ALBERT

Description: Dysfunctional T-cells associated with human tumors can be identified utilizing multi-parameter flow cytometry and studied for intracellular signalling checkpoints using phospho-flow or time-of-flight mass spectrometry. Whether tumor-induced hypo-responsive T-cells in patients with myeloma are anergic, exhausted or senescent has not yet been determined. Anergic T-cells are hypo-responsive with low cytokine production and induce tolerance to protect the host from autoimmune disease. T-cell exhaustion is usually associated with chronic viral infection such as CMV but also with some cancers. Both exhausted and anergic T-cells express PD-1, LAG-3, Tim-3, CD160 and CD28. Hypo-responsive T-cells associated with melanoma have the phenotype of exhausted T-cells which can be reactivated using targeted immune check point blockade, resulting in clinical benefit. Senescent T-cells accumulate in an oligoclonal manner with ageing and chronic antigen exposure. Senescence can be characterised by telomere shortening, the expression of CD57, KLRG-1, CD160 and the absence of CD28. However, not all senescent cells have shortened telomeres as telomere independent senescence involving the p21-p53 and p16-pRb pathways and senescence associated secretory phenotype (SASP) T-cells have been described. The aim of this study was to determine whether tumor-induced hypo-responsive T-cells in patients with myeloma are anergic, exhausted or senescent and to identify targetable checkpoints to restore T-cell function. Cytotoxic T-cell clones (CD57+ CD28- TCRVβ restricted) determined by BetaMark TCRVβ analysis were present in 51% of patients with myeloma (n=264), are protective (OS χ2=6.2; p

Description: Previous studies have suggested that expanded T-cell clones are found in the blood of 59% of patients with multiple myeloma. These expanded T-cell clones are associated with prolonged overall survival and thus it has been suggested that they may have anti-tumor activity. We have previously reported similar T-cell clones exist in the peripheral blood of patients with Waldenstrom’s Macroglobulinemia (WM) by using flow cytometry to determine the T cell receptor (TCR) Vβ repertoire. Expanded T-cell clones were detected in 9 of 15 (60%) patient samples. Of the nine patients with TCR Vβ clones, four patients had multiple clones. The TCR Vβ clones were not identical, representing a variety of families across the TCR Vβ repertoire. We have previously found that while the TCRVβ+CD8+CD57 negative subset represents polyclonal populations, the CD57 positive subset represents either monoclonal or biclonal populations. By comparing the genetic profiling of these two subsets from a statistically significant gene list, two genes have been found to be highly upregulated in the CD57 negative polyclonal subset. These two genes are i.) SESN3, a member in the Sorting Nexin (SNX) protein family which is implicated in regulating membrane traffic capable of interaction with phosphatidylinositol-3-phosphate (10.4 fold, p=0.0241); ii.) Epstein-Barr virus induced gene 2 (lymphocyte-specific G protein-coupled receptor) EBI2 (7.4 fold, p=0.0207): This finding is in contrast to previous report that EBI2 is expressed in B-lymphocyte cell lines and in lymphoid tissues but not in T-lymphocyte cell lines or peripheral blood T lymphocytes. For the CD57 positive clonal T cell expansions, consistent with our previous reports, CD28 expression was found to be down regulated by 2.6 fold. There are two genes found to be highly upregulated. They are i.) Granzyme B (4.3 fold, p=0.0337) also called Cytotoxic T-lymphocyte proteinase 2. This enzyme is necessary for target cell lysis in cell-mediated immune responses through caspase-dependent apoptosis; ii.) Granzyme H, also called Cytotoxic T-lymphocyte proteinase and probably necessary for target cell lysis in cell-mediated immune responses. In summary, we have shown that CD57 positive clonal T cell populations exist in some patients with WM. Importantly, microarray results have indicated some genes and proteins that may related to better patients survival as previously demonstrated in patients with Multiple Myeloma.

Description: A technic for the separation of leukocytes from whole blood, utilizing the known difference in density of leukocytes and erythrocytes, is described. Complete separation is effected by means of solutions of serum albumin adjusted to the correct physicochemical properties.

Description: By determining the percentage utilization of intravenously administered radioiron for hemoglobin production over a period of two to three weeks, certain measurements of internal iron metabolism can be made. With a normal rate of blood production, changes in per cent utilization reflect alteration in iron stores. Iron depletion is characterized by more rapid and more complete utilization of radioiron. States of iron excess in hemochromatosis can be identified by their profound depression of radioiron utilization. If, on the other hand, storage iron is not greatly altered, the percentage utilization is determined by the function of the erythropoietic tissue. In myelophthisic anemias, in uremia, and in infection, a similar depression of the curve is found. The rate of erythropoiesis may further be estimated by the slope of the utilization curve, and evidence of abnormal red cell destruction is found in early and abrupt plateau of the utilization curve. A correlation has been made in a variety of hematologic disorders between the radioiron utilization for hemoglobin production and the clinical factors which might be expected to affect iron metabolism in these patients.

Description: Although thalidomide has been reported to increase T cell activity and NK cell cytotoxicity in the blood of patients with multiple myeloma (MM) it is not known whether thalidomide stimulates tumour-specific T cells or causes a general lymphocytosis. We investigated the immunodulatory effects of thalidomide by studying peripheral blood samples from 64 patients in the Australian ALLG MM6 trial, a multicentre randomised phase III study of low-dose thalidomide, prednisolone and Zometa versus prednisolone and Zometa used as post-autologous stem cell transplant (ASCT) maintenance therapy in patients with MM. We have previously demonstrated that TCR Vβ clonal expansions are present in approximately 50% of patients with MM and that their presence correlates with a good prognosis. Sequencing has confirmed that these cells are a clonal expansion of CD3+CD8+CD57+CD28−CD27− late-differentiated cytotoxic effector cells. Flow cytometric analysis of TCR Vβ expansions (24 families) was performed on blood samples from 64 patients enrolled in the MM6 trial both prior to and 12 months post transplant. Prior to transplant, 59% of the 64 patients had TCR Vβ expansions. After thalidomide, T cell expansions were found in a further 8/34 patients compared with 3/30 in the control, no thalidomide group. In the thalidomide group, 70% of the patients had an expansion of more than one T cell clone compared with 32% in the control group (chi2=28.8; p

Description: Impact of donor characteristics is well described for standard intensity unrelated donor and matched sibling donor transplants but may differ in recipients of unrelated donor RIC transplants. Less immunosuppressive regimens at transplantation may lead to higher graft failure rates. We examined risk factors affecting graft failure, acute and chronic graft-versus-host disease (GVHD) and survival after RIC unrelated donor transplants in 715 patients with acute (n=394) and chronic leukemia (n=74), myelodysplastic syndrome (n=70) and non-Hodgkin lymphoma (n=177). Graft failure was defined as 95% donor chimerism. 63 patients had

Description: Red blood cells from patients with sickle cell disease (SCD) exhibit increased electrogenic cation permeability, particularly following deoxygenation and hemoglobin (Hb) polymerisation. This cation permeability, termed Psickle, contributes to cellular dehydration and sickling, and its inhibition remains a major goal for SCD treatment. Nevertheless, its characteristics remain poorly defined, its molecular identity is unknown, and effective inhibitors have not been established. Here, patch-clamp methodology was used to record whole-cell currents in single red blood cells from healthy individuals and patients with SCD. Oxygenated normal red blood cells had a low membrane conductance, unaffected by deoxygenation. Oxygenated HbS cells had significantly increased conductance and, on deoxygenation, showed a further rise in membrane conductance. The deoxygenation-induced pathway was variable in magnitude. It had equal permeability to Na+ and K+, but was less permeable to NMDG+ and Cl−. Conductance to Ca2+ was also of a similar magnitude to that of monovalent cations. It was inhibited by DIDS (100 μM), Zn2+ (100 μM), and by Gd3+ (IC50 of approximately 2 μM). It therefore shares some properties with Psickle. These findings represent the first electrical recordings of single HbS cells and will facilitate progress in understanding altered red blood cell cation transport characteristics of SCD.

Description: Background IL-6 receptor/JAK/STAT is a major signalling pathway associated with pleiotropic functions including cell transformation and proliferation. STAT3, one of a family of 7 STAT members, sits at the apex of the signalling cascade and is a potential target for cancer therapy. Small molecular weight inhibitors either prevent STAT3 phosphorylation or target high constitutive levels of phosphorylated pSTAT3 (pSTAT3). Increased constitutive expression of pSTAT3 is a hallmark of solid tumors and is associated with increased morbidity. However recent studies suggest that this may not apply to hematological malignancies. Phospho-flow cytometry offers a novel approach for the quantitation of constitutive and cytokine induced pSTAT3 expression in subsets of hemopoietic cells. Constitutive pSTAT3 expression is not a prognostic factor for patients with either multiple myeloma (MM) or acute myeloid leukemia (AML), though we recently demonstrated that IL-6 induced pSTAT3 was associated with better prognosis in MM (χ2 =13.06; p5% of normal lymphocytes) in the malignant plasma cells of 44% of patients with MM but by comparison was not increased in AML or B-CLL. Constitutive pSTAT5 was increased (〉5%) in 68% of MM plasma cells and 75% of AML cells and was either normal or decreased in CLL cells. Patients with increased pSTAT3 or 5 in MM cells also had high expression in autologous normal T cells. The level of constitutive pSTAT3 and 5 was not related to the disease status (relapse vs remission). In patients with MM, IL-6 induced an increased pSTAT3 expression (induced/constitutive ratio 〉1.5) in both MM cells and T cells in 88% of samples. IL-6 induced pSTAT3 expression in blasts of 89% of AML patients (median ratio = 79). There was no correlation between the level of pSTAT3 induced by IL-6 and IL-6 receptor expression. IL-6 did not increase pSTAT5 levels in MM, AML or CLL. SP cells in the MM cell line RPMI 8226 had lower constitutive pSTAT3 than the non-SP population and a lower IL-6 receptor expression, but had a greater response to cytokine stimulation, suggesting that SP cells may be more sensitive than non-SP cells to inhibitors of STAT phosphorylation. Addition of RPMI 8226 cells or supernatant to normal T cells caused an increase in pSTAT3, suggesting that the upregulation of pSTAT3 is at least partly due to a tumor-derived soluble agent in the microenvironment. Conclusions These results demonstrate the heterogeneity of constitutive and cytokine induced pSTAT3 and 5 expression in MM, AML and CLL. Elevated levels of constitutive pSTAT3 (approx 44% of MM) and pSTAT5 (88% of MM and AML) in the malignant clone were associated with high levels in autologous non-malignant T cells and are not of prognostic significance. Therefore elevated pSTAT3 and 5 may not be an exclusive hallmark of the malignant clone, as reported in solid tumors, but rather a result of the microenvironment and a high IL6-R expression. Phospho-flow cytometry enables precise quantitation of constitutive and cytokine-induced pSTAT3 and 5, and thus can identify the patients most likely to respond to direct STAT-3 or 5 inhibitor therapies. SP cells (putative MM stem cells) appear to be potentially targetable. Inhibitors targeting signalling pathways may be more efficacious if therapy is directed to patients with targetable checkpoints. Disclosures Hart: DendroCyte BioTech Pty Ltd: Equity Ownership.

Description: We have established a nonhuman primate (NHP) model to test novel agents for their ability to mobilize hemopoietic progenitors and stem cells. Both recombinant thrombopoietin and the truncated form, MGDF, have been shown in Phase 1 trials to increase the number of hemopoietic progenitors in the peripheral blood. We have explored this further in our NHP model by using the combination of pegMGDF with G-CSF and performed comparisons with other cytokines including G-CSF alone, pegylated G-CSF (pegG-CSF) and the combination of G-CSF + Stem Cell Factor (SCF). Male baboons aged between 7 and 14 years of age received cytokines as follows: 1. G-CSF alone 100mcg/kg/day S/C for 5 days; 2. pegG-CSF, single dose 300mcg/kg S/C; 3. G-CSF 100mcg/kg/day S/C + SCF 50mcg/kg/day S/C for 5 days; and 4. pegMGDF 1mcg/kg S/C second daily for 10 days + G-CSF 100mcg/kg/day S/C for 5 days starting 5 days after the pegMGDF. To control for inter-individual variation and allow a direct comparison, animals receiving G-CSF+pegMGDF were also mobilized with G-CSF+SCF with a minimum period of 12 weeks between mobilisations. Blood counts, peripheral blood (PB) CD34 positive cells and colony forming cells (CFC) were quantified at baseline and at day 5 after G-CSF. NOD/SCID repopulating cell (SRC) frequency was quantified at baseline from PBMNCs and on day 5 using cells harvested by leucapheresis. Baseline PB CD34 and CFC counts were 〈 2/μL and

Description: Aims: MSCs are cells being investigated for use in various therapies including facilitation of HSC transplantation and as gene therapy delivery vehicles. We have explored the potential to increase the number of bone marrow (BM) MSCs in vivo, induce mobilization using various cytokine regimens and improve gene transfer into these cells with adeno-associated virus (AAV) in a baboon model. Method: Baboons received cytokines as follows: 1. G-CSF 100mcg/kg/day for 5 days; 2. pegylated G-CSF (pegG-CSF), single dose 300mcg/kg day −5; 3. G-CSF 100mcg/kg/day + stem cell factor (SCF) 50mcg/kg/day for 5 days; and 4. pegylated megakaryocyte growth and development factor (pegMGDF) 1mcg/kg second daily for 10 days + G-CSF 100mcg/kg/day for 5 days starting day −5. Animals underwent BM aspiration at baseline and on the final day of cytokines along with leukapheresis to isolate PBMNCs for detection of peripheral blood (PB) CFU-F. The immunophenotype and differentiation potential of CFU-F derived from animals before and after cytokines was compared. The ability of AAV vectors pseudotyped with capsids derived from AAV of serotypes 1, 2, 3, 4, 5, 6, and 8 to mediate transduction of baboon and human MSCs was assessed. Results: Augmentation of bone marrow MSCs was observed with all cytokine regimens with the fold-increase compared to baseline as follows: 4.1, 2.1, 7.6 and 11.2 after G-CSF, pegG-CSF, G-CSF+SCF and G-CSF+pegMGDF respectively (see Figure 1). The immunophenotype of MSCs obtained after cytokines was identical to baseline cells as was their differentation potential. CFU-F were not detected in baseline PB however they were detected in 3/5 animals after G-CSF+SCF at a frequency of 0.8 to 1.5/mL, but no other cytokine regimen. A similar pattern of transduction efficiency using AAV was shared by human and baboon MSCs (see Figure 2) using control Ad293 cells. Specifically AAV vectors expressing capsids of serotypes 2, 3 and 5 were most efficient in transducing human and baboon MSCs. Those expressing capsids from serotypes 1, 4, 6, and 8 were much less efficient in transducing MSCs from either species. Baboon MSCs were able to be transduced by about 100-fold more than their human equivalent cells using AAV serotypes 2, 3, and 5. Conclusion: This is the first report of mobilization of primate MSCs and, together with the demonstration of in vivo augmentation and AAV gene transfer, offers increased therapeutic opportunities for their safe application in a burgeoning number of diseases. Figure 1: In vivo Bone Marrow CFU-F Augmentation following cytokines Figure 1:. In vivo Bone Marrow CFU-F Augmentation following cytokines Figure 2: Transduction profile of AAV vectors expressing capsids of various serotypes Figure 2:. Transduction profile of AAV vectors expressing capsids of various serotypes