Dulceaydee Gigliotti

Regulation and Role of IFN-γ in the Innate Resistance to Infection with Chlamydia pneumoniae

By using mice genomically lacking IFN-γR, IL-12, perforin, and recombination-activating gene-1 (RAG-1), we analyzed the regulation and importance of IFN-γ in the control of infection with Chlamydia pneumoniae. IL-12 participates in resistance of mice to C. pneumoniae, probably by regulating the protective levels of IFN-γ mRNA. In turn, IFN-γ is necessary for the increased IL-12p40 mRNA accumulation that occurs in lungs during infection with C. pneumoniae, suggesting a positive feedback regulation between these two cytokines. In experiments including RAG-1−/−/IFN-γR−/− mice we showed that IFN-γ produced by innate cells controls the bacterial load and is necessary for the increased accumulation of transcripts for enzymes controlling high output NO release (inducible NO synthase), superoxide production (gp-91 NADPH oxidase), and catalyzis of tryptophan (indoleamine 2,3-dioxygenase (IDO)), mechanisms probably related to bacterial killing. Adaptive immune reponses diminish the levels of IFN-γ and IL-12 mRNA and thereby the levels of inducible NO synthase, IDO, and gp91 NADPH oxidase trancripts. By using RAG-1−/−/perforin−/− mice, we excluded the overt participation of NK cell cytotoxicity in the control of C. pneumoniae. However, NK cells and probably other innate immune cells release IFN-γ during the bacterial infection.

IFN-αβ-Dependent, IFN-γ Secretion by Bone Marrow-Derived Macrophages Controls an Intracellular Bacterial Infection

Several reports have indicated that cell lineages apart from NK and T cells can also express IFN-γ. However, the biological relevance of this finding is uncertain. We show in this study that bone marrow-derived macrophages (BMMs) express IFN-γ at the mRNA and protein level early after infection with Chlamydia pneumoniae. Increased IFN-γ mRNA accumulation by infected BMMs is early, transient, and requires both bacterial and host protein synthesis. The induction of IFN-γ mRNA levels is independent of IL-12 and was dramatically enhanced in IL-10−/− BMMs. Such IL-10−/− BMMs contained less bacteria than the wild-type controls, whereas IFN-γR−/− BMMs showed increased C. pneumoniae load. Inducible NO synthase (iNOS) also participates in the control of bacterial load, as shown by the enhanced numbers of C. pneumoniae in iNOS−/− BMMs. However, the increased accumulation of iNOS mRNA and NO in C. pneumoniae-infected BMMs depended on the presence of IFN-αβ, but was independent of IFN-γ. Interestingly, IFN-αβ are also required for increased IFN-γ mRNA accumulation in C. pneumoniae-infected BMMs. Accordingly, IFN-αβR−/− BMMs showed higher levels of C. pneumoniae than wild-type BMMs. Our findings unravel an autocrine/paracrine macrophage activation pathway by showing an IFN-αβ-dependent IFN-γ and iNOS induction in response to infection, which protects macrophages against intracellular bacterial growth.

T-cell-epitope mapping of the idiotypic monoclonal IgG heavy and light chains in multiple myeloma

The idiotypic structures of the myeloma protein might be regarded as tumor‐specific antigens. The present study was designed to map T‐cell epitopes of the idiotypic myeloma protein to prove the existence of naturally occurring major‐histocompatibility‐complex‐dependent idiotype (peptide)‐specific T cells in multiple myeloma. The fine specificity of idiotype‐reactive, interferon‐γ‐producing blood T cells of a patient with multiple myeloma stage I was characterized by identification of idiotype (heavy and light chains)‐derived MHC‐restricted T‐cell epitopes. T cells specifically reacting with peptides corresponding to each of the 3 complementarity‐determining regions (CDRs) of the heavy‐chain variable part (VH) of the autologous idiotype were found. In contrast, none of the peptides corresponding to the 3 CDRs of the light chain (VL) induced a specific T‐cell response. The idiotype amino‐acid sequence corresponding to the junction of the VH, diversity (D), and joining (J) gene segments of the VH appeared to be an important target for T cells, since the sequence expressed MHC‐class‐I‐ as well as MHC‐class‐II‐restricted epitopes. The study provides further support for the existence of MHC‐restricted idiotype‐specific T cells, which may target immunogenic CDR peptides in multiple myeloma. Such T cells could be an important part of the specific anti‐tumor immune responses induced in idiotype vaccination protocols. Int. J. Cancer 80:671–680, 1999.

Apoptosis resistant bronchoalveolar lavage (BAL) fluid lymphocytes in sarcoidosis

Background: Sarcoidosis is a chronic granulomatous lung disease of unknown origin. The accumulation of activated T cells at sites of inflammation represents an early stage in granuloma formation. Since mechanisms governing the normal resolution of inflammatory processes are poorly understood, this study aimed to investigate the apoptotic phenotype of peripheral blood and lung T lymphocytes from patients with sarcoidosis. Methods: Bronchoalveolar lavage (BAL) was performed in 10 patients with active sarcoidosis and five healthy controls. Results: Virtually no lymphocyte apoptosis, as determined by annexin V or Hoechst staining, was seen in either patients or controls. Sustained caspase-3 activity in non-apoptotic BAL fluid lymphocytes of the patients was detected, however, in agreement with in vitro studies demonstrating caspase activation after T cell receptor (TCR) triggering as a physiological response required for efficient T cell activation. Only 11.0% (range 7.7–17.6) of the BAL lymphocytes from sarcoidosis patients were annexin V positive after exposure to the apoptotic stimulus tributyltin compared with 55.0% (range 42.0–62.0) of BAL lymphocytes from healthy controls (p<0.001). After anti-Fas treatment only 8.5% (range 6–10) of BAL fluid lymphocytes from patients but 45.5% (range 38–62) from healthy controls were apoptotic. Conclusion: BAL fluid lymphocytes from patients with sarcoidosis display a non-apoptotic morphology associated with endogenous caspase-3 activity. They seem to be resistant to apoptosis, which might contribute to the accumulation of inflammatory cells in the lungs, persistence of inflammation, and the development and maintenance of granuloma.

Characterization of the T-cell receptor V-beta repertoire in Kawasaki disease

Kawasaki disease (KD) is a paediatric multisystem necrotizing vasculitis constituting the most frequent cause of acquired heart disease in childhood. Conflicting data have been reported regarding expanded T‐cell populations using particular T‐cell receptor (TCR) β‐chain variable (BV) gene segments, suggesting either a superantigen‐ or a conventional antigen‐mediated immune response in this disease. In order to further investigate the role of T lymphocytes, cells were stained with an extensive panel of 21 different TCRBV specific monoclonal antibobies (MoAbs) covering almost 70% of all T‐cells. Flow cytometry was employed to analyse the expression of the TCRBV repertoire in the CD4+ and CD8+ subsets separately, and of activation markers, in freshly isolated peripheral blood lymphocytes of 25 Kawasaki disease patients during the acute and convalescent phases of the disease. No abnormal usage of any TCRBV family was found, neither acutely nor during convalescence, compared with a control group of healthy children. However, a significant increase in interleukin‐2 receptor (IL‐2R)‐expressing T lymphocytes restricted to the CD4+ subset was observed in KD patients. Our data confirm a strong immune activation in KD that might be of importance in the pathogenesis of the disease.

Detection of CD8 T-cell expansions with restricted T-cell receptor V gene usage in infants vertically infected by HIV-1.

Objective: To investigate the T‐cell receptor (TCR) repertoire usage in infants born to mothers infected with HIV‐1 in order to discern possible perturbations in TCR usage as a consequence of HIV‐1 infection. Design: Blood samples from five HIV‐1 ‐infected and six non‐infected children born to HIV‐1‐seropositive mothers were collected at two to three timepoints during the first and second year of life and the TCR variable gene usage was determined. Methods: Triple staining flow cytometry analysis using a panel of monoclonal antibodies (MAb) to TCR V&agr; and V&bgr; gene products and antibodies to CD4 and CD8 was performed. Results: Frequent large expansions of CD8+ lymphocyte subpopulations bearing distinct V&agr; and V&bgr; gene products was seen in HIV‐1‐infected children (four out of five) but was rarely detected in uninfected children. Conclusion: The study demonstrated the frequent occurrence of persistent and clonal expansions of CD8+ T cells bearing distinct V&agr;/V&bgr; gene products in some HIV‐1 vertically infected infants similar to those observed during primary infection in adults.

Cytokine expression in B-CLL in relation to disease progression andin vitro activation

Earlier, we reported an association between lowin vitro andin vivo IL-1 and IL-6 production, decreased IL-1β and IL-10 mRNA expression and B cell chronic lymphocytic leukemia (B-CLL) disease progression. We have now further investigated cytokine mRNA transcription in B-CLL cells and cytokine serum levels in B-CLL patients. Reverse transcriptase polymerase chain reaction (RT-PCR) amplification of tumor necrosis factor (TNFα), IFNγ, IL-6 and BCGF was equally often seen in non-progressive and progressive patients. However, 4 out of 23 non-progressive cases expressed mRNA for IL-12 while no IL-12 expression was seen in 15 progressive patients. No IL-12 was found in sera or supernatants fromin vitro stimulated B-CLL cells, whereas TNFα and IL-10 were detected in sera from 51 and 31 of 65 B-CLL patients, respectively. TNFα values were significantly high in sera from patients in stages III and IV with disease progression. TNFα and IL-10 were also detected in culture supernatants fromin vitro stimulated B-CLL cells, whereas IFNγ was undetectable in these cultures and rarely positive in serum. Although further investigations are required, our data and that from previous reports indicate that B-CLL-derived cytokines are involved in B-CLL disease progression.

Disturbances in the peripheral T-cell repertoire of patients with motor neuron disease: high levels of activation and indirect evidence of superantigen.

Our data on peripheral blood T cells from Motor neuron disease (MND) patients indicate major immunological disturbances linked to this disease. Both CD4+ and CD8+ T‐cell subsets display an increased fraction of cells showing activation markers compared to controls, indicating an unusually high level of activity in both populations. Likewise, an increased number of T‐cell expansions were noted in MND patients compared to controls, most dramatically observed in the CD4+ T‐cell subset, where 5/144 T‐cell V genes analyzed in eight subjects turned out to be expanded in the peripheral blood. In the CD8+ T‐cell subset, four out of eight MND patients had peripheral BV gene expansions, 9/144 V genes analyzed. However, the most interesting result was the observation that in three out eight MND patients, expansions concerning the same BV gene were present in both CD4+ and CD8+ subsets (BV8S1 in two and BV12S1 in one patient). Parallel expansions of BV‐gene restricted populations in both CD4+ and CD8+ subsets in the same individual, in an major histocompatibility complex (MHC)‐unrestricted manner, are normally limited to situations where superantigens are involved. No known superantigen has to date been described with the capacity to simultaneously stimulate both BV8S1 and BV12S1, suggesting that the postulated ‘MND‐associated’ superantigen is a hitherto undefined molecule.

Certain Human Gpl20-HIV Antibodies React with Anti-CD4 Antibodies

HIV+ human sera contain antibodies against most HIV proteins, including the envelope glycoprotein Gpl20. Some of these antibodies may have an epitope that sterically resembles the CD4 region to which the Gp120 molecule binds. Amongst 58 HIV+ sera tested, we found three with the capacity to block the binding of anti‐CD4 monoclonal antibodies to CD4+ cells. The serum with the highest blocking capacity was selected for further analysis. The inhibitor was shown to be an antibody that binds to the Gp120 molecule as well us to the anti‐CD4 monoclonal T4.2. These CD4‐mimicking antibodies were shown not to interfere with CD4‐dependent reactions in vitro. Virus neutralizing experiments in vitro could not show any neutralizing effect with these antibodies alone. The HIV+ individual providing this antibody is still healthy, although HIV since 1983.

T-Cell Activation and the Development of an Apoptosis-Resistant CD45RO+ T-Cell Population

Growing experimental evidence supports a broadening role for the caspases; not only do they participate in the process of apoptosis but also in the control of the cell cycle and cellular proliferation. The biological role of the caspases in the process of T‐cell activation and proliferation is still not defined. In the present study, we propose a potential role, by demonstrating an association of T‐cell receptor‐mediated caspase activity with the development of an apoptosis‐resistant memory CD45RO+ T‐cell population. As previously shown by us, a time‐dependent induction of caspase activity, in the absence of apoptosis, can be observed in CD3‐stimulated human peripheral blood lymphocytes. We here show that a population of CD45RO+ cells, with activated caspase‐3 and with resistance to tributyltin‐induced apoptosis, develops after 3 days of stimulation. A concomitant expression of the anti‐apoptotic protein Bcl‐xL accompanied the caspase activity and the development of the apoptosis‐resistant phenotype. Finally, upon co‐culturing with dexamethasone (DEX), the CD3‐induced caspase‐3 activity was blocked. During this condition, the expression of the activation marker HLA‐DR as well as the cellular proliferative response was strongly suppressed. The development of memory cells with a CD45RO+ phenotype was also blocked. Our data support the hypothesis that caspase‐3 activity, observed in CD3‐stimulated cells, may be an important component in the proliferation process and, furthermore, might play a role for the development of memory T cells, and DEX inhibits this process.