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Somatic hypermutation (SHM) is a pivotal process in adaptive immunity that occurs in the germinal centre and allows B cells to change their primary DNA sequence and diversify their antigen receptors. Here, we report that genome binding of Lamin B1, a component of the nuclear envelope involved in epigenetic chromatin regulation, is reduced during B-cell activation and formation of lymphoid germinal centres. Chromatin immunoprecipitation-Seq analysis showed that kappa and heavy variable immunoglobulin domains were released from the Lamin B1 suppressive environment when SHM was induced in B cells. RNA interference-mediated reduction of Lamin B1 resulted in spontaneous SHM as well as kappa-light chain aberrant surface expression. Finally, Lamin B1 expression level correlated with progression-free and overall survival in chronic lymphocytic leukaemia, and was strongly involved in the transformation of follicular lymphoma. In summary, here we report that Lamin B1 is a negative epigenetic regulator of SHM in normal B-cells and a ‘mutational gatekeeper’, suppressing the aberrant mutations that drive lymphoid malignancy.

ADMIRE was a multicenter, randomized-controlled, open, phase IIB superiority trial in previously untreated chronic lymphocytic leukemia. Conventional front-line therapy in fit patients is fludarabine, cyclophosphamide and rituximab (FCR). Initial evidence from non-randomized phase II trials suggested that the addition of mitoxantrone to FCR (FCM-R) improved remission rates. Two hundred and fifteen patients were recruited to assess the primary end point of complete remission (CR) rates according to International Workshop on Chronic Lymphocytic Leukemia criteria. Secondary end points were progression-free survival (PFS), overall survival (OS), overall response rate, minimal residual disease (MRD) negativity and safety. At final analysis, CR rates were 69.8 FCR vs 69.3% FCM-R (adjusted odds ratio (OR): 0.97; 95% confidence interval (CI): (0.53–1.79), P=0.932). MRD-negativity rates were 59.3 FCR vs 50.5% FCM-R (adjusted OR: 0.70; 95% CI: (0.39–1.26), P=0.231). During treatment, 60.0% (n=129) of participants received granulocyte colony-stimulating factor as secondary prophylaxis for neutropenia, a lower proportion on FCR compared with FCM-R (56.1 vs 63.9%). The toxicity of both regimens was acceptable. There are no significant differences between the treatment groups for PFS and OS. The trial demonstrated that the addition of mitoxantrone to FCR did not increase the depth of response. Oral FCR was well tolerated and resulted in impressive responses in terms of CR rates and MRD negativity compared with historical series with intravenous chemotherapy.

Relapse remains the most common cause of treatment failure in patients receiving autologous stem cell transplantation (ASCT) for follicular lymphoma (FL). The aim of this study was to evaluate the effect of adding radioimmunotherapy or rituximab (R) to BEAM (carmustine, etoposide, ara-c, melphalan) high-dose therapy for ASCT in patients with relapsed FL. Using the European Society for Blood and Marrow Transplantation registry, we conducted a cohort comparison of BEAM (n=1973), Zevalin-BEAM (Z-BEAM) (n=207) and R-BEAM (n=179) and also a matched-cohort analysis of BEAM vs Z-BEAM including 282 and 154 patients, respectively. BEAM, Z-BEAM and R-BEAM groups were well balanced for age, time from diagnosis to ASCT and disease status at ASCT. The cumulative incidences of relapse (IR) at 2 years were 34, 34 and 32% for Z-BEAM, R-BEAM and BEAM, respectively. By multivariate analysis, there were no significant differences with Z-BEAM or R-BEAM compared with BEAM for IR, non-relapse mortality, event-free survival or overall survival. With the caveat that the limitations of registry analyses have to be taken into account, this study does not support adding radioimmunotherapy or R to BEAM in ASCT for relapsed FL. However, we cannot rule out the existence a particular subset of patients who could benefit from Z-BEAM conditioning that cannot be identified in our series, and this should be tested in a randomized trial.

Compounds targeting phosphatidylinositol-3-kinase/mammalian target of rapamycin (PI3K/mTOR) signaling are being investigated in multiple clinical settings, but drug resistance may reduce their benefit. Compound rechallenge after drug holidays can overcome such resistance, yet little is known about the impact of drug holidays on cell biochemistry. We found that PI3K inhibitor (PI3Ki)-resistant cells cultured in the absence of PI3Ki developed a proliferative defect, increased oxygen consumption and accumulated reactive oxygen species (ROS), leading to lactate production through hypoxia-inducible factor-1α. This metabolic imbalance was reversed by mammalian target of rapamycin complex 1 (mTORC1) inhibitors. Interestingly, neither AKT nor c-MYC was involved in mediating the metabolic phenotype, despite the latter contributing to resistant cells’ proliferation. These data suggest that an AKT-independent PI3K/mTORC1 axis operates in these cells. The excessive ROS hampered cell division, and the metabolic phenotype made resistant cells more sensitive to hydrogen peroxide and nutrient starvation. Thus, the proliferative defect of PI3Ki-resistant cells during drug holidays is caused by defective metabolic adaptation to chronic PI3K/mTOR pathway inhibition. This metabolic imbalance may open the therapeutic window for challenge with metabolic drugs during drug holidays.

Chronic lymphocytic leukemia (CLL) is characterized by apoptosis resistance and a dysfunctional immune system. Previous reports suggested a potential role of myeloid cells in mediating these defects. However, the composition and function of CLL-associated myeloid cells have not been thoroughly investigated in vivo. Using the Eµ-TCL1 mouse model, we observed severe skewing of myeloid cell populations with CLL development. Monocytes and M2-like macrophages infiltrated the peritoneal cavity of leukemic mice. Monocytes also accumulated in the spleen in a CCR2-dependent manner, and were severely skewed toward Ly6Clowpatrolling or nonclassical phenotype. In addition, the percentage of MHC-IIhidendritic cells and macrophages significantly dropped in the spleen. Gene expression profiling of CLL-associated monocytes revealed aberrantly high PD-L1 expression and secretion of multiple inflammatory and immunosuppressive cytokines like interleukin-10, tumor necrosis factor-a and CXCL9. In vivomyeloid cell depletion using liposomal Clodronate resulted in a significant control of CLL development accompanied by a pronounced repair of innate immune cell phenotypes and a partial resolution of systemic inflammation. In addition, CLL-associated skewing of T cells toward antigen-experienced phenotypes was repaired. The presented data suggest that targeting nonmalignant myeloid cells might serve as a novel immunotherapeutical strategy for CLL.

Limited therapeutic options are available after relapse of acute leukaemia following first reduced intensity conditioning haematopoietic stem cell transplantation (RIC1). A retrospective study on European Society for Blood and Marrow Transplantation (EBMT) registry data was performed on 234 adult patients with acute leukaemia who received a second RIC transplantation (RIC2) from 2000 to 2012 as a salvage treatment for relapse following RIC1. At the time of RIC2, 167 patients (71.4%) had relapsed or refractory disease, 49 (20.9%) were in second CR and 18 (7.7%) in third or higher CR. With a median follow-up of 21 (1.5–79) months after RIC2, 51 patients are still alive. At 2 years, the cumulative incidence of non-relapse mortality (NRM), relapse incidence (RI), leukaemia-free survival (LFS) and overall survival (OS) were 22.4% (95% confidence interval (CI): 17–28.4), 63.9% (56.7–70.1), 14.6% (8.8–18.5) and 20.5% (14.9–26.1), respectively. In patients with acute myelogenous, biphenotypic and undifferentiated leukaemia (representing 89.8% of all patients), duration of remission following RIC1 >225 days, presence of CR at RIC2, patient’s Karnofsky performance status >80 at RIC2 and non-myeloablative conditioning were found to be the strongest predictors of patients’ favourable outcome.

Abstract: Detection of clonal tumor cells in leukemias and lymphomas by PCR in minimal residual disease (MRD) has been shown to be a valuable parameter for identifying patients who may require further treatment. Here we introduce the studies underway in our own and other institutions addressing the value of PCR technology in detecting residual CLL cells either in the autologous stem cell product or after induction of MRD in patients after autologous or allogeneic stem cell transplant. The PCR technology used for these questions and the results are discussed.

Signaling via the T-cell receptor complex is necessary but not sufficient to induce antigen-specific T lymphocytes to expand clonally. To proliferate, T cells must receive one or more costimulatory signals provided by antigen presenting cells (APCs). One such critical costimulatory signal is delivered by the CD28/CTLA-4 counterreceptor, B7, expressed on APCs. B7 costimulation induces CD28 signaling, resulting in interleukin 2 (IL-2) secretion, and T-cell proliferation. Conversely, T-cell receptor signaling in the absence of B7 costimulation results in induction of antigen-specific tolerance. Here, we show that activated human B lymphocytes express two additional CTLA-4 counterreceptors also capable of providing T-cell costimulation. At 24 hr postactivation, B cells express a CTLA-4 counterreceptor not recognized by anti-B7 or -BB-1 monoclonal antibodies (mAbs), which induces detectable IL-2 secretion and T-cell proliferation. At 48 and 72 hr postactivation, B cells express both B7 and a third CTLA-4 counterreceptor identified by the anti-BB-1 mAb. BB-1 appears to be a molecule distinct from B7 by its expression on B7- cells and its capacity to induce T cells to proliferate without significant accumulation of IL-2. As observed for B7, costimulatory signals mediated by these alternative CTLA-4/CD28 counterreceptors are likely to be essential for generation of an immune response and their absence may result in antigen-specific tolerance. We propose the following terminology for these CTLA-4 counterreceptors: (i) B7, B7-1; (ii) early CTLA-4 binding counterreceptor, B7-2; and (iii) BB-1, B7-3.

Induction and maintenance of a state of T cell unresponsiveness to specific alloantigen would have significant implications for human organ transplantation. Using human histocompatibility leukocyte antigen DR7-specific helper T cell clones, we demonstrate that blockade of the B7 family of costimulatory molecules is sufficient to induce alloantigen-specific T cell clonal anergy. Anergized cells do not respond to alloantigen and a variety of costimulatory molecules, including B7-1, B7-2, intercellular adhesion molecule-1 (ICAM-1), and lymphocyte function-associated molecule (LFA)-3. However, after culture in exogenous interleukin (IL)-2 for at least 7 d, anergized cells can respond to alloantigen in the presence of LFA-3. LFA-3 costimulation subsequently restores responsiveness to alloantigen in the presence of previously insufficient costimulatory signals. Expression of CD2R epitope is downregulated on anergic cells and is restored after 7 d of IL-2 culture. The loss of the CD2R is temporally associated with the inability of anergized cells to respond to LFA-3. These results suggest that in addition to blockade of B7 family members, inhibition of CD2 and, potentially, other costimulatory pathways that might reverse anergy will be necessary to maintain prolonged alloantigen-specific tolerance.

High dose therapy with the resulting myeloablation rescued by infusion of autologous bone marrow (ABMT) has become a major treatment option for an increasing number of patients with hematologic and solid tumors. ABMT has several potential advantages over allogeneic transplantation. However, the major obstacle to the use of ABMT is that the infusion of occult tumor cells harbored within the harvested marrow would result in more rapid relapse of disease. To minimize the effects of the infusion of significant numbers of malignant cells, marrow for ABMT is obtained when the patient is either in complete remission or when there is no histologic evidence of bone marrow infiltration of disease. There is increasing evidence that minimal numbers of malignant cells can be detected within the remission marrow of these patients, particularly when assessed by sensitive clonogenic assays or polymerase chain reaction amplification. A variety of methods, pharmacologic and immunologic have been developed to ““purge”” malignant cells from the marrow. The aim of purging is to eliminate any contaminating malignant cells and leave intact the hematopoietic stem cells that are necessary for engraftment. Although the rationale for removing any contaminating cells from the autologous marrow appears compelling, the issue of purging remains highly controversial. Intense argument persists as to whether attempts to remove residual tumor cells from the harvested bone marrow have contributed to improving disease-free survival in these patients. To date there have been no clinical trials testing the efficacy of purging by comparison of infusion of purged versus unpurged autologous bone marrow. This is due primarily to the large number of patients that would be required for such studies. In addition, the finding that the majority of patients who relapse after autologous BMT do so at sites of prior disease has led to the widespread view that purging of autologous marrow could contribute little to subsequent outcome after autologous BMT. The pre-clinical and clinical experience of in vitro marrow purging will be reviewed.

The outcome of conventional therapy in adult acute lymphoblastic leukaemia (ALL) is disappointing. Allogeneic bone-marrow transplantation may give improved results but has only limited applicability because of the lack of a suitable donor in most patients. We have therefore investigated intensive chemotherapy and chemo/radiotherapy protocols with autologous bone-marrow rescue. 31 patients have been treated, 14 beyond first remission and 17 in first remission. The result of this therapy in both groups is poor with only 2 longterm survivors in each group. There is no reason to believe from this study that ablative therapy with autologous bone-marrow rescue will yield superior results to conventional therapy in adult ALL but further experience with TBI containing regimes is required.

In the tumor-bearing host, T cells invariably fail to induce a clinically significant antitumor immune response. Although model systems support the existence of tumor peptide antigens, the molecular interactions critical for antigen presentation by the tumor cell remain unresolved. Here, we demonstrate that human follicular lymphoma cells are highly inefficient at presenting alloantigen despite their strong expression of major histocompatibility complex and low-to-intermediate expression of some adhesion and B7 costimulatory molecules. Activation of follicular lymphoma cells via CD40 induces or up-regulates both adhesion and B7 costimulatory molecules essential to repair this defect. More importantly, once primed, alloreactive T cells efficiently recognize unstimulated follicular lymphoma cells. Thus, correction of defective tumor immunity requires not only expression of major histocompatibility complex but also sufficient expression of multiple adhesion and costimulatory molecules.

Occupancy of the T-cell receptor complex does not appear to be a sufficient stimulus to induce a T-cell-mediated immune response. Increasing evidence suggests that cognate cell-cell interaction between an activated T cell and an antigen-presenting cell may provide such a stimulus. A candidate T-cell surface molecule for this costimulatory signal is the T-cell-restricted CD28 antigen. Following crosslinking with anti-CD28 mAb, suboptimally stimulated CD28+ T cells show increased proliferation and markedly increased secretion of a subset of lymphokines. Recently, the B-cell surface activation antigen B7 was shown to be a natural ligand for the CD28 molecule, and both B7 and CD28 are members of the immunoglobulin superfamily. Here we report that B7-transfected CHO cells can induce suboptimally activated CD28+ T cells to proliferate and secrete high levels of interleukin 2. The response is identical whether T cells are submitogenically stimulated with either phorbol myristate acetate or anti-CD3 to activate the T cells. This response is specific and can be totally abrogated with anti-B7 monoclonal antibody. As has previously been observed for anti-CD28 monoclonal antibody, B7 ligation induced secretion of interleukin 2 but not interleukin 4. We have previously demonstrated that B7 expression is restricted to activated B lymphocytes and interferon gamma-activated monocytes. Since these two cellular populations are involved in antigen presentation as well as cognate interaction with T lymphocytes, B7 is likely to represent a central constimulatory signal that is capable of amplifying an immune response.

The regulation of T cell-mediated immune responses requires a balance between amplification and generation of effector function and subsequent selective termination by clonal deletion. Although apoptosis of previously activated T cells can be induced by signaling of the tumor necrosis factor receptor family, these molecules do not appear to regulate T-cell clonal deletion in an antigen-specific fashion. We demonstrate that cross-linking of the inducible T-cell surface molecule CTLA4 can mediate apoptosis of previously activated human T lymphocytes. This function appears to be antigen-restricted, since a concomitant signal T-cell receptor signal is required. Regulation of this pathway may provide a novel therapeutic strategy to delete antigen-specific activated T cells.

The B7-1 molecule, expressed on antigen presenting cells (APC), provides a crucial costimulatory signal for T cell activation. Recent studies demonstrate the existence of alternative, non-B7-1 CTLA4 counter-receptors in mice and humans. Here, we describe the molecular cloning and demonstrate costimulatory function of the murine B7-2 (mB7-2) gene. Murine B7-2 cDNA encodes a member of the Ig supergene family that binds CTLA4-Ig and stains with the GL1 but not anti-mB7-1 mAb. Murine B7-2 costimulates the proliferation and interleukin 2 production of CD4+ T cells and this costimulation can be inhibited by either CTLA4-Ig or GL1 mAb. Identification of the B7-2 molecule will permit further manipulation of the B7:CD28/CTLA4 costimulatory pathway which has been shown to be involved in the prevention of tolerance, induction of tumor immunity, and most recently, in the pathogenesis of autoimmunity.

The maximal T-cell response to its antigen requires presentation of the antigen by a major histocompatibility complex class II molecule as well as the delivery of one or more costimulatory signals provided by the antigen-presenting cell (APC). Although a number of candidate molecules have been identified that are capable of delivering a costimulatory signal, increasing evidence suggests that one such critical pathway involves the interaction of the T-cell surface antigen CD28 with its ligand B7, expressed on APCs. In view of the number of potential costimulatory molecules that might be expressed on the cell surface of APCs, artificial APCs were constructed by stable transfection of NIH 3T3 cells with HLA-DR7, B7, or both. Here, we show that in a human antigen-specific model system, when tetanus toxoid peptide antigen is presented by cells cotransfected with HLA-DR7 and B7, optimal T-cell proliferation and interleukin 2 production result. In contrast, antigen presentation, in the absence of B7 costimulation, results in T-cell clonal anergy. These results demonstrate that it is possible to induce antigen-specific clonal tolerance in human T cells that have been previously sensitized to antigen. The artificial antigen-presenting system provides a useful model for the investigation of the biochemical events involved in the generation of tolerance and for the study of signals necessary to overcome tolerance.

Presentation of antigen by the major histocompatibility complex to T lymphocytes without the requisite costimulatory signals does not induce an immune response but rather results in a state of antigen-specific unresponsiveness, termed anergy. To determine which costimulatory signals are critical for the T cell commitment to activation or anergy, we developed an in vitro model system that isolated the contributions of alloantigen and each candidate costimulatory molecule. Here, we show that transfectants expressing HLA-DR7 and either B7 or intercellular adhesion molecule 1 (ICAM-1) deliver independent costimulatory signals resulting in alloantigen-induced proliferation of CD4-positive T lymphocytes. Although equivalent in their ability to costimulate maximal proliferation of alloreactive T cells, B7 but not ICAM-1 induced detectable interleukin 2 secretion and prevented the induction of alloantigen-specific anergy. These results are consistent with the hypothesis that blockade of the ICAM-1:lymphocyte function-associated 1 pathway results in immunosuppression, whereas blockade of the B7:CD28/CTLA4 pathway results in alloantigen-specific anergy. This approach, using this model system, should facilitate the identification of critical costimulatory pathways which must be inhibited in order to induce alloantigen-specific tolerance before human organ transplantation.

When stimulated through their antigen receptor, without costimulation, T cells enter a state of antigen-specific unresponsiveness, termed anergy. B7-mediated costimulation, signaling via CD28, is sufficient to prevent the induction of anergy. Here we show that ligation of T cell receptor (TCR) by alloantigen alone, which results in anergy, activates tyrosine phosphorylation of TCR zeta and its association with fyn. In contrast, TCR ligation in the presence of B7 costimulation, which results in productive immunity, activates tyrosine phosphorylation of TCR zeta and CD3 chains, which associate with activated lck and zeta-associated protein (ZAP) 70. Under these conditions, CD28 associates with activated lck and TCR zeta. These data suggest that the induction of anergy is an active signaling process characterized by the association of TCR zeta and fyn. In addition, CD28-mediated costimulation may prevent the induction of anergy by facilitating the effective association of TCR zeta and CD3 epsilon with the critical protein tyrosine kinase lck, and the subsequent recruitment of ZAP-70. Strategies to inhibit or activate TCR-associated, specific protein tyrosine kinase-mediated pathways may provide a basis for drug development with potential applications in the fields of transplantation, autoimmunity, and tumor immunity.

Following occupancy of the T cell receptor by antigen, T cell proliferation and lymphokine production are determined by a second costimulatory signal delivered by a ligand expressed on antigen presenting cells. The human B cell activation antigen B7, which is expressed on antigen presenting cells including activated B cells and gamma interferon treated monocytes, has been shown to deliver such a costimulatory signal upon attachment to its ligand on T cells, CD28. We have cloned and sequenced the murine homologue of the human B7 gene. The predicted murine protein has 44% amino acid identity with human B7. The greatest similarity is in the Ig-V and Ig-C like domains. Murine B7 mRNA was detected in murine hematopoietic cells of B cell but not T cell origin. Cells transfected with murine B7 provided a costimulatory signal to human CD28+ T lymphocytes. These results demonstrate the costimulatory activity of murine B7 and provide evidence that the ligand attachment site is conserved between the two species.

We report here a patient with acute mycloid leukaemia who relapsed 20 months after undergoing a double autograft procedure in first remission. He was reinduced and subsequently underwent a third autologous bone marrow transplantation in second remission using bone marrow harvested in second remission and a Busulphan and Cyclophosphamide conditioning regimen. Although the engraftment was very slow, he has remained in second remission for 34+ months. This case demonstrates that durable disease-free survival can be attained by a second preparative therapy, even in second remission, for patients relapsed after autologous bone marrow transplantation.

Multiresistant Pseudomonas infection remains a problem in hospital practice. We report a case of pseudomonas peritonitis successfully treated by a combination of azlocillin and amikacin. This is the first case report of the intraperitoneal use of azlocillin.

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