239

HIV-specific CD8+ T cells that secrete multiple cytokines in response to Ag stimulation are associated with the control of virus replication during chronic HIV infection. To determine whether the presence of polyfunctional CD8+ T cell responses distinguishes protected and unprotected monkeys in a live attenuated lentivirus model, SIV Gag peptide-specific CD8+ T cell responses of simian HIV (SHIV) 89.6-vaccinated, SIVmac239-challenged rhesus macaques were compared in two monkeys that controlled challenge virus replication and two that did not. The ratio of Bcl-2+ Gag-specific CD8+ T cells to caspase-3+ Gag-specific CD8+ T cells was higher in the vaccinated-protected animals compared with unprotected monkeys. In addition, polyfunctional SIV-specific CD8+ T cells were consistently detected through 12 wk postchallenge in the protected animals but not in the unprotected animals. In the unprotected monkeys, there was an increased frequency of CD8+ T cells expressing markers associated with effector memory T cells. Further, there was increased annexin V expression in central memory T cells of the unprotected animals before challenge. Thus, monkeys that control viral replication after live attenuated SHIV infection have polyfunctional SIV-specific CD8+ T cells with an increased survival potential. Importantly, the differences in the nature of the SIV-specific CD8+ T cell response in the protected and unprotected animals are present during acute stages postchallenge, before different antigenic levels are established. Thus, the polyfunctional capacity and increased survival potential of CD8+ SIV-specific T cells may account for live attenuated, SHIV89.6-mediated protection from uncontrolled SIV replication.     

CD4+ T cells from lupus-prone mice avoid antigen-specific tolerance induction in vivo

Activated T cells in spontaneous lupus presumably bypass normal tolerance mechanisms in the periphery, since thymic tolerance appears intact. To determine whether such T cells indeed avoid in vivo peripheral tolerance mechanisms, we assessed their activation and recall responses after in vivo Ag stimulation in the absence of exogenously supplied costimulatory signals. Naive CD4(+) AND (transgenic mice bearing rearranged TCR specific for pigeon cytochrome c, peptides 88-104) TCR-transgenic T cells, specific for pigeon cytochrome c, from lupus-prone Fas-intact MRL/Mp+(Fas-lpr) and from H-2(k)-matched control CBA/CaJ and B10.BR mice (MRL.AND, CBA.AND, and B10.AND, respectively) were adoptively transferred into (MRL x CBA)F(1) or (MRL x B10)F(1) recipients transgenically expressing membrane-bound pigeon cytochrome c as a self-Ag. MRL.AND and control CBA.AND and B10.AND-transgenic T cells were activated and divided after transfer, indicating encounter with their cognate Ag; however, T cells from CBA.AND and B10.AND mice were impaired in their ability to proliferate and produce IL-2 after challenge with pigeon cytochrome c in ex vivo recall assays, a typical phenotype of anergized cells. By contrast, MRL.AND T cells proliferated more, and a significantly higher percentage of such cells produced IL-2, compared with control T cells. This observation that MRL T cells avoided anergy induction in vivo was confirmed in an in vitro system where the cells were stimulated with an anti-CD3 in the absence of a costimulatory signal. These experiments provide direct evidence that CD4(+) T cells from Fas-intact lupus-prone MRL mice are more resistant than nonautoimmune control cells to anergy induction. Anergy avoidance in the periphery might contribute to the characteristic finding in lupus of inappropriate T cell activation in response to ubiquitous self-Ags.     

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.     

Gamma interferon-mediated inflammation is associated with lack of protection from intravaginal simian immunodeficiency virus SIVmac239 challenge in simian-human immunodeficiency virus 89.6-immunized rhesus macaques

Although gamma interferon (IFN-gamma) is a key mediator of antiviral defenses, it is also a mediator of inflammation. As inflammation can drive lentiviral replication, we sought to determine the relationship between IFN-gamma-related host immune responses and challenge virus replication in lymphoid tissues of simian-human immunodeficiency virus 89.6 (SHIV89.6)-vaccinated and unvaccinated rhesus macaques 6 months after challenge with simian immunodeficiency virus SIVmac239. Vaccinated-protected monkeys had low tissue viral RNA (vRNA) levels, vaccinated-unprotected animals had moderate tissue vRNA levels, and unvaccinated animals had high tissue vRNA levels. The long-term challenge outcome in vaccinated monkeys was correlated with the relative balance between SIV-specific IFN-gamma T-cell responses and nonspecific IFN-gamma-driven inflammation. Vaccinated-protected monkeys had slightly increased tissue IFN-gamma mRNA levels and a high frequency of IFN-gamma-secreting T cells responding to in vitro SIVgag peptide stimulation; thus, it is likely that they could develop effective anti-SIV cytotoxic T lymphocytes in vivo. In contrast, both high tissue IFN-gamma mRNA levels and strong in vitro SIV-specific IFN-gamma T-cell responses were detected in lymphoid tissues of vaccinated-unprotected monkeys. Unvaccinated monkeys had increased tissue IFN-gamma mRNA levels but weak in vitro anti-SIV IFN-gamma T-cell responses. In addition, in lymphoid tissues of vaccinated-unprotected and unvaccinated monkeys, the increased IFN-gamma mRNA levels were associated with increased Mig/CXCL9, IP-10/CXCL10, and CXCR3 mRNA levels, suggesting that increased Mig/CXCL9 and IP-10/CXCL10 expression resulted in recruitment of CXCR3(+) activated T cells. Thus, IFN-gamma-driven inflammation promotes SIV replication in vaccinated-unprotected and unvaccinated monkeys. Unlike all unvaccinated monkeys, most monkeys vaccinated with SHIV89.6 did not develop IFN-gamma-driven inflammation, but they did develop effective antiviral CD8(+)-T-cell responses.     

Human suppressor T cells induced by concanavalin A: suppressor T cells belong to distinctive T cell subclasses.

Human peripheral blood lymphocytes were stimulated by concanavalin A (Con A) and then evaluated by their suppressive activity for thymus-derived (T) cell- and bone marrow-derived (B) cell-proliferative responses to mitogen and allogeneic cells. Con A-activated T cells markedly suppressed these responses, but Con A-activated B cells failed to demonstrate suppressor activity. Discontinuous bovine serum albumin (BSA) density gradient separation of T cells which had been activated by Con A demonstrated that a fraction containing blast cells as well as fractions containing unproliferated cells manifest the same degree of suppressor capabilities. However, when density gradient separation of T cells followed by subsequent incubation with Con A was performed, fractions of proliferating cells of low density exhibited no suppression; a fraction containing high density T cells produced marked suppression, but this fraction incorporated only little thymidine in response to Con A. Thus, these studies indicate that Con A-induced suppressor T cells belong to a distinctive subpopulation which has already been programmed to express this function before exposure to Con A and that cell proliferation may not be a prerequisite for the development of such suppressor T cells.     

Suppressor cells in contact sensitivity. Effect on T cells helping antibody production to picryl chloride.

T cells from mice injected with picryl sulfonic acid have previously been shown to suppress the effector and possibly other phases of contact hypersensitivity reactions to picryl chloride. In this report we examine their effect on T cells helping the early direct anti-TNP plaque-forming cell response of mice painted with picryl chloride. They did not directly inhibit the activity of the helper cells but did inhibit the ability of mice to generate helper cells after skin painting. The suppressor cells were T cells as tested by passage through nylon wool columns and sensitivity to anti-θ serum. Viable syngeneic cells were required for suppression and their effect was specific. The suppressor cells could not be generated in adult thymectomized mice but could be produced in mice treated with high doses (200 mg/kg) of cyclophosphamide. These properties are distinct from those of suppressor T cells produced following immunization with picryl chloride but are the same as those of other suppressor cells induced by PSA which inhibit contact hypersensitivity.     

Activation of murine T cells by streptococcal pyrogenic exotoxin type A. Requirement for MHC class II molecules on accessory cells and identification of V beta elements in T cell receptor of toxin-reactive T cells

We investigated the mechanisms of murine T cell activation by streptococcal pyrogenic exotoxin type A (SPE A), focusing on the role of MHC class II molecules on accessory cells (AC) and V beta usage in alpha beta TCR of SPE A-reactive T cells in comparison with staphylococcal enterotoxin B-reactive T cells. L cells transfected with I-Ab genes functioned as effective AC for SPE A-induced responses by C57BL/6 T cells, proliferation, and IL-2 production, but control L cells were not effective AC. Anti-I-Ab mAb inhibited the SPE A-induced responses. Staphylococcal enterotoxin B-induced C57BL/6 T cell blasts were composed of cells bearing V beta 3, members of the V beta 8 family, and V beta 11. Most of the SPE A-induced T cell blasts (about 80%) bore V beta 8.2. mAb reactive to V beta 8.2 markedly inhibited SPE A-induced T cell responses. Apparently, SPE A activates mainly T cells bearing V beta 8.2 in physical association with MHC class II molecules expressed on AC. We also discuss the pathogenic activities of SPE A in relation to toxic shock syndrome.     

Early control of highly pathogenic simian immunodeficiency virus/human immunodeficiency virus chimeric virus infections in rhesus monkeys usually results in long-lasting asymptomatic clinical outcomes

In contrast to simian immunodeficiency viruses (SIVs), which induce immunodeficiency over a 1- to 2-year period, highly pathogenic simian-human immunodeficiency viruses (SHIVs) cause an irreversible and systemic depletion of CD4(+) T lymphocytes in macaque monkeys within weeks of inoculation. Nonetheless, the seemingly more aggressive SHIVs have proven to be easier to control by the same vaccine regimens which fail to contain SIV. Because early events during in vivo infections may determine both the pathogenic consequences of the challenge virus and its sensitivity to interventions that prevent disease, we have evaluated the effects of inoculum size and a potent antiretroviral drug on the development of disease in monkeys infected with SHIV(DH12R). The results obtained show that in a majority of inoculated animals, suppression of SHIV replication during the first 2 weeks of infection, which prevents complete loss of CD4(+) T cells, leads to very low to undetectable postpeak viremia and an asymptomatic clinical course for periods up to 4 years.     

Experimental analysis of the immunostimulant properties of vitamin A

The experiments on CBA mice and rabbits have shown that vitamin A injection to animals at a daily dose of 3000 and 200000 IU, respectively, stimulates the production of antibody-forming cells and antibodies upon immunization with sheep red blood cells, without affecting the immune response to Vi-antigen. Vitamin A blocks the generation of antigen-specific suppressors, having no effect on concanavalin A-induced suppressors.     

With minimal systemic T-cell expansion, CD8+ T Cells mediate protection of rhesus macaques immunized with attenuated simian-human immunodeficiency virus SHIV89.6 from vaginal challenge with simian immunodeficiency virus

The presence, at the time of challenge, of antiviral effector T cells in the vaginal mucosa of female rhesus macaques immunized with live-attenuated simian-human immunodeficiency virus 89.6 (SHIV89.6) is associated with consistent and reproducible protection from pathogenic simian immunodeficiency virus (SIV) vaginal challenge (18). Here, we definitively demonstrate the protective role of the SIV-specific CD8(+) T-cell response in SHIV-immunized monkeys by CD8(+) lymphocyte depletion, an intervention that abrogated SHIV-mediated control of challenge virus replication and largely eliminated the SIV-specific T-cell responses in blood, lymph nodes, and genital mucosa. While in the T-cell-intact SHIV-immunized animals, polyfunctional and degranulating SIV-specific CD8(+) T cells were present in the genital tract and lymphoid tissues from the day of challenge until day 14 postchallenge, strikingly, expansion of SIV-specific CD8(+) T cells in the immunized monkeys was minimal and limited to the vagina. Thus, protection from uncontrolled SIV replication in animals immunized with attenuated SHIV89.6 is primarily mediated by CD8(+) T cells that do not undergo dramatic systemic expansion after SIV challenge. These findings demonstrate that despite, and perhaps because of, minimal systemic expansion of T cells at the time of challenge, a stable population of effector-cytotoxic CD8(+) T cells can provide significant protection from vaginal SIV challenge.     

Quantitation of a lentivirus in its natural host: simian immunodeficiency virus in African green monkeys.

We have examined the viral load in the peripheral blood of simian immunodeficiency virus (SIV)-infected African green monkeys with a view to the unexplained apathogenicity of African green monkey SIV (SIVagm) in its natural host. By using polymerase chain reaction, viral DNA was detected in fresh peripheral blood mononuclear cells (PBMC) of each of nine seropositive animals. The virus DNA load was variable among the monkeys tested, ranging from 5 to 50 (mean = 15) copies per 10(5) PBMC, which is comparable to that of human immunodeficiency virus type 1 (HIV-1) in humans. The level of infectious SIVagm in PBMC was measured by endpoint dilution cultures. SIVagm was recovered from PBMC from 14 of 17 antibody-positive monkeys (82%), and the mean SIVagm titer in PBMC of seropositive African green monkeys was 10 tissue culture infectious doses per 10(6) cells, similar to the titer shown for HIV in asymptomatic carriers. Free infectious virus was isolated from the plasma of 4 of 17 monkeys (24%), and SIVagm expression in peripheral blood in vivo, as demonstrated by in situ hybridization, was detectable only in those animals which were viremic. SIVagm replication is therefore not totally suppressed in vivo, and SIVagm has a viral load equivalent to that seen for HIV-1 in asymptomatic humans.     

Genetic control of effector cell characteristics: con A-induced suppressor cells carry H-2 I region encoded determinants.

Activation of splenic lymphocytes with Con A leads to the formation of suppressor cells capable of interfering with the activity of several polyclonal B-cell-activating substances. Thus, these suppressor cells, or their products, most probably act directly on B cells. Suppressor cells could be recovered from the effluent cell population of nylon wool columns, and they were absent from the spleens of athymic nude mice. Furthermore, they were absent from the thymus of normal as well as cortison-treated mice. Cortisone treatment did not abolish the formation of Con A-induced suppressor cells in the spleen. Treatment of activated suppressor cells with antisera specific for distinct products of the H-2 I region revealed that they carried I-J cell surface antigens. We conclude that the suppressor cells in our test system, which unlike other Con A-induced suppressor cell populations have a direct effect on B cells, had antigenic characteristics similar to those previously described for I-J carrying suppressor cells.