Follicular lymphoma evolves with a surmountable dependency on acquired glycosylation motifs in the B-cell receptor.

ABSTRACT: An early event in the genesis of follicular lymphoma (FL) is the acquisition of new glycosylation motifs in the B-cell receptor (BCR) due to gene rearrangement and/or somatic hypermutation. These N-linked glycosylation motifs (N-motifs) contain mannose-terminated glycans and can interact with lectins in the tumor microenvironment, activating the tumor BCR pathway. N-motifs are stable during FL evolution, suggesting that FL tumor cells are dependent on them for their survival. Here, we investigated the dynamics and potential impact of N-motif prevalence in FL at the single-cell level across distinct tumor sites and over time in 17 patients. Although most patients had acquired at least 1 N-motif as an early event, we also found (1) cases without N-motifs in the heavy or light chains at any tumor site or time point and (2) cases with discordant N-motif patterns across different tumor sites. Inferring phylogenetic trees of the patients with discordant patterns, we observed that both N-motif-positive and N-motif-negative tumor subclones could be selected and expanded during tumor evolution. Comparing N-motif-positive with N-motif-negative tumor cells within a patient revealed higher expression of genes involved in the BCR pathway and inflammatory response, whereas tumor cells without N-motifs had higher activity of pathways involved in energy metabolism. In conclusion, although acquired N-motifs likely support FL pathogenesis through antigen-independent BCR signaling in most patients with FL, N-motif-negative tumor cells can also be selected and expanded and may depend more heavily on altered metabolism for competitive survival.

Targeted TLR9 Agonist Elicits Effective Antitumor Immunity against Spontaneously Arising Breast Tumors.

Spontaneous tumors that arise in genetically engineered mice recapitulate the natural tumor microenvironment and tumor-immune coevolution observed in human cancers, providing a more physiologically relevant preclinical model relative to implanted tumors. Similar to many cancer patients, oncogene-driven spontaneous tumors are often resistant to immunotherapy, and thus novel agents that can effectively promote antitumor immunity against these aggressive cancers show considerable promise for clinical translation, and their mechanistic assessment can broaden our understanding of tumor immunology. In this study, we performed extensive immune profiling experiments to investigate how tumor-targeted TLR9 stimulation remodels the microenvironment of spontaneously arising tumors during an effective antitumor immune response. To model the clinical scenario of multiple tumor sites, we used MMTV-PyMT transgenic mice, which spontaneously develop heterogeneous breast tumors throughout their 10 mammary glands. We found that i.v. administration of a tumor-targeting TLR9 agonist, referred to as PIP-CpG, induced a systemic T cell-mediated immune response that not only promoted regression of existing mammary tumors, but also elicited immune memory capable of delaying growth of independent newly arising tumors. Within the tumor microenvironment, PIP-CpG therapy initiated an inflammatory cascade that dramatically amplified chemokine and cytokine production, prompted robust infiltration and expansion of innate and adaptive immune cells, and led to diverse and unexpected changes in immune phenotypes. This study demonstrates that effective systemic treatment of an autochthonous multisite tumor model can be achieved using a tumor-targeted immunostimulant and provides immunological insights that will inform future therapeutic strategies.

CD81 costimulation skews CAR transduction toward naive T cells.

Adoptive cellular therapy using chimeric antigen receptors (CARs) has revolutionized our treatment of relapsed B cell malignancies and is currently being integrated into standard therapy. The impact of selecting specific T cell subsets for CAR transduction remains under investigation. Previous studies demonstrated that effector T cells derived from naive, rather than central memory T cells mediate more potent antitumor effects. Here, we investigate a method to skew CAR transduction toward naive T cells without physical cell sorting. Viral-mediated CAR transduction requires ex vivo T cell activation, traditionally achieved using antibody-mediated strategies. CD81 is a T cell costimulatory molecule that when combined with CD3 and CD28 enhances naive T cell activation. We interrogate the effect of CD81 costimulation on resultant CAR transduction. We identify that upon CD81-mediated activation, naive T cells lose their identifying surface phenotype and switch to a memory phenotype. By prelabeling naive T cells and tracking them through T cell activation and CAR transduction, we document that CD81 costimulation enhanced naive T cell activation and resultantly generated a CAR T cell product enriched with naive-derived CAR T cells.

Single-cell analysis can define distinct evolution of tumor sites in follicular lymphoma.

Tumor heterogeneity complicates biomarker development and fosters drug resistance in solid malignancies. In lymphoma, our knowledge of site-to-site heterogeneity and its clinical implications is still limited. Here, we profiled 2 nodal, synchronously acquired tumor samples from 10 patients with follicular lymphoma (FL) using single-cell RNA, B-cell receptor (BCR) and T-cell receptor sequencing, and flow cytometry. By following the rapidly mutating tumor immunoglobulin genes, we discovered that BCR subclones were shared between the 2 tumor sites in some patients, but in many patients, the disease had evolved separately with limited tumor cell migration between the sites. Patients exhibiting divergent BCR evolution also exhibited divergent tumor gene-expression and cell-surface protein profiles. While the overall composition of the tumor microenvironment did not differ significantly between sites, we did detect a specific correlation between site-to-site tumor heterogeneity and T follicular helper (Tfh) cell abundance. We further observed enrichment of particular ligand-receptor pairs between tumor and Tfh cells, including CD40 and CD40LG, and a significant correlation between tumor CD40 expression and Tfh proliferation. Our study may explain discordant responses to systemic therapies, underscores the difficulty of capturing a patient's disease with a single biopsy, and furthers our understanding of tumor-immune networks in FL.

Antigen presentation profiling reveals recognition of lymphoma immunoglobulin neoantigens.

Cancer somatic mutations can generate neoantigens that distinguish malignant from normal cells. However, the personalized identification and validation of neoantigens remains a major challenge. Here we discover neoantigens in human mantle-cell lymphomas by using an integrated genomic and proteomic strategy that interrogates tumour antigen peptides presented by major histocompatibility complex (MHC) class I and class II molecules. We applied this approach to systematically characterize MHC ligands from 17 patients. Remarkably, all discovered neoantigenic peptides were exclusively derived from the lymphoma immunoglobulin heavy- or light-chain variable regions. Although we identified MHC presentation of private polymorphic germline alleles, no mutated peptides were recovered from non-immunoglobulin somatically mutated genes. Somatic mutations within the immunoglobulin variable region were almost exclusively presented by MHC class II. We isolated circulating CD4+ T cells specific for immunoglobulin-derived neoantigens and found these cells could mediate killing of autologous lymphoma cells. These results demonstrate that an integrative approach combining MHC isolation, peptide identification, and exome sequencing is an effective platform to uncover tumour neoantigens. Application of this strategy to human lymphoma implicates immunoglobulin neoantigens as targets for lymphoma immunotherapy.

Fingolimod-Conjugated Charge-Altering Releasable Transporters Efficiently and Specifically Deliver mRNA to Lymphocytes In Vivo and In Vitro.

Charge-altering releasable transporters (CARTs) are a class of oligonucleotide delivery vehicles shown to be effective for delivery of messenger RNA (mRNA) both in vitro and in vivo. Here, we exploited the chemical versatility of the CART synthesis to generate CARTs containing the small-molecule drug fingolimod (FTY720) as a strategy to increase mRNA delivery and expression in lymphocytes through a specific ligand-receptor interaction. Fingolimod is an FDA-approved small-molecule drug that, upon in vivo phosphorylation, binds to the sphingosine-1-phosphate receptor 1 (S1P1), which is highly expressed on lymphocytes. Compared to its non-fingolimod-conjugated analogue, the fingolimod-conjugated CART achieved superior transfection of activated human and murine T and B lymphocytes in vitro. The higher transfection of the fingolimod-conjugated CARTs was lost when cells were exposed to a free fingolimod before transfection. In vivo, the fingolimod-conjugated CART showed increased mRNA delivery to marginal zone B cells and NK cells in the spleen, relative to CARTs lacking fingolimod. Moreover, fingolimod-CART-mediated mRNA delivery induces peripheral blood T-cell depletion similar to free fingolimod. Thus, we show that functionalization of CARTs with a pharmacologically validated small molecule can increase transfection of a cellular population of interest while conferring some of the targeting properties of the conjugated small molecule to the CARTs.

Single-cell RNA-Seq of follicular lymphoma reveals malignant B-cell types and coexpression of T-cell immune checkpoints.

Follicular lymphoma (FL) is a low-grade B-cell malignancy that transforms into a highly aggressive and lethal disease at a rate of 2% per year. Perfect isolation of the malignant B-cell population from a surgical biopsy is a significant challenge, masking important FL biology, such as immune checkpoint coexpression patterns. To resolve the underlying transcriptional networks of follicular B-cell lymphomas, we analyzed the transcriptomes of 34 188 cells derived from 6 primary FL tumors. For each tumor, we identified normal immune subpopulations and malignant B cells, based on gene expression. We used multicolor flow cytometry analysis of the same tumors to confirm our assignments of cellular lineages and validate our predictions of expressed proteins. Comparison of gene expression between matched malignant and normal B cells from the same patient revealed tumor-specific features. Malignant B cells exhibited restricted immunoglobulin (Ig) light chain expression (either Igκ or Igλ), as well the expected upregulation of the BCL2 gene, but also downregulation of the FCER2, CD52, and major histocompatibility complex class II genes. By analyzing thousands of individual cells per patient tumor, we identified the mosaic of malignant B-cell subclones that coexist within a FL and examined the characteristics of tumor-infiltrating T cells. We identified genes coexpressed with immune checkpoint molecules, such as CEBPA and B2M in regulatory T (Treg) cells, providing a better understanding of the gene networks involved in immune regulation. In summary, parallel measurement of single-cell expression in thousands of tumor cells and tumor-infiltrating lymphocytes can be used to obtain a systems-level view of the tumor microenvironment and identify new avenues for therapeutic development.

Distinct patterns of B-cell receptor signaling in non-Hodgkin lymphomas identified by single-cell profiling.

Kinases downstream of B-cell antigen receptor (BCR) represent attractive targets for therapy in non-Hodgkin lymphoma (NHL). As clinical responses vary, improved knowledge regarding activation and regulation of BCR signaling in individual patients is needed. Here, using phosphospecific flow cytometry to obtain malignant B-cell signaling profiles from 95 patients representing 4 types of NHL revealed a striking contrast between chronic lymphocytic leukemia (CLL) and mantle cell lymphoma (MCL) tumors. Lymphoma cells from diffuse large B-cell lymphoma patients had high basal phosphorylation levels of most measured signaling nodes, whereas follicular lymphoma cells represented the opposite pattern with no or very low basal levels. MCL showed large interpatient variability in basal levels, and elevated levels for the phosphorylated forms of AKT, extracellular signal-regulated kinase, p38, STAT1, and STAT5 were associated with poor outcome. CLL tumors had elevated basal levels for the phosphorylated forms of BCR-signaling nodes (Src family tyrosine kinase, spleen tyrosine kinase [SYK], phospholipase Cγ), but had low α-BCR-induced signaling. This contrasted MCL tumors, where α-BCR-induced signaling was variable, but significantly potentiated as compared with the other types. Overexpression of CD79B, combined with a gating strategy whereby signaling output was directly quantified per cell as a function of CD79B levels, confirmed a direct relationship between surface CD79B, immunoglobulin M (IgM), and IgM-induced signaling levels. Furthermore, α-BCR-induced signaling strength was variable across patient samples and correlated with BCR subunit CD79B expression, but was inversely correlated with susceptibility to Bruton tyrosine kinase (BTK) and SYK inhibitors in MCL. These individual differences in BCR levels and signaling might relate to differences in therapy responses to BCR-pathway inhibitors.

T-Cell Immunopeptidomes Reveal Cell Subtype Surface Markers Derived From Intracellular Proteins.

Immunopeptidomes promise novel surface markers as ideal immunotherapy targets, but their characterization by mass spectrometry (MS) remains challenging. Until recently, cell numbers exceeding 109 were needed to survey thousands of HLA ligands. Such limited analytical sensitivity has historically constrained the types of clinical specimens that can be evaluated to cell cultures or bulk tissues. Measuring immunopeptidomes from purified cell subpopulations would be preferable for many applications, particularly those evaluating rare, primary hematopoietic cell lineages. Here, we test the feasibility of immunopeptidome profiling from limited numbers of primary purified human regulatory T cells (TReg ), conventional T cells (Tconv ), and activated T cells. The combined T cell immunopeptide dataset reported here contains 13 804 unique HLA ligands derived from 5049 proteins. Of these, more than 700 HLA ligands were derived from 82 proteins that we exclusively identified from TReg -enriched cells. This study 1) demonstrates that primary, lineage-enriched T cell subpopulations recovered from single donors are compatible with immunopeptidome analysis; 2) presents new TReg -biased ligand candidates; and 3) supports immunopeptidome surveys' value for revealing T cell biology that may not be apparent from expression data alone. Taken together, these findings open up new avenues for targeting TReg and abrogating their suppressive functions to treat cancer.

Therapeutic antitumor immunity by checkpoint blockade is enhanced by ibrutinib, an inhibitor of both BTK and ITK.

Monoclonal antibodies can block cellular interactions that negatively regulate T-cell immune responses, such as CD80/CTLA-4 and PD-1/PD1-L, amplifying preexisting immunity and thereby evoking antitumor immune responses. Ibrutinib, an approved therapy for B-cell malignancies, is a covalent inhibitor of BTK, a member of the B-cell receptor (BCR) signaling pathway, which is critical to the survival of malignant B cells. Interestingly this drug also inhibits ITK, an essential enzyme in Th2 T cells and by doing so it can shift the balance between Th1 and Th2 T cells and potentially enhance antitumor immune responses. Here we report that the combination of anti-PD-L1 antibody and ibrutinib suppresses tumor growth in mouse models of lymphoma that are intrinsically insensitive to ibrutinib. The combined effect of these two agents was also documented for models of solid tumors, such as triple negative breast cancer and colon cancer. The enhanced therapeutic activity of PD-L1 blockade by ibrutinib was accompanied by enhanced antitumor T-cell immune responses. These preclinical results suggest that the combination of PD1/PD1-L blockade and ibrutinib should be tested in the clinic for the therapy not only of lymphoma but also in other hematologic malignancies and solid tumors that do not even express BTK.

Ibrutinib enhances the antitumor immune response induced by intratumoral injection of a TLR9 ligand in mouse lymphoma.

We have designed a novel therapeutic approach for lymphoma that combines targeted kinase inhibition with in situ vaccination. Intratumoral injection of an unmethylated cytosine guanine dinucleotide (CpG)-enriched oligodeoxynucleotide, an agonist for the toll-like receptor 9 (TLR9), induces the activation of natural killer cells, macrophages, and antigen presenting cells that control tumor growth at the local site. Ibrutinib, an irreversible inhibitor of Bruton's tyrosine kinase, a key enzyme in the signaling pathway downstream of B-cell receptor, is an effective treatment against many types of B-cell lymphomas. The combination of intratumoral injection of CpG with systemic treatment by ibrutinib resulted in eradication of the tumors not only in the injected site, but also at distant sites. Surprisingly, this combinatorial antitumor effect required an intact T-cell immune system since it did not occur in nude, severe combined immunodeficiency, or T-cell depleted mice. Moreover, T cells from animals treated with intratumoral CpG and ibrutinib prevented the outgrowth of newly injected tumors. This result suggests that ibrutinib can induce immunogenic cell death of lymphoma cells and that concomitant stimulation of antigen-presenting cells in the tumor microenvironment by toll-like receptor ligands can lead to a powerful systemic antitumor immune response.

Anti-KIR antibody enhancement of anti-lymphoma activity of natural killer cells as monotherapy and in combination with anti-CD20 antibodies.

Natural killer (NK) cells mediate antilymphoma activity by spontaneous cytotoxicity and antibody-dependent cell-mediated cytotoxicity (ADCC) when triggered by rituximab, an anti-CD20 monoclonal antibody (mAb) used to treat patients with B-cell lymphomas. The balance of inhibitory and activating signals determines the magnitude of the efficacy of NK cells by spontaneous cytotoxicity. Here, using a killer-cell immunoglobulin-like receptor (KIR) transgenic murine model, we show that blockade of the interface of inhibitory KIRs with major histocompatibility complex (MHC) class I antigens on lymphoma cells by anti-KIR antibodies prevents a tolerogenic interaction and augments NK-cell spontaneous cytotoxicity. In combination with anti-CD20 mAbs, anti-KIR treatment induces enhanced NK-cell-mediated, rituximab-dependent cytotoxicity against lymphoma in vitro and in vivo in KIR transgenic and syngeneic murine lymphoma models. These results support a therapeutic strategy of combination rituximab and KIR blockade through lirilumab, illustrating the potential efficacy of combining a tumor-targeting therapy with an NK-cell agonist, thus stimulating the postrituximab antilymphoma immune response.

High PD-1 expression and suppressed cytokine signaling distinguish T cells infiltrating follicular lymphoma tumors from peripheral T cells.

Defects in T-cell function in patients with cancer might influence their capacity to mount efficient antitumor immune responses. Here, we identified highly reduced IL-4-, IL-10-, and IL-21-induced phosphorylation of STAT6 and STAT3 in tumor-infiltrating T cells (TILs) in follicular lymphoma (FL) tumors, contrasting other non-Hodgkin lymphoma TILs. By combining phospho-protein-specific flow cytometry with several T-cell markers, we identified that CD4(+)CD45RO(+)CD62L(-) FL TILs were largely nonresponsive to cytokines, in contrast to the corresponding autologous peripheral blood subset. We observed differential expression of the inhibitory receptor PD-1 in FL TILs and peripheral blood T cells. Furthermore, CD4(+)PD-1(hi) FL TILs, containing T(FH) and non-T(FH) cells, had lost their cytokine responsiveness, whereas PD-1 TILs had normal cytokine signaling. However, this phenomenon was not tumor specific, because tonsil T cells were similar to FL TILs. FL tumor cells were negative for PD-1 ligands, but PD-L1(+) histiocytes were found within the T cell-rich zone of the neoplastic follicles. Disruption of the microenvironment and in vitro culture of FL TILs could restore cytokine signaling in the PD-1(hi) subset. Because FL TILs in vivo probably receive suppressive signals through PD-1, this provides a rationale for testing PD-1 Ab in combination with immunotherapy in patients with FL.

In situ vaccination against mycosis fungoides by intratumoral injection of a TLR9 agonist combined with radiation: a phase 1/2 study.

We have developed and previously reported on a therapeutic vaccination strategy for indolent B-cell lymphoma that combines local radiation to enhance tumor immunogenicity with the injection into the tumor of a TLR9 agonist. As a result, antitumor CD8(+) T cells are induced, and systemic tumor regression was documented. Because the vaccination occurs in situ, there is no need to manufacture a vaccine product. We have now explored this strategy in a second disease: mycosis fungoides (MF). We treated 15 patients. Clinical responses were assessed at the distant, untreated sites as a measure of systemic antitumor activity. Five clinically meaningful responses were observed. The procedure was well tolerated and adverse effects consisted mostly of mild and transient injection site or flu-like symptoms. The immunized sites showed a significant reduction of CD25(+), Foxp3(+) T cells that could be either MF cells or tissue regulatory T cells and a similar reduction in S100(+), CD1a(+) dendritic cells. There was a trend toward greater reduction of CD25(+) T cells and skin dendritic cells in clinical responders versus nonresponders. Our in situ vaccination strategy is feasible also in MF and the clinical responses that occurred in a subset of patients warrant further study with modifications to augment these therapeutic effects. This study is registered at www.clinicaltrials.gov as NCT00226993.

A clinical trial of therapeutic vaccination in lymphoma with serial tumor sampling and single-cell analysis.

ABSTRACT: In situ vaccination (ISV) triggers an immune response to tumor-associated antigens at 1 tumor site, which can then tackle the disease throughout the body. Here, we report clinical and biological results of a phase 1/2 ISV trial in patients with low-grade lymphoma, combining an intratumoral toll-like receptor 9 (TLR9) agonist with local low-dose radiation and ibrutinib (an inhibitor of B- and T-cell kinases). Adverse events were predominately low grade. The overall response rate was 50%, including 1 complete response. All patients experienced tumor reduction at distant sites. Single-cell analyses of serial fine needle aspirates from injected and uninjected tumors revealed correlates of clinical response, such as lower CD47 and higher major histocompatibility complex class II expression on tumor cells, enhanced T-cell and natural killer cell effector function, and reduced immune suppression from transforming growth factor ß and inhibitory T regulatory 1 cells. Although changes at the local injected site were more pronounced, changes at distant uninjected sites were more often associated with clinical responses. Functional immune response assays and tracking of T-cell receptor sequences provided evidence of treatment-induced tumor-specific T-cell responses. Induction of immune effectors and reversal of negative regulators were both important in producing clinically meaningful tumor responses. The trial was registered at www.clinicaltrials.gov as #NCT02927964.

CD137 is expressed in follicular dendritic cell tumors and in classical Hodgkin and T-cell lymphomas: diagnostic and therapeutic implications.

CD137 (also known as 4-1BB and TNFRSF9) is a member of the tumor necrosis factor receptor superfamily. Originally identified as a costimulatory molecule expressed by activated T cells and NK cells, CD137 is also expressed by follicular dendritic cells, monocytes, mast cells, granulocytes, and endothelial cells. Anti-CD137 immunotherapy has recently shown promise as a treatment for solid tumors and lymphoid malignancies in preclinical models. We defined the expression of CD137 protein in both normal and neoplastic hematolymphoid tissue. CD137 protein is expressed by follicular dendritic cells in the germinal center and scattered paracortical T cells, but not by normal germinal-center B cells, bone marrow progenitor cells, or maturing thymocytes. CD137 protein is expressed by a select group of hematolymphoid tumors, including classical Hodgkin lymphoma, T-cell and NK/T-cell lymphomas, and follicular dendritic cells neoplasms. CD137 is a novel diagnostic marker of these tumors and suggests a possible target for tumor-directed antibody therapy.

A CpG-loaded tumor cell vaccine induces antitumor CD4+ T cells that are effective in adoptive therapy for large and established tumors.

We designed a whole tumor cell vaccine by "loading" lymphoma tumor cells with CG-enriched oligodeoxynucleotide (CpG), a ligand for the Toll-like receptor 9 (TLR9). CpG-loaded tumor cells were phagocytosed, delivering both tumor antigen(s) and the immunostimulatory CpG molecule to antigen-presenting cells (APCs). These APCs then expressed increased levels of costimulatory molecules and induced T-cell immunity. TLR9 was required in the APCs but not in the CpG-loaded tumor cell. We demonstrate that T cells induced by this vaccine are effective in adoptive cellular therapy for lymphoma. T cells from vaccinated mice transferred into irradiated, syngeneic recipients protected against subsequent lymphoma challenge and, remarkably, led to regression of large and established tumors. This therapeutic effect could be transferred by CD4(+) but not by CD8(+) T cells. A CpG-loaded whole-cell vaccination is practical and has strong potential for translation to the clinical setting. It is currently being tested in a clinical trial of adoptive immunotherapy for mantle-cell lymphoma.

Donor immunization with WT1 peptide augments antileukemic activity after MHC-matched bone marrow transplantation.

The curative potential of MHC-matched allogeneic bone marrow transplantation (BMT) is in part because of immunologic graft-versus-tumor (GvT) reactions mediated by donor T cells that recognize host minor histocompatibility antigens. Immunization with leukemia-associated antigens, such as Wilms Tumor 1 (WT1) peptides, induces a T-cell population that is tumor antigen specific. We determined whether allogeneic BMT combined with immunotherapy using WT1 peptide vaccination of donors induced more potent antitumor activity than either therapy alone. WT1 peptide vaccinations of healthy donor mice induced CD8(+) T cells that were specifically reactive to WT1-expressing FBL3 leukemia cells. We found that peptide immunization was effective as a prophylactic vaccination before tumor challenge, yet was ineffective as a therapeutic vaccination in tumor-bearing mice. BMT from vaccinated healthy MHC-matched donors, but not syngeneic donors, into recipient tumor-bearing mice was effective as a therapeutic maneuver and resulted in eradication of FBL3 leukemia. The transfer of total CD8(+) T cells from immunized donors was more effective than the transfer of WT1-tetramer(+)CD8(+) T cells and both required CD4(+) T-cell help for maximal antitumor activity. These findings show that WT1 peptide vaccination of donor mice can dramatically enhance GvT activity after MHC-matched allogeneic BMT.

CD137 stimulation enhances the antilymphoma activity of anti-CD20 antibodies.

Antibody-dependent cell-mediated cytotoxicity (ADCC), which is largely mediated by natural killer (NK) cells, is thought to play an important role in the efficacy of rituximab, an anti-CD20 monoclonal antibody (mAb) used to treat patients with B-cell lymphomas. CD137 is a costimulatory molecule expressed on a variety of immune cells after activation, including NK cells. In the present study, we show that an anti-CD137 agonistic mAb enhances the antilymphoma activity of rituximab by enhancing ADCC. Human NK cells up-regulate CD137 after encountering rituximab-coated tumor B cells, and subsequent stimulation of these NK cells with anti-CD137 mAb enhances rituximab-dependent cytotoxicity against the lymphoma cells. In a syngeneic murine lymphoma model and in a xenotransplanted human lymphoma model, sequential administration of anti-CD20 mAb followed by anti-CD137 mAb had potent antilymphoma activity in vivo. These results support a novel, sequential antibody approach against B-cell malignancies by targeting first the tumor and then the host immune system.