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Angioimmunoblastic lymphadenopathy with dysproteinemia (AILD) is defined in the current lymphoma classifications as a T-cell non-Hodgkin's lymphoma. However, in approximately one third of the cases of this lymphoproliferative disease rearrangements of T-cell receptor (TCR) genes indicating clonal expansion of T cells are not detectable. It is currently believed that these cases may represent early stages of a lymphoma with a minor oligoclonal T-cell population. In the present study, 18 lymph nodes with the characteristic histology of AILD were investigated for clonal T-cell receptor gene rearrangements by analysis of DNA extracted from whole tissue sections. Dominant T-cell clones were detected in 12 of these cases. Single CD4(+) and CD8(+) T cells and proliferating Ki67(+) cells of seven cases were micromanipulated from frozen tissue sections. TCRbeta gene rearrangements were amplified from these cells by polymerase chain reaction and sequenced. In all informative cases, the clonal gene rearrangements were only detected among CD4(+), and not among CD8(+) T cells, indicating that the tumor clones in AILD usually derive from CD4(+) T cells. Minor clonal T-cell populations in those cases in which no clone was found by whole-tissue DNA analysis were not detectable even at single cell resolution. T-cell clones in 4 of 10 cases were found to express similar TCRbeta chains, indicating a potential role of (super) antigen triggering in at least some cases of AILD.

In the immunobiological characterization of lymph node cells, sinus-lining cells (SLCs) have been given little attention mainly due to the difficulties in their recognition. Ki-M9 is a new monoclonal antibody (MAb) selected for its unique capability to visualize SLCs in human lymph nodes. The details were established by light and electron microscopy and immunoprecipitation of the corresponding biosynthetically labeled antigen. Ki-M9 recognizes a 70-kd protein localized on the surface membrane of SLCs. In the lymphoid tissue, a mild reactivity was exclusively encountered on follicular dendritic reticulum cells in the germinal centers of secondary lymphoid follicles. In other organs, some squamous epithelial and myoepithelial cells were recognized by this antibody. Immunomonitoring of SLCs on light and electron microscopic levels revealed their dendritic morphology, lack of phagosomes, and their close association with type IV collagen fibers. Considering the occurrence of typical dendritic SLCs on the front line of antigen flood, we propose that SLCs be investigated for a possible antigen-binding property.

To study the distribution pattern of interleukin-6 (IL-6)-producing cells in normal human lymph nodes, we applied the in situ reverse transcription-polymerase chain reaction technique. We describe a new modification of this technique for monitoring small amounts of specific nucleotide sequences in conventional paraffin sections. This technique differs in at least two respects from those described earlier. The two decisive steps are: 1) the reverse transcription of mRNA and the subsequent amplification of cDNA by polymerase chain reaction are performed by a new single enzyme capable of both reaction types in one and the same medium without buffer exchange; and 2) for the specific detection of the amplified cDNA, a modified version of the primed in situ labeling technique was used. The technique, carried out on normal human lymph nodes, traces a low load of IL-6 mRNA in fibroblasts, endothelial cells, and a minor population of T lymphocytes in the pulp region. High levels of expression were encountered in about 20% of perisinusoidal pulp macrophages. In addition, moderate activity was detectable in sinus lining cells. Because no major activity was found in the germinal centers of the lymphoid B follicles and in the T zone, it is suggested that the plasma cell differentiation ensuing from primary and secondary B-cell immunization is mainly effected by the sinus lining cells as well as perifollicular and perisinusoidal pulp macrophages capable of producing high amounts of IL-6.

Relevant prognostic information can be added to the histological classification of non-Hodgkin's lymphoma (NHL) by the determination of the growth fraction. In unfixed fresh tissue samples the Ki-67 antigen has proven its utility as an operational marker of proliferative activity in NHL. We have generated a monoclonal antibody directed against a formalin-resistant epitope of the Ki-67 antigen, designated Ki-S5. The immunoreactivity of Ki-S5 is confined to the nuclei of proliferating cells and, unlike Ki-67, no cross-reactivity with cytoplasmic antigens of epithelial cells occurs. In 100 cases of different NHL types, parallel staining of Ki-67 and Ki-S5 antigen yielded almost identical results in fresh tissues and fixed tissues, respectively (P < 0.00001; r = 0.95). In some cases, the Ki-S5 labeling index exceeded that of Ki-67, most probably due to a partial degradation of the epitope recognized by Ki-67 in frozen stored tissue samples. Significant differences in the median Ki-S5 labeling index could be demonstrated between high grade (> 50%) and low grade malignant NHL (< or = 30%), although the proliferative activity varied to a considerable range within identical histological categories (P < 0.001). We conclude that Ki-S5 provides a reliable means to assess proliferation in paraffin-embedded NHL specimens. Retrospective studies relating the proliferative activity to clinical outcome are thus rendered possible using archival routinely processed tissue samples.

A monoclonal antibody (Ki-S1) has been raised that reacts with the nuclei of proliferating cells. The antigen recognized is resistant to formalin fixation and can be detected in frozen tissues as well as in routinely processed specimens. In immunohistochemistry, nuclear staining can be seen in those tissues and cellular compartments known to be actively proliferating. Peripheral blood lymphocytes are negative but show a strong increase in antigen expression after mitogen stimulation. Flow cytometric determination of DNA content and antigen expression revealed negativity of G0 cells and positivity of G1 to G2/M cells. A cytoplasmic co-reactivity, not associated with proliferation, was confined to Langerhans islands of the pancreas. The nuclear localized antigen has a molecular mass of 160 kd and therefore seems to be different from all other known immunohistochemical markers of proliferating cells. We conclude that the monoclonal antibody Ki-S1 might provide a useful tool for studying cell proliferation in situ under normal and pathological circumstances.

T cell receptor beta (TcR beta) chain gene rearrangements have been reported in cases of angioimmunoblastic lymphadenopathy (AILD) and provided evidence for the presence of clonal T cell proliferations in this disorder. Twenty-three cases of AILD and two cases of hyperimmune reaction (HR) were investigated. In the two HR cases, essentially the same histologic pattern was present as in AILD but lymph node follicles were hyperplastic. Both HR cases showed germline configuration for the TcR and immunoglobulin heavy chain (IgH) genes. All other patients diagnosed with AILD had clonal rearrangements for TcR gamma and beta chain genes. In addition, seven out of these cases had clonally rearranged their IgH genes. These two different rearrangement patterns (TcR with or without Ig gene rearrangement) correlated to immunohistochemical and clinical data. Cases with TcR but without Ig gene rearrangements (group I) exclusively showed CD4+ proliferating T cells, whereas those cases with TcR and Ig gene rearrangements had significantly elevated numbers of CD8+ proliferating cells (group II). Group II patients significantly more often presented with hemolytic anemia and went into transient remission spontaneously or under steroid treatment. Group I patients, however, had a higher response to chemotherapy and a longer survival time. These data show that, based on different rearrangement patterns, it is possible to divide AILD into two different groups with distinct immunophenotypic properties and differences in clinical parameters. Immunogenotyping in AILD thus will have prognostic and therapeutic implications.