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Recent reports have described complete or major molecular remission in patients with polycythemia vera after long-term treatment with the immunomodulatory agent IFN-α2. Accordingly, there are reasons to believe that the immune system is a key player in eradicating the JAK2 mutated clone in these patients. Foxp3+regulatory T cells play a pivotal role in maintaining immune homeostasis and, importantly, preventing immune reactivity to self-antigens; however, their suppressive activity can compromise an effective antitumor immune response, and high frequencies of regulatory T cells in peripheral blood have been reported in both hematologic and solid cancers. We have analyzed the number, phenotype, and function of circulating CD4+CD25+Foxp3+T cells in patients with chronic myeloproliferative neoplasms. Surprisingly, we found a marked expansion of this subset of lymphocytes in patients treated with IFN-α2 (13.0%; 95% confidence interval [CI] 10.8% to 15.2%) compared with healthy donors (6.1%; 95% CI 4.9% to 7.2%), patients with untreated chronic myeloproliferative neoplasms (6.9%; 95% CI 5.8% to 7.4%), or patients treated with hydroxyurea (5.8%; 95% CI 4.3% to 7.4%; P< .0001).

RhoH is a small GTPase expressed only in the hematopoietic system. With the use of mice with targeted disruption of the RhoH gene, we demonstrated that RhoH is crucial for thymocyte maturation during DN3 to DN4 transition and during positive selection. Furthermore, the differentiation and expansion of DN3 and DN4 thymocytes in vitro were severely impaired. These defects corresponded to defective TCR signaling. Although RhoH is not required for TCR-induced activation of ZAP70 and ZAP70-mediated activation of p38, it is crucial for the tyrosine phosphorylation of LAT, PLCγ1, and Vav1 and for the activation of Erk and calcium influx. These data suggest that RhoH is important for pre–TCR and TCR signaling because it allows the efficient interaction of ZAP70 with the LAT signalosome, thus regulating thymocyte development.

Fas (APO-1/CD95) is a cell-surface receptor involved in cell death signaling. Germline mutations in the Fasgene have been associated with autoimmune lymphoproliferative syndrome, and somaticFasmutations have been found in multiple myeloma. We have examined the entire coding region and all splice sites of theFasgene in 150 cases of non-Hodgkin's lymphoma. Overall, mutations were identified in 16 of the tumors (11%). Missense mutations within the death domain of the receptor were associated with retention of the wild-type allele, indicating a dominant-negative mechanism, whereas missense mutations outside the death domain were associated with allelic loss. Fasmutations were identified in 3 (60%) MALT-type lymphomas, 9 (21%) diffuse large B-cell lymphomas, 2 (6%) follicle center cell lymphomas, 1 (50%) anaplastic large cell lymphoma, and 1 unusual case of B-cell chronic lymphocytic leukemia with a marked tropism for skin. Among the 16 patients with somaticFasmutations, 15 showed extranodal disease at presentation, and 6 relapsed in extranodal areas. Ten of 13 evaluable patients showed features suggestive of autoreactive disease. Our data indicate that somatic disruption of Fasmay play a role in the pathogenesis of some lymphomas, and suggest a link between Fasmutation, cancer and autoimmunity.

Allogeneic hematopoietic cell transplantation (HCT) has a well-documented ability to cure a number of malignant hematological diseases. The curative principle in allogeneic HCT is the Graft-versus-Leukemia (GVL) effect and nonmyeloablative (NMA) conditioning HCT relies exclusively on this anti-tumor effect to eliminate tumor cells. Donor T-cells are documented to be responsible for the GVL effect, however, often they also cause Graft-versus-Host disease (GVHD) which is associated with high morbidity and mortality. Characterization of cells and molecules involved in both GVL and GVHD would potentially set the stage for separation of GVL and GVHD in order to augment GVL in the absence of GVHD. In the present study, we analyzed the clonotype composition of CD8+T cells following NMA conditioning and HCT, in two patients with chronic lymphocytic leukemia (CLL). T-cell receptor (TCR) clonotype mapping (RT-PCR combined with denaturing gradient gel electrophoresis (DGGE)) was used to identify clonally expanded CD8+T cells in blood samples. This method provides a “molecular fingerprint” of each unique T cell based on junctional diversity of the TCR CDR3 region and, thus, offers the means to track T-cell clonotypes in time and space. Longitudinal comparative analyses showed that CD8+T-cell clonality was highly dynamic during early phases after transplantation with various clonotypes emerging and disappearing. However, clonal diversity decreased after 4–5 months and stable CD8+T-cell clonotypes appeared and persisted throughout the analyzed period (up to two years). One patient received donor lymphocyte infusion (DLI) due to disease progression and this was shown to lead to establishment of recurrent (detected prior to DLI) CD8+T-cell clonotypes as well as new CD8+T-cell clonotypes. The appearance of these cells correlated with disease remission strongly suggesting their engagement in anti-CLL reactivity. To examine the functional capacities of clonally expanded T cells after HCT, recipient CD8+T cells were stimulated ex vivowith pre-transplant patient CLL cells and/or normal hematopoietic cells and the T-cell surface expression of CD107a (marker for cytotoxicity) was detected by FACS. Clonotype mapping analyses of FACS sorted CD107a positive CD8+T cells after stimulation with CLL cells demonstrated that such cytotoxic CD8+T cells were present as stable clonally expanded T cells in vivostrongly implying their involvement in an ongoing anti-CLL-response. Furthermore, co-culture with normal hematopoietic cells resulted in a unique CD107a positive expanded T-cell clonotype. Our results strongly suggest that clonally expanded CD8+T cells are involved in an ongoing tumor response and support data which demonstrate that GVL and GVHD are the result of distinct responses.