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Cross-linking of Fas by antibodies to a peculiar domain of gp120 V3 loop can enhance T cell apoptosis in HIV-1-infected patients.

Silvestris F, Nagata S, Cafforio P, Silvestris N, Dammacco F - J. Exp. Med. (1996)

Bottom Line: In addition, anti-Fas immunoglobulin G strongly inhibited the [3H]thymidine uptake of CEM cells in proliferative assays, inducing a suppression as high as provoked by both CH11 mAb and recombinant human Fas ligand.Since anti-Fas were reactive to gp120, it is conceivable that antibodies binding that domain within the V3 region are effective cross-linkers of Fas and increase apoptosis in peripheral T cells.These results suggest that autologous stimulation of the Fas pathway, rather than of lymphocytotoxic antibodies, may aggravate lymphopenia in a number of HIV-1+ subjects.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomedical Sciences and Human Oncology, University of Bari, Italy.

ABSTRACT
Previous studies have demonstrated that T cell-reactive antibodies in HIV-1 infection contribute to lymphocyte depletion by cytotoxicity that involves differential membrane targets, such as the 43.5-kD receptor on CEM cells. Here, we show that these antibodies bind Fas as result of a molecular mimicry of the gp120. Both flow cytometry and immunoblotting using the human Fas-transfected mouse WC8 lymphoma revealed positive binding of immunoglobulin G from several patients to a 43.8-kD membrane receptor that also reacts with the CH11 anti-Fas monoclonal antibody. Specificity to Fas was further confirmed to chimeric recombinant human Fas-Fc by ELISA, whereas overlapping peptide mapping of a Fas domain (VEINCTR-N) shared by gp120 V3 loop demonstrated a predominant affinity to the full-length 10-mer peptide. Four anti-Fas affinity preparations greatly increased the subdiploid DNA peak of CEM cells similar to agonist ligands of Fas. In addition, anti-Fas immunoglobulin G strongly inhibited the [3H]thymidine uptake of CEM cells in proliferative assays, inducing a suppression as high as provoked by both CH11 mAb and recombinant human Fas ligand. Since anti-Fas were reactive to gp120, it is conceivable that antibodies binding that domain within the V3 region are effective cross-linkers of Fas and increase apoptosis in peripheral T cells. These results suggest that autologous stimulation of the Fas pathway, rather than of lymphocytotoxic antibodies, may aggravate lymphopenia in a number of HIV-1+ subjects.

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(A) Epitope mapping of a domain  of Fas (aa position 90–117), including an 8-mer  stretch (VEINCTR—N) shared by the V3 loop  of gp120. A panel of 17 overlapping 10-mer  peptides used as antigens in ELISA revealed a  predominant affinity to that domain in sera 3,  41, and 54 although IgG from patient 54  showed a slight increase of reactivity to a subsequent peptide constructed with (N) Asn in  the 108 aa position. Specificity to the  VEINCTR peptide was almost undetectable in  sera from patients unreactive to the CEM membrane. (B) ELISA reactivity to synthetic peptides resembling the homology sequence of the  aa 99–108 domain of Fas in the V3 loop of  HIV-1iiib (VEINCTRPNN) and HIV-1mn  (VQINCTRPNY) strains. OD values to both  peptides were apparently equivalent in most of  the anti-Fas IgG in groups A and C. In contrast,  sera 3, 21, and 41 showed a slight difference in  their affinity to the peptides, suggesting that the  replacement of E (Glu) by Q (Gln) was influencing the antigenicity of the epitope.
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Figure 5: (A) Epitope mapping of a domain of Fas (aa position 90–117), including an 8-mer stretch (VEINCTR—N) shared by the V3 loop of gp120. A panel of 17 overlapping 10-mer peptides used as antigens in ELISA revealed a predominant affinity to that domain in sera 3, 41, and 54 although IgG from patient 54 showed a slight increase of reactivity to a subsequent peptide constructed with (N) Asn in the 108 aa position. Specificity to the VEINCTR peptide was almost undetectable in sera from patients unreactive to the CEM membrane. (B) ELISA reactivity to synthetic peptides resembling the homology sequence of the aa 99–108 domain of Fas in the V3 loop of HIV-1iiib (VEINCTRPNN) and HIV-1mn (VQINCTRPNY) strains. OD values to both peptides were apparently equivalent in most of the anti-Fas IgG in groups A and C. In contrast, sera 3, 21, and 41 showed a slight difference in their affinity to the peptides, suggesting that the replacement of E (Glu) by Q (Gln) was influencing the antigenicity of the epitope.

Mentions: The next set of experiments investigated whether anti-Fas IgG in sera from groups A and C were reactive to the aa 99–108 stretch of Fas, which is also shared by the V3 loop of gp120. To this purpose, the ELISA was prepared with overlapping peptides of this domain and those resembling the V3-related homology sequence from both HIV-1iiib and HIV-1mn as the antigen. The results obtained with the most reactive sera (Nos. 3, 41, and 54) showed significant binding starting with peptide 96GLEVEINCTR105 (Fig. 5 A) and stable until 100EINCTRTQNT109, supporting the evidence that the shared stretch alone could constitute the reactive epitope. Interestingly, construction of the peptide with N (Asn) in aa position 108 induced a small but definite increment of reactivity in IgG from serum 54. This was consistent with a potential contribution of Asn to a conformational rather than a linear domain reacting with those antibodies. Moderate and low IgM reactivities (OD <0.31) to the full-length peptide were observed in two sera from group B (Nos. 34 and 78), whereas parallel ELISA tests searching for potential specificities to the peptide in sera from patients who were unreactive to CEM membrane and from uninfected donors revealed a prevalent lack of reactivity in both control groups (data not shown).


Cross-linking of Fas by antibodies to a peculiar domain of gp120 V3 loop can enhance T cell apoptosis in HIV-1-infected patients.

Silvestris F, Nagata S, Cafforio P, Silvestris N, Dammacco F - J. Exp. Med. (1996)

(A) Epitope mapping of a domain  of Fas (aa position 90–117), including an 8-mer  stretch (VEINCTR—N) shared by the V3 loop  of gp120. A panel of 17 overlapping 10-mer  peptides used as antigens in ELISA revealed a  predominant affinity to that domain in sera 3,  41, and 54 although IgG from patient 54  showed a slight increase of reactivity to a subsequent peptide constructed with (N) Asn in  the 108 aa position. Specificity to the  VEINCTR peptide was almost undetectable in  sera from patients unreactive to the CEM membrane. (B) ELISA reactivity to synthetic peptides resembling the homology sequence of the  aa 99–108 domain of Fas in the V3 loop of  HIV-1iiib (VEINCTRPNN) and HIV-1mn  (VQINCTRPNY) strains. OD values to both  peptides were apparently equivalent in most of  the anti-Fas IgG in groups A and C. In contrast,  sera 3, 21, and 41 showed a slight difference in  their affinity to the peptides, suggesting that the  replacement of E (Glu) by Q (Gln) was influencing the antigenicity of the epitope.
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Related In: Results  -  Collection

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Figure 5: (A) Epitope mapping of a domain of Fas (aa position 90–117), including an 8-mer stretch (VEINCTR—N) shared by the V3 loop of gp120. A panel of 17 overlapping 10-mer peptides used as antigens in ELISA revealed a predominant affinity to that domain in sera 3, 41, and 54 although IgG from patient 54 showed a slight increase of reactivity to a subsequent peptide constructed with (N) Asn in the 108 aa position. Specificity to the VEINCTR peptide was almost undetectable in sera from patients unreactive to the CEM membrane. (B) ELISA reactivity to synthetic peptides resembling the homology sequence of the aa 99–108 domain of Fas in the V3 loop of HIV-1iiib (VEINCTRPNN) and HIV-1mn (VQINCTRPNY) strains. OD values to both peptides were apparently equivalent in most of the anti-Fas IgG in groups A and C. In contrast, sera 3, 21, and 41 showed a slight difference in their affinity to the peptides, suggesting that the replacement of E (Glu) by Q (Gln) was influencing the antigenicity of the epitope.
Mentions: The next set of experiments investigated whether anti-Fas IgG in sera from groups A and C were reactive to the aa 99–108 stretch of Fas, which is also shared by the V3 loop of gp120. To this purpose, the ELISA was prepared with overlapping peptides of this domain and those resembling the V3-related homology sequence from both HIV-1iiib and HIV-1mn as the antigen. The results obtained with the most reactive sera (Nos. 3, 41, and 54) showed significant binding starting with peptide 96GLEVEINCTR105 (Fig. 5 A) and stable until 100EINCTRTQNT109, supporting the evidence that the shared stretch alone could constitute the reactive epitope. Interestingly, construction of the peptide with N (Asn) in aa position 108 induced a small but definite increment of reactivity in IgG from serum 54. This was consistent with a potential contribution of Asn to a conformational rather than a linear domain reacting with those antibodies. Moderate and low IgM reactivities (OD <0.31) to the full-length peptide were observed in two sera from group B (Nos. 34 and 78), whereas parallel ELISA tests searching for potential specificities to the peptide in sera from patients who were unreactive to CEM membrane and from uninfected donors revealed a prevalent lack of reactivity in both control groups (data not shown).

Bottom Line: In addition, anti-Fas immunoglobulin G strongly inhibited the [3H]thymidine uptake of CEM cells in proliferative assays, inducing a suppression as high as provoked by both CH11 mAb and recombinant human Fas ligand.Since anti-Fas were reactive to gp120, it is conceivable that antibodies binding that domain within the V3 region are effective cross-linkers of Fas and increase apoptosis in peripheral T cells.These results suggest that autologous stimulation of the Fas pathway, rather than of lymphocytotoxic antibodies, may aggravate lymphopenia in a number of HIV-1+ subjects.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomedical Sciences and Human Oncology, University of Bari, Italy.

ABSTRACT
Previous studies have demonstrated that T cell-reactive antibodies in HIV-1 infection contribute to lymphocyte depletion by cytotoxicity that involves differential membrane targets, such as the 43.5-kD receptor on CEM cells. Here, we show that these antibodies bind Fas as result of a molecular mimicry of the gp120. Both flow cytometry and immunoblotting using the human Fas-transfected mouse WC8 lymphoma revealed positive binding of immunoglobulin G from several patients to a 43.8-kD membrane receptor that also reacts with the CH11 anti-Fas monoclonal antibody. Specificity to Fas was further confirmed to chimeric recombinant human Fas-Fc by ELISA, whereas overlapping peptide mapping of a Fas domain (VEINCTR-N) shared by gp120 V3 loop demonstrated a predominant affinity to the full-length 10-mer peptide. Four anti-Fas affinity preparations greatly increased the subdiploid DNA peak of CEM cells similar to agonist ligands of Fas. In addition, anti-Fas immunoglobulin G strongly inhibited the [3H]thymidine uptake of CEM cells in proliferative assays, inducing a suppression as high as provoked by both CH11 mAb and recombinant human Fas ligand. Since anti-Fas were reactive to gp120, it is conceivable that antibodies binding that domain within the V3 region are effective cross-linkers of Fas and increase apoptosis in peripheral T cells. These results suggest that autologous stimulation of the Fas pathway, rather than of lymphocytotoxic antibodies, may aggravate lymphopenia in a number of HIV-1+ subjects.

Show MeSH
Related in: MedlinePlus