Hodgkin lymphoma HL

Depending on the geography and histological type the frequency of EBV-positive HLs vary. The classical HL tissue contains only a few (about 1-10%) Hodgkin-Reed Sternberg (H-RS) cells [29]. Whether these H-RS cells carry or not the viral genome, the lymphoma tissues are made up by T-, B-cells, macrophages, eosinophils, and plasma cells, thus they have an inflammatory character. The EBV gene expression pattern in the H-RS cells is regularly Type Ila with abundant LMP-1. While commonly the H-RS cells belong to the B-lymphoid lineage [30], they do not express all B-cell-specific genes, such as transcription factors and B-cell-associated markers, like CD19, CD20 and immunoglobulins [31, 32]. Detailed analysis of the gene expression profile of the HL lines combined with immunohistochemical analysis of tumor samples showed considerable deviations from the B-lymphocyte pattern, showing that the differentiation program of the H-RS cell is impaired. Since the regular EBV latent program in B lymphocytes is Type III or Type I, this reflects also that the cells deviate from normal B-cells. EBNA-2 expression requires B-cell-specific transcription factors such as Oct-2 and BSAP (PAX-5) [33, 34]. In their absence the Wp and Cp promoters that regulate EBNA-1, -2, -3, -4, -5, -6 expression during type III latency is inactive.

HL has been studied in great detail and its literature is abundant. As mentioned above the characteristics of the HL tissues are similar in the EBV-negative and -positive cases. Special properties that could be ascribed to the presence of the virus in the H-RS cells have not been discovered yet; however some studies concluded that the prognosis of EBV-positive cases is more favorable for certain age groups and histologic subtypes.

LMP-1 may not only function in the rescue of the faulty differentiated B lymphocytes, but could contribute to the establishment of the granulomatous tissue as well. For the phenotype and survival of the H-RS cell activation of the nuclear factor (NF)-kappa B, that is the hallmark of these cells, is decisive [35]. In the EBV-carrying cases LMP-1, in the EBV-negative cases mutations in the NF-kappa B inhibitors (IkB-alpha) [36], amplification of the REL gene (encodes an NF-kB family member) [37] or signals acting on surface receptors CD30 and CD40 [38, 39], may activate NF-kappa B.

The HL granuloma is a complex interactive environment to which a multitude of cellular and soluble factors contribute. Secretion of the CCL17/ TARC and CCL22/ MDC chemokines by the H-RS cells can attract CCR4-expressing Th2 and regulatory T-cells [40, 41]. The H-RS cells may get survival signals from the infiltrating Th-2 cells, while the regulatory T-cells may protect them from the EBV-specific immune response [42, 43]. Furthermore the H-RS cells secrete immunosuppressive Cytokines, such as TGF-beta and IL-10. Though LMP-1 could impose immuno-genicity on the cell, the same molecule released from cells has been shown to be immunosuppressive [44].

The difficulty to establish cell lines from EBV-carrying HL is in line with the complex structure of the lymphoma tissue. In spite of considerable effort, HL-derived cell lines with type Ila EBV latency have not been established [45]. With one exception - the Type III L591 line, that however does not represent the in vivo H-RS cell - the few available HL-derived cell lines are all EBV-negative [46]. This shows that the Type IIa EBV expression does not induce cell proliferation, unless additional cellular changes or signals from the microenvironment contribute.

The phenotype of the H-RS cells deviates thus from the mature B lymphocytes. Its particular phenotype and the lack of in vitro type IIa latency model motivated our studies for the cell-virus interaction. With the help of a recombinant EBV strain containing the neomycin resistance gene, we established a virus-carrying subline of the EBV-negative KM-H2 line [47]. For maintenance of the virus, the cells had to be carried in the selective medium. Importantly, only the EBNA-1 protein was expressed in these cells, thus they were Type I. Similar results were reported by Baumforth and co-workers [48]. Thus, the in vitro-established EBV carrier state of the KM-H2 cells did not correspond to the in vivo expression of viral proteins in HRS cells. However, LMP-1 became induced when IL-4 and contact with CD40-ligand (CD40L) were provided to the cells, and this was not accompanied by EBNA-2 expression [47]. We showed thus for the first time that the EBV gene expression pattern can be influenced by cytokines and LMP-1 can be induced in HL-derived cells in the absence of EBNA-2 expression [47]. This condition can prevail in vivo because in the lymphogranuloma the H-RS are surrounded by activated CD4-positive T-cells that produce a variety of cytokines and express CD40L [38, 49].

Similarly to the experimentally created KMH2-EBV cells, EBNA-2 could not be induced in the EBV-carrying cell lines derived from body cavity lymphoma (pleural effusion lymphoma, PEL) [50] that have also lost several B-cell characteristics [51].

On the basis of our results showing cytokine-induced expression of LMP-1 we modify the scheme proposed by Thorley-Lawson and discussed above. We introduce the contribution of the microenvironment for the generation of Type IIa cells, in that we propose that LMP-1 expression is induced by extracellular signals in the GC, provided by cytokines and cell contacts. (Fig. 10.1). This is in line also with the observations that EBV-positive memory B-cells isolated from tonsils express

Fig. 10.1 Development of the EBV-positive H-RS cell in the germinal center, emphasizing the development of Type IIa EBV latency under the influence of the microenvironment. EBV carrier normal B-cells in GC express the Type I viral pattern. When they leave the GC, the virally encoded proteins are turned off. During the process of somatic hypermutation occasional cells acquire non-functional mutations, they do not express sIg, and succumb to apoptosis. EBV can rescue these cells. Signals provided by T-cells through the CD40L and cytokines, particularly IL-4, IL-10 and IL-21, induce the expression of LMP-1. Interaction of these cells with the environment, cell-cell contacts and cytokines leads to their survival and entry to the cell cycle. Constitutive activation of NF-kB, induced by LMP-1 has a key role in the malignant property of the H-RS cell. The circles in the nucleus of the cells represent the viral episome. sIg denotes surface immunoglobulin

Fig. 10.1 Development of the EBV-positive H-RS cell in the germinal center, emphasizing the development of Type IIa EBV latency under the influence of the microenvironment. EBV carrier normal B-cells in GC express the Type I viral pattern. When they leave the GC, the virally encoded proteins are turned off. During the process of somatic hypermutation occasional cells acquire non-functional mutations, they do not express sIg, and succumb to apoptosis. EBV can rescue these cells. Signals provided by T-cells through the CD40L and cytokines, particularly IL-4, IL-10 and IL-21, induce the expression of LMP-1. Interaction of these cells with the environment, cell-cell contacts and cytokines leads to their survival and entry to the cell cycle. Constitutive activation of NF-kB, induced by LMP-1 has a key role in the malignant property of the H-RS cell. The circles in the nucleus of the cells represent the viral episome. sIg denotes surface immunoglobulin

LMP-1, while the ones in blood are LMP-1 negative [52]. Upon exit from the GC, the long-lived, resting memory B-cells do not express the viral proteins. The H-RS cells originate from "crippled "B-cells that are normally eliminated [28]. When the cells acquire EBV infection, their fate changes. LMP-1 (and LMP-2a) is induced and they escape apoptosis. Occasional cells enter in a mutual stimulatory interaction with the normal cellular components of the lymph-node, persist, and can enter the mitotic cycle.

Elimination of the EBV-carrying type III latent B-cells is critical for the infected individual since these cells have a proliferative potential. LMP-1 and LMP-2a expression together with the co-stimulatory molecules and intact antigen-presenting machinery would be expected to elicit an immune response in HL as well. In the HL granuloma, however, the immune response seems to be locally inhibited [53] by immunosuppressive cytokines (IL10, TGF-beta) and by regulatory T cells [42, 43].

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