Lymphoma cell survival and progression are putatively dependent on a specific microanatomic localization within secondary lymphoid organs. Despite compelling data correlating homeostatic chemokine receptor expression and human lymphoma pathogenesis, genetic models that either mimic lymphoma dissemination or dissect a crosstalk of lymphoma and stromal cells are missing. Applying the genetically tractable EMyc transgenic mouse model, we show that the chemokine receptor CCR7 regulates EMyc lymphoma homing to lymph nodes and distinctive microanatomic sites of the spleen. CCR7-controlled access of lymphoma cells to the splenic T-cell zone led to a significant survival advantage compared with CCR7-deficient lymphoma cells, which were excluded from this zone. Within the niche, lymphoma cells stimulated a reciprocal cross-talk with gp38 ؉ fibroblastic reticular cells. This reciprocal cooperation program was mediated by lymphoma B cellpresented lymphotoxin, which acted on lymphotoxin-␤-receptor-bearing stromal cells followed by alteration of stromal cellular composition. Cross-talk inhibition by lymphotoxin-␣ deletion and using a lymphotoxin-␤ receptor-immunoglobulin fusion protein impaired lymphoma growth. Thus, abrogation of CCR7-governed migration and of sustained lymphotoxin signaling could provide new targets in lymphoma therapy. (Blood. 2011;118(4):1020-1033) IntroductionThe crosstalk between lymphoid tumor cells and their microenvironment provides pivotal signals for the localization and progression of lymphoid malignancies. Thus, it has become increasingly important to define which molecular mechanisms allow the interactions between accessory cells and malignant B-cells and to identify the stromal cells that mediate such signals. 1,2 In E-Myc transgenic mice, a mouse model of Myc-driven aggressive human B-cell lymphoma, the precancerous state is characterized by excessive proliferation in conjunction with the onset of an apoptotic program within the bone marrow precursor and immature B-cell compartment. 3,4 Although the role of genetic lesions as steps toward cell autonomy and tumor growth has been appreciated, 5,6 in vivo the conditions for lymphoma cell lodging within secondary lymphoid organs (SLOs) remain to be addressed. Several in vitro studies or transfer models of human lymphoma cell lines into immunodeficient mice could demonstrate that a variety of cytokines, adhesion molecules, and growth factors were involved in the cross-talk at the stroma-lymphoma cell interface. 7,8 Clinical studies have correlated the involvement of chemokine receptors with nodal homing of B-cell non-Hodgkin (NHL) and Hodgkin lymphoma. 9,10 Conversely, lack of the major lymph node (LN) addressins, foremost the homeostatic chemokine receptors CXCR5 and CCR7, may predispose those tumor cells for extranodal dissemination instead. 11 In vivo, the dissemination of primary central nervous system lymphoma toward brain-expressed chemokine ligand CCL21 has been shown to be controlled by CCR7 expression. 12 Although this investigation consi...

Key Points• Donor T-cell infiltration of the bone marrow is associated with impaired B-cell immunity after allogeneic HSCT.• Quantification of k-deleting recombination excision circles as a biomarker for bone marrow B-cell output in different clinical episodes.B-cell immune dysfunction contributes to the risk of severe infections after allogeneic hematopoietic stem cell transplantation (allo-HSCT). Delayed B-cell regeneration is found in patients with systemic graft-versus-host disease (GVHD) and is often accompanied by bone marrow (BM) suppression. Little is known about human BM GVHD. We analyzed the reconstitution kinetics of B-cell subsets in adult leukemic patients within 6 months after allo-HSCT. B-cell deficiency already existed before transplant and was aggravated after transplant. Onset of B-cell reconstitution characterized by transitional B-cell recovery occurred either early (months 2-3) or late (from month 6 on) and correlated highly positively with reverse transcription-polymerase chain reaction quantified numbers of k-deleting recombination excision circles (KRECs). Delayed recovery was associated with systemic acute GVHD and full-intensity conditioning therapy. Histological analysis of BM trephines revealed increased T-cell infiltration in late recovering patients, which was associated with reduced numbers of osteoblasts. Functionally, late recovering patients displayed less pneumococcal polysaccharide-specific immunoglobin M-producing B cells on ex vivo B-cell activation than early recovering patients. Our results provide evidence for acute BM GVHD in allo-HSCT patients with infiltrating donor T cells and osteoblast destruction. This is associated with delayed B-cell reconstitution and impaired antibody response. Herein, KREC appears suitable to monitor BM B-cell output after transplant. (Blood. 2014;124(6):963-972)

BackgroundAfter hematopoietic stem cell transplantation (HSCT) T- and B-cell reconstitution from primary lymphoid organs are a prerequisite for an effective early lymphocyte reconstitution and a long-term survival for adult patients suffering from acute leukemia. Here, we asked whether quantification of T cell receptor excision circle, (TREC) and kappa-deleting recombination excision circle (KREC) before and within six month after allogeneic HSCT could be used to measure the thymic and bone marrow outputs in such patients.MethodsWe used a duplex real time PCR assay to quantify the absolute copy counts of TREC and KREC, and correlated the data with absolute cell counts of CD3+CD4+ T-cell and CD19+ B-cell subsets determined by flow cytometry, respectively.ResultsBy comparing two recently proposed naïve T cell subsets, CD31+ naive and CD31- naive T cells, we found a better correlation for the CD31+ subset with TREC level post alloHSCT, in line with the assumption that it contained T cells recently derived from the thymus, indicating that TREC levels reflected real thymic de novo production. Transitional as well as naïve B cells highly correlated with KREC levels, which suggested an association of KREC levels with ongoing bone marrow B cell output. CD45RO+ memory T cells and CD27+ memory B cells were significantly less correlated with TREC and KREC recovery, respectively.ConclusionWe conclude that simultaneous TREC/ KREC quantification is as a suitable and practicable method to monitor thymic and bone marrow output post alloHSCT in adult patients diagnosed with acute leukemia.

Rabbit antithymocyte globulin-GenzymeTM is used to prevent graft-versus-host disease after allogeneic hematopoietic stem cell transplantation. Common disadvantages of treatment are infectious complications. The effects of rabbit antithymocyte globulin-Genzyme TM on thymic function have not been well-studied. Multicolor flow cytometry was used to analyze the kinetics of conventional and regulatory T cells in adult patients treated (n=12) or not treated (n=8) with rabbit antithymocyte globulin-Genzyme TM during the first 6 months after allogeneic hematopoietic stem cell transplantation. Patients treated with rabbit antithymocyte globulin-Genzyme TM had almost undetectable levels of recent thymic emigrants (CD45RA + CD31 + ) of both conventional and regulatory CD4 T cells throughout the 6 months after allogeneic hematopoietic stem cell transplantation whereas CD4 + CD45RA -memory T cells were less affected, but their levels were also significantly lower than in patients not treated with rabbit antithymocyte globulin-Genzyme TM . In vitro, rabbit antithymocyte globulin-Genzyme TM induced apoptosis and cytolysis of human thymocytes, and its cytotoxic effects were greater than those of rabbit antithymocyte globulin-Fresenius TM . Rabbit antithymocyte globulin-Genzyme TM in combination with a conditioning regimen strongly impairs thymic recovery of both conventional and regulatory CD4 + T cells. The sustained depletion of conventional and regulatory CD4 + T cells carries a high risk of both infections and graft-versus-host disease. Our data indicate that patients treated with rabbit antithymocyte globulin-Genzyme TM could benefit from thymus-protective therapies and that trials comparing this product with other rabbit antithymocyte globulin preparations or lymphocyte-depleting compounds would be informative.

The capacity of dendritic cells (DCs) to regulate tumour-specific adaptive immune responses depends on their proper differentiation and homing status. Whereas DC-associated tumour-promoting functions are linked to T-cell tolerance and formation of an inflammatory milieu, DC-mediated direct effects on tumour growth have remained unexplored. Here we show that deletion of DCs substantially delays progression of Myc-driven lymphomas. Lymphoma-exposed DCs upregulate immunomodulatory cytokines, growth factors and the CCAAT/enhancer-binding protein b (C/EBPb). Moreover, Em-Myc lymphomas induce the preferential translation of the LAP/LAP* isoforms of C/EBPb. C/EBPb À/ À DCs are unresponsive to lymphoma-associated cytokine changes and in contrast to wild-type DCs, they are unable to mediate enhanced Em-Myc lymphoma cell survival. Antigen-specific T-cell proliferation in lymphoma-bearing mice is impaired; however, this immune suppression is reverted by the DC-restricted deletion of C/EBPb. Thus, we show that C/EBPb-controlled DC functions are critical steps for the creation of a lymphoma growth-promoting and -immunosuppressive niche.