Assessment of T cell chemotaxis to S1P

Introduction: Migration of leukocytes in response to chemical gradients, chemotaxis, is dependent on many factors, including cell type, surface markers, the chemoattractant, etc. Sphinogsine-1-phosphate (S1P) is a chemoattractant playing a large role in migrating activated T cells out of lymph nodes by binding to their S1P receptor, S1P1. The importance of the egress in T cells from lymph nodes is highlighted by pharmacological disruption of this migration can lead to immune dampening and thus control of multiple sclerosis, an autoimmune disease. In the case of human immunodeficiency virus (HIV), it has been shown that HIV downregulates S1P1 surface expression, effectively inhibiting chemotaxis. Our experiments attempt to study a particular HIV-encoded protein, Nef in S1P-elicited T cell migration, and to optimize the conditions for assessing T cell migration in response to S1P. Methods: In our Transwell migration assays, migration of serum-starved SupT1 cells was induced using various concentrations of S1P bound to delipidated bovine serum albumin (BSA). Before migration, cells were labeled using Calcein AM. Cells were allowed to migrate for 2-4 hours at 37°C in serum-free media. After migration, fluorescence intensity was measured using a CLARIOstar microplate reader. Results: S1P showed a direct dose-dependent response to SupT1 cell migration from 0 to 100 nM S1P. Optimization of the migration showed that both number of trans-migrated cells and those still present within the transwell filter were significant indicators of SupT1 migration. Conclusion: S1P’s chemoattractant ability is prevalent in the migration of SupT1 cells in concentrations lower than 125nM. Because we have inducible systems for HIV-Nef expression established in this cell line, these data are useful for testing the role of Nef in HIV-mediated T cell retention.

2LTRZFP Interacts Specifically to HIV-1 DNA without Off-Target Effects as Determined by Biolayer Interferometry

Protein and DNA interactions are crucial for many cellular processes. Biolayer Interferometry (BLI) is a label-free technology for determining kinetic biomolecular interactions with high accuracy results. In the present study, we determined the kinetic binding of a zinc finger scaffold, 2LTRZFP, which formerly constructed the interfering effect on HIV-1 integration process using BLI. The competitive Enzyme-linked immunosorbent assay (ELISA) was used to initially show the specific binding of 2LTRZFP. The percentages of inhibition were 62% and 22% in double-stranded 2LTR (ds2LTR) and irrelevant DNA (dsNeg), respectively. Consequently, the binding affinity of 2LTRZFP against ds2LTR target analyzed by BLI was 40 nM, which is stronger than the interaction of HIV-1 integrase (IN) enzyme to the 2LTR circle junction. Additionally, the 2LTRZFP did not interact with the genomic DNA extracted from SupT1 cell line. This result indicates that 2LTRZFP did not exhibit off-target effects against human genome. The knowledge obtained from this study supports the prospect of using 2LTRZFP in HIV-1 gene therapy.

Generation of a Highly Sensitive CD22 CAR

Background: CD22 is an attractive target for Chimeric Antigen Receptor (CAR) since it is expressed by most B-cell malignancies. However, CAR targeting of CD22 is challenging since CD22 has a long ectodomain of 300 Å, which is several times wider than the optimal immune synapse. Further, the ectodomain is rigid, so even targeting of membrane proximal domains may not allow effective synapse formation. Further still, CD22 is expressed at low density and dropping of antigen density has been described as a mode of escape. A previously described CD22 CAR based on the M971 antibody required a very short linker between single-chain VH-VL for sensitive function, presumably to allow CAR concatenation but which comes at the cost of basal signalling. Our aim was to engineer an anti-CD22 CAR which triggered not only killing, but proliferation in response to less than 1000 CD22 molecules per cell, without basal signalling. We conducted a functional screen for stable / high affinity binders recognizing the CD22 membrane proximal domains that function in a CAR against cells expressing low levels of CD22 target. Methods: Binders were generated in two campaigns. Hyperimmune mice were vaccinated with recombinant CD22 protein lacking the highly immunogenic N-terminal V-type domain. Similarly, Wistar rats were immunized via genetic vaccination using a modified gene encoding the 4 membrane proximal domains of CD22, cloned into a DNA vaccine plasmid designed to stimulate a humoral immune response. A total of 18 binders passed biophysical screening properties of stability, affinity and proximal binding. Binding affinity ranged between 1-36.5nM; the binders were cloned into 41BB-Zeta signalling CARs. We generated a panel of CD22 expressing SupT1 cell lines. Using expression cassettes with amber stop codons preceding the CD22 frame, we generated stable SuptT1 cell lines which express CD22 below the limit of amplified flow cytometry detection (<100 copies, CD22Und) and a cell line with 255 copies / cell (CD22Low). In addition, using conventional means, we generated cell lines expressing intermediate and high levels of CD22 expression (CD22Mid=3,444, CD22High=78,916 copies/cell). Results: The 18 CAR constructs went through an initial round of screening using primary T-cells as effectors and engineered SupT1 cells as targets. A threshold of >50% killing of CD22Und target cells at an effector:target (E:T) ratio of 1:4 at 72 hour was set for further characterization. Only one CAR derived from the hyperimmune murine binders, and two derived from the rat binders met this threshold. Further characterization of killing at lower E:T ratios, cytokine release and proliferation revealed that CAR based on the rat binder 9A8 had superior function in response to low density target cells compared with the other two CARs and was characterized further and compared with the M971 CAR. This comparison showed approximately 2-fold better killing of CD22Und 9A8, and approximately 4-fold increased proliferation with no basal signalling. Next, we tried to correlate binder characteristics with sensitivity to low CD22 density. No correlation was found between either affinity, epitope, or stability and sensitivity. In fact, two of the best candidates could be paired with CARs which recognized the same CD22 domain and had similar binding kinetics and stability but had much lower sensitivity to CD22. This suggests that some other as yet unappreciated binder characteristic leads to sensitivity and we are currently investigating this via a structural analysis of binder / target interaction. Finally, we are currently testing 9A8 co-expressed with the CAT19 CD19 CAR (AUTO1) (Blood 2017 130:806) in small animal models of B-ALL. Conclusion: A highly sensitive CD22 CAR could be selected by empiric screening of CARs derived from a set of binders which target the proximal domains of CD22. CAR concatenation was not required for a high-degree of sensitivity to CD22 antigen. No correlation was found between binder characteristics and function. Disclosures Thomas: Autolus: Employment, Equity Ownership. Cordoba:Autolus: Employment, Equity Ownership. Pule:Autolus: Employment, Equity Ownership, Patents & Royalties.

CD4-Dependent Modulation of HIV-1 Entry by LY6E

ABSTRACTLymphocyte antigen 6E (LY6E) is a GPI-anchored, interferon-inducible protein that has been shown to modulate viral infection in a cell type-dependent manner. Our recent work showed that LY6E promotes HIV-1 infection in some high-CD4-expressing cells, including human peripheral blood mononuclear cells (PBMCs) and the SupT1 cell line. In this work, we provide evidence that LY6E inhibits HIV-1 entry and spread in low-CD4-expressing Jurkat cells and human monocyte-derived macrophages (MDMs) through downregulation of the viral receptor CD4. We found that knockdown of LY6E in Jurkat cells and MDMs increases HIV-1 infection, yet overexpression of LY6E in Jurkat cells inhibits HIV-1 entry and replication. LY6E was found to be colocalized with CD4 on the plasma membrane of Jurkat cells and MDMs and enhances CD4 internalization. We artificially manipulated the CD4 level in Jurkat and SupT1 cells and found that overexpression of CD4 in Jurkat cells overcomes the inhibitory effect of LY6E; conversely, blocking the function of CD4 in SupT1 with a neutralizing antibody eliminates the enhancement of LY6E on HIV-1 entry. The CD4-dependent inhibitory phenotype of LY6E in low-CD4-expressing human MDMs can be recapitulated for a panel of transmitted founder viruses and laboratory-adapted HIV-1 strains. Given that HIV-1 can target low-CD4-expressing cells during acute infection yet replicates efficiently in high-CD4-expressing T cells at the late stage of disease, our observation that LY6E differentially modulates HIV-1 replication in a CD4-dependent manner has implications for understanding the complex roles of interferon (IFN)-induced proteins in AIDS pathogenesis.IMPORTANCEThe role of IFN-induced genes (ISGs) in viral infection remains incompletely understood. While most ISGs are antiviral, some ISGs have been shown to promote viral infection, including HIV-1 infection. We previously showed that IFN-inducible LY6E protein promotes HIV-1 infection in human PMBCs and high-CD4-expressing SupT1 cells. Here we found that LY6E inhibits HIV-1 entry and replication in low-CD4-expressing MDMs and Jurkat cells. Mechanistically, we demonstrated that LY6E downregulates the cell surface receptor CD4, thus impairing the virus binding to target cells. This is in contrast to the situation of high-CD4-expressing cells, where LY6E predominantly promotes viral membrane fusion. The opposing role of IFN-inducible LY6E in modulating HIV-1 infection highlights the complex roles of ISGs in viral infection and viral pathogenesis.

Autocrine proliferative effect of ghrelin on leukemic HL-60 and THP-1 cells

Ghrelin is a 28 amino acid peptide hormone that is mainly produced by the stomach, but also by several tissues and tumors. Ghrelin is octanoylated on the Ser3, but is also detected as a des-acylated form. Only the acylated ghrelin activates the GH secretagogue receptor (GHS-R) type 1a to stimulate GH release, and regulate food intake and energy metabolism. For the first time, we report that ghrelin and des-acyl ghrelin are present in human promyelocytic HL-60, monocytic THP-1 and lymphoblastic SupT1 cell lines. The human leukemic cell lines did not express the functional GHS-R 1a, whereas they expressed GHS-R 1b, a truncated variant of the receptor. Leukemic cell proliferation was not modified by the addition of octanoylated or des-acyl ghrelins. However, THP-1 and HL-60 cell proliferations were inhibited by SB801, an antibody directed against the N-terminal octanoylated portion of ghrelin, suggesting that octanoylated ghrelin stimulates cell proliferation via an autocrine pathway involving an as yet unidentified ghrelin receptor. Both octanoylated and des-acyl ghrelins did not alter the basal adenylate cyclase activity. Treatments of THP-1 and SupT1 cells by both octanoylated and des-acyl ghrelins did not modify the adenylate cyclase activity in response to vasoactive intestinal peptide, suggesting that ghrelin is unlikely to modulate the anti-inflammatory and differentiating properties of vasoactive intestinal peptide.

NOTCH1 Is Targeted by Ig Translocations in NHL.

NOTCH1 signaling is required for normal T cell development. Its aberrant activation by either the rare t(7;9) translocation or frequent mutation(s) in T-ALL points to an important role of this gene in T cell lymphomagenesis. So far, a pathogenetic role for NOTCH1 in B-cell malignancies is unknown. We report here evidence for NOTCH1 rearrangements in B-cell lymphoma. Two novel 9q34 translocations involving either the 14q32/IGH or the 22q11/IGL locus were identified in 2 patients with respectively, Richter syndrome (RS) and follicular lymphoma (FL). The first case showed a clone with a sole trisomy 12 detected by FISH in 25% of interphase cells and a subclone with an additional dic(9;14)(q34;q32) in 60% of cells from a lymph node sample. The karyotype of the FL case was characterized by the typical t(14;18)(q32;q21)/IGH-BCL2 accompanied by additional t(8;14)(q24;q32)/IGH-CMYC and t(9;22)(q34;q11). FISH analysis of dic(9;14) and t(9;22) using a set of BAC clones mapped both 9q34 breakpoints to the 5′end of NOTCH1. These breakpoints were different from the breakpoint of t(7;9)(q35;q34) (intron 24), as demonstrated by FISH analysis of the T-ALL SUPT1 cell line. NOTCH1 rearrangements showed to be rare in B-NHL. Any of the additional 30 lymphoma cases with structural aberrations of 9q34 analyzed by FISH showed rearrangement of this gene. So far, aberrant activation of NOTCH1 in the reported cases with dic(9;14) and t(9;22) could not be documented: both lymphomas but also 9 control CLL and FL cases without 9q34 aberrations revealed expression of NOTCH1 mRNA by RT-PCR, its ligand JAGGED2 and its transcriptional target HES1. This expression pattern indicates a ligand-driven NOTCH1 signaling in all analyzed cases, possibly reflecting its physiological activation in progenitor B cells and thus, masking the presumed aberrant activation of NOTCH1 in present lymphomas. Whether IGH/L-NOTCH1 translocations in B-NHL lead to the generation of truncated/activated NOTCH1 proteins, similar to these found in T-ALL, remains to be determined. Particularly interesting is the association of the dic(9;14) with Richter transformation in the first case suggesting that rearrangement of NOTCH1 could be responsible for a rapid progression of the underlied CLL. In the second case of FL, t(9;22) seemed to be associated with the evolution of t(14;18)-positive karyotype. These findings contrast with t(7;9), considered as a primary oncogenic event in development of T-ALL. Further molecular and immunohistochemical investigations of the reported cases are in progress.