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There is increasing appreciation of the important role of B cells in many autoimmune diseases and consequently, increasing interest in treating these disorders through B cell-depletion therapy with rituximab, an anti-CD20 monoclonal antibody. Yet, precisely how this and related drugs exert their therapeutic effects remains controversial. In particular, it is unclear how, in a number of contexts, rituximab can greatly reduce the titer of serum autoantibodies without substantially altering the overall antibody titer. We have studied the action of this drug in the K/BxN mouse model of inflammatory arthritis after first crossing in a human CD20 transgene. Rituximab treatment of these mice led to a decrease in the titer of serum antibodies targeting glucose-6-phosphate isomerase, the relevant autoantigen, but not in the total antibody titer. Glucose-6-phosphate isomerase-specific plasma cells did not reside primarily in the bone marrow as expected but rather in the spleen and lymph nodes, where they had short lives, expressed CD20, and were rapidly depleted by rituximab. These data support a model whereby autoreactive plasma cells (at least certain specificities thereof) are intrinsically different from protective antimicrobial plasma cells in their differentiation, migration, and survival properties. Rituximab targets the former and spares the latter.

Background: The MS4A gene family in humans includes CD20 (MS4A1), FcRbeta (MS4A2), Htm4 (MS4A3), and at least 13 other syntenic genes encoding membrane proteins, most having characteristic tetraspanning topology. Expression of MS4A genes is variable in tissues throughout the body; however, several are limited to cells in the hematopoietic system where they have known roles in immune cell functions. Genes in the small TMEM176 group share significant sequence similarity with MS4A genes and there is evidence of immune function of at least one of the encoded proteins. In this study, we examined the evolutionary history of the MS4A/TMEM176 families as well as tissue expression of the phylogenetically earliest members, in order to investigate their possible origins in immune cells., Principal Findings: Orthologs of human MS4A genes were found only in mammals; however, MS4A gene homologs were found in most jawed vertebrates. TMEM176 genes were found only in mammals and bony fish. Several unusual MS4A genes having 2 or more tandem MS4A sequences were identified in the chicken (Gallus gallus) and early mammals (opossum, Monodelphis domestica and platypus, Ornithorhyncus anatinus). A large number of highly conserved MS4A and TMEM176 genes was found in zebrafish (Danio rerio). The most primitive organism identified to have MS4A genes was spiny dogfish (Squalus acanthus). Tissue expression of MS4A genes in S. acanthias and D. rerio showed no evidence of expression restricted to the hematopoietic system., Conclusions/significance: Our findings suggest that MS4A genes first appeared in cartilaginous fish with expression outside of the immune system, and have since diversified in many species into their modern forms with expression and function in both immune and nonimmune cells.

The prognostic significance of CD20 expression in B-cell precursor acute lymphoblastic leukemia (BCP-ALL) has been mostly studied in children and yielded conflicting results. In 143 adults with Philadelphia chromosome-negative BCP-ALL treated in the multicentric GRAALL 2003 trial, CD20 positivity over 20% was observed in 32% of patients. While not influencing complete remission achievement, CD20 expression was associated with a higher cumulative incidence of relapse (CIR) at 42 months (P=0.04), independently of the ALL high-risk subset (P=0.025). Notably, the negative impact of CD20 expression on CIR was only observed in patients with a white blood cell count (WBC) over 30x10(9)/L (P=0.006), while not in those with a lower WBC. In the former subgroup, this impact translated into lower event-free survival (15% vs. 59% at 42 months, P=0.003). CD20 expression thus appears to be associated with a worse outcome, which reinforces the interest of evaluating rituximab combined to chemotherapy in CD20-positive adult BCP-ALL. ClinicalTrials.gov ID, NCT00222027.

A pilot study previously demonstrated that thrice-weekly, fractionated-dose intravenous rituximab (RTX) limits CD20 loss from chronic lymphocytic leukemia (CLL) B cells, thereby enhancing immunotherapeutic targeting. Here, we investigated the feasibility of giving 20 mg rituximab subcutaneously thrice weekly for up to 12 weeks in 4 previously treated CLL patients. Subcutaneous rituximab was well-tolerated with minimal injection site reactions; a variable degree of efficacy was observed, likely influenced by the size of the patients' B cell/CD20 burden. Subcutaneous RTX largely preserved CD20 expression on leukemic cells but the most effective therapeutic dosing regimen needs to be established (ClinicalTrials.gov Identifier: NCT00366418).

Antibodies to CD20 have confirmed the hypothesis that monoclonal reagents can be given in vivo to alleviate human diseases. The targeting of CD20 on normal, malignant and auto-immune B-lymphocytes by rituximab has demonstrated substantial benefits for patients with a variety of B-cell lymphomas, as well as some with autoimmune disorders. There has been a notable increase in the survival rates from B-cell lymphoma in the decade since anti-CD20 therapy was introduced.

CD20 was the first B cell differentiation antigen identified, and CD20-specific mAbs are commonly used for the treatment of B cell malignancies and autoantibody-mediated autoimmune diseases. Despite this the role of CD20 in human B cell physiology has remained elusive. We describe here a juvenile patient with CD20 deficiency due to a homozygous mutation in a splice junction of the CD20 gene (also known as MS4A1) that results in "cryptic" splicing and nonfunctional mRNA species. Analysis of this patient has led us to conclude that CD20 has a central role in the generation of T cell-independent (TI) antibody responses. Key evidence to support this conclusion was provided by the observation that although antigen-independent B cells developed normally in the absence of CD20 expression, antibody formation, particularly after vaccination with TI antigens, was strongly impaired in the patient. Consistent with this, TI antipolysaccharide B cell responses were severely impeded in CD20-deficient mice. Our study therefore identifies what we believe to be a novel type of humoral immunodeficiency caused by CD20 deficiency and characterized by normal development of antigen-independent B cells, along with a reduced capacity to mount proper antibody responses.

Background/aims: Protocol biopsies offer new possibilities to predict kidney allograft outcome. The aim of this study was to find clinical, laboratory, morphological and molecular predictors of short-term renal graft survival., Methods: Three-month protocol kidney graft biopsy was carried out on 257 patients. The real-time RT-PCR was used to identify intragraft mRNA expression of several cytokines and chemokines and predictive statistics was performed to find markers connected with the risk of premature graft failure., Results: Compared to patients with normal morphology at 3 months, patients with subclinical rejection including borderline changes had experienced more frequent (p < 0.001) acute rejections before 3-month biopsy, serum creatinine >or=170 micromol/l (p < 0.01), and higher intrarenal expression of RANTES, IP-10 (p < 0.001), C3, CD3, IgJ (p < 0.01) and CD20 (p < 0.05). There was a significant correlation between subclinical rejection and the occurrence of late acute rejection and graft failure at the first year after transplantation. Moreover, higher RANTES and IP-10 expressions in subclinical rejection predicted graft loss at one year after transplantation in the univariate analysis., Conclusions: Patients with subclinical rejection including borderline changes in 3-month biopsy and particularly those with higher intrarenal expression of RANTES and IP-10 mRNA were found to be at risk for premature kidney graft loss., (Copyright 2010 S. Karger AG, Basel.)

Earlier detection of antibody-mediated rejection of kidney allografts may improve graft outcomes. Profiling of gene expression holds promise for the diagnosis and prognosis of antibody-mediated rejection. Here, we identified 730 patients who received kidney transplants during 2002-2005, including 21 patients (2.9%) who experienced early acute antibody-mediated rejection. We also identified a matched group of 43 patients with early acute T cell-mediated rejection to serve as controls. Compared with patients with T cell-mediated rejection, those with antibody-mediated rejection had significantly higher intrarenal mRNA expression of the cytoprotective heme oxygenase-1 but had lower expression of the regulatory T cell marker forkhead box P3 (FoxP3), the B cell marker CD20, and the chemokine regulated upon activation, normal T cell expressed and secreted (RANTES). T cell infiltration was similar in both groups of patients. Compared with grafts that had a favorable course, those that failed as a result of antibody-mediated rejection had expression profiles suggesting a lack of regulation (less FoxP3, TGF-beta1, RANTES, and CD20). Grafts that failed as a result of T cell-mediated rejection only revealed lower expression of CD20 mRNA. In summary, these data suggest that severe antibody-mediated rejection and T cell-mediated rejection result in graft loss by distinct mechanisms. Molecular phenotypes of early acute rejection might help to identify grafts with poor prognosis, allowing earlier application of additional therapies.

We have established a model of leukemia immunotherapy using T cells expressing chimeric T-cell receptors (cTCRs) targeting the CD20 molecule expressed on normal and neoplastic B cells. After transfer into human CD20 (hCD20) transgenic mice, cTCR(+) T cells showed antigen-specific delayed egress from the lungs, concomitant with T-cell deletion. Few cTCR(+) T cells reached the bone marrow (BM) in hCD20 transgenic mice, precluding effectiveness against leukemia. Anti-hCD20 antibody-mediated B-cell depletion before adoptive T-cell therapy permitted egress of mouse CD20-specific cTCR(+) T cells from the lungs, enhanced T-cell survival, and promoted cTCR(+) T cell-dependent elimination of established mouse CD20(+) leukemia. Furthermore, CD20-specific cTCR(+) T cells eliminated residual B cells refractory to depletion with monoclonal antibodies. These findings suggest that combination of antibody therapy that depletes antigen-expressing normal tissues with adoptive T-cell immunotherapy enhances the ability of cTCR(+) T cells to survive and control tumors.

Although rituximab is a critical monoclonal antibody therapy for CD20-positive B-cell lymphomas, rituximab resistance showing a CD20-negative phenotypic change has been a considerable clinical problem. Here we demonstrate that CD20 mRNA and protein expression is repressed by recruitment of a histone deacetylase protein complex to the MS4A1 (CD20) gene promoter in CD20-negative transformed cells after treatment with rituximab. CD20 mRNA and protein expression were stimulated by decitabine (5-Aza-dC) in CD20-negative transformed cells, and was enhanced by trichostation A (TSA). Immunoblotting indicated that DNMT1 expression was first downregulated 1 day after treatment with 5-Aza-dC, but IRF4 and Pu.1, the transcriptional regulators of MS4A1, were still expressed with or without 5-Aza-dC. Interestingly, CpG methylation of the MS4A1 promoter was not observed in CD20-negative transformed cells without 5-Aza-dC. A chromatin immunoprecipitation (ChIP) assay indicated that the Sin3A-HDAC1 co-repressor complex was recruited to the promoter and dissociated from the promoter with 5-Aza-dC and TSA, resulting in histone acetylation. Under these conditions, IRF4 and Pu.1 were continually recruited to the promoter with or without 5-Aza-dC and TSA. These results suggest that recruitment of the Sin3A-HDAC1 complex is related to downregulation of CD20 expression in CD20-negative B-cells after treatment with rituximab.

Lipopolysaccharide (LPS), CpG-containing phosphothioate oligonucleotides (CpG) and various cytokines impact chronic lymphocytic leukemia (CLL) B cells. For example, they influence cell cycle entry, expression of co-receptors, and CD20. Rituximab (RTX), for which CD20 molecule is the target, proved to be less efficient in CLL than in lymphoma. This is accounted for by a lower CD20 level in the former than in the latter B lymphocytes. CD20 transcription is mediated by four transcription factors, of which only purine-rich box-1 (PU.1) is reduced in CLL. We thus examined the effects of LPS, CpG, tumor necrosis factor-alpha, interferon-alpha, interleukin (IL)-3, IL-4, IL-21, granulocyte macrophage-colony stimulating factor (CSF), and granulocyte-CSF on the transcription of PU.1, and the subsequent expression of CD20. It appeared that CpG was unique in that it raised the membrane expression of CD20 on malignant B cells, owing to a PU.1 independent increase in its gene transcription. Moreover, RTX-induced complement-mediated lysis was also ameliorated. Thus, CpG accelerates the transcription of CD20 independently of PU.1, and thereby improves the efficacy of RTX in CLL.

Immunophenotypic classification of acute lymphoblastic leukemia (ALL) has well-recognized prognostic implications. The significance of CD20 expression has been evaluated in childhood precursor B-lineage ALL with conflicting results. We retrospectively analyzed the influence of CD20 expression on outcome in 253 adults with de novo precursor B-lineage ALL treated with either conventional (VAD/CVAD) or intensive (hyper-CVAD) frontline chemotherapy regimens in the pre-rituximab era. Overall, CD20 positivity of at least 20% was associated with lower 3-year rates of complete remission duration (CRD; 20% vs 55%, P < .001) and overall survival (OS; 27% vs 40%, p = .03). In the CD20 negative subset, the 3-year rates for CRD (58% vs 42%, p = .04) and OS (60% vs 28%, P < .001) were superior for hyper-CVAD compared with VAD/CVAD; rates were particularly favorable for the CD20 negative younger age group (68% and 85%, respectively). In contrast, 3-year CRD and OS rates were uniformly poor for the CD20-positive group regardless of therapy (27% or less). Multivariate analysis for event-free survival identified older age, leukocyte count higher than 30 x 10(9)/L, presence of Philadelphia chromosome, high systemic risk classification, and CD20 positivity as independent predictors of worse outcome. In conclusion, CD20 expression in de novo adult precursor B-lineage ALL appears to be associated with a poor prognosis. Incorporation of monoclonal antibodies directed against CD20 into frontline chemotherapy regimens warrants investigation.

Background: Rituximab, a chimeric antibody targeted to the human CD20 molecule, is a major component of the R-CHOP protocol used to treat patients with diffuse large B cell lymphoma (DLBCL). It is also a very expensive drug. Though the response rate of R-CHOP is higher than that of CHOP alone, patients may still experience a poor response. One possible mechanism that may mediate the poor response to R-CHOP is poor binding of the drug to the CD20 molecule, perhaps due to mutations or polymorphisms of the CD20 gene that affect its structure. To test this hypothesis and perhaps define a new predictor of R-CHOP response, our pilot study evaluated the CD20 gene sequence from patients exhibiting a poor outcome to R-CHOP., Methods: Eleven patients with DLBCL who exhibited a recurrence of their disease and/or died within 24 months of R-CHOP treatment were categorized as showing poor progression-free survival (poor outcomes). Paraffin-embedded tissue specimens from the original tumors were evaluated for mutations or polymorphisms of the CD20 gene. Furthermore, sections from these patients and as well as from matched control patients who were categorized as good outcome patients (no progression of their disease within 36 months) were stained with anti-CD20 antibody and compared for CD20 protein expression., Results: DNA sequence analyses revealed that no mutations were observed in DNA from the coding region of the CD20 gene in any of the initial tumors from 11 patients who showed poor outcomes with R-CHOP therapy. One case showed a synonymous single nucleotide polymorphism in exon 2 (C216T; rs2070770; Ile>>Ile). No significant differences in CD20 expression was observed between good and poor outcome patients with R-CHOP., Conclusions: Our results do not support association of CD20 mutations in specimens of the initial tumor or structural polymorphisms of the CD20 gene with patients who exhibited poor outcomes to R-CHOP.

Although rituximab is a key molecular targeting drug for CD20-positive B-cell lymphomas, resistance to rituximab has recently been recognized as a considerable problem. Here, we report that a CD20-negative phenotypic change after chemotherapies with rituximab occurs in a certain number of CD20-positive B-cell lymphoma patients. For 5 years, 124 patients with B-cell malignancies were treated with rituximab-containing chemotherapies in Nagoya University Hospital. Relapse or progression was confirmed in 36 patients (29.0%), and a rebiopsy was performed in 19 patients. Of those 19, 5 (26.3%; diffuse large B-cell lymphoma [DLBCL], 3 cases; DLBCL transformed from follicular lymphoma, 2 cases) indicated CD20 protein-negative transformation. Despite salvage chemotherapies without rituximab, all 5 patients died within 1 year of the CD20-negative transformation. Quantitative reverse-transcription-polymerase chain reaction (RT-PCR) showed that CD20 mRNA expression was significantly lower in CD20-negative cells than in CD20-positive cells obtained from the same patient. Interestingly, when CD20-negative cells were treated with 5-aza-2'-deoxycytidine in vitro, the expression of CD20 mRNA was stimulated within 3 days, resulting in the restoration of both cell surface expression of the CD20 protein and rituximab sensitivity. These findings suggest that some epigenetic mechanisms may be partly related to the down-regulation of CD20 expression after rituximab treatment.

Purpose: Rituximab is commonly incorporated into CD20-positive B-cell lymphoma therapy to improve response and prognosis. With increasing use, resistance to rituximab is a continuing concern, but CD20 mutation as a cause of resistance has not previously been reported., Experimental Design: Freshly collected lymphoma cells from 50 patients with previously untreated or relapsed/resistant non-Hodgkin's B-cell lymphomas (diffuse large B cell, n = 22; follicular, n = 7; mucosa associated lymphoid tissue, n = 16; chronic lymphocytic leukemia, n = 2; small lymphocytic lymphoma, n = 1; lymphoplasmacytic, n = 1; mantle cell lymphoma, n = 1) were assessed for CD20 expression by flow cytometry, and CD20 gene sequencing was done on extracted DNA., Results: CD20 mutations were found in 11 (22.0%) of 50 patients and could be grouped as C-terminal deletion (8.0%), early termination (10.0%), and extracellular domain (2.0%) or transmembrane domain (2.0%) mutations. The mean fluorescence intensity of CD20 on fresh lymphoma cells was significantly lower for the C-terminal deletion mutation [3.26; 95% confidence interval (95% CI), 0.09-6.89] compared with wild type (30.8; 95% CI, 22.4-39.2; P < 0.05). In contrast, early termination mutations did not show significant differences in CD20 expression compared with wild type (19.5; 95% CI, 10.7-28.4; P > 0.05)., Conclusions: It is possible that C-terminal deletion mutations of CD20 may be related to relapse/resistance after rituximab therapy. These mutations should be examined in patients showing progression of disease after partial remission.

Rituximab binds an epitope on the CD20 antigen, encompassed in exon 5 of the MS4A1 gene. We sequenced this region and correlated the presence of mutations with CD20 protein expression and response to R-CHOP in patients with diffuse large B-cell lymphoma: 264 diagnostic biopsies and 15 biopsies taken at the time of relapse were successfully sequenced. CD20 mutations involving the rituximab epitope were detected in only 1/264 (0.4%) and 1/15 (6%) of the biopsies taken at diagnosis and relapse, respectively. No polymorphic sequence variants were detected in this region. Three patients had malignant cells that were CD20 protein-positive at diagnosis but CD20-negative at relapse. Thus, CD20 mutations involving the rituximab epitope are rare in both de novo and relapsed diffuse large B-cell lymphoma, and do not represent a significant cause of R-CHOP resistance. CD20 protein-negative relapses occur after R-CHOP therapy but their clinical relevance is unknown.

Background: Antibody-dependent cellular cytotoxicity (ADCC) has recently been identified as one of the critical mechanisms underlying the clinical efficacy of therapeutic antibodies, especially anticancer antibodies. Therapeutic antibodies fully lacking the core fucose of the Fc oligosaccharides have been found to exhibit much higher ADCC in humans than their fucosylated counterparts. However, data which show how fully non-fucosylated antibodies achieve such a high ADCC in human whole blood have not yet been disclosed. The precise mechanisms responsible for the high ADCC mediated by fully non-fucosylated therapeutic antibodies, even in the presence of human plasma, should be explained based on direct evidence of non-fucosylated antibody action in human blood., Methods: Using a human ex vivo B-cell depletion assay with non-fucosylated and fucosylated anti-CD20 IgG1s rituximab, we monitored the binding of the therapeutic agents both to antigens on target cells (target side interaction) and to leukocyte receptors (FcgammaR) on effector cells (effector side interaction), comparing the intensities of ADCC in human blood., Results: In the target side interaction, down-modulation of CD20 on B cells mediated by anti-CD20 was not observed. Simple competition for binding to the antigens on target B cells between fucosylated and non-fucosylated anti-CD20s was detected in human blood to cause inhibition of the enhanced ADCC of non-fucosylated anti-CD20 by fucosylated anti-CD20. In the effector side interaction, non-fucosylated anti-CD20 showed sufficiently high FcgammaRIIIa binding activity to overcome competition from plasma IgG for binding to FcgammaRIIIa on natural killer (NK) cells, whereas the binding of fucosylated anti-CD20 to FcgammaRIIIa was almost abolished in the presence of human plasma and failed to recruit NK cells effectively. The core fucosylation levels of individual serum IgG1 from healthy donors was found to be so slightly different that it did not affect the inhibitory effect on the ADCC of fucosylated anti-CD20., Conclusion: Our results demonstrate that removal of fucosylated antibody ingredients from antibody therapeutics elicits high ADCC in human blood by two mechanisms: namely, by evading the inhibitory effects both of plasma IgG on FcgammaRIIIa binding (effector side interaction) and of fucosylated antibodies on antigen binding (target side interaction).

Among the heterogeneous population of circulating hematopoietic and endothelial progenitors, we identified a subpopulation of CD133(+) cells displaying myogenic properties. Unexpectedly, we observed the expression of the B-cell marker CD20 in blood-derived CD133(+) stem cells. The CD20 antigen plays a role in the modulation of intracellular calcium homeostasis through signaling pathways activation. Several observations suggest that an increase in intracellular calcium concentration ([Ca(2+)](i)) could be involved in the etiology of the Duchenne muscular dystrophy (DMD). Here, we show that a CD20-related signaling pathway able to induce an increase in [Ca(2+)](i) is differently activated after brain derived neurotrophic factor (BDNF) stimulation of normal and dystrophic blood-derived CD133(+) stem cells, supporting the assumption of a "CD20-related calcium impairment" affecting dystrophic cells. Presented findings represent the starting point toward the expansion of knowledge on pathways involved in the pathology of DMD and in the behavior of dystrophic blood-derived CD133(+) stem cells.

Veltuzumab is a humanized anti-CD20 monoclonal antibody with complementarity-determining regions (CDRs) identical to rituximab, except for one residue at the 101st position (Kabat numbering) in CDR3 of the variable heavy chain (V(H)), having aspartic acid (Asp) instead of asparagine (Asn), with framework regions of epratuzumab, a humanized anti-CD22 antibody. When compared with rituximab, veltuzumab has significantly reduced off-rates in 3 human lymphoma cell lines tested, as well as increased complement-dependent cytotoxicity in 1 of 3 cell lines, but no other in vitro differences. Mutation studies confirmed that the differentiation of the off-rate between veltuzumab and rituximab is related to the single amino acid change in CDR3-V(H). Studies of intraperitoneal and subcutaneous doses in mouse models of human lymphoma and in normal cynomolgus monkeys disclosed that low doses of veltuzumab control tumor growth or deplete circulating or sessile B cells. Low- and high-dose veltuzumab were significantly more effective in vivo than rituximab in 3 lymphoma models. These findings are consistent with activity in patients with non-Hodgkin lymphoma given low intravenous or subcutaneous doses of veltuzumab. Thus, changing Asn(101) to Asp(101) in CDR3-V(H) of rituximab is responsible for veltuzumab's lower off-rate and apparent improved potency in preclinical models that could translate into advantages in patients.

Objective: The immunologic events that lead to persistent joint inflammation in certain patients with Lyme arthritis post-antibiotic treatment have been elusive so far. The prevalence of this condition is highest in individuals with rheumatoid arthritis-associated HLA-DR alleles. This study was undertaken to generate a murine model with persistent arthritis post-antibiotic treatment., Methods: We have previously shown that CD28(-/-) mice develop intermittent monarticular Lyme arthritis that is responsive to antibiotics. Since there seems to be a link in humans between persistent arthritic manifestations post-antibiotic treatment and the HLA-DR4 allele, we generated DR4+/+CD28(-/-)MHCII(-/-) mice, infected them with Borrelia burgdorferi, and subsequently treated them with antibiotics., Results: Thirty-eight percent of the B burgdorferi-infected DR4+/+CD28(-/-)MHCII(-/-) mice, but none of the B burgdorferi-infected CD28(-/-)MHCII(-/-) mice, remained arthritic post-antibiotic treatment. A significant fraction (36%) of these mice, but none of the mice in which arthritis resolved, had serum antibodies to outer surface protein A of B burgdorferi. After abrogation of active B burgdorferi infection, the inflammatory reaction in mice with persistent joint inflammation was restricted to the joints, since their draining lymph nodes were no longer enlarged. Increased CD20 and interferon-gamma messenger RNA expression in the inflamed joints of these mice suggested a possible role of B cells and inflammatory cytokines in the pathogenesis of persistent arthritis post-antibiotic treatment., Conclusion: The establishment of this murine model allows, for the first time, the elucidation of the immunologic events that lead to persistent Lyme arthritis post-antibiotic therapy in genetically susceptible individuals.

IgG4-related disease is a recently proposed clinical entity with several unique clinicopathological features. A chronic inflammatory state with marked fibrosis, which can often be mistaken for malignancy, especially by clinical imaging analyses, unifies these features. Little is known about lymphomagenesis in the context of IgG4-related disease, we recently first reported the ocular adnexal marginal zone B-cell lymphomas arising from IgG4-related disease. To the best of our knowledge, no existing study has ever established the neoplastic potential of IgG4-producing cells. In the present report, we describe the first IgG4-producing lymphoma. The patient was a 72-year-old male who was being followed for an asbestos-related pleural plaque. During follow-up, computed tomography revealed bilateral renal masses and multiple swollen retroperitoneal lymph nodes. A retroperitoneal lymph node biopsy was performed. Histologically, the interfollicular areas were expanded by medium to large plasmacytoid cells. These plasmacytoid cells showed nuclear pleomorphism and had prominent Russell bodies. Immunohistochemistry and double immunofluorescence staining of these cells revealed IgG4 positivity and monotypic lambda-light chain predominance. A portion of these cells were partially positive for CD20, negative for CD3, and somewhat faintly positive for CD138. In addition, serum IgG4 was elevated. Southern blot analysis of the lymph node specimen detected immunoglobulin heavy chain gene rearrangement. The present study indicates that, not only can malignant lymphomas occur in the setting of IgG4-related disease, but IgG4-producing cells can also be neoplastic.

Development of methods to engineer gamma-retroviral vectors capable of transducing target cells in a cell-specific manner could impact the future of the clinical application of gene therapy as well as the understanding of the biology of transfer gene vectors. Two molecular events are critical for controlling the entry of gamma-retroviral vectors to target cells: binding to cell-surface receptors and the subsequent fusion of viral vector membrane and cellular membrane. In this report, we evaluated a method to incorporate a membrane-bound antibody and a fusogenic molecule to provide binding and fusion functions respectively, into gamma-retroviral vectors for targeted gene delivery. An anti-CD20 antibody and a fusogenic protein derived from Sindbis virus glycoprotein could be efficiently co-displayed on the surface of viral vectors. Vectors bearing anti-CD20 antibody conferred their binding specificity to cells expressing CD20. Enhanced in vitro transduction towards CD20-expressing cells was observed for gamma-retroviral vectors displaying both an antibody and a fusogen. We found that the biological activity of the fusogen played an important role on the efficiency of such a targeting strategy and were able to engineer several mutant forms of the fusogen exhibiting elevated fusion function to improve the overall efficiency of targeted transduction. We devised an animal model to show that subcutaneous injection of such engineered vectors to the areas xenografted with target cells could achieve targeted gene delivery in vivo. Taken together, we demonstrated as proof-of-principle a flexible and modular two-molecule strategy for engineering targeting gamma-retroviral vectors.

The anti-CD20 monoclonal antibody rituximab has been less successful in treating chronic lymphocytic leukemia (CLL) than lymphoma, possibly due to the lower density of CD20 on B lymphocytes from CLL patients than on those from lymphoma patients. This lowering may result from insufficiency of one of the transcription factors of cd20. Of these, purine-rich box-1 (PU.1) is poorly expressed in CLL. To estimate its weight in CD20 expression, pu.1 cDNA was transfected into CLL B cells and shown to raise the membrane expression of CD20 and to improve the rituximab-induced lysis of transfected cells. Granulocyte macrophage colony-stimulating factor and all-trans-retinoic acids were not involved in the defective expression of PU.1 or the excessive methylation of the pu.1 gene, because 6 of 14 CLL samples tested were normally methylated. This was confirmed by the failure of DNA methyltransferase inhibitors to restore pu.1 transcription in hypermethylated CLL, and, in fact, the expression of PU.1 was down-regulated by excessive expression of the FMS proto-oncogene-like tyrosine kinase 3 (Flt3) receptor. This abnormality is consistent with our finding of elevated levels of Flt3 ligand (FL) in 20 of 23 CLL sera tested. We propose that FL-dependent increased Flt3 signaling prevents the expression of PU.1, which down-regulates that of CD20, and accounts for resistance of leukemic B cells to rituximab-induced lysis.

Adoptive immunotherapy with T cells expressing a tumor-specific chimeric T-cell receptor is a promising approach to cancer therapy that has not previously been explored for the treatment of lymphoma in human subjects. We report the results of a proof-of-concept clinical trial in which patients with relapsed or refractory indolent B-cell lymphoma or mantle cell lymphoma were treated with autologous T cells genetically modified by electroporation with a vector plasmid encoding a CD20-specific chimeric T-cell receptor and neomycin resistance gene. Transfected cells were immunophenotypically similar to CD8(+) effector cells and showed CD20-specific cytotoxicity in vitro. Seven patients received a total of 20 T-cell infusions, with minimal toxicities. Modified T cells persisted in vivo 1 to 3 weeks in the first 3 patients, who received T cells produced by limiting dilution methods, but persisted 5 to 9 weeks in the next 4 patients who received T cells produced in bulk cultures followed by 14 days of low-dose subcutaneous interleukin-2 (IL-2) injections. Of the 7 treated patients, 2 maintained a previous complete response, 1 achieved a partial response, and 4 had stable disease. These results show the safety, feasibility, and potential antitumor activity of adoptive T-cell therapy using this approach. This trial was registered at www.clinicaltrials.gov as #NCT00012207.

Gene transfer into B cells by lentivectors can provide an alternative approach to managing B lymphocyte malignancies and autoreactive B cell-mediated autoimmune diseases. These pathogenic B cell populations can be distinguished by their surface expression of monospecific immunoglobulin. Development of a novel vector system to deliver genes to these specific B cells could improve the safety and efficacy of gene therapy. We have developed an efficient method to target lentivectors to monospecific immunoglobulin-expressing cells in vitro and in vivo. We were able to incorporate a model antigen CD20 and a fusogenic protein derived from the Sindbis virus as two distinct molecules into the lentiviral surface. This engineered vector could specifically bind to cells expressing surface immunoglobulin recognizing CD20 (alphaCD20), resulting in efficient transduction of target cells in a cognate antigen-dependent manner in vitro, and in vivo in a xenografted tumor model. Tumor suppression was observed in vivo, using the engineered lentivector to deliver a suicide gene to a xenografted tumor expressing alphaCD20. These results show the feasibility of engineering lentivectors to target immunoglobulin- specific cells to deliver a therapeutic effect. Such targeting lentivectors also could potentially be used to genetically mark antigen-specific B cells in vivo to study their B cell biology.

Aim: To construct recombinant retroviruses expressing anti-CD20 scFv/CD80/CD28/zeta gene and detect its expression in Jurkat cells., Method: CD28-zetacDNA were amplified from plasmids pBULLET and inserted into pLNCX vector that contained anti-CD20 scFv/CD80 gene. The recombinant plasmids were transfected into PA 317 cells. Retroviruses were harvested from culture medium of PA 317 cells. Then NIH 3T3 were transfected with retroviruses. Objective gene expression was determined by PCR and FACS. Jurkat cells were transfected with high titer of retroviruses and resistant clones were obtained by G418 selection. Objective mRNA was determined by RT- PCR., Results: The recombinant eukaryotic vector was constructed successfully by PCR and enzyme digestion analysis and objective gene was amplified from NIH 3T3 cells transfected with retroviruses by PCR; FACS showed that objective protein could be expressed in NIH 3T3 cells. Objective gene was amplified from Jurkat cells transfected with retroviruses by RT-PCR., Conclusion: Recombinant retrovirus expressing anti-CD20 scFv/CD80/CD28/zeta gene was successfully constructed and objective protein could be expressed in Jurkat cells.

Aim: To construct and express human 4-1BBL/anti-CD20 bispecific fusion protein and identify its biological activity., Methods: PCR and overlapping PCR were used to construct human 4-1BBL/anti-CD20 bispecific fusion protein. DNA sequencing was performed by the terminus of the fusion protein nucleotide.The product was purified by affinity chromatography and analyzed by Western blot and its antigen-binding activity was examined by FACS., Results: The data of DNA sequence showed that human 4-1BBL/anti-CD20 bispecific fusion protein was correct. The fusion protein was recovered in high yield (up to 4 mg/L) after E-tag purification and predominantly(90%) as a dimer. The fusion protein could bind to Raji cells(CD20(+)) and A549 cells(4-1BB(+)), respectively., Conclusion: The human 4-1BBL/anti-CD20 bispecific fusion protein with high level expression was successfully obtained and could bind to Raji ceIls and A549 cells.

We have reported earlier that the non-viral Sleeping Beauty (SB) transposon system can mediate genomic integration and long-term reporter gene expression in human primary peripheral blood (PB) T cells. In order to test whether this system can be used for genetically modifying both PB T cells and umbilical cord blood (UCB) T cells as graft-versus-leukemia effector cells, an SB transposon was constructed to coexpress a single-chain chimeric antigen receptor (CAR) for human CD19 and CD20. PB and UCB were nucleofected with the transposon and a transposase plasmid, activated and then expanded in culture using anti-CD3/CD28 beads. Stable dual-gene expression was confirmed in both T-cell types, permitting enrichment by positive selection with Rituxan. The engineered CD4(+) T cells and CD8(+) T cells both exhibited specific cytotoxicity against CD19(+) leukemia and lymphoma cell lines, as well as against CD19 transfectants, and produced high-levels of antigen-dependent Th1 (but not Th2) cytokines. The in vivo adoptive transfer of genetically engineered T cells significantly reduced tumor growth and prolonged the survival of the animal. Taken together, these data indicate that T cells from PB and UCB can be stably modified using a non-viral DNA transfer system, and that such modified T cells may be useful in the treatment of refractory leukemia and lymphoma.

Acquirement of resistance to rituximab has been observed in lymphoma patients. To define mechanisms associated with rituximab resistance, we developed various rituximab-resistant cell lines (RRCL) and studied changes in CD20 expression/structure, lipid raft domain (LRD) reorganization, calcium mobilization, antibody-dependent cellular cytotoxicity, and complement-mediated cytotoxicity (CMC) between parental and RRCL. Significant changes in surface CD20 antigen expression were shown in RRCL. Decreased calcium mobilization and redistribution of CD20 into LRD were found in RRCL. Western blotting identified a unique 35 kDa protein band in RRCL, which was not seen in parental cells and was secondary to an increase in surface and cytoplasmic expression of IgM light chains. CD20 gene expression was decreased in RRCL. In vitro exposure to PS341 increased CD20 expression in RRCL and minimally improved the sensitivity to rituximab-associated CMC. Our data strongly suggest that the acquisition of rituximab resistance is associated with global gene and protein down-regulation of the CD20 antigen affecting LRD organization and downstream signaling. CD20 expression seems to be regulated at the pretranscriptional and posttranscriptional levels. Proteasome inhibition partially reversed rituximab resistance, suggesting the existence of additional mediators of rituximab resistance. Future research is geared to identify drugs and/or biological agents that are effective against RRCL.

Mantle cell lymphoma (MCL) is defined pathologically by the detection of CD20, CD5, and most importantly cyclin D1 (CCND1). Its distinction from other lymphomas is important for prognosis and appropriate therapy, but occasional cases may fail to express CCND1 and morphologic simulators may express CD20 and CD5 but not CD23. In this study, we show that the transcription factor Sox11 is specifically expressed in the nucleus of MCL compared with other lymphomas and benign lymphoid tissue. Although the role of Sox11 presently is not known in lymphocyte ontogeny, it is normally expressed in the developing central nervous system in the embryo and shows sequence homology with Sox4, a transcription factor crucial for B lymphopoiesis. Sox11 mRNA is increased in gliomas compared with healthy brain tissue, suggesting a role in malignant transformation and/or cell survival. Our novel finding of specific overexpression of Sox11 mRNA and nuclear protein in both cyclin D1-positive and - negative MCL may be useful for the diagnosis of MCL as a complement to cyclin D1 and also suggests a functional role for Sox11 in MCL.

Primary cutaneous B-cell lymphoma is a B-cell lymphoma of the skin with no evidence of extracutaneous involvement at the time of diagnosis. In this report, we describe an 8-year-old boy who presented with a firm, alopecic, skin-colored, smooth nodule over the right frontal scalp. Histological examination revealed a mid-to deep-dermal mononuclear lesion. Immunohistochemical staining revealed a B-cell population that was CD10(+), CD5(-), CD21(+), and bcl2(-). This pattern of reactivity is characteristic of primary cutaneous B-cell lymphoma of follicle-center subtype. To the best of our knowledge, this is the first report of this type of cutaneous lymphoma in a child.

Autoimmune diabetes is believed to be mediated primarily by T cells. However, B cells have been implicated in the pathogenesis of the disease in NOD mice. Although preclinical studies have been limited by the absence of anti-CD20 reagents that can induce B cell depletion in mice, a clinical trial using the B cell-depleting anti-CD20 monoclonal antibody rituximab (Rituxan) is underway in type 1 diabetes patients. In this issue of the JCI, Hu et al. describe the generation of transgenic NOD mice that express human CD20 on B cells (see the related article beginning on page 3857). They show that anti-CD20 therapy induces B cell depletion in these mice and offers some level of protection against diabetes. Although many questions remain unanswered, this mouse model represents the first opportunity to evaluate the potential value of rituximab as a novel therapy for autoimmune diabetes.

The precise roles of B cells in promoting the pathogenesis of type 1 diabetes remain undefined. Here, we demonstrate that B cell depletion in mice can prevent or delay diabetes, reverse diabetes after frank hyperglycemia, and lead to the development of cells that suppress disease. To determine the efficacy and potential mechanism of therapeutic B cell depletion, we generated a transgenic NOD mouse expressing human CD20 (hCD20) on B cells. A single cycle of treatment with an antibody specific for hCD20 temporarily depleted B cells and significantly delayed and/or reduced the onset of diabetes. Furthermore, disease established to the point of clinical hyperglycemia could be reversed in over one-third of diabetic mice. Why B cell depletion is therapeutic for a variety of autoimmune diseases is unclear, although effects on antibodies, cytokines, and antigen presentation to T cells are thought to be important. In B cell-depleted NOD mice, we identified what we believe is a novel mechanism by which B cell depletion may lead to long-term remission through expansion of Tregs and regulatory B cells. Our results demonstrate clinical efficacy even in established disease and identify mechanisms for therapeutic action that will guide design and evaluation of parallel studies in patients.

In mice, genetic deletion of B cells strongly suppresses systemic autoimmunity, providing a rationale for depleting B cells to treat autoimmunity. In fact, B cell depletion with rituximab is approved for rheumatoid arthritis patients, and clinical trials are underway for systemic lupus erythematosus. Yet, basic questions concerning mechanism, pathologic effect, and extent of B cell depletion cannot be easily studied in humans. To better understand how B cell depletion affects autoimmunity, we have generated a transgenic mouse expressing human CD20 on B cells in an autoimmune-prone MRL/MpJ-Fas(lpr) (MRL/lpr) background. Using high doses of a murine anti-human CD20 mAb, we were able to achieve significant depletion of B cells, which in turn markedly ameliorated clinical and histologic disease as well as antinuclear Ab and serum autoantibody levels. However, we also found that B cells were quite refractory to depletion in autoimmune-prone strains compared with non-autoimmune-prone strains. This was true with multiple anti-CD20 Abs, including a new anti-mouse CD20 Ab, and in several different autoimmune-prone strains. Thus, whereas successful B cell depletion is a promising therapy for lupus, at least some patients might be resistant to the therapy as a byproduct of the autoimmune condition itself.

With the introduction of rituximab to chemotherapy in lymphoma, CHOP changed to R-CHOP in elderly, intermediate risk DLBCL (diffuse large B-cell lymphoma) patients. Although the treatment is not yet standard, due to insufficient evidence, in clinical practice it is an R-containing regimen, for example, in mantle cell lymphoma, such as HyperCVAD/MA to R-HyperCVAD/MA. Recently, another group and ours reported the presence of rituximab resistance during R-containing chemotherapy. If the lymphoma is bulky,the overexpression of CD 55 (complement regulatory molecule) leads to resistance to rituximab. When the patients evidenced the loss of CD 20 antigen in refractory/relapsed lymphoma after R-containing therapy, some patients showed the presence of CD 20 point mutation. In the cases of refractory/relapsed cases, radioimmunotherapy or other monoclonal antibodies are prepared, including Zevalin and CD 22, CD 40, CD 74, and HLA-DR targeting antibodies. Not only monoclonal antibodies but also HDACI or bortezomib (NF-kappaB) and other signal inhibitors (for Akt, ERK/MAPK) have been developed. In Japan, we must consider the higher speed of infusion rituximab and we must prepare standard therapy for lymphoma because of recruiting phase I/II clinical trials after use of rituximab for easy entry.

Rituximab has been established as an effective and safe therapy for cutaneous B-cell lymphoma (CBCL). Different survival pathways, that is the Raf/MEK/Erk- or the p38MAPK cascade, have been suggested as downstream mediators of rituximab and may be involved in treatment failure. Biopsies from four patients, suffering from different subtypes of CBCL, which were obtained at various time points of relapse during or after therapy with 375 mg rituximab per m2 of body surface area, were analysed for the expression of CD20, CD3, Ki-67, Raf-kinase inhibitory protein (RKIP) and bcl-2 by immunohistochemistry. No CD20-loss variants, that is the suggested main tumour escape mechanism to rituximab therapy, were observed in any specimen of relapsing CBCL. Notably, the expression of proapoptotic RKIP remained increased in these tumour samples. This was concomitated by a constant to slightly reduced proliferation status as demonstrated by Ki-67 staining. However, relapsing CBCL exhibited a strong upregulation of the antiapoptotic molecule bcl-2 in comparison to pretherapeutic levels. The immunohistochemical analyses of this case series of rituximab refractory CBCL suggest that upregulation of bcl-2 may play a major role in therapy resistance.

Objective: To construct anti-CD20scFv/CD80/CD28/zeta recombinant gene modified T cells, test its effectiveness of eradicating CD20+ lymphoma cells and provide a probably new approach to tumor adoptive immunotherapy., Methods: CD28-zeta cDNA were amplified from vector pBULLET and inserted into pLNCX vector that contained anti-CD20scFv/CD80 gene. The recombinant vectors were transduced into PA317 cells and high titer retroviruses were obtained to infect human peripheral blood T lymphocytes. Resistant T cells were obtained by G418 selection at one week. Then transduced T lymphocytes and lymphoma cell lines Daudi Raji were cocultured. The cytotoxicity and cytokine production of transduced T cells were determined by non-radio-activation cytotoxicity assay and ELISA respectively., Results: The recombinant eukaryotic vector was constructed successfully as proved by enzyme digestion analysis and sequencing. These T cells were able to lyse CD20+ target cells and secrete high levels of IL-2 and IFN-gamma in vitro., Conclusion: Recombinant gene modified T cells can be constructed successfully. It can specially kill CD20 positive lymphoma cells in vitro.

Objective: A dietary compound curcumin hardwires to multiple cellular processes, with suppression of cell proliferation, induction of apoptosis, and inhibition of metastasis considered as the major mechanisms underlying its anticancer properties. Based on our recent evidence that curcumin triggers cell demise in follicular lymphoma (FL) cells, we aimed to identify curcumin-regulated genes of utmost importance for the treatment of follicular lymphoma., Materials and Methods: Large-scale gene-expression profiling was performed during curcumin-triggered apoptosis (8-36 hours) in follicular lymphoma HF4.9 cells using Sentrix Human WG-6 BeadChips. Expression levels of selected differentially expressed genes were verified by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) and immunoblotting. Chemical inhibitor studies (cyclosporin A and AMD3100) were performed to provide further insights into the functional significance of selected genes., Results: Comprehensive transcriptional response is associated with curcumin treatment in HF4.9 cells, including differential expression of genes encoding apoptotic signaling proteins, tumor and metastasis suppressors, transcription and splicing factors, proteins involved in regulation of cell adhesion, migration (e.g., CXCR4), lymphoid development, or B-cell activation (e.g. CD20), and others. CXCR4 downregulation was confirmed by both qRT-PCR and immunoblotting. Importantly, curcumin induced downregulation of CXCR4 protein also in other FL cell lines, and similar effect was observed upon prolonged incubation with low concentration of curcumin. AMD3100 (a selective CXCR4 antagonist) alone enhanced neither spontaneous nor serum-starvation-induced death at 24 hours of treatment, but impaired long-term cell growth in a cell line-dependent fashion., Conclusions: To our knowledge this is the first study showing curcumin-induced downregulation of CXCR4, and at attainable in vivo concentration of the polyphenol. Other curcumin-regulated genes identified herein, e.g., CD20, are also seemingly pertinent to the pathophysiology of follicular lymphoma.

Background: Polymorphisms in FcgammaRIIa and FcgammaRIIIa receptors are associated with responses to the CD20-directed immunoglobulin G1 (IgG1) monoclonal antibody rituximab among patients with indolent lymphoma. At odds with the aforementioned clinical observations has been the finding that IgG1 binding is impacted by polymorphisms in FcgammaRIIIa but not FcgammaRIIa. One possibility for this discrepancy might involve linkage of polymorphisms between FcgammaRIIa and FcgammaRIIIa., Materials and Methods: As such, we performed allelespecific polymerase chain reaction and directed sequencing of the genomic DNA coding region of FcgammaRIIA and FcgammaRIIIA for 52 healthy individuals., Results: Two common polymorphisms were observed for FcgammaRIIA (at positions 27 and 131) and FcgammaRIIIA (at positions 48 and 158). Importantly, we observed linkage among polymorphisms within and between FcgammaRIIa and FcgammaRIIIa, including the expression of histidine at FcgammaRIIa-131 and valine at FcgammaRIIIa, both of which are associated with enhanced responses to rituximab. The results of these studies demonstrate that there is wide linkage within and between polymorphisms in FcgammaRIIa and FcgammaRIIIa and might provide an explanation for why polymorphisms at FcgammaRIIa are associated with rituximab responses despite a lack of impact on IgG1 binding., Conclusion: Knowledge of such linkages could facilitate the development of diagnostic tests aimed at identifying patients who might be more suitable for treatment with rituximab and possibly other therapeutic antibodies.

We report the first case of diffuse large B-cell lymphoma (DLBCL) of the stomach displaying CD20-negative relapse after rituximab-containing treatment and the re-appearance of CD20 expression at the second failure. The loss of CD20 expression in B-cell lymphomas relapsing after rituximab is a well-known phenomenon, but its actual impact in DLBCL is difficult to estimate. This paradigmatic case suggests that CD20-expression reappearance after purging of CD20-positive clones with rituximab might be an underestimated occurrence in B-cell lymphomas. Accordingly, every relapse, whenever possible, should be histologically assessed with diagnostic and immunophenotyping purposes.

There has been interest in generating T cells expressing chimeric artificial receptors (CARs) targeting CD19/CD20 antigens to treat B-cell lymphomas. If successful, however, this approach would likely impair humoral immunity because T cells may persist long-term. Most low-grade lymphoma and chronic lymphocytic leukemia (B-CLL) cells express monoclonal immunoglobulins carrying either kappa or lambda light chains. We, therefore, explored whether T lymphocytes could be genetically modified to target the tumor-associated light chain, sparing B lymphocytes expressing the reciprocal light chain, and consequently reduce impairment of humoral immunity. We found that T lymphocytes expressing the anti-kappa light chain CAR showed cytotoxic activity against Igkappa(+) tumor cell lines and B-CLL cells both in vitro and in vivo. We also found that the incorporation of the CD28 endodomain within the CAR enhanced the in vitro and in vivo expansion of transgenic T cells after tumor-associated antigen stimulation. Free Igkappa(+) did not compromise the ability of redirected T lymphocytes to eliminate Igkappa(+) tumors because these free immunoglobulins served to sustain proliferation of CAR-CD28 transgenic T cells. Thus, adoptive transfer of T lymphocytes targeting the appropriate light chain could be a useful immunotherapy approach to treat B-lymphocyte malignancies that clonally express immunoglobulin without entirely compromising humoral immunity.

Rituximab is a monoclonal antibody widely used in the treatment of malignant lymphoma and autoimmunity. Its epitope within the B-cell antigen CD20 is largely unknown. We used phage display libraries to select peptides binding to rituximab. Enriched peptides showed 2 sequence patterns: one motif (CALMIANSC) is related to (170)ANPS(173) within CD20, while another motif (WEWTI) may mimic the CD20 segment (182)YCYSI(185). Phages displaying either motif specifically bound rituximab. Binding to rituximab by the CD20 peptides ANPS and YCYSI was weak when used separately and enhanced when both peptides were linked. Recombinant CD20 extracellular loop proteins blocked binding of the selected CWWEWTIGC phage to rituximab, suggesting that CWWEWTIGC mimics the epitope. Blocking capacity was strongly reduced upon mutation of the CD20 strings ANPS or YCYSI. We conclude that rituximab binds a discontinuous epitope in CD20, comprised of (170)ANPS(173) and (182)YCYSI(185), with both strings brought in steric proximity by a disulfide bridge between C(167) and C(183).

Long-term acceptance of solid organ allografts remains a challenge. While many acute rejection episodes can be treated, new mechanisms of allograft damage are now being defined especially in kidney transplantation. Unexpected clusters of CD20(+) cells have been discovered in renal biopsies performed for clinical rejection. C4d deposition is now routinely seen in refractory rejection. Despite the rapid introduction of new immunosuppressive agents in transplantation, the search for an efficacious anti-B-cell agent remains. With novel mechanisms of allograft damage now being defined, it is important to consider how an anti-B-cell agent might fit into an immunosuppressive regimen. Rituximab is a high-affinity CD20 specific antibody that depletes the B-cell compartment by inducing cellular apoptosis. Thus, it is a rational choice for therapy in transplantation to abrogate B-cell mediated events. In this review, we will discuss the mechanisms of action of rituximab, and its use in for a variety of indications in solid organ transplantation. There are emerging case reports that show that rituximab may be an effective agent to treat antibody-mediated rejection, and post-transplant lymphoproliferative disorder. Rituximab has been frequently cited as an important adjunct therapy in desensitization protocols for highly sensitized transplant recipients as well as recipients of ABO incompatible transplants. Rituximab demonstrates promise in this regard and warrants additional consideration in prospective clinical trials.

We have developed an efficient method to target lentivirus-mediated gene transduction to a desired cell type. It involves incorporation of antibody and fusogenic protein as two distinct molecules into the lentiviral surface. The fusogen is constructed by modifying viral envelope proteins, so that they lack the ability to bind to their cognate receptor but still retain the ability to trigger pH-dependent membrane fusion. Thus, the specificity of such a lentiviral vector is solely determined by the antibody, which is chosen to recognize a specific surface antigen of the desired cell type. This specific binding then induces endocytosis of the surface antigen, bringing the lentivirus into an endosome. There, the fusogen responds to the low pH environment and mediates membrane fusion, allowing the virus core to enter the cytosol. Using CD20 as a target antigen for human B cells, we have demonstrated that this targeting strategy is effective both in vitro and in intact animals. This methodology is flexible and can be extended to other forms of cell type-specific recognition to mediate targeting. The only requirement is that the antibody (or other binding protein) must be endocytosed after interaction with its cell surface-binding determinant.