Xiao-Jie Yan

CD137 costimulatory T cell receptor engagement reverses acute disease in lupus-prone NZB × NZW F1 mice

Systemic lupus erythematosus (SLE) is a CD4(+) T cell-dependent, immune complex-mediated, autoimmune disease that primarily affects women of childbearing age. Generation of high-titer affinity-matured IgG autoantibodies, specific for double-stranded DNA and other nuclear antigens, coincides with disease progression. Current forms of treatment of SLE including glucocorticosteroids are often inadequate and induce severe side effects. Immunological approaches for treating SLE in mice using anti-CD4 mAbs or CTLA4-Ig and anti-CD154 mAbs have proven to be effective. However, like steroid treatment, these regimens induce global immunosuppression, and their withdrawal allows for disease progression. In this report we show that lupus-prone NZB x NZW F(1) mice given three injections of anti-CD137 (4-1BB) mAbs between 26 and 35 weeks of age reversed acute disease, blocked chronic disease, and extended the mices lifespan from 10 months to more than 2 years. Autoantibody production in recipients was rapidly suppressed without inducing immunosuppression. Successful treatment could be traced to the fact that NZB x NZW F(1) mice, regardless of their age or disease status, could not maintain pathogenic IgG autoantibody production in the absence of continuous CD4(+) T cell help. Our data support the hypothesis that CD137-mediated signaling anergized CD4(+) T cells during priming at the DC interface.

Chronic lymphocytic leukemia antibodies with a common stereotypic rearrangement recognize nonmuscle myosin heavy chain IIA

Leukemic B lymphocytes of a large group of unrelated chronic lymphocytic leukemia (CLL) patients express an unmutated heavy chain immunoglobulin variable (V) region encoded by IGHV1-69, IGHD3-16, and IGHJ3 with nearly identical heavy and light chain complementarity-determining region 3 sequences. The likelihood that these patients developed CLL clones with identical antibody V regions randomly is highly improbable and suggests selection by a common antigen. Monoclonal antibodies (mAbs) from this stereotypic subset strongly bind cytoplasmic structures in HEp-2 cells. Therefore, HEp-2 cell extracts were immunoprecipitated with recombinant stereotypic subset-specific CLL mAbs, revealing a major protein band at approximately 225 kDa that was identified by mass spectrometry as nonmuscle myosin heavy chain IIA (MYHIIA). Reactivity of the stereotypic mAbs with MYHIIA was confirmed by Western blot and immunofluorescence colocalization with anti-MYHIIA antibody. Treatments that alter MYHIIA amounts and cytoplasmic localization resulted in a corresponding change in binding to these mAbs. The appearance of MYHIIA on the surface of cells undergoing stress or apoptosis suggests that CLL mAb may generally bind molecules exposed as a consequence of these events. Binding of CLL mAb to MYHIIA could promote the development, survival, and expansion of these leukemic cells.

A novel adoptive transfer model of chronic lymphocytic leukemia suggests a key role for T lymphocytes in the disease.

Chronic lymphocytic leukemia (CLL) is an incurable adult disease of unknown etiology. Understanding the biology of CLL cells, particularly cell maturation and growth in vivo, has been impeded by lack of a reproducible adoptive transfer model. We report a simple, reproducible system in which primary CLL cells proliferate in nonobese diabetes/severe combined immunodeficiency/γc(null) mice under the influence of activated CLL-derived T lymphocytes. By co-transferring autologous T lymphocytes, activated in vivo by alloantigens, the survival and growth of primary CFSE-labeled CLL cells in vivo is achieved and quantified. Using this approach, we have identified key roles for CD4(+) T cells in CLL expansion, a direct link between CD38 expression by leukemic B cells and their activation, and support for CLL cells preferentially proliferating in secondary lymphoid tissues. The model should simplify analyzing kinetics of CLL cells in vivo, deciphering involvement of nonleukemic elements and nongenetic factors promoting CLL cell growth, identifying and characterizing potential leukemic stem cells, and permitting preclinical studies of novel therapeutics. Because autologous activated T lymphocytes are 2-edged swords, generating unwanted graph-versus-host and possibly autologous antitumor reactions, the model may also facilitate analyses of T-cell populations involved in immune surveillance relevant to hematopoietic transplantation and tumor cytoxicity.

Chronic Lymphocytic Leukemia B Cells Can Undergo Somatic Hypermutation and Intraclonal Immunoglobulin VHDJH Gene Diversification

Chronic lymphocytic leukemia (CLL) arises from the clonal expansion of a CD5+ B lymphocyte that is thought not to undergo intraclonal diversification. Using VHDJH cDNA single strand conformation polymorphism analyses, we detected intraclonal mobility variants in 11 of 18 CLL cases. cDNA sequence analyses indicated that these variants represented unique point-mutations (1–35/patient). In nine cases, these mutations were unique to individual submembers of the CLL clone, although in two cases they occurred in a large percentage of the clonal submembers and genealogical trees could be identified. The diversification process responsible for these changes led to single nucleotide changes that favored transitions over transversions, but did not target A nucleotides and did not have the replacement/silent nucleotide change characteristics of antigen-selected B cells. Intraclonal diversification did not correlate with the original mutational load of an individual CLL case in that diversification was as frequent in CLL cells with little or no somatic mutations as in those with considerable mutations. Finally, CLL B cells that did not exhibit intraclonal diversification in vivo could be induced to mutate their VHDJH genes in vitro after stimulation. These data indicate that a somatic mutation mechanism remains functional in CLL cells and could play a role in the evolution of the clone.

Anti-CD20/CD3 T cell–dependent bispecific antibody for the treatment of B cell malignancies

Anti-CD20/CD3 T cell–dependent bispecific antibodies may be useful for the treatment of B cell malignancies. Two-headed cancer therapy Immunotherapeutic approaches harness either humoral (antibody-mediated) or cellular (T cell–mediated) immunity to fight cancer. Sun et al. combine these approaches by designing a CD3/CD20 TDB (T cell–dependent bispecific), a dual-targeted antibody that recruits T cells to CD20-expressing cells. Their humanized TDB induces T cells to kill primary patient leukemia and lymphoma cells both in vitro and in a mouse model and can deplete CD20-expressing B cells in a macaque model with similar properties as conventional antibodies. If these data hold true in clinical studies, this CD20/CD3 TDB could add to our expanding arsenal of cancer immunotherapeutics. Bispecific antibodies and antibody fragments in various formats have been explored as a means to recruit cytolytic T cells to kill tumor cells. Encouraging clinical data have been reported with molecules such as the anti-CD19/CD3 bispecific T cell engager (BiTE) blinatumomab. However, the clinical use of many reported T cell–recruiting bispecific modalities is limited by liabilities including unfavorable pharmacokinetics, potential immunogenicity, and manufacturing challenges. We describe a B cell–targeting anti-CD20/CD3 T cell–dependent bispecific antibody (CD20-TDB), which is a full-length, humanized immunoglobulin G1 molecule with near-native antibody architecture constructed using “knobs-into-holes” technology. CD20-TDB is highly active in killing CD20-expressing B cells, including primary patient leukemia and lymphoma cells both in vitro and in vivo. In cynomolgus monkeys, CD20-TDB potently depletes B cells in peripheral blood and lymphoid tissues at a single dose of 1 mg/kg while demonstrating pharmacokinetic properties similar to those of conventional monoclonal antibodies. CD20-TDB also exhibits activity in vitro and in vivo in the presence of competing CD20-targeting antibodies. These data provide rationale for the clinical testing of CD20-TDB for the treatment of CD20-expressing B cell malignancies.

Functional loss of IκBε leads to NF-κB deregulation in aggressive chronic lymphocytic leukemia

Mansouri et al. applied targeted deep sequencing to identify mutations within NF-κB core complex genes in CLL. NFKBIE, the gene encoding the inhibitory IκBε molecule, was most frequently mutated, especially in poor-prognostic subgroups of CLL. The authors show that NFKBIE mutations were associated with significantly reduced IkBε expression and p65 inhibition, ultimately leading to NF-κB activation and a more aggressive disease.

B Cell Proliferation, Somatic Hypermutation, Class Switch Recombination, and Autoantibody Production in Ectopic Lymphoid Tissue in Murine Lupus

Intraperitoneal exposure of nonautoimmune mice to 2,6,10,14-tetramethylpentadecane (TMPD) causes lupus and the formation of ectopic lymphoid tissue. Although associated with humoral autoimmunity, it is not known whether Ab responses develop within ectopic lymphoid tissue or if B cells only secondarily migrate there. We show that ectopic lymphoid tissue induced by TMPD not only resembles secondary lymphoid tissue morphologically, but it also displays characteristics of germinal center reactions. Proliferating T and B lymphocytes were found within ectopic lymphoid tissue, activation-induced cytidine deaminase was expressed, and class-switched B cells were present. The presence of circular DNA intermediates, a hallmark of active class switch recombination, suggested that class switching occurs within the ectopic lymphoid tissue. Individual collections of ectopic lymphoid tissue (“lipogranulomas”) from the same mouse contained different B cell repertoires, consistent with local germinal center-like reactions. Class-switched anti-RNP autoantibody-producing cells were also found in the lipogranulomas. Somatic hypermutation in the lipogranulomas was T cell-dependent, as was the production of isotype-switched anti-Sm/RNP autoantibodies. Thus, ectopic lymphoid tissue induced by TMPD recapitulates many of the functional characteristics of secondary lymphoid tissue and contains autoantibody-secreting cells, which may escape from normal censoring mechanisms in this location.

Not all IGHV3-21 chronic lymphocytic leukemias are equal: Prognostic considerations

An unresolved issue in chronic lymphocytic leukemia (CLL) is whether IGHV3-21 gene usage, in general, or the expression of stereotyped B-cell receptor immunoglobulin defining subset #2 (IGHV3-21/IGLV3-21), in particular, determines outcome for IGHV3-21-utilizing cases. We reappraised this issue in 8593 CLL patients of whom 437 (5%) used the IGHV3-21 gene with 254/437 (58%) classified as subset #2. Within subset #2, immunoglobulin heavy variable (IGHV)-mutated cases predominated, whereas non-subset #2/IGHV3-21 was enriched for IGHV-unmutated cases (P = .002). Subset #2 exhibited significantly shorter time-to-first-treatment (TTFT) compared with non-subset #2/IGHV3-21 (22 vs 60 months, P = .001). No such difference was observed between non-subset #2/IGHV3-21 vs the remaining CLL with similar IGHV mutational status. In conclusion, IGHV3-21 CLL should not be axiomatically considered a homogeneous entity with adverse prognosis, given that only subset #2 emerges as uniformly aggressive, contrasting non-subset #2/IGVH3-21 patients whose prognosis depends on IGHV mutational status as the remaining CLL.

Autoantigen can promote progression to a more aggressive TCL1 leukemia by selecting variants with enhanced B-cell receptor signaling

Significance These studies indicate that autoantigen-reactivity plays a role in the progression of a murine leukemia that models human chronic lymphocytic leukemia. This indication is consistent with the notion that chronic lymphocytic leukemia evolves by selection of normal B cells that bind autoantigen via the B-cell antigen receptor. (Auto)antigen engagement by the B-cell receptor (BCR) and possibly the sites where this occurs influence the outcome of chronic lymphocytic leukemia (CLL). To test if selection for autoreactivity leads to increased aggressiveness and if this selection plays out equally in primary and secondary tissues, we used T-cell leukemia (TCL)1 cells reactive with the autoantigen phosphatidylcholine (PtC). After repeated transfers of splenic lymphocytes from a single mouse with oligoclonal PtC-reactive cells, outgrowth of cells expressing a single IGHV-D-J rearrangement and superior PtC-binding and disease virulence occurred. In secondary tissues, increased PtC-binding correlated with enhanced BCR signaling and cell proliferation, whereas reduced signaling and division of cells from the same clone was documented in cells residing in the bone marrow, blood, and peritoneum, even though cells from the last site had highest surface membrane IgM density. Gene-expression analyses revealed reciprocal changes of genes involved in BCR-, CD40-, and PI3K-signaling between splenic and peritoneal cells. Our results suggest autoantigen-stimulated BCR signaling in secondary tissues promotes selection, expansion, and disease progression by activating pro-oncogenic signaling pathways, and that—outside secondary lymphoid tissues—clonal evolution is retarded by diminished BCR-signaling. This transferrable, antigenic-specific murine B-cell clone (TCL1-192) provides a platform to study the types and sites of antigen-BCR interactions and genetic alterations that result and may have relevance to patients.

Common nonmutational NOTCH1 activation in chronic lymphocytic leukemia

Significance A pathogenetic role of NOTCH1 in chronic lymphocytic leukemia (CLL) has been implied by the presence of deregulating mutations in a relatively small fraction of cases. Our results now indicate that ∼50% of CLL cases devoid of mutations express the active form of NOTCH1 ICN1 (intracellular portion of NOTCH1), thus implicating a much broader role of this transcription factor in the disease. ICN1+ CLL cases display equivalent NOTCH1-dependent transcriptional responses regardless of the gene mutation status, indicating that the detection of ICN1 represents a reliable biomarker of NOTCH1 activation for diagnostic and therapeutic targeting. Finally, our results identify the NOTCH1-dependent transcriptional program in CLL cells, thus providing direct insights into the pathogenesis of a large fraction of CLL cases. Activating mutations of NOTCH1 (a well-known oncogene in T-cell acute lymphoblastic leukemia) are present in ∼4–13% of chronic lymphocytic leukemia (CLL) cases, where they are associated with disease progression and chemorefractoriness. However, the specific role of NOTCH1 in leukemogenesis remains to be established. Here, we report that the active intracellular portion of NOTCH1 (ICN1) is detectable in ∼50% of peripheral blood CLL cases lacking gene mutations. We identify a “NOTCH1 gene-expression signature” in CLL cells, and show that this signature is significantly enriched in primary CLL cases expressing ICN1, independent of NOTCH1 mutation. NOTCH1 target genes include key regulators of B-cell proliferation, survival, and signal transduction. In particular, we show that NOTCH1 transactivates MYC via binding to B-cell–specific regulatory elements, thus implicating this oncogene in CLL development. These results significantly extend the role of NOTCH1 in CLL pathogenesis, and have direct implications for specific therapeutic targeting.