ALBERT

Description: 5271 Cold agglutinin disease is an autoimmune hemolytic anemia mediated by cold reactive autoantibodies triggering a complement mediated hemolysis. This condition, when not associated with infection, is characterized by clonal proliferation of CD20+ B cells that produce monoclonal IgM cold agglutinins. Conventional therapies for primary cold agglutinin disease (CAD) are ineffective, but case reports suggest that rituximab, an anti-CD20 monoclonal antibody, may be effective. In this retrospective single institution study, we evaluated the use of rituximab therapy in 6 patients (pts) [1 male, 5 female; median age 70 years (range 62 – 89)]. Three pts had primary CAD, 2 pts had mixed CAD and warm AIHA, and 2 pts had CAD in the setting of CLL. Five pts had received steroid therapy, 2 pts IVIG, 1 pt azathioprine, and 5 pts PRBC transfusion (2–10 units). Five received induction therapy with rituximab 375 mg/m2 IV weekly for four weeks and 1 for seven weeks. Four patients received maintenance rituximab 375 mg/m2 IV every two months (4+ to 12 cycles). All responded to therapy with a median rise in hemoglobin of 1.8 g/dl at 2 months from initiation of induction with further improvement over time (figure 1). Re-induction was performed in 2 pts; both had an initial one year duration of response and both responded following re-treatment. Median duration of response is 3+ yrs (range 1–8+ yrs); the 8+ yr response was in pt 2 who received only induction therapy. Two pts have completed two years of maintenance therapy and remain in remission at 4 months and 2 years post, respectively. Cold agglutinin titers decreased by 4 fold in 2 patients, 1 fold in 1 pt and remained stable in 1. Despite the improvement in hgb in all pts, laboratory evidence of low grade hemolysis persisted in 4 pts. All pts were able to be tapered off steroids and all remain transfusion free. No unexpected adverse events were noted. Rituximab appears to be a well tolerated and effective therapy for cold agglutinin disease. The need for and length of maintenance therapy remains to be determined. Disclosures: No relevant conflicts of interest to declare.

Description: BACKGROUND: LGL leukemia is a neoplasm arising from either CD3+ T-cells or CD3− NK-cells. Autoimmune-mediated anemia, neutropenia, and rheumatoid arthritis occur frequently in these patients and immunosuppressive agents are used for these associated clinical syndromes. In our previous studies, we found that patients display a constitutively activated Ras and MAPK/ERK signaling cascade that may drive leukemia survival. A multicenter phase 2 clinical trial was initiated to treat LGL leukemia patients with the farnysltransferase-inhibitor R115777 (tipifarnib, Zarnestra®, Johnson & Johnson) that inhibits Ras and other farnesylated proteins. One of the goals of this study was to determine the shifts in cytokine production during therapy. We found that LGL cells treated with tipifarnib in vitro displayed a switch to Th2 (IL-4 and IL-10)-polarized differentiation. After tipifarnib treatment of LGL patients, antigen-activated T-cells produced greater amounts of Th2 (IL4/IL-10) cytokines but less Th1 (IFNγ/TNFα). In this study, we determined the mechanism governing tipifarnib-mediated Th2 polarization in T-cells. METHODS: PBMCs were isolated from 10 healthy donors and from seven patients with T-LGL leukemia before and after treatment with tipifarnib 300 mg twice daily for 21 days of a 28- day cycle. LGL leukemia patients had increased numbers of αβ T lymphocytes and evidence of clonality in association with either neutropenia or transfusion-dependent anemia. Th1 and Th2 cytokines were determined by intracellular staining and flow cytometry after activation with anti-CD3 plus anti-CD28. In some experiments, Th1 polarization was induced by IL-12; whereas, Th2 was induced by IL-4. Expression of T-bet and GATA-3 transcription factors that regulate Th1 and Th2 polarization, respectively, phosphorylated (active) MAPK (ERK1 and ERK2), and total MAPK were measured by Western blots. FTI2153, tipifarnib, and geranylgeranyl transferase inhibitor(GGTI)-2417 were used compared to DMSO control. RESULTS: PBMCs from patients with T-LGL leukemia displayed a dose and time-dependent increase in IL-4 and IL-10 production after drug treatment (average increase to 100% and 43%, respectively). A dose-dependent increase in these Th2 cytokines in T-cells from healthy donors showed that the farnesylated protein targeted by tipifarnib was not selectively expressed in LGL leukemia. Culture with IL-12 induced Th1 differentiation associated with ERK phosphorylation and increased T-bet expression. Pre-treatment with tipifarnib and FTI2153 but not GGTI2417 prior to IL-12 inhibited T-bet induction with no change in anti-CD3-induced MAPK leading to enhanced IL-4 signaling and greater Th2 polarization. CONCLUSIONS: Our data reveal unique, previously unreported effects of FTIs on cytokine signaling in T-cells by inhibiting the induction of T-bet and blocking Th1 differentiation. These results are critical to determine the mechanism of action of tipifarnib in LGL leukemia and suggest that FTIs may be useful for autoimmune or lymphocyte-mediated disorders associated with excessive Th1 cytokine production.

Description: Low-dose methotrexate (MTX) is used as an immunosuppressive agent for the treatment of rheumatoid arthritis (RA), Large Granular Lymphocyte (LGL) leukemia, Cutaneous T Cell Lymphoma (CTCL), autoimmune diseases, and prevention of GvHD during bone marrow transplants. The mechanism for immunosuppression is not clearly understood but most data suggests that apoptosis of activated lymphocytes plays a critical role. In this study, we wanted to define the MTX-sensitive population and to determine the apoptotic pathway activated by MTX. Using a clinically relevant dosage range (8 nM- 1 μM), MTX-mediated apoptosis was first examined in a T lymphoblastic leukemia cell line (CEM). The apoptotic pathway induced by MTX included phosphotidylinositol externalization and caspase-3 activation along with a slight increase in mitochondrial membrane depolarization. We next examined a series of tumor cell lines and normal cells for evidence of MTX-induced apoptosis. Using the same clinically relevant dosage range, we found that MTX-induced apoptosis was primarily observed in the four T cell leukemia cell lines including CEM, Jurkat, MT-2, and HUT78 and in normal PBMCs activated with mitogens and IL-2. Less MTX-induced apoptosis was observed in two myeloid leukemia cell lines including HL-60 and K562 and in a B cell leukemia cell line Raji, and the multiple myeloma cell line 8226. Unactivated PBMCs were resistant to MTX-mediated apoptosis. T cells that are clonally expanded in patients with T-LGL leukemia have a CD8+ cytotoxic phenotype, whereas other diseases that are treated with low-dose MTX, such as CTCL and RA, are characterized by the expansion of CD4+ T cells. We found that both freshly sorted CD4+ and CD8+ cells were MTX resistant. In contrast, PHA plus IL-2 treatment induced MTX sensitivity in T cell with both immunophenotypes. We also examined clinical samples from patients with LGL leukemia. We found that freshly isolated PBMCs from T-LGL leukemia patients were resistant to MTX. Clonal cells from the peripheral blood of LGL leukemia patients are in G0/G1 phase of the cell cycle. Interestingly, we found that PHA plus IL-2 treatment induced the cells to enter S-phase and to become MTX sensitive. These results suggest that only fully activated, proliferating T cells from patients with LGL leukemia undergo apoptosis in response to low-dose MTX. Because there was only minor depolarization of mitochondria after MTX treatment in both CEM cells and normal activated PBMCs, we wanted to examine upstream apoptotic events after MTX treatment. We found that caspase-8 cleavage and enzymatic activity was induced by MTX in both CD95 Type I (HUT78) and Type II (CEM and Jurkat) cells but that there was a differential requirement for caspase-8 activity for apoptosis. We found that caspase-8 activation was independent of the Fas receptor as shown by immunoprecipitation experiments and MTX apoptotic assays in the JM3A5 Fas-receptor mutant Jurkat cell line. Using a Jurkat cell line with a homozygous deletion of the FADD gene, we found that caspase-8 activation, caspase-3 activation, and apoptosis in response to MTX were dependent on the adaptor protein FADD. These findings have important implications for understanding the mechanism of MTX for immunosuppressive therapy.

Description: We previously reported that the lectin domain of αMβ2 receptors on hepatic macrophages mediates rapid clearance of washed murine platelets transfused after refrigeration for 2 hours, recognizing exposed βN-acetylglucosamine (βGlcNAc) residues of N-linked glycans on clustered platelet GPIbα molecules and that the same receptors elicit phagocytosis of refrigerated human platelets human macrophages in vitro. A platelet-associated galactosyltransferase catalyzes the covering of βGlcNAc residues with galactose in the presence of UDP-galactose, thereby blocking clearance of cold mouse platelets in vivo and phagocytosis of human platelets in vitro. These intriguing findings contradicted earlier evidence that refrigeration of human platelets procured for transfusion only promotes their rapid clearance after prolonged (〉8h) incubation and also are inconsistent with the well-known recognition system for exposed galactose residues through asialoglycoprotein (ASGP) receptors. Reconciling these contradictions, we report that the absence of plasma during storage accounts for the differences in time of exposure to cold to promote clearance and that mouse platelets cold-stored in plasma also only clear rapidly after long-term (48h) storage. We also found that hepatic clearance of long-term cold-stored (LTCS) mouse platelets occurs in hepatocytes. Streptavidin-POD staining revealed abundant LTCS biotinylated platelets in hepatocyte phagosomes. Furthermore, cells of the hepatocyte HepG2 line avidly ingest fluorescently-labeled LTCS human platelets (7-fold above the baseline of room-temperature-stored platelets), as evidenced by flow cytometry, fluorescent microscopy and by time-laps video microscopy. Long-term cold storage increases by ~1.7-fold platelet binding of the galactose-specific lectin RCA I, implying that with long-term cold storage, exposed galactose residues cluster sufficiently to induce recognition by hepatocyte ASGPR receptors. The results define a new clearance mechanism, representing the first example of blood cell removal by a non-myeloid cell. Since we find that human platelets also express a cell surface sialotransferase that adds sialic acid to galactose residues, we suggest that a combination of sialylation and glactosylation, achievable by addition of sugar substrates alone, might accommodate long-term cold storage of platelets for transfusion.

Description: Key Points TRAIL is upregulated in LGL leukemia and TRAIL-DcR2 signaling drives constitutive activation of NF-κB. Proteasome inhibitors (bortezomib and ixazomib) effectively interrupt TRAIL-induced activation of NF-κB and induce apoptosis.

Description: Clonal T-cell expansion in patients with T-large-granular lymphocyte (LGL) leukemia occurs by an undefined mechanism that may be related to Fas apoptosis resistance. Here, we demonstrate polarized expansion of CD8+ terminal-memory differentiation in such patients, as demonstrated by CD45RA expression and absence of CD62L expression, suggesting repeated stimulation by antigen in vivo. Elimination of antigen-stimulated T cells normally occurs through Fas-mediated apoptosis. We show that cells from LGL leukemia patients express increased levels of c-FLIP and display resistance to Fas-mediated apoptosis and abridged recruitment of proteins that comprise the death-inducing signaling complex (DISC), including the Fas-associated protein with death-domain (FADD) and caspase-8. Exposure to interleukin-2 (IL-2) for only 24 hours sensitized leukemic LGL to Fas-mediated apoptosis with enhanced formation of the DISC, and increased caspase-8 and caspase-3 activities. We observed dysregulation of c-FLIP by IL-2 in leukemic LGL, suggesting a role in Fas resistance. Our results demonstrate that expanded T cells in patients with LGL leukemia display both functional and phenotypic characteristics of prior antigen activation in vivo and display reduced capacity for Fas-mediated DISC formation.

Description: The idiopathic pneumonia syndrome (IPS) represents a common and often fatal complication of hematopoietic stem cell transplantation (HSCT). Gelsolin is a highly conserved actin-binding protein normally present in plasma that may serve a basic physiological role in limiting acute lung injury of diverse etiologies. We hypothesized that depletion of circulating gelsolin following HSCT might play a permissive role in the pathogenesis of IPS. Plasma gelsolin levels were measured by immunoblotting in frozen samples obtained weekly from 24 patients undergoing allogeneic HSCT. Patients with and without IPS were similar with respect to age, diagnosis, histocompatibility differences between donor and recipient, and conditioning regimen. Mean gelsolin levels in the 9 patients with rapidly fatal IPS were significantly lower than those in patients without this complication by week 3 after HSCT (101 ± 61 mg/L versus 221 ± 54 mg/L; P = .0002). Seven (88%) of the 8 patients with gelsolin levels of less than 100 mg/L in the first month after HSCT died from IPS within 3 months; conversely, gelsolin levels fell to less than 100 mg/L in 7 (78%) of the 9 patients who died from IPS within 3 months of HSCT (P = .0007). These findings suggest that gelsolin levels shortly after allogeneic HSCT can predict the later development of fatal IPS. Gelsolin replacement in selected transplant patients may offer a novel strategy to prevent or reverse IPS.

Description: NK-LDGL is associated with the expansion of CD3−, CD16+ and/or CD56+ lymphocytes, which express a skewed repertoire of NK receptors. (Epling-Burnette et al, Blood103:3431, 2004) Single anti-KIR antibody reactivity was noted in seven patients (54%) and “defective” KIR reactivity in four patients (31%) (n=13). Defective KIR expression was characterized by the loss of reactivity to all available antibodies. These patients expressed a large number of activating KIRs by genotype analysis. Semi-quantitative RT-PCR demonstrated that lower than normal levels of RNA of the inhibitory KIR was present in some patients in contrast to normal NK cells. Consistent with a high level of activating receptors, we found the NK-LDGL cells have potent cytolytic function in both direct and redirected cytotoxicity assays. Overexpression of activating receptors in the absence of the appropriate inhibitory receptors may contribute to the bone marrow suppression commonly observed in these patients. We first performed Class I typing of the HLA-C locus of five patients with NK-LDGL using PCR-SSP (One Lambda, Inc, Canoga Park, CA). Phenotypes included Cw05/Cw16, Cw15/15, Cw07/12, Cw04/08, and Cw02/03. Each of the five patients had lower then normal levels of inhibitory KIR (KIR2DL1 and KIR2DL2/3) by flow-cytometry and by RT-PCR. In one patient, we examined the functional ability of these inhibitory receptors to block killing of tumor cells expressing cross-reactive Class-I ligands. Homozygous Cw15/15 was expressed by NK-LDGL5, which is a cross-reactive epitope to Cw3 and recognized by KIR2DL2/3. NK cells from NK-LDGL5 were capable of killing both the class I-negative tumor target 721.221 cells and HLA-Cw3-transfected 721.221 cells suggesting that deficient levels of inhibitory KIR were expressed on the patients NK cells to block activating NK receptor-mediated killing. These results suggest that NK cells from patients with NK-LDGL could mount dysregulated autoimmune-mediated killing, which may impact disease pathogenesis.

Description: αMβ2 integrin receptors on myeloid cells mediate the adhesion or uptake of diverse ligands, and particular sequences on this integrin’s alpha subunit are responsible for recognition specificity. Inhibition of neutrophil adhesion to platelet monolayers by αM-derived peptides has implicated an αM sequence (I-domain) that also binds iC3b, fibrinogen, ICAM-1 and other ligands. However, αMβ2 also recognizes βN-acetylglucosamine (βGlcNAc) residues on the platelet von Willebrand factor receptor (GPIbα) that are clustered after cooling, suggesting that the αM lectin domain is important for this recognition. The phagocytosis of chilled platelets can be reconstituted when CHO cells are transfected with αMβ2. Removal of the N-T portion of GPIbα, containing the exposed βGlcNAc residues, from the chilled platelet surface using the snake venom mocarhagin inhibits this phagocytosis, as does the addition of 1 mM β-methyl-N-acetyl-D-glucosamine. Replacement of the I domain, or the lectin domain of the αM-chain with the corresponding domain from the αx-chain (p150), reveals that the activity of the αMβ2 integrin toward chilled platelets resides within the lectin-domain, and does not require the I domain. Other evidences for this conclusion include: (1) Sf9 cells expressing αM lectin domain but not β2 bind 3-4 x more chilled platelets than control cells; βGlcNAc inhibits this binding. (2) Soluble recombinant αM lectin domain inhibits the phagocytosis of chilled platelets by αMβ2-expressing THP-1 cells while I-domain substrates (fibrinogen or LPM19c), recombinant I-domain protein, or a synthetic peptide that inhibits neutrophil adhesion to platelet monolayers, are without inhibitory effect. We conclude that chilled platelets are removed from blood by an interaction between βGlcNAc residues on clustered GPIbα and the lectin domain of αM-chain of the αMβ2 integrin. Since the phagocytosis and clearance of chilled platelets by hepatic macrophages has precluded refrigeration of platelets procured for transfusion, a more precise molecular definition of the recognition mechanism could inform strategies for its inhibition, thereby accommodating cold platelet storage.

Description: The natural killer (NK) type of aggressive large granular lymphocytic (LGL) leukemia is a fatal illness that pursues a rapid clinical course. There are no effective therapies for this illness, and pathogenetic mechanisms remain undefined. Here we report that the survivin was highly expressed in both aggressive and chronic leukemic NK cells but not in normal NK cells. In vitro treatment of human and rat NK-LGL leukemia cells with cell-permeable, short-chain C6-ceramide (C6) in nanoliposomal formulation led to caspase-dependent apoptosis and diminished survivin protein expression, in a time- and dose-dependent manner. Importantly, systemic intravenous delivery of nanoliposomal ceramide induced complete remission in the syngeneic Fischer F344 rat model of aggressive NK-LGL leukemia. Therapeutic efficacy was associated with decreased expression of survivin in vivo. These data suggest that in vivo targeting of survivin through delivery of nanoliposomal C6-ceramide may be a promising therapeutic approach for a fatal leukemia.