The entire DNA sequence of chromosome III of the yeast Saccharomyces cerevisiae has been determined. This is the first complete sequence analysis of an entire chromosome from any organism. The 315-kilobase sequence reveals 182 open reading frames for proteins longer than 100 amino acids, of which 37 correspond to known genes and 29 more show some similarity to sequences in databases. Of 55 new open reading frames analysed by gene disruption, three are essential genes; of 42 non-essential genes that were tested, 14 show some discernible effect on phenotype and the remaining 28 have no overt function.

Checkpoint blockade can reverse T-cell exhaustion and promote antitumor responses. Although blocking the PD-1 pathway has been successful in Hodgkin lymphoma, response rates have been modest in B-cell non-Hodgkin lymphoma (NHL). Coblockade of checkpoint receptors may therefore be necessary to optimize antitumor T-cell responses. Here, characterization of coinhibitory receptor expression in intratumoral T cells from different NHL types identified TIGIT and PD-1 as frequently expressed coinhibitory receptors. Tumors from NHL patients were enriched in CD8+ and CD4+ T effector memory cells that displayed high coexpression of TIGIT and PD-1, and coexpression of these checkpoint receptors identified T cells with reduced production of IFNγ, TNFα, and IL2. The suppressed cytokine production could be improved upon in vitro culture in the absence of ligands. Whereas PD-L1 was expressed by macrophages, the TIGIT ligands CD155 and CD112 were expressed by lymphoma cells in 39% and 50% of DLBCL cases and in some mantle cell lymphoma cases, as well as by endothelium and follicular dendritic cells in all NHLs investigated. Collectively, our results show that TIGIT and PD-1 mark dysfunctional T cells and suggest that TIGIT and PD-1 coblockade should be further explored to elicit potent antitumor responses in patients with NHL.

T cells infiltrating follicular lymphoma (FL) tumors are considered dysfunctional, yet the optimal target for immune checkpoint blockade is unknown. Characterizing coinhibitory receptor expression patterns and signaling responses in FL T-cell subsets might reveal new therapeutic targets. Surface expression of 9 coinhibitory receptors governing T-cell function was characterized in T-cell subsets from FL lymph node tumors and from healthy donor tonsils and peripheral blood samples, using high-dimensional flow cytometry. The results were integrated with T-cell receptor (TCR)-induced signaling and cytokine production. Expression of T-cell immunoglobulin and ITIM domain (TIGIT) ligands was detected by immunohistochemistry. TIGIT was a frequently expressed coinhibitory receptor in FL, expressed by the majority of CD8 T effector memory cells, which commonly coexpressed exhaustion markers such as PD-1 and CD244. CD8 FL T cells demonstrated highly reduced TCR-induced phosphorylation (p) of ERK and reduced production of IFNγ, while TCR proximal signaling (p-CD3ζ, p-SLP76) was not affected. The TIGIT ligands CD112 and CD155 were expressed by follicular dendritic cells in the tumor microenvironment. Dysfunctional TCR signaling correlated with TIGIT expression in FL CD8 T cells and could be fully restored upon culture. The costimulatory receptor CD226 was downregulated in TIGIT compared with TIGIT CD8 FL T cells, further skewing the balance toward immunosuppression. TIGIT blockade is a relevant strategy for improved immunotherapy in FL. A deeper understanding of the interplay between coinhibitory receptors and key T-cell signaling events can further assist in engineering immunotherapeutic regimens to improve clinical outcomes of cancer patients. .

T cells modified to express chimeric antigen receptor (CAR) targeting CD19 (CD19CAR) have produced remarkable clinical responses in patients with relapsed/refractory B-cell acute lymphoblastic leukemia. CD19CAR T-cell therapy has also demonstrated prominent effects in B-cell non-Hodgkin lymphoma (B-NHL) patients. However, a subset of patients who relapse after CD19CAR T-cell therapy have outgrowth of CD19− tumor cells. Hence, development of alternative CARs targeting other B-cell markers represents an unmet medical need for B-cell acute lymphoblastic leukemia and B-NHL. Here, we confirmed previous data by showing that, overall, B-NHL has high expression of CD37. A second-generation CD37CAR was designed, and its efficacy in T cells was compared with that of CD19CAR. In vitro assessment of cytotoxicity and T-cell function upon coculture of the CAR T cells with different target B-cell lymphoma cell lines demonstrated comparable efficacy between the 2 CARs. In an aggressive B-cell lymphoma xenograft model, CD37CAR T cells were as potent as CD19CAR T cells in controlling tumor growth. In a second xenograft model, using U2932 lymphoma cells containing a CD19− subpopulation, CD37CAR T cells efficiently controlled tumor growth and prolonged survival, whereas CD19CAR T cells had limited effect. We further show that, unlike CD19CAR, CD37CAR was not sensitive to antigen masking. Finally, CD37CAR reactivity was restricted to B-lineage cells. Collectively, our results demonstrated that CD37CAR T cells also can effectively eradicate B-cell lymphoma tumors when CD19 antigen expression is lost and support further clinical testing for patients with relapsed/refractory B-NHL.

BackgroundCytokines of the transforming growth factor β (TGF-β) superfamily exert effects on proliferation, apoptosis and differentiation in various cell types. Cancer cells frequently acquire resistance to the anti-proliferative signals of TGF-β, which can be due to mutations in proteins of the signalling cascade. We compared the TGF-β-related signalling properties in B-cell lymphoma cell lines that were sensitive or resistant to TGF-β-induced anti-proliferative effects.ResultsTGF-β sensitive cell lines expressed higher cell surface levels of the activin receptor-like kinase 5 (Alk-5), a TGF-β receptor type 1. The expression levels of the other TGF-β and bone morphogenetic protein receptors were comparable in the different cell lines. TGF-β-induced phosphorylation of Smad2 was similar in TGF-β sensitive and resistant cell lines. In contrast, activation of Smad1/5 was restricted to cells that were sensitive to growth inhibition by TGF-β. Moreover, with activin A we detected limited anti-proliferative effects, strong phosphorylation of Smad2, but no Smad1/5 phosphorylation. Up-regulation of the TGF-β target genes Id1 and Pai-1 was identified in the TGF-β sensitive cell lines. Constitutive phosphorylation of MAPK p38 was restricted to the TGF-β sensitive cell lines. Inhibition of p38 MAPK led to reduced sensitivity to TGF-β.ConclusionsWe suggest that phosphorylation of Smad1/5 is important for the anti-proliferative effects of TGF-β in B-cell lymphoma. Alk-5 was highly expressed in the sensitive cell lines, and might be important for signalling through Smad1/5. Our results indicate a role for p38 MAPK in the regulation of TGF-β-induced anti-proliferative effects.

Bone morphogenetic proteins (BMP) are multifunctional cytokines that belong to the TGF-b superfamily. BMP have been shown to regulate haematopoietic stem cells, B lymphopoiesis and early thymocyte differentiation. In the present study we explored the role of BMP-6 in Jurkat TAg cells. BMP-6 rapidly induced phosphorylation of Smad1/5/8, p38 and ERK1/2, followed by a potent up-regulation of ID1, ID2 and ID3. ID1 and ID3 were also induced at the protein level. Genome-wide expression profiling of cells treated with BMP-6 compared to medium confirmed that ID1-ID3 were target genes of BMP-6 together with Noggin and Smad6. Furthermore, several genes involved in transcriptional regulation were also identified, including NFKBIA, HEY1, DLX2, KLF10 and early growth response 1. Stimulation with BMP-6 exerted an antiproliferative effect that was counteracted by inhibitor of DNA binding (Id)1 siRNA, indicating that Id1 is an important downstream mediator in Jurkat TAg cells. A subset of CD4 + T cells were found to express the BMP receptors Alk-2 and Alk-3 (type I), in addition to BMPRII (type II). BMP-6 also induced phosphorylation of Smad1/5/8, followed by transcriptional increase in ID1-ID3 mRNA expression. However, we did not observe significant changes in Id protein expression in CD4 + T cells. Altogether, the data indicate a role for BMP-6 in human T lineage cells.

Differences in the quality of B-cell antigen receptor (BCR) signaling control key steps of B cell maturation and differentiation. Endogenously produced H2O2 is thought to fine tune the level of BCR signaling by reversibly inhibiting phosphatases. However, relatively little is known about how B cells at different stages sense and respond to such redox cues. Here, we used phospho-specific flow cytometry and high-dimensional mass cytometry (CyTOF) to compare BCR signaling responses in mature human tonsillar B cells undergoing germinal center (GC) reactions. GC B cells, in contrast to mature naïve B cells, memory B cells, and plasmablasts, were hypersensitive to a range of H2O2 concentrations and responded by phosphorylating SYK and other membrane proximal BCR effectors in the absence of BCR engagement. These findings reveal that stage specific redox responses distinguish human GC B cells.

Background Follicular lymphoma (FL) is an indolent non-Hodgkin lymphoma that has a risk of transformation to more aggressive lymphoma. Relatively little is known about the non-malignant B-cell and T-cell subset composition within the tumor microenvironment and whether altered phenotypes are associated with patterns of lymphoma B-cell heterogeneity. Methods Two mass cytometry (CyTOF) panels were designed to immunophenotype B and T cells in FL tumors. Populations of malignant B cells, non-malignant B cells, and T cells from each FL tumor were identified and their phenotypes compared to B and T cells from healthy human tonsillar tissue. Results Diversity in cellular phenotype between tumors was greater for the malignant B cells than for non-malignant B or T cells. The malignant B-cell population bore little phenotypic similarity to any healthy B-cell subset, and unexpectedly clustered closer to naïve B-cell populations than GC B-cell populations. Among the non-malignant B cells within FL tumors, a significant lack of GC and plasmablast B cells was observed relative to tonsil controls. In contrast, non-malignant T cells in FL tumors were present at levels similar to their cognate tonsillar T-cell subsets. Conclusion Mass cytometry revealed that diverse HLA-DR expression on FL cells within individual tumors contributed greatly to tumor heterogeneity. Both malignant and non-malignant B cells in the tumor bore little phenotypic resemblance to healthy GC B cells despite the presence of T follicular helper cells in the tumor. These findings suggest that ongoing signaling interactions between malignant B cells and intra-tumor T cells shape the tumor microenvironment.