RUNX1 regulates promoter activity in the absence of cognate DNA binding motifs.

Runt-related transcription factor 1 (RUNX1) plays an important role in normal haematopoietic cell development and function, and its function is frequently disrupted in leukaemia. RUNX1 is widely recognised as a sequence-specific DNA binding factor that recognises the motif 5'-TG(T/C)GGT-3' in promoter and enhancer regions of its target genes. Moreover, RUNX1 fusion proteins, such as RUNX1-ETO formed by the t(8;21) translocation, retain the ability to recognise and bind to this sequence to elicit atypical gene regulatory effects on bona fide RUNX1 targets. However, our analysis of publicly available RUNX1 chromatin immunoprecipitation sequencing (ChIP-Seq) data has provided evidence challenging this dogma, revealing that this motif-specific model of RUNX1 recruitment and function is incomplete. Our analyses revealed that the majority of RUNX1 genomic localisation occurs outside of promoters, that 20% of RUNX1 binding sites lack consensus RUNX motifs, and that binding in the absence of a cognate binding site is more common in promoter regions compared to distal sites. Reporter assays demonstrate that RUNX1 can drive promoter activity in the absence of a recognised DNA binding motif, in contrast to RUNX1-ETO. RUNX1-ETO supresses activity when it is recruited to promoters containing a sequence specific motif, while interestingly, it binds but does not repress promoters devoid of a RUNX1 recognition site. These data suggest that RUNX1 regulation of target genes occurs through multiple mechanisms depending on genomic location, the type of regulatory element and mode of recruitment.

Selective Targeting of Protein Kinase C (PKC)-θ Nuclear Translocation Reduces Mesenchymal Gene Signatures and Reinvigorates Dysfunctional CD8+ T Cells in Immunotherapy-Resistant and Metastatic Cancers.

Protein kinase C (PKC)-θ is a serine/threonine kinase with both cytoplasmic and nuclear functions. Nuclear chromatin-associated PKC-θ (nPKC-θ) is increasingly recognized to be pathogenic in cancer, whereas its cytoplasmic signaling is restricted to normal T-cell function. Here we show that nPKC-θ is enriched in circulating tumor cells (CTCs) in patients with triple-negative breast cancer (TNBC) brain metastases and immunotherapy-resistant metastatic melanoma and is associated with poor survival in immunotherapy-resistant disease. To target nPKC-θ, we designed a novel PKC-θ peptide inhibitor (nPKC-θi2) that selectively inhibits nPKC-θ nuclear translocation but not PKC-θ signaling in healthy T cells. Targeting nPKC-θ reduced mesenchymal cancer stem cell signatures in immunotherapy-resistant CTCs and TNBC xenografts. PKC-θ was also enriched in the nuclei of CD8+ T cells isolated from stage IV immunotherapy-resistant metastatic cancer patients. We show for the first time that nPKC-θ complexes with ZEB1, a key repressive transcription factor in epithelial-to-mesenchymal transition (EMT), in immunotherapy-resistant dysfunctional PD1+/CD8+ T cells. nPKC-θi2 inhibited the ZEB1/PKC-θ repressive complex to induce cytokine production in CD8+ T cells isolated from patients with immunotherapy-resistant disease. These data establish for the first time that nPKC-θ mediates immunotherapy resistance via its activity in CTCs and dysfunctional CD8+ T cells. Disrupting nPKC-θ but retaining its cytoplasmic function may offer a means to target metastases in combination with chemotherapy or immunotherapy.

Targeting Nuclear LSD1 to Reprogram Cancer Cells and Reinvigorate Exhausted T Cells via a Novel LSD1-EOMES Switch.

Lysine specific demethylase 1 (LSD1) is a key epigenetic eraser enzyme implicated in cancer metastases and recurrence. Nuclear LSD1 phosphorylated at serine 111 (nLSD1p) has been shown to be critical for the development of breast cancer stem cells. Here we show that circulating tumor cells isolated from immunotherapy-resistant metastatic melanoma patients express higher levels of nLSD1p compared to responders, which is associated with co-expression of stem-like, mesenchymal genes. Targeting nLSD1p with selective nLSD1 inhibitors better inhibits the stem-like mesenchymal signature than traditional FAD-specific LSD1 catalytic inhibitors such as GSK2879552. We also demonstrate that nLSD1p is enriched in PD-1+CD8+ T cells from resistant melanoma patients and 4T1 immunotherapy-resistant mice. Targeting the LSD1p nuclear axis induces IFN-γ/TNF-α-expressing CD8+ T cell infiltration into the tumors of 4T1 immunotherapy-resistant mice, which is further augmented by combined immunotherapy. Underpinning these observations, nLSD1p is regulated by the key T cell exhaustion transcription factor EOMES in dysfunctional CD8+ T cells. EOMES co-exists with nLSD1p in PD-1+CD8+ T cells in resistant patients, and nLSD1p regulates EOMES nuclear dynamics via demethylation/acetylation switching of critical EOMES residues. Using novel antibodies to target these post-translational modifications, we show that EOMES demethylation/acetylation is reciprocally expressed in resistant and responder patients. Overall, we show for the first time that dual inhibition of metastatic cancer cells and re-invigoration of the immune system requires LSD1 inhibitors that target the nLSD1p axis.

Lysine-Specific Histone Demethylase 1A Regulates Macrophage Polarization and Checkpoint Molecules in the Tumor Microenvironment of Triple-Negative Breast Cancer.

Macrophages play an important role in regulating the tumor microenvironment (TME). Here we show that classical (M1) macrophage polarization reduced expression of LSD1, nuclear REST corepressor 1 (CoREST), and the zinc finger protein SNAIL. The LSD1 inhibitor phenelzine targeted both the flavin adenine dinucleotide (FAD) and CoREST binding domains of LSD1, unlike the LSD1 inhibitor GSK2879552, which only targeted the FAD domain. Phenelzine treatment reduced nuclear demethylase activity and increased transcription and expression of M1-like signatures both in vitro and in a murine triple-negative breast cancer model. Overall, the LSD1 inhibitors phenelzine and GSK2879552 are useful tools for dissecting the contribution of LSD1 demethylase activity and the nuclear LSD1-CoREST complex to switching macrophage polarization programs. These findings suggest that inhibitors must have dual FAD and CoREST targeting abilities to successfully initiate or prime macrophages toward an anti-tumor M1-like phenotype in triple-negative breast cancer.

Epigenetic programming of T cells impacts immune reconstitution in hematopoietic stem cell transplant recipients.

Immune reconstitution following hematopoietic stem cell transplantation (HSCT) is critical in preventing harmful sequelae in recipients with cytomegalovirus (CMV) infection. To understand the molecular mechanisms underlying immune reconstitution kinetics, we profiled the transcriptome-chromatin accessibility landscape of CMV-specific CD8+ T cells from HCST recipients with different immune reconstitution efficiencies. CMV-specific T cells from HSCT recipients with stable antiviral immunity expressed higher levels of interferon/defense response and cell cycle genes in an interconnected network involving PI3KCG, STAT5B, NFAT, RBPJ, and lower HDAC6, increasing chromatin accessibility at the enhancer regions of immune and T-cell receptor signaling pathway genes. By contrast, the transcriptional and epigenomic signatures of CMV-specific T cells from HSCT recipients with unstable immune reconstitution showed commonalities with T-cell responses in other nonresolving chronic infections. These signatures included higher levels of EGR and KLF factors that, along with lower JARID2 expression, maintained higher accessibility at promoter and CpG-rich regions of genes associated with apoptosis. Furthermore, epigenetic targeting via inhibition of HDAC6 or JARID2 enhanced the transcription of genes associated with differential responses, suggesting that drugs targeting epigenomic modifiers may have therapeutic potential for enhancing immune reconstitution in HSCT recipients. Taken together, these analyses demonstrate that transcription factors and chromatin modulators create different chromatin accessibility landscapes in T cells of HSCT recipients that not only affect immediate gene expression but also differentially prime cells for responses to additional signals. Epigenetic therapy may be a promising strategy to promote immune reconstitution in HSCT recipients.

Distinct mechanisms of regulation of the ITGA6 and ITGB4 genes by RUNX1 in myeloid cells.

Integrins are transmembrane adhesion receptors that play an important role in hematopoiesis by facilitating interactions between hematopoietic cells and extracellular matrix components of the bone marrow and hematopoietic tissues. These interactions are important in regulating the function, proliferation, and differentiation of hematopoietic cells, as well as their homing and mobilization in the bone marrow. Not surprisingly altered expression and function of integrins plays a key role in the development and progression of cancer including leukemias. However, the regulation of integrin gene expression is not well characterized and the mechanisms by which integrin genes are disrupted in cancer remain unclear. Here we demonstrate for the first time that a key regulator of hematopoiesis, RUNX1, binds to and regulates the promoters of both the ITGA6 and ITGB4 genes in myeloid cells. The ITGA6 and ITGB4 integrin genes form the α6ß4 integrin receptor. However, our data indicate that RUNX1 functions differently at these two promoters. RUNX1 regulates ITGA6 through a consensus RUNX1 binding motif in its promoter. In contrast, although the ITGB4 promoter is also activated by RUNX1, it does so in the absence of a recognized consensus RUNX1 binding motif. Furthermore, our data suggest that regulation of ITGB4 may involve interactions between the promoter and upstream regulatory elements.

Nuclear PKC-θ facilitates rapid transcriptional responses in human memory CD4+ T cells through p65 and H2B phosphorylation.

Memory T cells are characterized by their rapid transcriptional programs upon re-stimulation. This transcriptional memory response is facilitated by permissive chromatin, but exactly how the permissive epigenetic landscape in memory T cells integrates incoming stimulatory signals remains poorly understood. By genome-wide ChIP-sequencing ex vivo human CD4(+) T cells, here, we show that the signaling enzyme, protein kinase C theta (PKC-θ) directly relays stimulatory signals to chromatin by binding to transcriptional-memory-responsive genes to induce transcriptional activation. Flanked by permissive histone modifications, these PKC-enriched regions are significantly enriched with NF-κB motifs in ex vivo bulk and vaccinia-responsive human memory CD4(+) T cells. Within the nucleus, PKC-θ catalytic activity maintains the Ser536 phosphorylation on the p65 subunit of NF-κB (also known as RelA) and can directly influence chromatin accessibility at transcriptional memory genes by regulating H2B deposition through Ser32 phosphorylation. Furthermore, using a cytoplasm-restricted PKC-θ mutant, we highlight that chromatin-anchored PKC-θ integrates activating signals at the chromatin template to elicit transcriptional memory responses in human memory T cells.

Dichloroacetate Prevents Cisplatin-Induced Nephrotoxicity without Compromising Cisplatin Anticancer Properties.

Cisplatin is an effective anticancer drug; however, cisplatin use often leads to nephrotoxicity, which limits its clinical effectiveness. In this study, we determined the effect of dichloroacetate, a novel anticancer agent, in a mouse model of cisplatin-induced AKI. Pretreatment with dichloroacetate significantly attenuated the cisplatin-induced increase in BUN and serum creatinine levels, renal tubular apoptosis, and oxidative stress. Additionally, pretreatment with dichloroacetate accelerated tubular regeneration after cisplatin-induced renal damage. Whole transcriptome sequencing revealed that dichloroacetate prevented mitochondrial dysfunction and preserved the energy-generating capacity of the kidneys by preventing the cisplatin-induced downregulation of fatty acid and glucose oxidation, and of genes involved in the Krebs cycle and oxidative phosphorylation. Notably, dichloroacetate did not interfere with the anticancer activity of cisplatin in vivo. These data provide strong evidence that dichloroacetate preserves renal function when used in conjunction with cisplatin.

Differential Roles for DUSP Family Members in Epithelial-to-Mesenchymal Transition and Cancer Stem Cell Regulation in Breast Cancer.

Dual-specificity phosphatases (DUSPs) dephosphorylate threonine/serine and tyrosine residues on their substrates. Here we show that DUSP1, DUSP4, and DUSP6 are involved in epithelial-to-mesenchymal transition (EMT) and breast cancer stem cell (CSC) regulation. DUSP1, DUSP4, and DUSP6 are induced during EMT in a PKC pathway signal-mediated EMT model. We show for the first time that the key chromatin-associated kinase PKC-θ directly regulates a subset of DUSP family members. DUSP1, DUSP4, and DUSP6 globally but differentially co-exist with enhancer and permissive active histone post-translational modifications, suggesting that they play distinct roles in gene regulation in EMT/CSCs. We show that nuclear DUSP4 associates with the key acetyltransferase p300 in the context of the chromatin template and dynamically regulates the interplay between two key phosphorylation marks: the 1834 (active) and 89 (inhibitory) residues central to p300's acetyltransferase activity. Furthermore, knockdown with small-interfering RNAs (siRNAs) shows that DUSP4 is required for maintaining H3K27ac, a mark mediated by p300. DUSP1, DUSP4, and DUSP6 knockdown with siRNAs shows that they participate in the formation of CD44hi/CD24lo/EpCAM+ breast CSCs: DUSP1 knockdown reduces CSC formation, while DUSP4 and DUSP6 knockdown enhance CSC formation. Moreover, DUSP6 is overexpressed in patient-derived HER2+ breast carcinomas compared to benign mammary tissue. Taken together, these findings illustrate novel pleiotropic roles for DUSP family members in EMT and CSC regulation in breast cancer.

The role of protein kinase-C theta in control of epithelial to mesenchymal transition and cancer stem cell formation.

The protein kinase C (PKC) activator phorbol 12-myristate 13-acetate (PMA) induces transition of the epithelial MCF-7 cell line to a mesenchymal phenotype. A subset of the resulting mesenchymal cells has surface markers characteristics of a cancer stem cell (CSC) population. We profiled the transcriptome changes associated with the epithelial to mesenchymal transition and those that occurred in the CSC subset. Using a siRNA knockdown strategy, we examined the extent to which these changes were dependent on the PKC family member, PKC-θ. The importance of the cytoplasmic signaling role of this kinase is well established and in this study, we have shown by PKC-θ ChIP-sequencing analysis that this kinase has a dual role with the ability to also associate with chromatin on a subset of PKC-θ dependent genes. In the associated manuscript (Zafar et al., 2014 [5]) we presented evidence for the first time showing that this nuclear role of PKC-θ is also important for gene induction and mesenchymal/CSC phenotype. Here we describe the analysis associated with the transcriptome and ChIP-seq data presented in Zafar et al. (2014) [5] and uploaded to NCBI Gene Expression Omnibus (GSE53335).

Multi-layered epigenetic mechanisms contribute to transcriptional memory in T lymphocytes.

BACKGROUND: Immunological memory is the ability of the immune system to respond more rapidly and effectively to previously encountered pathogens, a key feature of adaptive immunity. The capacity of memory T cells to "remember" previous cellular responses to specific antigens ultimately resides in their unique patterns of gene expression. Following re-exposure to an antigen, previously activated genes are transcribed more rapidly and robustly in memory T cells compared to their naïve counterparts. The ability for cells to remember past transcriptional responses is termed "adaptive transcriptional memory". RESULTS: Recent global epigenome studies suggest that epigenetic mechanisms are central to establishing and maintaining transcriptional memory, with elegant studies in model organisms providing tantalizing insights into the epigenetic programs that contribute to adaptive immunity. These epigenetic mechanisms are diverse, and include not only classical acetylation and methylation events, but also exciting and less well-known mechanisms involving histone structure, upstream signalling pathways, and nuclear localisation of genomic regions. CONCLUSIONS: Current global health challenges in areas such as tuberculosis and influenza demand not only more effective and safer vaccines, but also vaccines for a wider range of health priorities, including HIV, cancer, and emerging pathogens such as Ebola. Understanding the multi-layered epigenetic mechanisms that underpin the rapid recall responses of memory T cells following reactivation is a critical component of this development pathway.

Chromatinized protein kinase C-θ directly regulates inducible genes in epithelial to mesenchymal transition and breast cancer stem cells.

Epithelial to mesenchymal transition (EMT) is activated during cancer invasion and metastasis, enriches for cancer stem cells (CSCs), and contributes to therapeutic resistance and disease recurrence. Signal transduction kinases play a pivotal role as chromatin-anchored proteins in eukaryotes. Here we report for the first time that protein kinase C-theta (PKC-θ) promotes EMT by acting as a critical chromatin-anchored switch for inducible genes via transforming growth factor ß (TGF-ß) and the key inflammatory regulatory protein NF-κB. Chromatinized PKC-θ exists as an active transcription complex and is required to establish a permissive chromatin state at signature EMT genes. Genome-wide analysis identifies a unique cohort of inducible PKC-θ-sensitive genes that are directly tethered to PKC-θ in the mesenchymal state. Collectively, we show that cross talk between signaling kinases and chromatin is critical for eliciting inducible transcriptional programs that drive mesenchymal differentiation and CSC formation, providing novel mechanisms to target using epigenetic therapy in breast cancer.

Chromatinized Protein Kinase C-θ: Can It Escape the Clutches of NF-κB?

We recently provided the first description of a nuclear mechanism used by Protein Kinase C-theta (PKC-θ) to mediate T cell gene expression. In this mode, PKC-θ tethers to chromatin to form an active nuclear complex by interacting with proteins including RNA polymerase II, the histone kinase MSK-1, the demethylase LSD1, and the adaptor molecule 14-3-3ζ at regulatory regions of inducible immune response genes. Moreover, our genome-wide analysis identified many novel PKC-θ target genes and microRNAs implicated in T cell development, differentiation, apoptosis, and proliferation. We have expanded our ChIP-on-chip analysis and have now identified a transcription factor motif containing NF-κB binding sites that may facilitate recruitment of PKC-θ to chromatin at coding genes. Furthermore, NF-κB association with chromatin appears to be a prerequisite for the assembly of the PKC-θ active complex. In contrast, a distinct NF-κB-containing module appears to operate at PKC-θ targeted microRNA genes, and here NF-κB negatively regulates microRNA gene transcription. Our efforts are also focusing on distinguishing between the nuclear and cytoplasmic functions of PKCs to ascertain how these kinases may synergize their roles as both cytoplasmic signaling proteins and their functions on the chromatin template, together enabling rapid induction of eukaryotic genes. We have identified an alternative sequence within PKC-θ that appears to be important for nuclear translocation of this kinase. Understanding the molecular mechanisms used by signal transduction kinases to elicit specific and distinct transcriptional programs in T cells will enable scientists to refine current therapeutic strategies for autoimmune diseases and cancer.

The NF-κB transcription factor c-Rel is required for Th17 effector cell development in experimental autoimmune encephalomyelitis.

Experimental autoimmune encephalomyelitis (EAE) is a T cell-mediated autoimmune disease involving effector Th subsets such as Th1 and Th17. In this study, we demonstrate that mice lacking the NF-κB transcription factor family member c-Rel (rel(-/-)), which are known to be resistant to EAE, show impaired Th17 development. Mixed bone marrow chimeras and EAE adoptive transfer experiments show that the deficiency of effector Th17 cells in rel(-/-) mice is T cell intrinsic. Consistent with this finding, c-Rel was activated in response to TCR signaling in the early stages of Th17 development and controlled the expression of Rorc, which encodes the Th17 transcription factor retinoic acid-related orphan receptor γt. CD28, but not IL-2, repression of Th17 development was dependent on c-Rel, implicating a dual role for c-Rel in modulating Th17 development. Adoptive transfer experiments also suggested that c-Rel control of regulatory T cell differentiation and homeostasis influences EAE development and severity by influencing the balance between Th17 and regulatory T cells. Collectively, our findings indicate that in addition to promoting Th1 differentiation, c-Rel regulates the development and severity of EAE via multiple mechanisms that impact on the generation of Th17 cells.

Regulation of the IL-21 gene by the NF-κB transcription factor c-Rel.

IL-21 is a member of the common gamma-chain-dependent cytokine family and is a key modulator of lymphocyte development, proliferation, and differentiation. IL-21 is highly expressed in activated CD4(+) T cells and plays a critical role in the expansion and differentiation of the Th cell subsets, Th17 and follicular helper T (T(FH)) cells. Because of its potent activity in both myeloid and lymphoid cell immune responses, it has been implicated in a number of autoimmune diseases and has also been used as a therapeutic agent in the treatment of some cancers. In this study, we demonstrate that c-Rel, a member of the NF-kappaB family of transcription factors, is required for IL-21 gene expression in T lymphocytes. IL-21 mRNA and protein levels are reduced in the CD4(+) cells of rel(-/-) mice when compared with rel(+/+) mice in both in vitro and in vivo models. A c-Rel binding site identified in the proximal promoter of il21 is confirmed to bind c-Rel in vitro and in vivo and to regulate expression from the il21 promoter in T cells. Downstream of IL-21 expression, Th17, T(FH), and germinal center B cell development are also impaired in rel(-/-) mice. The administration of IL-21 protein rescued the development of T(FH) cells but not germinal center B cells. Taken together, c-Rel plays an important role in the expression of IL-21 in T cells and subsequently in IL-21-dependent T(FH) cell development.

Genome-wide analysis of gene expression in T cells to identify targets of the NF-kappa B transcription factor c-Rel.

It is well established that the NF-kappaB family of transcription factors serves a major role in controlling gene expression in response to T cell activation, but the genome-wide roles of individual family members remain to be determined. c-Rel, a member of the NF-kappaB family, appears to play a specific role in T cell function because T cells from c-Rel(-/-) animals are defective in their response to immune signals. We have used expression profiling to identify sets of genes that are affected by either deletion or overexpression of c-Rel in T cells. Very few of these genes exhibit a strong requirement for c-Rel; rather, c-Rel appears to modulate the expression of a large number of genes in these cells. The sets of c-Rel-affected genes are significantly enriched for genes containing consensus NF-kappaB/Rel sites in their proximal promoter regions. In addition, their promoters contain a higher average density of NF-kappaB/Rel sites compared with all genes represented on the microarrays. A transcriptional module comprised of two closely spaced c-Rel consensus sites is found with higher frequency in the c-Rel-affected gene sets and may represent an important control module for genes regulated by c-Rel or other NF-kappaB family members. We confirmed the importance of these findings on a subgroup of genes by using quantitative PCR to monitor gene expression as well as in vitro c-Rel/DNA binding assays and luciferase reporter assays. The c-Rel-regulated genes identified here support a role for c-Rel in inflammatory responses as well as in the promotion of cell growth and survival.

Glucose induces increases in levels of the transcriptional repressor Id2 via the hexosamine pathway.

Changes in glucose levels are known to directly alter gene expression. A number of previous studies have found that these effects are in part mediated by modulating the levels and the activity of transcription factors. We have investigated an alternative mechanism by which glucose might regulate gene expression by modulating levels of a transcriptional repressor. We have focused on Id2, which is a protein that indirectly regulates gene expression by sequestering certain transcription factors and preventing them from forming functional dimers. Id2 targets include the class A basic helix-loop-helix transcription factors and the sterol regulatory element-binding protein (SREBP)-1. We demonstrate that increases in glucose levels cause a rapid increase in levels of Id2 in J774.2 macrophages, and a number of lines of evidence indicate that this is via the hexosamine pathway because 1) the effect of glucose requires glutamine; 2) the effect of glucose is mimicked by low levels of glucosamine; 3) the effect of glucose is inhibited by azaserine, an inhibitor of glutamine:fructose-6-phosphate amidotransferase (GFAT); and 4) adenoviral mediated overexpression of GFAT increases levels of Id2. We go on to show that increases in Id2 can have functional effects on metabolic genes, because Id2 blocked the SREBP-1-induced induction of hormone-sensitive lipase (HSL) promoter activity, whereas Id2 alone does not modulate activity of the HSL promoter. In summary, these studies define a new mechanism by which glucose uses the hexosamine pathway to regulate gene expression by increasing levels of a transcriptional repressor.

Differential effects of the antioxidant alpha-lipoic acid on the proliferation of mitogen-stimulated peripheral blood lymphocytes and leukaemic T cells.

The effects of the antioxidant alpha-lipoic acid (LA) on the proliferation of mitogen-stimulated human peripheral blood lymphocytes (HPBL) were investigated in comparison to its effects on the proliferation of two leukaemic T cell lines, Jurkat and CCRF-CEM. At low mM concentrations, LA inhibited in a dose-dependent manner DNA synthesis of HPBL stimulated with either phorbol myristate acetate (PMA) in combination with ionomycin (IoM), or phytohaemagglutinin (PHA). At similar concentrations, LA inhibited the proliferation of Jurkat and CCRF-CEM cells. However, LA was preferentially cytotoxic to the leukaemic cell lines. The selective toxicity of LA to Jurkat cells was shown by electron microscopy (EM) to be due to the induction of apoptosis. Furthermore, LA had different effects on the secretion of interleukin-2 (IL-2) and steady-state levels of IL-2 mRNA in mitogen-stimulated HPBL depending on the mitogens used. LA dramatically increased the induction of IL-2 mRNA and IL-2 protein secretion in PMA/IoM-stimulated HPBL, whereas it inhibited these in HPBL stimulated with PHA. The differential effects of LA on normal and leukaemic T lymphocytes may indicate a new route towards development of therapeutic agents.