ALBERT

Description: Kaposi's sarcoma-associated herpes virus (KSHV)/human herpes virus 8 (HHV8) DNA sequences have been demonstrated in Kaposi's sarcoma (KS), as well as in some acquired immunodeficiency syndrome (AIDS)-related non-Hodgkin's lymphomas (NHL) and in multicentric Castleman's disease. Although KSHV DNA generally is abundant in KSHV-associated lymphomas, few copies of the virus are present in KS, a property that confounds detection by in situ methods. Previous in situ studies, which identified KSHV in lesions of KS, relied on the use of polymerase chain reaction (PCR) to amplify target DNA sequences before in situ hybridization (ISH) for localization or used ISH with radioactively-labeled probes to obtain adequate levels of detection sensitivity. In this study, a novel nonisotopic nucleic acid ISH method using catalyzed signal amplification and colorimetric detection without PCR-dependent target amplification was used to identify KSHV-specific sequences. The level of sensitivity was increased further by using a probe that detects viral cyclin D homolog transcripts, which are expressed at significant levels during latent viral infection. Thirty cutaneous lesions of KS (25 AIDS-related and five classical European type) were evaluated. AIDS-related NHL and cell lines derived from patients with AIDS-related NHL, all of which were known to harbor KSHV by Southern blot analysis, were used as positive controls. NHL and benign cutaneous vascular lesions not associated with AIDS were used as negative controls. For each of the 30 KS lesions studied, hybridization signals were detected in most of the spindle cells surrounding the atypical slit-like vascular channels and also were detected in some endothelial cells in well-formed blood vessels in the perilesional dermis. Plaque and nodular lesions generally contained more labeled cells than did early patch lesions. All AIDS-related NHL and cell lines contained KSHV-specific sequences; however, the non-AIDS–related NHLs and benign vascular lesions were negative. These results confirm the presence of KSHV sequences in cutaneous KS and provide in situ evidence of infection by this virus in early patch-stage lesions. This study also defines the in situ expression of the KSHV cyclin D homolog viral oncogene in cutaneous KS. The use of this sensitive nonisotopic ISH method should allow detection of other KSHV-specific gene products, further defining the pathobiology of this virus.

Description: The immunoglobulin (Ig) variable region (V) genes expressed by IgM chronic lymphocytic leukemia (CLL) B cells display little or no somatic mutations. However, preliminary findings have shown that Ig V genes of IgA and IgG CLLs may be somatically mutated, suggesting that isotype-switched CLLs may represent a “subtype” of the disease. To investigate the degree and nature of somatic mutations and the role of antigen (Ag) in the clonal selection and expansion of isotype-switched CLLs, and to determine whether specific oncogene or tumor suppressor gene mutations are associated with isotype-switched CLLs, we analyzed the expressed Ig VH gene, bcl-1 and bcl-2 proto-oncogene, and p53 tumor suppressor gene configurations of 3 IgA-, 1 IgG-, and 1 IgA/IgG-expressing CLLs. These isotype-switched CLL B cells expressed surface HLA-DR, CD19, CD23, and CD5, and displayed no alterations of the bcl-1 and bcl-2 oncogenes and the p53 tumor-suppressor gene. The cDNA VH -D-JH gene sequence was joined with that of the Cα gene in the B cells of the three IgA CLLs, and with that of the Cγ gene in the IgG CLL B cells. In the IgA/IgG-coexpressing CLL B cells, identical VH -D-JH cDNA sequences were spliced to either Cα or Cγ genes. In all five CLLs, the pattern of Cμ DNA probe hybridization to the digested genomic DNAs was consistent with deletion of the Cμ exon from the rearranged Ig gene locus, suggesting that these CLL B cells had undergone DNA switch recombination. In one IgA CLL, the expressed VH gene was unmutated. In all other class-switched CLLs, the Ig VH segment gene was mutated, but the point mutations were not associated with intraclonal diversification. In one IgA and in the IgA/IgG-coexpressing CLL, the nature and distribution of the mutations were consistent with Ag selection. These findings suggest that IgA- and/or IgG-expressing CLLs represent, in their VH gene structure, transformants of B cells at different stages of ontogeny. They also suggest that Ag may play a role in the clonal selection of some of these isotype-switched leukemic cells, but bcl-1 and bcl-2 oncogene rearrangements and p53 tumor suppressor gene mutation are not associated with the pathogenesis of isotype-switched CLLs.

Description: We used a model of combined bone marrow and heart transplantation, in which tolerance and stable chimerism is induced after conditioning with fractionated irradiation of the lymphoid tissues and anti–T-cell antibodies. Graft acceptance and chimerism required host CD4+CD25+ Treg production of IL-10 that was in-turn enhanced by host invariant natural killer (NK) T-cell production of IL-4. Up-regulation of PD-1 on host Tregs, CD4+CD25− conventional T (Tcon) cells, and CD8+ T cells was also enhanced by NKT cell production of IL-4. Up-regulated PD-1 expression on Tregs was linked to IL-10 secretion, on CD8+ T cells was linked to Tim-3 expression, and on CD4+ Tcon cells was associated with reduced IFNγ secretion. Changes in the expression of PD-1 were induced by the conditioning regimen, and declined after bone marrow transplantation. In conclusion, NKT cells in this model promoted changes in expression of negative costimulatory receptors and anti-inflammatory cytokines by Tregs and other T-cell subsets in an IL-4–dependent manner that resulted in tolerance to the bone marrow and organ grafts.

Description: Kaposi's sarcoma-associated herpes virus (KSHV)/human herpes virus 8 (HHV8) DNA sequences have been demonstrated in Kaposi's sarcoma (KS), as well as in some acquired immunodeficiency syndrome (AIDS)-related non-Hodgkin's lymphomas (NHL) and in multicentric Castleman's disease. Although KSHV DNA generally is abundant in KSHV-associated lymphomas, few copies of the virus are present in KS, a property that confounds detection by in situ methods. Previous in situ studies, which identified KSHV in lesions of KS, relied on the use of polymerase chain reaction (PCR) to amplify target DNA sequences before in situ hybridization (ISH) for localization or used ISH with radioactively-labeled probes to obtain adequate levels of detection sensitivity. In this study, a novel nonisotopic nucleic acid ISH method using catalyzed signal amplification and colorimetric detection without PCR-dependent target amplification was used to identify KSHV-specific sequences. The level of sensitivity was increased further by using a probe that detects viral cyclin D homolog transcripts, which are expressed at significant levels during latent viral infection. Thirty cutaneous lesions of KS (25 AIDS-related and five classical European type) were evaluated. AIDS-related NHL and cell lines derived from patients with AIDS-related NHL, all of which were known to harbor KSHV by Southern blot analysis, were used as positive controls. NHL and benign cutaneous vascular lesions not associated with AIDS were used as negative controls. For each of the 30 KS lesions studied, hybridization signals were detected in most of the spindle cells surrounding the atypical slit-like vascular channels and also were detected in some endothelial cells in well-formed blood vessels in the perilesional dermis. Plaque and nodular lesions generally contained more labeled cells than did early patch lesions. All AIDS-related NHL and cell lines contained KSHV-specific sequences; however, the non-AIDS–related NHLs and benign vascular lesions were negative. These results confirm the presence of KSHV sequences in cutaneous KS and provide in situ evidence of infection by this virus in early patch-stage lesions. This study also defines the in situ expression of the KSHV cyclin D homolog viral oncogene in cutaneous KS. The use of this sensitive nonisotopic ISH method should allow detection of other KSHV-specific gene products, further defining the pathobiology of this virus.

Description: Primary effusion (body cavity–based) lymphoma (PEL) is a recently recognized subtype of malignant lymphoma that exhibits distinctive clinical and biological features, most notably its usual infection with the Kaposi's sarcoma–associated herpesvirus (KSHV). The vast majority of cases also contain Epstein-Barr virus (EBV). This dual viral infection is the first example of a consistent dual herpesviral infection in a human neoplasm and provides a unique model to study viral interactions. We analyzed the pattern of EBV latent gene expression to determine the pathogenic role of this agent in PELs. We examined five PELs coinfected with EBV and KSHV by reverse transcription-polymerase chain reaction (RT-PCR), in situ hybridization, and immunohistochemistry. EBER1 mRNA, a consistent marker of viral latency, was positive in all PEL cases, although at lower levels than in the non-PEL controls due to EBER1 expression by only a variable subset of lymphoma cells. Qp-initiated mRNA, encoding only EBNA1 and characteristic of latencies I and II, was positive in all PEL cases. Wp- and Cp-initiated mRNAs, encoding all EBNAs and characteristic of latency III, were negative in all cases. LMP1 mRNA, expressed in latencies II and III, was present in three cases of PEL, although at very low levels that were not detectable at the protein level by immunohistochemistry. Low levels of LMP2A mRNA were detected in all cases. BZLF1, an early-intermediate lytic phase marker, was weakly positive in four cases, suggesting a productive viral infection in a very small proportion of cells, which was confirmed by ZEBRA antigen expression. Therefore, PELs exhibit a restricted latency pattern, with expression of EBNA1 in all cases, and low LMP1 and LMP2A levels.

Description: Primary effusion (body cavity–based) lymphoma (PEL) is a recently recognized subtype of malignant lymphoma that exhibits distinctive clinical and biological features, most notably its usual infection with the Kaposi's sarcoma–associated herpesvirus (KSHV). The vast majority of cases also contain Epstein-Barr virus (EBV). This dual viral infection is the first example of a consistent dual herpesviral infection in a human neoplasm and provides a unique model to study viral interactions. We analyzed the pattern of EBV latent gene expression to determine the pathogenic role of this agent in PELs. We examined five PELs coinfected with EBV and KSHV by reverse transcription-polymerase chain reaction (RT-PCR), in situ hybridization, and immunohistochemistry. EBER1 mRNA, a consistent marker of viral latency, was positive in all PEL cases, although at lower levels than in the non-PEL controls due to EBER1 expression by only a variable subset of lymphoma cells. Qp-initiated mRNA, encoding only EBNA1 and characteristic of latencies I and II, was positive in all PEL cases. Wp- and Cp-initiated mRNAs, encoding all EBNAs and characteristic of latency III, were negative in all cases. LMP1 mRNA, expressed in latencies II and III, was present in three cases of PEL, although at very low levels that were not detectable at the protein level by immunohistochemistry. Low levels of LMP2A mRNA were detected in all cases. BZLF1, an early-intermediate lytic phase marker, was weakly positive in four cases, suggesting a productive viral infection in a very small proportion of cells, which was confirmed by ZEBRA antigen expression. Therefore, PELs exhibit a restricted latency pattern, with expression of EBNA1 in all cases, and low LMP1 and LMP2A levels.

Description: The immunoglobulin (Ig) variable region (V) genes expressed by IgM chronic lymphocytic leukemia (CLL) B cells display little or no somatic mutations. However, preliminary findings have shown that Ig V genes of IgA and IgG CLLs may be somatically mutated, suggesting that isotype-switched CLLs may represent a “subtype” of the disease. To investigate the degree and nature of somatic mutations and the role of antigen (Ag) in the clonal selection and expansion of isotype-switched CLLs, and to determine whether specific oncogene or tumor suppressor gene mutations are associated with isotype-switched CLLs, we analyzed the expressed Ig VH gene, bcl-1 and bcl-2 proto-oncogene, and p53 tumor suppressor gene configurations of 3 IgA-, 1 IgG-, and 1 IgA/IgG-expressing CLLs. These isotype-switched CLL B cells expressed surface HLA-DR, CD19, CD23, and CD5, and displayed no alterations of the bcl-1 and bcl-2 oncogenes and the p53 tumor-suppressor gene. The cDNA VH -D-JH gene sequence was joined with that of the Cα gene in the B cells of the three IgA CLLs, and with that of the Cγ gene in the IgG CLL B cells. In the IgA/IgG-coexpressing CLL B cells, identical VH -D-JH cDNA sequences were spliced to either Cα or Cγ genes. In all five CLLs, the pattern of Cμ DNA probe hybridization to the digested genomic DNAs was consistent with deletion of the Cμ exon from the rearranged Ig gene locus, suggesting that these CLL B cells had undergone DNA switch recombination. In one IgA CLL, the expressed VH gene was unmutated. In all other class-switched CLLs, the Ig VH segment gene was mutated, but the point mutations were not associated with intraclonal diversification. In one IgA and in the IgA/IgG-coexpressing CLL, the nature and distribution of the mutations were consistent with Ag selection. These findings suggest that IgA- and/or IgG-expressing CLLs represent, in their VH gene structure, transformants of B cells at different stages of ontogeny. They also suggest that Ag may play a role in the clonal selection of some of these isotype-switched leukemic cells, but bcl-1 and bcl-2 oncogene rearrangements and p53 tumor suppressor gene mutation are not associated with the pathogenesis of isotype-switched CLLs.