Regulatory T cells in Hodgkin lymphoma

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2004 103: 1565-1566 doi:10.1182/blood-2003-12-4285

Regulatory T cells in Hodgkin lymphoma Sibrand Poppema

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“Complex” receptor for vitamin B12-intrinsic factor Patients with Imerslund-Gra¨sbeck syndrome (I-GS, megaloblastic anemia 1 [MGA1], hereditary megaloblastic anemia, Online Mendelian Inheritance in Man [OMIM] no. 2611001) usually present with megaloblastic anemia between 1 and 5 years of age. They have decreased levels of serum vitamin B12 (cobalamin) in the presence of normal levels of intrinsic factor (IF), and many patients have proteinuria of the tubular type. The Schilling test result is characteristic of the inability of enterocytes to absorb the intrinsic factor–cobalamin complex. Patients in the original studies were described as being Finnish or Norwegian. Currently, more than 250 patients have been identified, many of whom are of Middle Eastern descent. I-GS has locus heterogeneity; in most Finnish families, the disease is caused by mutations in the cubilin (CUBN) gene on chromosome 10p12.1.2 However, mutations in CUBN were not found in Norwegian patients

showing the same phenotype. Through linkage studies, a candidate gene was located on the long arm of chromosome 14q32, and mutations were found in the AMN gene in the Norwegian patients.3 This gene emerged as a candidate because of an expression pattern similar to that of CUBN, with high levels of expression in both small intestine and kidney. Ever since the unexpected discovery of mutations in either the CUBN or the AMN gene in patients with I-GS, it has become essential to address the interaction of the 2 gene products:

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cubilin and amnionless. Fyfe and colleagues (page 1573) show colocalization of cubilin and amnionless proteins in the apical membranes and endocytic apparatus of renal proximal tubule cells. They also demonstrate physical interaction between these 2 proteins following sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) affinity purification. Cotransfection of Chinese hamster ovary (CHO) cells with CUBN and AMN constructs alters the exclusively intracellular locations seen with transfection of CUBN alone, to include the plasma membrane, and allows the endocytosis of IF-cobalamin. These data reinforce findings in the canine model of I-GS in which cubilin is not expressed on the surface of intestinal and renal cells and is retained in an early biosynthetic compartment. Canine I-GS maps to a region orthologous to human chromosome 14 and presumably mutations in AMN will soon be found in affected dogs. A number of questions are still unanswered. Megalin, a member of the lowdensity lipoprotein (LDL) receptor family, has been postulated to be involved in cubilin function. Previous work has shown the colocalization of cubilin with megalin, and megalin-deficient mice have decreased cubilin expression and uptake of cubilin ligands. The case for amnionless has been made much stronger than that for megalin because of the finding of mutations in AMN in I-GS and by the studies of Fyfe and colleagues. What, if anything, then is the physiologic role of megalin in cobalamin absorption? Also, both cubilin and amnionless are implicated in early embryonic development in rodents. Little is known about the mechanisms involved; the only known phenotype resulting from mutations in the human CUBN and AMN genes is I-GS. Finally, which ligands other than cobalamin–intrinsic factor interact with the new complex of cubilin and amnionless for which Fyfe and colleague have coined the name “cubam”? —David Rosenblatt McGill University

1.

Online Mendelian Inheritance in Man, OMIM [database online]. Baltimore, MD: Johns Hopkins University; 2003. Available at: http://www.ncbi. nlm.nih.gov/omim/. Accessed February 20, 2003.

2.

Aminoff M, Carter JE, Chadwick RB, et al. Mutations in CUBN, encoding the intrinsic factorvitamin B12 receptor, cubilin, cause hereditary megaloblastic anaemia 1. Nat Genet. 1999;21: 309-313.

3.

Tanner SM, Aminoff M, Wright FA, et al. Amnionless, essential for mouse gastrulation, is mutated in recessive hereditary megaloblastic anemia. Nat Genet. 2003;33:426-429.

Regulatory T cells in Hodgkin lymphoma The presence of an extensive lymphoid infiltrate distinguishes Hodgkin lymphoma (HL) from most other lymphomas. With respect to this infiltrate, a number of questions can be asked. The first one is what causes such an extensive infiltrate in the presence of only a few tumor cells. The explanation may well be that Reed-Sternberg cells produce and secrete high amounts of chemokines, in particular thymus and activation-regulated chemokine (TARC) and macrophage-derived chemokine (MDC), that attract cells expressing the CCR4 receptor, such as the T-helper 2 (Th2) lymphocyte.1 Another important question is why there is no effective immune response against the tumor cells. Marshall and colleagues (page 1755) confirm previous findings that the HLinfiltrating lymphocytes are anergic to stimulation with some mitogens and primary as well as recall antigens but also demonstrate that these cells suppress peripheral blood mononuclear cell (PBMC) responses. They identify the presence of interleukin-10 (IL-10)–secreting cells and CD4⫹CD25⫹ regulatory T cells. The immunosuppressive effect of the HL-infiltrating cells could be neutralized with anti–IL-10, by preventing cell-to-cell contact, and by anti– cytotoxic T-lymphocyte–associated antigen 4 (anti–CTLA-4). Marshall and colleagues also conclude that the lymphocytes in HL do not produce

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cytokines, such as IL-2, IL-4, and interferon-␥ (IFN-␥), with primary (keyhole limpet hemocyanin [KLH]) and recall (purified protein derivative [PPD]) antigens and the mitogen concanavalin A (ConA). However, in previous studies the lymphocytes were found to produce these cytokines when stimulated with phytohemagglutinin (PHA) or with phorbolester (PMA)–ionomycin. Specifically, when the CD26⫺ CD4 cells immediately surrounding the Reed-Sternberg cells were purified and stimulated with PMA ionomycin, they produced IL-4 and IFN-␥. The potential to produce IL-4 was the reason why these cells were previously considered Th2-like.2 The absence of IL-2 production upon stimulation is also associated with anergy. The exact nomenclature of these cells is thus a matter of semantics. In addition to the IL-10–producing cells (Tr1), there are also transforming growth factor ␤ (TGF-␤)–producing cells present in the infiltrate, and these have been termed Th3. The findings by Marshall and colleagues indicate that there are variations in the populations involved in different cases. It can be concluded that, as an overall population, the infiltrating lymphocytes do not have Th1 type functions and are probably attracted into the tissues by chemokines TARC and MDC as CCR4-expressing Th2 cells. Although these cells do not spontaneously produce IL-2 or IL-4, they produce IL-10, despite not being fully activated, and therefore function as Tr1 cells. The major remaining question is what causes the predominance of T cells with suppressor activity in Hodgkin lymphoma. It appears that Reed-Sternberg cells, although they have the genotype of B cells, execute a functional program that is similar to antigen-presenting cells but results in tolerance. Mechanisms include the production of immunosuppressive cytokines like IL-10, especially in Epstein-Barr virus–positive cases, and TGF-␤, especially in nodular sclerosis cases, as well as the expression of FAS ligand that induces cell death in FASexpressing activated T cells, while the ReedSternberg cells themselves are protected by overexpression of cFLIP (Fas-associating

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protein with death domain–like IL-1␤– converting enzyme [FLICE]–inhibitory protein) or infrequently by FAS mutation.3 The relevance of these findings is that they may allow a better design of new treatment modalities. There are indications that the infiltrating cells in fact support the growth and survival of the Reed-Sternberg cells, and therefore blocking chemokines to prevent the influx of T cells may be effective. On the other hand, blocking of the immunosuppressive signals, such as IL-10 and TGF-␤ cytokines, or the removal of the suppressor regulatory T cells may enhance the effect of adoptive transfer of cytotoxic T cells.4 —Sibrand Poppema University Medical Center Groningen 1.

Van den Berg A, Visser L, Poppema S. High expression of CC chemokine TARC in Reed-Sternberg cells: a possible explanation for the characteristic lymphocytic infiltrate in Hodgkin’s disease. Am J Pathol. 1999;154:1685-1691.

2.

Poppema S, van den Berg A. Interaction between host T-cells and Reed-Sternberg cells in Hodgkin Lymphomas. Semin Cancer Biol. 2000;10:345-350.

3.

Maggio E, van den Berg A, de Jong D, Diepstra A, Poppema S. Low frequency of FAS mutations in Reed-Sternberg cells of Hodgkin lymphoma. Am J Pathol. 2003;162:29-35.

4.

Bollard CM, Rossig C, Calonge MJ, et al. Adapting a transforming growth factor beta-related tumor protection strategy to enhance antitumor immunity. Blood. 2002;99:3179-3187.

More complexity in MLL-associated leukemias The mixed lineage leukemia gene (MLL, also known as ALL-1, and HRX) has rightly attracted much interest as a major player in leukemia.1 MLL’s central role is clear by its involvement with over 30 different partner genes in recurrent translocations. As if this were not enough, MLL is also implicated in leukemia by overexpression in the absence of overt mutations or by acquisition of partial tandem MLL duplications. What, then, accounts for the leukemogenicity of MLL? Might there be some common functional thread tying together many of the fusion genes and MLL overexpression? At least one strong clue has emerged from the recognition that a major function of MLL,

like its Drosophila homolog Trithorax, is to serve as a maintenance factor for the expression of many members of the Hox family of transcription factors. Hox genes are now recognized as major components of the regulatory machinery of primitive hematopoietic cells. Strikingly, multiple lines of evidence link Hox genes directly to leukemic transformation.2,3 This evidence includes induction of leukemia in mice following engineered overexpression of certain Hox genes (eg, HOXA9 and HOXA10) and the observed overexpression of multiple Hox genes in human leukemia and, notably, in MLL-associated leukemias. Perhaps most convincingly, multiple members of the Hox family, HOXA9 for one, have been identified as translocation partners in leukemias with the common partner Nucleoporin 98.4 Thus, a satisfying model for some if not all MLL-induced leukemias would be through induced deregulation of key Hox target genes. Strong support for this has recently been reported by Cleary and colleagues, who found that MLL-ENL lost leukemogenicity in bone marrow cells taken from Hoxa7 or Hoxa9 knockout mice.5 The jump to a unifying model involving MLL and HOXA9, however, is not without a tumble or two as indicated in the article by Kumar and colleagues (page 1823) in this issue of Blood. Their studies reveal unabated leukemogenicity by the fusion gene MLL-AF9 in the absence of Hoxa9. While there were clear influences of Hoxa9 on the phenotype of the leukemia, the essential transformation was not altered by the presence or absence of Hoxa9. The striking differences between these 2 recent studies involving related partner genes may be a consequence of several experimental and biologic differences. Kumar and colleagues have used a gene knock-in model of MLL-AF9 fusion rather than retroviral overexpression; the fusion genes may indeed have differential effects on Hox targets, rendering other members of the cluster more or less important. Indeed, multiple members of the Hox A cluster were observed to be up-regulated by MLL-AF9, making it possible that additive levels of Hox gene expression may be

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