Survivin is expressed on CD40 stimulation and interfaces proliferation and apoptosis in B-cell chronic lymphocytic leukemia

In B-cell chronic lymphocytic leukemia (B-CLL), defective apoptosis causes the accumulation of mature CD5(+) B cells in lymphoid organs, bone marrow (BM), and peripheral blood (PB). These cells are the progeny of a proliferating pool that feeds the accumulating compartment. The authors sought to determine which molecular mechanisms govern the proliferating pool, how they relate to apoptosis, and what the role is of the microenvironment. To begin to resolve these problems, the expression and modulation of the family of inhibitor of apoptosis proteins (IAPs) were investigated, with consideration given to the possibility that physiological stimuli, such as CD40 ligand (CD40L), available to B cells in the microenvironment, might modulate IAP expression. The in vitro data on mononuclear cells from PB or BM of 30 patients demonstrate that B-CLL cells on CD40 stimulation express Survivin and that Survivin is the only IAP whose expression is induced by CD40L. Through immunohistochemistry, in vivo Survivin expression in lymph node (LN) and BM biopsies was evaluated. In reactive LN, Survivin was detected only in highly proliferating germinal center cells. In LN from patients with B-CLL, Survivin was detected only in pseudofollicles. Pseudofollicle Survivin(+) cells were actively proliferating and, in contrast to Survivin(+) B cells found in normal GC, were Bcl-2(+). In B-CLL BM biopsies, CD5(+), Survivin(+) cells were observed in clusters interspersed with T cells. These findings establish that Survivin controls the B-CLL proliferative pool interfacing apoptosis and that its expression may be modulated by microenvironmental stimuli.     

Bone marrow-based homeostatic proliferation of mature T cells in nonhuman primates: implications for AIDS pathogenesis

Bone marrow (BM) is the key hematopoietic organ in mammals and is involved in the homeostatic proliferation of memory CD8(+) T cells. Here we expanded on our previous observation that BM is a preferential site for T-cell proliferation in simian immunodeficiency virus (SIV)-infected sooty mangabeys (SMs) that do not progress to AIDS despite high viremia. We found high levels of mature T-cell proliferation, involving both naive and memory cells, in healthy SMs and rhesus macaques (RMs). In addition, we observed in both species that lineage-specific, BM-based T-cell proliferation follows antibody-mediated in vivo CD4(+) or CD8(+) T-cell depletion, thus indicating a role for the BM in maintaining T-cell homeostasis under depleting circumstances. We also observed that, in SIV-infected SMs, but not RMs, the level of proliferation of BM-based CD4(+) T cells is higher than that of circulating CD4(+) T cells. Interestingly, limited BM-based CD4(+) T-cell proliferation was found in SIV-infected SMs with low CD4(+) T-cell counts, suggesting a regenerative failure in these animals. Collectively, these results indicate that BM is involved in maintaining T-cell homeostasis in primates and suggest a role for BM-based CD4(+) T-cell proliferation in determining the benign nature of natural SIV infection of SMs.     

T cells selectively infiltrate bone marrow areas with residual haemopoiesis of patients with acquired aplastic anaemia

Aplastic anaemia (AA) is characterized by pancytopenia and bone marrow (BM) hypocellularity. In some patients AA may be mediated by T cells. To localize inflammatory cell infiltrates, we carried out a quantitative immunohistochemical analysis of BM biopsies of AA patients. In five out of eight biopsies, significantly higher numbers T cells were found in the areas with residual haemopoiesis (RH). The significantly increased numbers of CD3⁺ T cells in areas with RH supports the hypothesis of a site-directed infiltration and/or a local proliferation of T cells in the BM of patients with AA.     

T cells selectively infiltrate bone marrow areas with residual haemopoiesis of patients with acquired aplastic anaemia

Aplastic anaemia (AA) is characterized by pancytopenia and bone marrow (BM) hypocellularity. In some patients AA may be mediated by T cells. To localize inflammatory cell infiltrates, we carried out a quantitative immunohistochemical analysis of BM biopsies of AA patients. In five out of eight biopsies, significantly higher numbers T cells were found in the areas with residual haemopoiesis (RH). The significantly increased numbers of CD3⁺ T cells in areas with RH supports the hypothesis of a site-directed infiltration and/or a local proliferation of T cells in the BM of patients with AA.     

Vaccination with autologous tumor-loaded dendritic cells induces clinical and immunologic responses in indolent B-cell lymphoma patients with relapsed and measurable disease: a pilot study

Eighteen relapsed patients with measurable indolent non-Hodgkin lymphoma (NHL) were vaccinated with dendritic cells (DCs) loaded with killed autologous tumor cells. Six patients had objective clinical responses including 3 continuous complete responses (CRs) and 3 partial responses (PRs), with a median follow up of 50.5 months. Eight patients had stable disease, whereas 4 had progressive disease. Clinical responses were significantly associated with a reduction in CD4(+)CD25(+)FOXP3(+) regulatory T cells, an increase in CD3(-)CD56(dim)CD16(+) natural killer (NK) cells, and maturation of lymphocytes to the effector memory stage in either postvaccination peripheral blood or tumor specimen samples. In partial responding patients, vaccination significantly boosted the IFN-gamma-producing T-cell response to autologous tumor challenge. In one HLA-A*0201(+) patient who achieved CR, IL-4 release by circulating T cells in response to tumor-specific IgH-encoded peptides was also documented. Immunohistochemical analysis of tumor biopsies using biotin-conjugated autologous serum samples revealed a tumor-restricted humoral response only in the postvaccination serum from responding patients. Collectively these results demonstrate that vaccination with tumor-loaded DCs may induce both T- and B-cell responses and produces clinical benefits in indolent NHL patients with measurable disease. This study is registered with the Istituto Superiore di Sanità: http://www.iss.it with protocol number 7578-PRE 21-801.     

Circulating memory B-cell subpopulations are affected differently by HIV infection and antiretroviral therapy

OBJECTIVE: To determine if the depletion of IgM memory B cells might contribute to the increased susceptibility of HIV patients to pneumococcal infection, memory B-cell subpopulations were investigated in HIV patients, including patients receiving antiretroviral therapy (ART). METHODS: Blood B cells with the phenotype of IgM memory B cells (CD27, IgM) and switched memory B cells (CD27, IgM) were measured in antiretroviral-treated (n = 32) and untreated (n = 24) HIV patients and non-HIV controls (n = 35). Serum levels of IgG and IgG2 antibodies to pneumococcal polysaccharides, IgG, IgG subclasses, IgM and IgA were also assayed in HIV patients. RESULT: Switched memory B-cell counts were lower than controls in HIV patients (P < 0.01) irrespective of antiretroviral status and correlated with CD4 T-cell counts (r = 0.56, P = 0.001) in treated patients. In untreated patients, IgM memory B-cell counts correlated with CD4 T-cell counts (r = 0.73, P < 0.0001) reflecting higher values than controls in patients with CD4 T-cell counts greater than 300 cells/microl (P = 0.004) and lower values than controls in patients with CD4 T-cell counts below 300 cells/microl (P = 0.0001). There was no relationship between serum levels of pneumococcal antibodies and IgM or switched memory B cells. CONCLUSION: The depletion of IgM memory B cells in untreated HIV patients with a CD4 T-cell count below 300 cells/microl might be a risk factor for pneumococcal infection. The depletion of switched memory B cells is a complication of HIV infection irrespective of ART and might contribute to impaired IgG antibody responses. Memory B-cell subpopulations might predict the risk of pneumococcal sepsis more accurately than the CD4 T-cell count or pneumococcal antibody levels.     

B-cell depletion and remissions of malignancy along with cytokine-associated toxicity in a clinical trial of anti-CD19 chimeric-antigen-receptor-transduced T cells

We conducted a clinical trial to assess adoptive transfer of T cells genetically modified to express an anti-CD19 chimeric Ag receptor (CAR). Our clinical protocol consisted of chemotherapy followed by an infusion of anti-CD19-CAR-transduced T cells and a course of IL-2. Six of the 8 patients treated on our protocol obtained remissions of their advanced, progressive B-cell malignancies. Four of the 8 patients treated on the protocol had long-term depletion of normal polyclonal CD19(+) B-lineage cells. Cells containing the anti-CD19 CAR gene were detected in the blood of all patients. Four of the 8 treated patients had prominent elevations in serum levels of the inflammatory cytokines IFNγ and TNF. The severity of acute toxicities experienced by the patients correlated with serum IFNγ and TNF levels. The infused anti-CD19-CAR-transduced T cells were a possible source of these inflammatory cytokines because we demonstrated peripheral blood T cells that produced TNF and IFNγ ex vivo in a CD19-specific manner after anti-CD19-CAR-transduced T-cell infusions. Anti-CD19-CAR-transduced T cells have great promise to improve the treatment of B-cell malignancies because of a potent ability to eradicate CD19(+) cells in vivo; however, reversible cytokine-associated toxicities occurred after CAR-transduced T-cell infusions.     

Long-term restoration of the human T-cell compartment after thymectomy during infancy: a role for thymic regeneration?

Thymectomy during early childhood is generally thought to have serious consequences for the establishment of the T-cell compartment. In the present study, we investigated the composition of the T-cell pool in the first 3 decades after thymectomy during infancy due to cardiac surgery. In the first 5 years after thymectomy, naive and total CD4(+) and CD8(+) T-cell numbers in the blood and T-cell receptor excision circle (TREC) levels in CD4(+) T cells were significantly lower than in healthy age-matched controls. In the first years after thymectomy, plasma IL-7 levels were significantly elevated and peripheral T-cell proliferation levels were increased by ～ 2-fold. From 5 years after thymectomy onward, naive CD4(+) and CD8(+) T-cell counts and TRECs were within the normal range. Because TREC levels are expected to decline continuously in the absence of thymic output, we investigated whether normalization of the naive T-cell pool could be due to regeneration of thymic tissue. In the majority of individuals who had been thymectomized during infancy, thymic tissue could indeed be identified on magnetic resonance imaging scans. Whereas thymectomy has severe effects on the establishment of the naive T-cell compartment during early childhood, our data suggest that functional regrowth of thymic tissue can limit its effects in subsequent years.     

Immunoglobulin M levels inversely correlate with CD40 ligand promoter methylation in patients with primary biliary cirrhosis

The cross-talk of cluster of differentiation (CD)40/CD40 ligand (CD40L) plays a key role in CD4(+) T-cell priming, B-cell terminal maturation, and immunoglobulin (Ig) class-switch recombination. Genetic defects in the CD40L lead to a disorder characterized by elevated concentrations of serum IgM and immunodeficiency. Patients with primary biliary cirrhosis (PBC) characteristically show circulating antimitochondrial antibodies (AMAs), liver-infiltrating autoreactive T lymphocytes against mitochondrial antigens, and high levels of IgM. We hypothesized that CD40L may play a key role in the pathogenesis of the elevated serum IgM and analyzed genetic and epigenetic modifications of the gene coding for CD40L in CD4(+) and CD8(+) T cells isolated from circulating mononuclear cells from PBC patients and healthy controls. We herein demonstrate significantly lower levels of DNA methylation of the CD40L promoter in CD4(+) T cells from PBC patients, as compared with controls, and this decreased methylation was inversely correlated with levels of serum IgM in PBC patients. CONCLUSION: The findings of an absence of genetic modifications of the CD40L gene, in concert with decreased DNA methylation of the CD40L promoter in PBC patients, suggests that environmental factors, rather than genetics, must play a major role in the pathogenesis of elevated serum IgM in PBC.     

A novel Notch ligand, Dll4, induces T-cell leukemia/lymphoma when overexpressed in mice by retroviral-mediated gene transfer.

Notch receptors mediate cell-fate decisions through interaction with specific ligands during development. The biological role of a novel Notch ligand, Dll4, in mice was explored by reconstituting lethally irradiated mice with bone marrow (BM) cells transduced with Dll4 retroviral vector. White blood cell and lymphocyte counts in Dll4-overexpressing mice were reduced at the early stage of reconstitution but increased significantly at approximately 10 weeks after BM transplantation. BM, spleen, lymph nodes, and peripheral blood of Dll4-overexpressing mice contained predominantly CD4(+)CD8(+) T cells and virtually lacked B cells. The Dll4-overexpressing mice eventually developed a lethal phenotype that was characterized by the progression of a T-cell lymphoproliferative disease (restricted to BM and lymphoid tissues) to transplantable monoclonal T-cell leukemia/lymphoma scattered to multiple organs. Results suggest that the interaction of Dll4 with Notch1 may provide key signals for T-cell development.     

Recruitment of T cells into bone marrow of ITP patients possibly due to elevated expression of VLA-4 and CX3CR1

In idiopathic thrombocytopenic purpura (ITP), platelets are destroyed in the spleen, liver, and bone marrow (BM) by autoantibodies and cytotoxic T cells. In a DNA microarray screen of peripheral blood T cells, we found that VLA-4, CX3CR1, and CXCR4, involved in T-cell homing, had increased expression in ITP patients compared with controls. However, we only found increased protein expression of VLA-4 on T cells from peripheral blood by flow cytometry. To address a possible recruitment of T cells into the organs involved in platelet destruction, we analyzed T cells in BM. In BM, T-cell surface expression of VLA-4 and CX3CR1 was increased in ITP patients compared with controls. Furthermore, the number of CD3(+) T cells in BM, but not in blood, was increased in ITP patients compared with controls. This finding was confirmed by immunohistochemistry of BM biopsies. The number of regulatory T cells (CD4(+)/CD25(bright)) was decreased in the BM of ITP patients, whereas Fas expression was increased. In conclusion, ITP is associated with accumulation and activation of T cells in the BM. Recruitment of T cells into the target organ (eg, BM) is plausible and may be facilitated through increased VLA-4 and CX3CR1 expression. These molecules might serve as new treatment targets in ITP.     

Immunophenotypic profile of T cells in common variable immunodeficiency: is there an association with different clinical findings?

BackgroundA system based on the B-cell phenotype has recently been proposed to classify patients suffering from common variable immunodeficiency (CVID). Immunophenotypic T-cell abnormalities have also been correlated with clinical findings, although they have never been used in classification strategies.ObjectiveTo simultaneously assess T and B-cell subset abnormalities in CVID patients and their relationship with clinical findings. To identify potential immunophenotypic T-cell abnormalities that could be further evaluated in multicenter studies.Patients and MethodsPeripheral blood lymphocytes from 21 CVID patients and 21 healthy donors were stained for T and B-cell subsets, analyzed by flow cytometry, and correlated with clinical characteristics.ResultsPatients classified as MB0 (CD19/CD27+ < 11 %) showed higher percentages of CD4/CD45RA? (87 % vs 67 %, p = 0.028) and lower percentages of CD8/CD45RA+CCR7+ (10 % vs 26 %, p = 0.028) and CD4/CD25+ T-cells (36 % vs 62 %, p = 0.034) than MB2 patients. Even though our cohort was small, we observed a higher prevalence of distinct clinical complications of CVID in patients with B and T-cell abnormalities. Nonmalignant lymphoproliferative disorders and IgG hypercatabolism were more frequently observed in MB0 patients. A higher prevalence of splenomegaly was observed among CVID patients with increased levels of CD4/CD45RA?, activated CD4/CD38+DR+, CD8/DR+, and CD8/CD38+ T-cells, as well as in those with lower percentages of CD4/CD45RA+CCR7+ and CD4/CD25+ T-cells. Lymphoproliferative disorders were more prevalent among CVID patients with higher CD4/CD45RA? percentages.ConclusionThe study of T-cell subsets warrants further evaluation as a potential tool to better identify CVID patients with distinct clinical profiles.     

Severe deficiency of switched memory B cells (CD27(+)IgM(-)IgD(-)) in subgroups of patients with common variable immunodeficiency: a new approach to classify a heterogeneous disease

Hypogammaglobulinemia is the hallmark of common variable immunodeficiency (CVID) syndrome, a heterogeneous disorder predisposing patients to recurrent bacterial infections. In this study, we investigated the peripheral B-cell compartment of 30 well-characterized CVID patients in comparison to 22 healthy controls. Flow cytometric analysis of peripheral blood lymphocytes revealed a reduction of class-switched CD27(+)IgM(-)IgD(-) memory B cells below 0.4% in 77% of our patients (group I), while this B-cell subpopulation exceeded 0.5% in all healthy donors and in 23% of CVID patients (group II). These results correlate well with the capacity of peripheral blood lymphocytes to produce immunoglobulins in vitro upon stimulation with Staphylococcus aureus Cowan I (SAC) plus interleukin-2 because the production of immunoglobulin G in vitro is entirely dependent on the presence of switched memory B cells. The subdivision of group I into patients with an increased proportion of CD21(-) peripheral B cells (> 20%; group Ia) and patients with normal percentages of CD21(-) B cells (< 20%; group Ib) revealed a significant clustering of patients with splenomegaly and autoimmune cytopenias in group Ia. Based on these observations, we propose a fast and reliable new classification for CVID patients by flow cytometric quantification of class-switched memory and immature B cells in the peripheral blood of patients. Our results point toward defects at various stages of B-cell differentiation in CVID subgroups and support the value of a B-cell-oriented classification principle. A consensus on this new classification system will hopefully provide a tool for rapidly defining homogeneous subgroups of CVID for functional studies and genetic linkage analysis.     

Epstein-Barr virus persistence in the absence of conventional memory B cells: IgM+IgD+CD27+ B cells harbor the virus in X-linked lymphoproliferative disease patients

Epstein-Barr virus (EBV) persists in healthy virus carriers within the immunoglobulin (Ig)D(-)CD27(+) (class-switched) memory B-cell compartment that normally arises through antigen stimulation and germinal center transit. Patients with X-linked lymphoproliferative disease (XLP) lack such class-switched memory B cells but are highly susceptible to EBV infection, often developing fatal symptoms resembling those seen in EBV-associated hemophagocytic syndrome (EBV-AHS), a disease caused by aberrant virus entry into the NK- or T-cell system. Here we show that XLP patients who survive primary EBV exposure carry relatively high virus loads in the B-cell, but not the NK- or T-cell, compartment. Interestingly, in the absence of conventional class-switched memory B cells, the circulating EBV load was concentrated within a small population of IgM(+)IgD(+)CD27(+) (nonswitched) memory cells rather than within the numerically dominant naive (IgM(+)IgD(+)CD27(-)) or transitional (CD10(+)CD27(-)) subsets. In 2 prospectively studied patients, the circulating EBV load was stable and markers of virus polymorphism detected the same resident strain over time. These results provide the first definitive evidence that EBV can establish persistence in the B-cell system in the absence of fully functional germinal center activity and of a class-switched memory B-cell compartment.     

Idiopathic CD4+ T lymphocytopenia is associated with increases in immature/transitional B cells and serum levels of IL-7

Idiopathic CD4+ T lymphocytopenia (ICL) is a rare heterogeneous disorder defined by CD4+ T-cell counts below 300 cells/muL in the absence of human immunodeficiency virus (HIV) infection or other known immune deficiency disorders. Here, we report the expansion of immature/transitional B cells in patients with ICL, which is associated with elevated serum levels of IL-7. Both the percentage of immature/transitional B cells and levels of IL-7 were inversely correlated with levels of CD4+ T-cell counts and directly correlated to each other. Further analyses of B cells indicated that, in contrast to the activating effects of HIV disease on mature B cells, the expansion of immature/transitional B cells in patients with ICL occurred at the expense of memory B cells. These findings extend previous reports on primary immunodeficiencies as well as HIV disease by suggesting that CD4+ T-cell lymphopenia has an impact on human B-cell development either directly or indirectly via the associated elevation of IL-7 levels.     

CD4+ and CD8+ T cell receptor repertoire perturbations with normal levels of T cell receptor excision circles in HIV-infected,therapy-naive adolescents

Our objective was to determine whether treatment-naive HIV-infected adolescents manifest abnormalities in thymus function and peripheral T cell repertoire, and to assess relationships of these immunologic characteristics with each other, with plasma HIV virus load, and T cell surface markers. TCR Vbeta repertoire was determined by CDR3 length spectratyping in purified CD4(+) and CD8(+) T cells of high-risk, HIV-negative adolescents and of treatment-naive, HIV-infected adolescents. Thymus function was investigated by the simultaneous examination of T cell receptor excision circles (TRECs) in the CD4(+) and CD8(+) T cell subsets. HIV-infected adolescents exhibited significantly greater perturbations in their TCR Vbeta repertoire in comparison with HIV-negative subjects. Perturbations in the CD8(+) T cell compartment were more profound in comparison with CD4(+) T cells. The CD4(+) TCR Vbeta perturbations were negatively correlated with the total and phenotypically naive CD4(+) T cells, and with CD4(+) TRECs. CD8(+) TRECs, although not correlated with CD8(+) TCR Vbeta perturbations, showed negative correlation with memory and activated CD8(+) T cells. Interestingly, TRECs in CD4(+) and CD8(+) T cells were not significantly different between HIV-infected and uninfected adolescents. The TCR Vbeta repertoire in adolescents is profoundly perturbed even in early stages of HIV infection, when total CD4(+) cell counts in most subjects are within normal limits. The correlative analyses demonstrating negative association of CD4(+) cell TRECs with CD4(+) TCR Vbeta perturbations and of CD8(+) TRECs with CD8(+) cell activation markers provide evidence of the intense activation of the central and peripheral immune compartments in this study population.     

Low B-cell and monocyte counts on day 80 are associated with high infection rates between days 100 and 365 after allogeneic marrow transplantation

To ascertain which mononuclear cell subset deficiency plays a role in the marrow transplant recipient's susceptibility to infections, mononuclear cell subset counts were prospectively determined in 108 patients on day 80. Infections occurring between day 100 and 365 were recorded by an investigator blinded to the subset counts. In univariate analyses, the counts of the following subsets showed a significant inverse correlation with infection rates: total B cells, IgD(+) B cells, IgD(-) B cells, total CD4 T cells, CD28(+) CD4 T cells, CD28(-) CD4 T cells, CD45RA(low/-) CD4 T cells and monocytes. In multivariate analyses, the counts of the following subsets remained significantly inversely correlated with the infection rates: total B cells (P =.0004) and monocytes (P =.009). CD28(-) CD8 T-cell counts showed no correlation with infection rates. In conclusion, the susceptibility of patients to infections late posttransplant may be due in part to the slow reconstitution of B cells and monocytes.     

Lymphocytes and B-cell abnormalities in patients with common variable immunodeficiency (CVID)

Background and aimsCommon variable immunodeficiency (CVID) is a primary antibody deficiency characterised by decreased antibody production and low or normal B-cell numbers. To elucidate the clinical and immunological heterogeneity of CVID, we studied 16 patients diagnosed with CVID.MethodsWe analysed B, T and NK cell populations. We also assessed CD27 expression to define B-cell subsets and examined the expression of molecules important in B-cell proliferation and differentiation, such as the transmembrane activator and CALM interactor (TACI), inducible costimulator (ICOS), CD154 and CD40.ResultsWe observed reduced B and T-cell numbers in CVID patients; this reduction was more pronounced in adults. While one group of patients (group I) showed a significant reduction in CD27+ memory B-cells, another group (group II) of patients exhibited numbers of CD27+ memory B-cells similar to the healthy donor. The frequency of B-cells and T-cells expressing CD40 and ICOS, respectively, was significantly lower in all CVID patients compared with healthy donors. Finally, a correlation between the frequency of CD27+ memory B-cells and clinical features was observed in CVID patients.ConclusionThese results suggest that in some patients, the combined defects in both T and B-cells may account for CVID. Additionally, patients in group I exhibited an increased frequency of pneumonia and chronic diarrhoea.     

Definition and characterization of the systemic T-cell dysregulation in untreated indolent B-cell lymphoma and very early CLL

Epidemiologic data show that the immune system may control or promote the emergence and growth of neoplastic lymphomatous clones. Conversely, systemic lymphomas, especially myeloma and chronic lymphocytic leukemia (CLL), are associated with clinical immunodeficiency. This prospective controlled study demonstrates substantially reduced circulating T helper cells, predominantly naive CD4(+) cells, in patients with nonleukemic follicular lymphoma and extranodal marginal zone lymphoma, but not in monoclonal gammopathy and early CLL. These changes were correlated with a preactivated phenotype, hyperreactivity in vitro, pre-senescence, and a T helper 2 shift of peripheral T helper cells. No prominent alterations existed in the regulatory T-cell compartment. Gene expression profiling of in vitro-stimulated CD4(+) cells revealed an independent second alteration of T helper cell physiology, which was most pronounced in early CLL but also detectable in follicular lymphoma/extranodal marginal zone lymphoma. This pattern consisted of down-regulation of T-cell receptor signaling cascades and globally reduced cytokine secretion. Both types of T-cell dysfunction may contribute to significant immunodeficiency in nonleukemic indolent B-cell lymphomas as demonstrated by unresponsiveness to hepatitis B vaccination. The precise definition of systemic T-cell dysfunction serves as the basis to study its prognostic impact, its relationship to the established influence of the lymphoma microenvironment, and its therapeutic manipulation.