Modified cell trace violet proliferation assay preserves lymphocyte viability and allows spectral flow cytometry analysis.

In this study we describe three different methods for labeling T lymphocytes with cell trace violet (CTV), in order to track cell division in mouse and human cells, in both the in vitro and in vivo setting. We identified a modified method of CTV labeling that can be applied directly to either conventional or spectral flow cytometry, that maintained lymphocyte viability and function, yet minimized dye spill-over into other fluorochrome channels. Our optimized method for CTV labeling allowed us to identify up to eight cell divisions and the replication index for in vitro-stimulated mouse and human lymphocytes, and the co-expression of T-cell subset markers. Furthermore, the homeostatic trafficking, expansion and division of CTV-labeled congenic donor T cells could be detected using spectral cytometry, in an adoptive T-cell transfer mouse model. Our optimized CTV method can be applied to both in vitro and in vivo settings to examine the behavior and phenotype of activated T cells.

Ibrutinib protects T cells in patients with CLL from proliferation-induced senescence.

BACKGROUND: The development of Bruton's tyrosine kinase inhibitors (BTKi) for the treatment of chronic lymphocytic leukaemia (CLL) has provided a highly effective and relatively non-toxic alternative to conventional chemotherapy. Some studies have shown that BTKi can also lead to improvements in T cell immunity in patients despite in vitro analyses suggesting an immunosuppressive effect of BTKi on T cell function. METHODS: In this study, we examined both the in vitro effect and long-term in vivo effect of two clinically available BTKi, ibrutinib and zanubrutinib. Additional in vitro assessments were undertaken for a third BTKi, acalabrutinib. Immune subset phenotyping, cytokine secretion, T cell degranulation and proliferation assays were performed on peripheral blood mononuclear cells isolated from untreated CLL patients, and CLL patients on long-term (> 12 months) BTKi treatment. RESULTS: Similar to prior studies we observed that long-term BTKi treatment normalises lymphocyte subset frequency and reduces PD-1 expression on T cells. We also observed that T cells from patients taken prior to BTKi therapy showed an abnormal hyper-proliferation pattern typical of senescent T cells, which was normalised by long-term BTKi treatment. Furthermore, BTKi therapy resulted in reduced expression of the T cell exhaustion markers PD-1, TIM3 and LAG3 in late generations of T cells undergoing proliferation. CONCLUSIONS: Collectively, these findings indicate that there are critical differences between the in vitro effects of BTKi on T cell function and the effects derived from long-term BTKi exposure in vivo. Overall long-term exposure to BTKi, and particularly ibrutinib, resulted in improved T cell fitness in part due to suppressing the abnormal hyper-proliferation of CLL T cells and the associated development of T cell senescence.

Venetoclax or Ruxolitinib in Pre-Transplant Conditioning Lowers the Engraftment Barrier by Different Mechanisms in Allogeneic Stem Cell Transplant Recipients.

Allogeneic stem cell transplantation (alloSCT) is utilised to cure haematological malignancies through a combination of conditioning regimen intensity and immunological disease control via the graft versus tumour (GVT) effect. Currently, conventional myeloablative chemotherapeutic or chemoradiation conditioning regimens are associated with significant side effects including graft versus host disease (GVHD), infection, and organ toxicity. Conversely, more tolerable reduced intensity conditioning (RIC) regimens are associated with unacceptably higher rates of disease relapse, partly through an excess incidence of mixed chimerism. Improvement in post-alloSCT outcomes therefore depends on promotion of the GVT effect whilst simultaneously reducing conditioning-related toxicity. We have previously shown that this could be achieved through BCL-2 inhibition, and in this study, we explored the modulation of JAK1/2 as a strategy to lower the barrier to donor engraftment in the setting of RIC. We investigated the impact of short-term treatment of BCL2 (venetoclax) or JAK1/2 (ruxolitinib) inhibition on recipient natural killer and T cell immunity and the subsequent effect on donor engraftment. We identified striking differences in mechanism of action of these two drugs on immune cell subsets in the bone marrow of recipients, and in the regulation of MHC class-II and interferon-inducible gene expression, leading to different rates of GVHD. This study demonstrates that the repurposed use of ruxolitinib or venetoclax can be utilised as pre-transplant immune-modulators to promote the efficacy of alloSCT, whilst reducing its toxicity.

Immune recovery in patients with mantle cell lymphoma receiving long-term ibrutinib and venetoclax combination therapy.

Combination venetoclax plus ibrutinib for the treatment of mantle cell lymphoma (MCL) has demonstrated efficacy in the relapsed or refractory setting; however, the long-term impact on patient immunology is unknown. In this study, changes in immune subsets of MCL patients treated with combination venetoclax and ibrutinib were assessed over a 4-year period. Multiparameter flow cytometry of peripheral blood mononuclear cells showed that ≥12 months of treatment resulted in alterations in the proportions of multiple immune subsets, most notably CD4+ and CD8+ effector and central memory T cells and natural killer cells, and normalization of T-cell cytokine production in response to T-cell receptor stimulation. Gene expression analysis identified upregulation of multiple myeloid genes (including S100 and cathepsin family members) and inflammatory pathways over 12 months. Four patients with deep responses stopped study drugs, resulting in restoration of normal immune subsets for all study parameters except myeloid gene/pathway expression, suggesting long-term combination venetoclax and ibrutinib irreversibly affects this population. Our findings demonstrate that long-term combination therapy is associated with immune recovery in MCL, which may allow responses to subsequent immunotherapies and suggests that this targeted therapy results in beneficial impacts on immunological recovery. This trial was registered at www.clinicaltrials.gov as #NCT02471391.

Strategies to enhance the graft versus tumour effect after allogeneic haematopoietic stem cell transplantation.

Relapse of haematological malignancies after allogeneic haematopoietic stem cell transplant is a major cause of mortality. The immunological mechanisms that may lead to disease relapse may include immunological immaturity prior to reconstitution of the allogeneic immune system, tumour antigen downregulation or promotion of T-cell exhaustion by interactions with the tumour microenvironment. Current therapeutic strategies for post-transplant relapse are limited in their efficacy and alternative approaches are required. In this review, we discuss the mechanisms of T and NK-cell immune evasion that facilitate relapse of haematological malignancies after allogeneic stem cell transplantation, and explore emerging strategies to augment the allogeneic immune system in order to construct a more potent graft versus tumour response.

Recipient BCL2 inhibition and NK cell ablation form part of a reduced intensity conditioning regime that improves allo-bone marrow transplantation outcomes.

Allogeneic hematopoietic stem cell transplantation (alloSCT) is used to treat over 15,000 patients with acute myeloid leukemia (AML) per year. Donor graft-versus-leukemia (GVL) effect can prevent AML relapse; however, alloSCT is limited by significant toxicity related to conditioning intensity, immunosuppression, opportunistic infections, and graft-versus-host disease (GVHD). Reducing the intensity of conditioning regimens prior to alloSCT has improved their tolerability, but does not alter the pattern of GVHD and has been associated with increased rates of graft rejection and relapse. Here, using a murine pre-clinical model, we describe a novel recipient conditioning approach combining reduced intensity conditioning with either genetic or pharmacological inhibition of NK cell numbers that permits efficient donor engraftment and promotes GVL without inducing GVHD. We show that NK cell-specific deletion of Bcl2 or Mcl1 in mice, or pharmacological inhibition of BCL2 impairs radio-resistant NK cell-mediated rejection of allogeneic engraftment and allows reduction of conditioning intensity below that associated with GVHD priming. The combination of reduced intensity conditioning and NK cell targeting in mice allowed successful donor T cell engraftment and protective immunity against AML while avoiding GVHD. These findings suggest that reduced conditioning in combination with targeted therapies against recipient NK cells may allow the delivery of effective alloSCT against AML while reducing the toxicities associated with more intensive conditioning including GVHD.

A radio-resistant perforin-expressing lymphoid population controls allogeneic T cell engraftment, activation, and onset of graft-versus-host disease in mice.

Immunosuppressive pretransplantation conditioning is essential for donor cell engraftment in allogeneic bone marrow transplantation (BMT). The role of residual postconditioning recipient immunity in determining engraftment is poorly understood. We examined the role of recipient perforin in the kinetics of donor cell engraftment. MHC-mismatched BMT mouse models demonstrated that both the rate and proportion of donor lymphoid cell engraftment and expansion of effector memory donor T cells in both spleen and BM were significantly increased within 5 to 7 days post-BMT in perforin-deficient (pfn(-/-)) recipients, compared with wild-type. In wild-type recipients, depletion of natural killer (NK) cells before BMT enhanced donor lymphoid cell engraftment to that seen in pfn(-/-) recipients. This demonstrated that a perforin-dependent, NK-mediated, host-versus-graft (HVG) effect limits the rate of donor engraftment and T cell activation. Radiation-resistant natural killer T (NKT) cells survived in the BM of lethally irradiated mice and may drive NK cell activation, resulting in the HVG effect. Furthermore, reduced pretransplant irradiation doses in pfn(-/-) recipients permitted long-term donor lymphoid cell engraftment. These findings suggest that suppression of perforin activity or selective depletion of recipient NK cells before BMT could be used to improve donor stem cell engraftment, in turn allowing for the reduction of pretransplant conditioning.

Cytotoxic T cell adoptive immunotherapy as a treatment for nasopharyngeal carcinoma.

Epstein-Barr virus (EBV) is associated with nasopharyngeal carcinoma (NPC). We assess the safety and tolerability of adoptive transfer of autologous cytotoxic T lymphocytes (CTLs) specific for the EBV latent membrane protein (LMP) in a patient with recurrent NPC. After infusion, the majority of pulmonary lesions were no longer evident, although the primary tumor did not regress.

The anti-cancer drug, phenoxodiol, kills primary myeloid and lymphoid leukemic blasts and rapidly proliferating T cells.

BACKGROUND: The redox-active isoflavene anti-cancer drug, phenoxodiol, has previously been shown to inhibit plasma membrane electron transport and cell proliferation and promote apoptosis in a range of cancer cell lines and in anti-CD3/anti-CD28-activated murine splenocytes but not in non-transformed WI-38 cells and human umbilical vein endothelial cells. DESIGN AND METHODS: We determined the effects of phenoxodiol on plasma membrane electron transport, MTT responses and viability of activated and resting human T cells. In addition, we evaluated the effect of phenoxodiol on the viability of leukemic cell lines and primary myeloid and lymphoid leukemic blasts. RESULTS: We demonstrated that phenoxodiol inhibited plasma membrane electron transport and cell proliferation (IC(50) 46 microM and 5.4 microM, respectively) and promoted apoptosis of rapidly proliferating human T cells but did not affect resting T cells. Phenoxodiol also induced apoptosis in T cells stimulated in HLA-mismatched allogeneic mixed lymphocyte reactions. Conversely, non-proliferating T cells in the mixed lymphocyte reaction remained viable and could be restimulated in a third party mixed lymphocyte reaction, in the absence of phenoxodiol. In addition, we demonstrated that leukemic blasts from patients with primary acute myeloid leukemia (n=22) and acute lymphocytic leukemia (n=8) were sensitive to phenoxodiol. The lymphocytic leukemic blasts were more sensitive than the myeloid leukemic blasts to 10 muM phenoxodiol exposure for 24h (viability of 23+/-4% and 64+/-5%, respectively, p=0.0002). CONCLUSIONS: The ability of phenoxodiol to kill rapidly proliferating lymphocytes makes this drug a promising candidate for the treatment of pathologically-activated lymphocytes such as those in acute lymphoid leukemia, or diseases driven by T-cell proliferation such as auto-immune diseases and graft-versus-host disease.

Optimization of LMP-specific CTL expansion for potential adoptive immunotherapy in NPC patients.

Nasopharyngeal carcinoma (NPC) is Epstein-Barr virus (EBV) positive in all undifferentiated cases, expressing the latency II phenotype of latent membrane proteins (LMPs) 1 and 2, in addition to EBV nuclear antigen (EBNA) 1. Several studies have attempted to treat NPC with EBV-specific cytotoxic T lymphocyte (CTL) with a partial response. To improve this therapy, there is a need to expand CTL targeted to the latency II antigens of EBV, rather than the immunodominant EBV nuclear antigens 3-6 peptides typically expanded by lymphoblastoid cells. In order to maximize the expansion of LMP-specific CTL in vitro for use in adoptive immunotherapy of nasopharyngeal carcinoma patients, we used lymphoblastoid cell lines coated with synthetic peptides corresponding to CTL determinants from the LMP proteins. We investigated several issues pertaining to the expansion of an immunologically weak CTL response, including peptide and interleukin-2 concentration, and screening assays for selecting the optimal peptide for use in expansion of LMP-specific CTL. Although screening of ex vivo peripheral blood mononuclear cells did not prove to be useful in the selection of an LMP peptide for use in CTL cultures, the peptide and interleukin-2 concentrations were critical for the maximum expansion of CTL. Therefore, it is imperative that stimulation protocols are optimized for the expansion of LMP-specific CTL.

Clonal population structure of the chestnut blight fungus in expanding ranges in southeastern Europe.

Expanding populations are often less genetically diverse at their margins than at the centre of a species' range. Established, older populations of the chestnut blight fungus, Cryphonectria parasitica, are more variable for vegetative compatibility (vc) types than in expanding populations in southeastern Europe where C. parasitica has colonized relatively recently. To test whether vc types represent clones, we genotyped 373 isolates of C. parasitica from southern Italy, Romania, Bulgaria, Macedonia, Greece and Turkey using 11 sequence-characterized amplified region (SCAR) markers. Ten SCAR loci and six vegetative incompatibility (vic) loci were polymorphic in these samples. These populations are clonal by all criteria tested: (i) among 373 isolates, we found only eight multilocus haplotypes, and the same haplotypes were found in multiple countries, sometimes separated in time by as much as 12 years; (ii) the number of haplotypes observed was significantly less than expected under random mating; (iii) populations are in linkage disequilibrium; (iv) the two sets of independent markers, SCARs and vc types, are highly correlated; and (v) sexual structures of C. parasitica were found only in Bulgaria and Romania. One mating type (MAT-1) was found in 98% of the isolates sampled. In contrast, a population in northern Italy, in the central part of the range in Europe, had 12 multilocus haplotypes among 19 isolates. The spread of a few clones could be the result either of founder effect and restricted migration, or these clones have greater fitness than others and spread because they are better adapted to conditions in southeastern Europe.

Determining virological, serological and immunological parameters of EBV infection in the development of PTLD.

Post-transplant lymphoproliferative disease (PTLD) in Epstein-Barr virus (EBV) seronegative solid organ transplant recipients remains a significant problem, particularly in the first year post-transplant. Immune monitoring of a cohort of high-risk patients indicated that four EBV seronegative transplant recipients developed early-onset PTLD prior to evidence of an EBV humoral response. EBV status has been classically defined serologically, however these patients demonstrated multiple parameters of EBV infection, including the generation of EBV-specific CTL, outgrowth of spontaneous lymphoblastoid cell lines, and elevated EBV DNA levels, despite the absence of a classic EBV antibody response. As EBV serology is influenced by both immunosuppression and cytomegalovirus immunoglobulin treatment, both the EBV-specific CTL response and elevated EBV levels are more reliable indicators of EBV infection post-transplant.

Reconstitution of the latent T-lymphocyte response to Epstein-Barr virus is coincident with long-term recovery from posttransplant lymphoma after adoptive immunotherapy.

BACKGROUND: Adoptive transfer of Epstein-Barr virus (EBV)-specific cytotoxic T lymphocytes (CTLs) has been used to treat EBV-induced posttransplant lymphoproliferative disease (PTLD) in solid-organ recipients. This study defines, in detail, the temporal relationship between adoptive transfer and the clinical response, EBV DNA load, and CTL response to EBV latent and lytic antigens in a patient with a subcutaneous PTLD presentation treated with adoptive transfer of autologous CTL. METHODS: A heart transplant patient developed multiple subcutaneous PTLD deposits and was treated with a total of six doses (20 x 106 CTL per dose) of cultured autologous polyclonal EBV-specific CTL by adoptive transfer. RESULTS: Complete regression occurred after the sixth CTL dose, and the patient has remained disease-free from 47 weeks to the present (136 weeks). Real-time polymerase chain reaction analysis showed a reduction in viral load after therapy. Enzyme-linked immunospot analysis using defined EBV CTL epitopes showed that the CTL precursor frequency (pCTL) toward a lytic antigen epitope was elevated early in the course of disease but tended to decrease to lower levels after long-term regression of PTLD. The most dramatic result was seen in relation to three latent CTL epitopes studied. Long-term regression of PTLD was characterized by high pCTL toward the latent antigens. CONCLUSIONS: Increased pCTL reactivity to latent EBV CTL epitopes is coincident with recovery from disease after adoptive transfer of autologous CTL. Furthermore, the results are compatible with the belief that activation of a sustained CTL response to EBV latent epitopes is protective and may be a characteristic of patients in long-term remission from PTLD.

Purification of natural killer cell cytotoxic granules for assaying target cell apoptosis.

We compared two methods originally devised to purify cytoplasmic granules from granulocytes for their capacity to produce cytotoxic granules from natural killer cell lines, suitable for use in target cell apoptosis assays. Both methods utilised nitrogen cavitation to efficiently lyse cells, followed by density gradient fractionation on Percoll to separate the granules from other organelles and granule debris. The first method, originally described by Millard and colleagues, employed DNase I to reduce the viscosity of the initial cell lysate, but the resulting granule fractions were found to contain residual nuclease activity that made them unsuitable for use in apoptosis assays that measure DNA fragmentation. An alternative method described by Borregaard and colleagues utilised a cell relaxation buffer without DNase I. Cytotoxic granules isolated from the NK tumor cell line YT by this protocol were localised predominantly to the densest Percoll fractions, with a density of approximately 1.13 g/ml. These granule fractions were rich in perforin and enzymatically active granzyme B, and induced potent Ca(2+)-dependent lysis and DNA fragmentation of Jurkat cells. Corresponding fractions from non-cytolytic cells, or YT granule extracts incubated with EGTA were unable to mediate significant target cell damage. Cytotoxic granule extracts purified through the Borregaard method were therefore free of nonspecific nuclease activity, and most suitable for studying the mechanism of target cell death induced through the perforin/Ca(2+)-dependent granule pathway.