Matthew J. Ahearne

Enhancement of CD154/IL4 proliferation by the T follicular helper (Tfh) cytokine, IL21 and increased numbers of circulating cells resembling Tfh cells in chronic lymphocytic leukaemia.

Chronic lymphocytic leukaemia (CLL) cells encounter T‐cells and proliferate in response to T‐cell signals in the lymph node microenvironment. In this report we determined interleukin 21 (IL21) function in CLL and showed that IL21 and interleukin 4 (IL4) act co‐operatively to promote leukaemic cell proliferation without apoptosis or differentiation We further show that IL21 increased side population (SP) cells, which are associated with resistance to chemotherapy and increased self‐renewal capacity in CLL. IL21 and IL4 are the major cytokines produced by the recently described CD4+ T follicular helper (Tfh) cell subset. Determination of Tfh cells in peripheral blood showed that patients had significantly increased numbers as compared to normal subjects although no association was found between Tfh numbers and IGHV gene mutational status or clinical stage. Our data suggests that the Tfh cytokines, IL4 and IL21, contribute to driving leukaemic cell proliferation in the lymph node microenvironment, and may contribute to the specific production of cells resistant to conventional chemotherapy. We suggest that increased circulating Tfh cells is a component of T‐cell dysregulation in CLL. Our findings have implications for the therapeutic use of IL21.

The role of BCL6 in lymphomas and routes to therapy

BCL6 is a transcription factor that has essential B‐cell and T‐cell roles in normal antibody responses. It is involved in chromosomal translocations in diffuse large B‐cell lymphoma (DBCL; including primary mediastinal B‐cell lymphoma) and nodular lymphocyte predominant Hodgkin lymphoma, and is expressed in follicular lymphoma and Burkitts lymphoma. The neoplastic T‐cells of angioimmunoblastic T‐cell lymphoma also express BCL6. BCL6 prevents terminal B‐cell differentiation largely through repression of PRDM1. In the “cell of origin” classification of DLBCL BCL6 is associated with the germinal centre subtype, which carries a good response to modern treatments. More recently, specific BCL6 antagonists, including small molecule inhibitors, have been developed. These antagonists have demonstrated that DLBCL cells, in which BCL6 is transcriptionally active, are dependent on this gene for survival. BCL6 antagonists are active against primary DLBCL and may find future application in the treatment of lymphomas.

The role of BCL6 in lymphomas and routes to therapy.

BCL6 is a transcription factor that has essential B‐cell and T‐cell roles in normal antibody responses. It is involved in chromosomal translocations in diffuse large B‐cell lymphoma (DBCL; including primary mediastinal B‐cell lymphoma) and nodular lymphocyte predominant Hodgkin lymphoma, and is expressed in follicular lymphoma and Burkitts lymphoma. The neoplastic T‐cells of angioimmunoblastic T‐cell lymphoma also express BCL6. BCL6 prevents terminal B‐cell differentiation largely through repression of PRDM1. In the “cell of origin” classification of DLBCL BCL6 is associated with the germinal centre subtype, which carries a good response to modern treatments. More recently, specific BCL6 antagonists, including small molecule inhibitors, have been developed. These antagonists have demonstrated that DLBCL cells, in which BCL6 is transcriptionally active, are dependent on this gene for survival. BCL6 antagonists are active against primary DLBCL and may find future application in the treatment of lymphomas.

Follicular helper T-cells: expanding roles in T-cell lymphoma and targets for treatment

Follicular helper T‐cells (Tfh cells) are a subset of CD4+ T‐cells that are essential for normal production of high affinity antibodies. Tfh cells characteristically produce IL21 and IL4 and show high expression of surface markers CXCR5, ICOS, PDCD1 (PD‐1) and the chemokine CXCL13. In this review we will focus on the emerging links between Tfh cells and subtypes of T‐cell non‐Hodgkin lymphoma: angioimmunoblastic T‐cell lymphoma (AITL) and ~20% of peripheral T‐cell lymphoma not otherwise specified (PTCL‐NOS) have surface marker features of Tfh cells and share a spectrum of genetic abnormalities. The recurrent genetic abnormalities associated with AITL include mutations in epigenetic modifiers such as TET2 and DNMT3A and the motility and adhesion gene, RHOA, is mutated in up to 70% of cases. ~20% of PTCL‐NOS demonstrate RHOA mutations and have other characteristics suggesting an origin in Tfh cells. The recognition that specific genetic and surface markers are associated with malignant Tfh cells suggests that the next few years will bring major changes in diagnostic and treatment possibilities. For example, antibodies against IL21, PDCD1 and ICOS are already in clinical trials for autoimmune disease or other malignancies and antibodies against CXCL13 are in pre‐clinical development.

Cap-Translation Inhibitor, 4EGI-1, Restores Sensitivity to ABT-737 Apoptosis through Cap-Dependent and -Independent Mechanisms in Chronic Lymphocytic Leukemia

Purpose: The lymph node microenvironment promotes resistance to chemotherapy in chronic lymphocytic leukemia (CLL), partly through induction of BCL2 family prosurvival proteins. Currently available inhibitors do not target all BCL2 family prosurvival proteins and their effectiveness is also modified by proapoptotic BCL2 homology domain 3 (BH3) only protein expression. The goal of this study was to evaluate synergy between the eIF4E/eIF4G interaction inhibitor, 4EGI-1, and the BH3 mimetic, ABT-737. Experimental Design: CLL cells were cultured in conditions to mimic the lymph node microenvironment. Protein synthesis and cap-complex formation were determined. Polysome association of mRNAs from BCL2 family survival genes was analyzed by translational profiling. The effects of 4EGI-1 and the BCL2/BCL2L1 antagonist, ABT-737, on CLL cell apoptosis were determined. Results: Protein synthesis was increased approximately 6-fold by stromal cell/CD154 culture in a phosphoinositide 3-kinase α (PI3Kα)–specific manner and was reduced by 4EGI-1. PI3K inhibitors and 4EGI-1 also reduced cap-complex formation but only 4EGI-1 consistently reduced BCL2L1 and BCL2A1 protein levels. 4EGI-1, but not PI3K inhibitors or rapamycin, induced an endoplasmic reticulum stress response including proapoptotic NOXA and the translation inhibitor phosphorylated eIF2α. 4EGI-1 and ABT-737 synergized to cause apoptosis, independent of levels of prosurvival protein expression in individual patients. Conclusions: Overall protein synthesis and cap-complex formation are induced by microenvironment stimuli in CLL. Inhibition of the cap-complex was not sufficient to repress BCL2 family prosurvival expression, but 4EGI-1 inhibited BCL2A1 and BCL2L1 while inducing NOXA through cap-dependent and -independent mechanisms. 4EGI-1 and ABT-737 synergized to produce apoptosis, and these agents may be the basis for a therapeutically useful combination. Clin Cancer Res; 19(12); 3212–23. ©2013 AACR.

Circulating Tfh1 (cTfh1) cell numbers and PD1 expression are elevated in low-grade B-cell non-Hodgkin’s lymphoma and cTfh gene expression is perturbed in marginal zone lymphoma

CD4+ T-cell subsets are found in the tumour microenvironment (TME) of low-grade B-cell non-Hodgkin’s lymphomas such as marginal zone lymphoma (MZL) or follicular lymphoma (FL). Both numbers and architecture of activating follicular helper T-cells (Tfh) and suppressive Treg in the TME of FL are associated with clinical outcomes. There has been almost no previous work on CD4+ T-cells in MZL. It is now recognised that circulating CD4+CXCR5+ T-cells are the memory compartment of Tfh cells. We determined differences in number of circulating Tfh (cTfh) cells and cTfh subsets between normal subjects and patients with FL or MZL. Lymphoma patients showed increased numbers of cTfh1 and reduced cTfh17 cells due to decreased expression of the subset-defining marker CCR6 in patients. PD1, a surface marker associated with Tfh cells, showed increased expression on cTfh subsets in patients. Focusing on MZL we determined expression of 96 T-cell associated genes by microfluidic qRT-PCR. Analysis of differentially expressed genes showed significant differences between normal subjects and patients both for bulk cTfh (CCL4) and the cTfh1 subset (JAK3). While our findings require confirmation in larger studies we suggest that analysis of number and gene expression of circulating T-cells might be a source of clinically useful information as is the case for T-cells within lymphoma lymph nodes.

Comparison of interleukin-2-inducible kinase (ITK) inhibitors and potential for combination therapies for T-cell lymphoma

Patients with peripheral T-cell lymphomas generally have poor clinical outcomes with conventional chemotherapy. Recent advances have demonstrated that a large subgroup of PTCL are derived from follicular helper (Tfh) T-cells. These cases show a characteristic pattern of gene expression, which includes high-level protein expression of interleukin-2-inducible kinase (ITK). ITK is a member of the TEC family of kinases and normally has essential functions in regulating T-cell receptor signalling and T-cell differentiation. Here we report a side-by-side comparison of four ITK inhibitors. We investigate effects on apoptosis, phosphorylation of signaling molecules, calcium flux and migration. In line with a specific mechanism of action ONO7790500 and BMS509744 did not inhibit MEK1/2 or AKT phosphorylation although other ITK inhibitors, ibrutinib and PF-06465469, did have this effect. Specific ITKi had modest effects on apoptosis alone but there was definite synergy with doxorubicin, pictilisib (PI3Ki) and idelalisib (PI3Kδi). ITKi repressed migration of Jurkat cells caused by CXCL12 and the CXCR4 antagonist, plerixafor enhanced this effect. Overall ITKi may have several mechanisms of action that will be therapeutically useful in PTCL including reduction in survival and perturbation of trafficking.

Clonal evolution in the transition from cutaneous disease to acute leukaemia suggested by liquid biopsy in blastic plasmacytoid dendritic cell neoplasm

We have detected novel mutations in TET2 and RHOA genes in a case of blastic plasmacytoid dendritic cell neoplasm (BPDCN). Analysis of peripheral blood mononuclear cell (PBMNC) samples at two timepoints allowed inference of subclonal variation associated with the evolution of acute leukemia from

PD1 hi cells associate with clusters of proliferating B-cells in marginal zone lymphoma

BackgroundAbnormally sustained immune reactions drive B-cell proliferation in some cases of marginal zone lymphoma but the CD4+ T-cell subsets, which are likely to contribute to the B-cell responses in the tumour microenvironment, are not well characterised and neither has the spatial distribution of the different subsets in involved lymph nodes been investigated.MethodsEmploying a workflow of multiplex semi-automated immunohistochemistry combined with image processing we investigated association between infiltrating T-cells and proliferating lymphoma B-cells.ResultsBoth total numbers of activating follicular helper (Tfh) cells (defined by high expression of PD1) and suppressive regulatory (Treg) T-cells (defined by FOXP3+ expression) and the Tfh:Treg ratio, assessed over relatively large areas of tissue, varied among cases of marginal zone lymphoma. We determined spatial distribution and demonstrated that PD1hi cells showed significantly more clustering than did FOXP3+. To investigate the association of infiltrating T-cells with lymphoma B-cells we employed Pearson correlation and Morisita-Horn index, statistical measures of interaction. We demonstrated that PD1hi cells were associated with proliferating B-cells and confirmed this by nearest neighbour analysis.ConclusionsThe unexpected architectural complexity of T-cell infiltration in marginal zone lymphoma, revealed in this study, further supports a key role for Tfh cells in driving proliferation of lymphoma B-cells. We demonstrate the feasibility of digital analysis of spatial architecture of T-cells within marginal zone lymphoma and future studies will be needed to determine the clinical importance of these observations.