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Animal models of lymphoma should reflect their counterparts in humans; however, it can be difficult to ascertain whether an induced disease is intralymphatic or extralymphatic based on direct visualization. Current imaging methods are insufficient for identifying lymphatic and intralymphatic involvement. To differentiate intralymphatic from extralymphatic involvement, we have developed a magnetic resonance imaging-based lymphangiography method and tested it on two animal models of lymphoma. A gadolinium (Gd)-labeled dendrimer nanoparticle (generation-6; approximately 220 kDa/ approximately 10 nm) was injected interstitially into mice bearing hematologic malignancies to perform dynamic micromagnetic resonance lymphangiography (micro-MRL). Both a standard T1-weighted 3D fast spoiled gradient echo and a T2/T1-weighted 3D fast imaging employing steady-state acquisition (3D-FIESTA-C) were compared in an imaging study to differentiate intralymphatic from extralymphatic involvement of tumors. The lymphatics and lymph nodes were visualized with both methods in all cases. In addition, 3D-FIESTA-C depicted both the lymphatic system and the extralymphatic tumor. In an animal model, 3D-FIESTA-C demonstrated that the bulk of the tumor thought to be intralymphatic was actually extralymphatic. In conclusion, micro-MRL, using Gd-labeled dendrimer nanoparticles with the combined method, can define both the normal and abnormal lymphatics and can distinguish intralymphatic from extralymphatic diseases in mouse models of malignant lymphoma.

DNA vaccines are a promising technology for the induction of Ag-specific immune responses, and much recent attention has gone into improving their immune potency. In this study we test the feasibility of delivering a plasmid encoding IL-15 as a DNA vaccine adjuvant for the induction of improved Ag-specific CD8+ T cellular immune responses. Because native IL-15 is poorly expressed, we used PCR-based strategies to develop an optimized construct that expresses 80-fold higher than the native IL-15 construct. Using a DNA vaccination model, we determined that immunization with optimized IL-15 in combination with HIV-1gag DNA constructs resulted in a significant enhancement of Ag-specific CD8+ T cell proliferation and IFN-γ secretion, and strong induction of long-lived CD8+ T cell responses. In an influenza DNA vaccine model, coimmunization with plasmid expressing influenza A PR8/34 hemagglutinin with the optimized IL-15 plasmid generated improved long term CD8+ T cellular immunity and protected the mice against a lethal mucosal challenge with influenza virus. Because we observed that IL-15 appeared to mostly adjuvant CD8+ T cell function, we show that in the partial, but not total, absence of CD4+ T cell help, plasmid-delivered IL-15 could restore CD8 secondary immune responses to an antigenic DNA plasmid, supporting the idea that the effects of IL-15 on CD8+ T cell expansion require the presence of low levels of CD4 T cells. These data suggest a role for enhanced plasmid IL-15 as a candidate adjuvant for vaccine or immunotherapeutic studies.

The survival of CD8 + /CD44 hi memory phenotype T cells depends on an IL-15 activity on nonlymphoid cells. Here, we report that IL-15 and its receptor were induced on dendritic cells (DCs) by a combination of IFN-γ and NF-κB relA inducers. IL-15 conferred in an autocrine loop resistance to apoptosis that accompanied the maturation process in DCs in vitro . As an apparent result in vivo , mice deficient in IL-15 or its receptor harbor few DCs. Injecting DCs into IL-15 -/- mice was associated with the appearance of CD8 + /CD44 hi T cells that depended on IL-15 expression but also correlated with the longevity of the DCs. These findings support the hypothesis that DCs mediate the effect of IL-15 on CD8 + /CD44 hi memory phenotype T cells.

Fluorescence energy homotransfer offers a powerful tool for the investigation of the state of oligomerization of cell surface receptors on a cell-by-cell basis by measuring the polarized components of fluorescence intensity of cells labeled with fluorescently stained antibodies. Here we describe homotransfer-based methods for the flow cytometric detection and analysis of hetero- and homo-associations of cell surface receptors. Homotransfer efficiencies for two- and three-body energy transfer interactions are defined and their frequency distribution curves are computed from the fluorescence anisotropy distributions of multiple-labeled cells. The fractions of receptors involved in homo-clustering is calculated based on the dependence of the fluorescence anisotropy on the surface concentration of the fluorescently stained antibodies. A homotransfer analysis of the homo- and hetero-clustering of the MHCI and MHCII glycoproteins, the cytokine receptor IL-2Rα, transferrin receptor and the receptor-type tyrosine phosphatase CD45 on JY B and Kit-225-K6 T cells is presented. We investigated how various factors such as the type of dye, rotational mobility of the dye and dye-targeting antibody, as well as the wavelength of the exciting light affect the homotransfer. We show that the homotransfer technique combined with the high statistical resolution of flow cytometry is an effective tool for detecting different oligomeric states of receptors by using fluorophores having restricted rotational mobility on the time scale of fluorescence.

Adult T-cell leukemia (ATL) develops in a small proportion of individuals infected with human T-cell lymphotrophic virus-1. The leukemia consists of an overabundance of activated T cells, which express CD25 on their cell surfaces. Presently, there is no accepted curative therapy for ATL. Flavopiridol, an inhibitor of cyclin-dependent kinases, has potent antiproliferative effects and antitumor activity. We investigated the therapeutic efficacy of flavopiridol alone and in combination with humanized anti-Tac antibody (HAT), which recognizes CD25, in a murine model of human ATL. The ATL model was established by intraperitoneal injection of MET-1 leukemic cells into nonobese diabetic/severe combined immunodeficient mice. Either flavopiridol, given 2.5 mg/kg body weight daily for 5 days, or HAT, given 100 μg weekly for 4 weeks, inhibited tumor growth as monitored by serum levels of human β-2-microglobulin (β2μ; P < .01), and prolonged survival of the leukemia-bearing mice (P < .05) as compared with the control group. Combination of the 2 agents dramatically enhanced the antitumor effect, as shown by both β2μ levels and survival of the mice, when compared with those in the flavopiridol or HAT alone group (P < .01). The significantly improved therapeutic efficacy by combining flavopiridol with HAT provides support for a clinical trial in the treatment of ATL.

Gold nanoparticles of 30 nm diameter bound to cell-surface receptor major histocompatibility complex glycoproteins (MHCI and MHCII), interleukin-2 receptor alpha subunit (IL-2Ralpha), very late antigen-4 (VLA-4) integrin, transferrin receptor, and the receptor-type protein tyrosin phosphatase CD45 are shown by the patch-clamp technique to selectively modulate binding characteristics of Pi(2) toxin, an efficient blocker of K(v)1.3 channels. After correlating the electrophysiological data with those on the underlying receptor clusters obtained by simultaneously conducted flow cytometric energy transfer measurements, the modulation was proved to be sensitive to the density and size of the receptor clusters, and to the locations of the receptors as well. Based on the observation that engagement of MHCII by a monoclonal antibody down-regulates channel current and based on the close nanometer-scale proximity of the MHCI and MHCII glycoproteins, an analogous experiment was carried out when gold nanoparticles bound to MHCI delayed down-regulation of the K(v)1.3 current initiated by ligation of MHCII. Localization of K(v)1.3 channels in the nanometer-scale vicinity of the MHC-containing lipid rafts is demonstrated for the first time. A method is proposed for detecting receptor-channel or receptor-receptor proximity by observing nanoparticle-induced increase in relaxation times following concentration jumps of ligands binding to channels or to receptors capable of regulating channel currents.

Background: The presence of lymph node metastases is an important factor in breast cancer patient prognosis. Therefore, the precise identification of sentinel lymph nodes in these patients is critical. Improving current magnetic resonance (MR) imaging methods using a newly synthesized nano-size paramagnetic molecule, G6, as a contrast agent, provides an attractive means toward attaining this goal. Methods: A four-dimensional method of micro-MR lymphangiography using G6 (9 nm/240 kd) was developed to visualize the lymphatic ducts and lymph nodes draining mouse mammary tumors over time. The ability of micro-MR lymphangiography with the G6 contrast agent to visualize lymphatic drainage of normal mouse mammary tissue was compared with that of the conventional MR contrast agent, Gd-[DTPA]-dimeglumine (

Purpose: The use of an α emitter for radioimmunotherapy has potential advantages compared with β emitters. When administered systemically optimal targeting of intact antibodies requires >24 h, therefore limiting the use of short-lived α emitters. This study investigated the biodistribution of bismuth-labeled biotin in A431 tumor-bearing mice pretargeted with antibody B3-streptavidin (B3-SA) and examined the therapeutic efficacy of the α emitter, 213Bi-labeled biotin.Experimental Design: Biotinidase-resistant 7,10-tetraazacyclododecane-N,N′,N″,N‴-tetraacetic acid (DOTA)-biotin was radiolabeled with 205,206Bi or 213Bi. Treatment of tumor-bearing mice began by administration of B3-SA (400 μg) to target the tumor sites for 24 h. Then, an agent containing biotin and galactose groups was used to clear the conjugate from the circulation. Four h later, bismuth-radiolabeled DOTA-biotin was given, and biodistribution or therapy was evaluated. Dose escalation treatment from 3.7–74 MBq was performed, and the effects on tumors of different sizes were investigated. Tumor growth, complete blood cell counts, toxicity, and survival were monitored.Results: Radiolabeled biotin cleared rapidly. Rapid tumor uptake resulted in much higher tumor:nontumor targeting ratios than achieved with the directly labeled monoclonal antibody. Dose escalation revealed that 74 MBq caused acute death of mice, whereas 0.37–37 MBq doses inhibited tumor growth and prolonged survival significantly. Evidence of mild hematological toxicity was noted. At therapeutically effective doses renal toxicity was observed.Conclusions: 213Bi-DOTA-biotin, directed by the Pretarget method to tumor-targeted B3-SA, showed a therapeutic effect, although the therapeutic index was low. The source of the toxicity was most likely related to the renal toxicity.

Micro-MRI methods to detect renal cysts in mice. Background Mouse models of disease, especially using transgenic and knockout technologies, are powerful tools to analyze the molecular basis of disease. We recently reported that a new dynamic micro-MRI method with dendrimer-based contrast agents can visualize renal structure and function in normal living mice and mice with acute renal failure. While MRI contrast enhancement is useful for detecting functional impairment of the kidneys, this technology has limitations in assessing morphologic changes, particularly cystic disease, because contrast-enhanced micro-MRI depicts cysts as low-intensity areas that cannot be distinguished from fibrotic foci. Methods In the current study, we evaluated if micro-MRI employing a new three-dimensional MR hydrography signal sequence [three-dimensional fast imaging employing steady-state acquisition (3D-FIESTA)] can visualize chronic cystic changes without any contrast agents. Results We were able to positively depict multiple renal cortical cysts of ∼0.2mm diameter in a mouse model of sickle cell disease and observe serial changes of renal cysts (>0.2mm diameter) in cyclooxygenase-2 (COX-2) knockout mice during a 2 1 / 2 -month period. Some cysts decreased in size over time. Conclusions Micro-MRI with 3D-FIESTA can depict cyst formation in the diseased kidneys of living mice without injection of contrast agents.

The efficacy of monoclonal antibodies (mAbs) as single agents in targeted cancer therapy has proven to be limited. Arming mAbs with a potent toxic drug could enhance their activity. Here we report that conjugating geldanamycin (GA) to the anti-HER2 mAb Herceptin improved the activity of Herceptin. The IC50s of the immunoconjugate H-GA were 10–200-fold lower than that of Herceptin in antiproliferative assays, depending on the cell line. The H-GA mode of action involved HER2 degradation, which was partially lactacystin sensitive and thus proteasome dependent. The linkage between GA and Herceptin remained stable in the circulation, as suggested by the pharmacokinetics of Herceptin and conjugated GA, which were almost identical and significantly different from that of free GA. Tumor uptake of Herceptin and H-GA were similar (52 ± 7 and 43 ± 7% of the initial injected dose per gram tissue, respectively; P = 0.077), indicating no apparent damage attributable to conjugation. Therapy experiments in xenograft-bearing mice consisted of weekly i.p. doses, 4 mg/kg for 4 months. H-GA showed a greater antitumor effect than Herceptin because it induced tumor regression in 69% of the recipients compared with 7% by Herceptin alone. Median survival time was 145 days as opposed to 78 days, and 31% of the recipients remained tumor free 2 months after therapy was terminated versus 0% in the Herceptin group. Enhancement of Herceptin activity could be of significant clinical value. In addition, the chemical linkage and the considerations in therapeutic regimen described here could be applied to other immunoconjugates for targeted therapy of a broad spectrum of cancers.

Our emerging understanding of the IL-2/IL-2R system opens the possibility for more specific immunotherapy of CTCL. This understanding, taken in conjunction with the ability to produce humanized antibodies to the IL-2R subunit by genetic engineering; to arm these antibodies, as well as IL-2 itself with toxins or with alpha- and beta-emitting radionuclides; and to modulate IL-2R subunits to optimize targeting of these agents provides a rational therapeutic strategy for the treatment of IL-2R-expressing CTCL. Although most of these studies were conducted in HTLV-1-associated T-cell lymphomas or CTCL, it is likely that these agents may be applicable to other T-cell lymphomas, including the anaplastic large cell lymphomas, peripheral T-cell lymphomas, and the natural killer lymphomas, because these cells express the IL-2 receptor.

Although the introduction of the monoclonal antibody rituximab 5 years ago led to a marked improvement in the treatment of non-Hodgkin's lymphoma (NHL), most patients do not experience a complete response to therapy, and many who do respond relapse. One way of improving the efficacy of monoclonal antibodies is to use them to deliver cytotoxic agents, such as radionuclides, to the tumor. Monoclonal antibodies armed with radionuclides provide a means of targeting radiation therapy specifically to tumor cells that express the antigen to which the antibody was originally raised. Subsequently, in 2002, the first radiolabeled monoclonal antibody, 90Y-ibritumomab tiuxetan was approved for the treatment of patients with relapsed or refractory low-grade follicular or transformed B-cell NHL, including patients with follicular lymphoma refractory to rituximab. Attempts to optimize the efficacy of radioimmunotherapy are ongoing, however, and there are three factors that need to be considered: choice of antibody/antigen, choice of delivery of system to be used, and choice of radionuclide. CD25 (IL-2R alpha) is an ideal choice for a target antigen as it is over-expressed by a number of tumor cells, including adult T-cell leukemia (ATL); 9 of 16 patients with ATL responded to treatment with anti-Tac (which targets the interleukin-2 receptor-alpha [IL-2R alpha]), conjugated to 90Y. The dose of radionuclide that can be delivered to a tumor can be increased dramatically by using a three-step process in which the antibody and radioactivity are delivered separately to the antigen in order to improve tumor-to-normal tissue ratios. The most commonly used radionuclides in radioimmunotherapy to date are beta-emitters. However, the pretargeting process makes the use of short-lived alpha-emitters more feasible. The results of experiments involving this pretargeting process and alpha- and beta-emitting radionuclides in leukemia and lymphoma models suggest that alpha-emitters may be more effective in the treatment of small tumors, micrometastases and isolated cells, and that beta-emitters may be more suitable for use in large tumor masses, such as lymphomas.

Therapy for severe uveitis is frequently long-term immunosuppression using systemic corticosteroids and cytotoxic agents, but side effects make long-term therapy difficult. A long-term (>4 year) Phase I/II single armed interventional study using intravenous anti-IL-2 receptor alpha treatments (daclizumab) and a short-term Phase II study evaluating the use of a subcutaneous daclizumab formulation were conducted. Patients were tapered off their systemic immunosuppressive therapy and received daclizumab infusions or subcutaneous injections at intervals varying from 2 to 6 weeks. In the long-term study, seven of ten enrolled patients were tapered from their original immunosuppressive medications and maintained exclusively on repeated daclizumab infusions for control of their uveitis for over 4 years. No patient was permanently removed from therapy for an adverse event ascribed to the medication. The use of 6-week infusion intervals led to recurrence of uveitis, while 2- to 4-week intervals did not. Only one patient developed measurable anti-daclizumab antibodies but this disappeared when subcutaneous therapy was begun. In the short-term study, four of the five patients receiving the subcutaneous formulation met the study endpoints for success within the first 12 weeks. All five were successful by 26 weeks. These studies provide preliminary evidence that regularly administered long-term daclizumab therapy can be given in lieu of standard immunosuppression for years to treat severe uveitis and that subcutaneously administered daclizumab appeared to be a clinically viable treatment strategy. These studies suggest that anti-IL-2 receptor blockade could be useful in the treatment of Th1-mediated autoimmune conditions.

Adult T-cell leukemia (ATL) develops in a small proportion of individuals infected with the retrovirus human T-cell leukemia virus (HTLV-1). We evaluated the efficacy of MEDI-507 (a humanized monoclonal antibody directed against CD2) alone and in combination with humanized anti-Tac (HAT) directed toward CD25, the interleukin-2 receptor α (IL-2Rα) using a human adult T-cell leukemia xenograft model. Weekly treatments (4) with HAT significantly prolonged the survival of the ATL-bearing mice when compared with phosphate-buffered saline (PBS)–treated controls (P < .0001). Mice treated with MEDI-507 (100 μg/wk for 4 weeks) survived longer than those treated with HAT (P < .0025). Furthermore, prolonged treatment (6 months) of ATL with MEDI-507 significantly improved the outcome when compared with a short course (4 weeks) of therapy (P < .0036). Such treatment with weekly MEDI-507 for 6 months led to a prolonged survival of the ATL-bearing mice that was comparable with the survival observed in the control group of mice that did not receive a tumor or therapeutic agent. We also found that the expression of Fcγ receptors (FcRγ) on polymorphonuclear leukocytes and monocytes was required for MEDI-507–mediated tumor killing in vivo. Thus, the tumor-killing mechanism with MEDI-507 in vivo required the expression of the receptor FcRγIII on polymorphonuclear leukocytes and monocytes, suggesting that it is mediated by a form of antibody-dependent cellular cytotoxicity. These results demonstrate that MEDI-507 has therapeutic efficacy on ATL in vivo and provides support for a clinical trial involving this monoclonal antibody in the treatment of patients with CD2-expressing leukemias and lymphomas. (Blood. 2003; 102:284-288)

Vaccine efficacy is determined largely by cellular and humoral immunity as well as long-lasting immunological memory. IL-2 and IL-15 were evaluated in vaccinia vectors expressing HIV gp160 for the establishment of an effective vaccine strategy. Both IL-2 and IL-15 in the vaccinia vector induced strong and long-lasting antibody-mediated immunity as well as a short-term cytotoxic T cell response against HIV gp120. In addition, IL-15 also supported robust CD8 + T cell-mediated long-term immunity, whereas the CD8 + T cell-mediated immunity induced by IL-2 was short-lived. Moreover, we found that the cytokine milieu at the time of priming had surprisingly persistent effects on the character of the memory CD8 T cells long afterward with respect to their fate, functional activities, cytokine receptor expression, and antigen-independent proliferation.

We report intriguing aspects of the contribution of IL-15Ralpha to IL-15 functions. Consistent with high-affinity interactions between IL-15 and IL-15Ralpha, these two molecules form stable complexes on the cell surface of activated monocytes. The formation of IL-15/IL-15Ralpha complexes on cell surfaces induces a trans-endosomal recycling of IL-15 leading to the persistence of surface-bound IL-15 due to the constant reappearance of IL-15 on plasma membranes. This complex contributes to the long survival of T cells expressing IL-15Ralpha after IL-15 withdrawal. Finally, these complexes on activated monocytes present IL-15 in trans to target cells such as CD8(+) T cells that express only IL-2/15Rbeta and gammac upon cell-cell interaction.

Subunits (α, β and γ) of the interleukin-2 receptor complex (IL-2R) are involved in both proliferative and activation-induced cell death (AICD) signaling of T cells. In addition, the signaling β and γ chains are shared by other cytokines (e.g. IL-7, IL-9, IL-15). However, the molecular mechanisms responsible for recruiting/sorting the α chains to the signaling chains at the cell surface are not clear. Here we show, in four cell lines of human adult T cell lymphoma/leukemia origin, that the three IL-2R subunits are compartmented together with HLA glycoproteins and CD48 molecules in the plasma membrane, by means of fluorescence resonance energy transfer (FRET), confocal microscopy and immuno-biochemical techniques. In addition to the β and γc chains constitutively expressed in detergent-resistant membrane fractions (DRMs) of T cells, IL-2Rα (CD25) was also found in DRMs, independently of its ligand-occupation. Association of CD25 with rafts was also confirmed by its colocalization with GM-1 ganglioside. Depletion of membrane cholesterol using methyl-β-cyclodextrin substantially reduced co-clustering of CD25 with CD48 and HLA-DR, as well as the IL-2 stimulated tyrosine-phosphorylation of STATs (signal transducer and activator of transcription). These data indicate a GPI-microdomain (raft)-assisted recruitment of CD25 to the vicinity of the signaling β and γc chains. Rafts may promote rapid formation of a high affinity IL-2R complex, even at low levels of IL-2 stimulus, and may also form a platform for the regulation of IL-2 induced signals by GPI-proteins (e.g. CD48). Based on these data, the integrity of these GPI-microdomains seems critical in signal transduction through the IL-2R complex.

Quantitative serial serum measurements of human chorionic gonadotropin (hCG) and alpha-fetoprotein (AFP) levels using sensitive double-antibody radioimmunoassays were performed in nine patients with germinal cell tumors before and during treatment. The sera of eight of the nine were found to have a hCG marker and five of the nine an AFP marker. The sera of four patients were found to have both. Serial serum levels of hCG, of AFP, or both were useful for monitoring disease activity during therapy in all nine patients. In two patients tumor masses failed to diminish during chemotherapy, but the tumor markers fell appropriately. At surgery one patient had a mature teratoma, the other a mature teratoma with a microscopic focus of an embryonal cell tumor. In one patient tumor reactivation was reflected by the emergence of only one of two previously elevated tumor markers. One patient had a rise in hCG, another a rise in both markers coincident with recurrence of tumor. Serial measurements of AFP and hCG are useful for following the response to therapy of germinal tumors, and can assist in making therapeutic decisions.

Although interleukin-2 (IL-2) and -15 (IL-15) share two receptor subunits and many functions, at times they provide contrasting contributions to T-cell-mediated immune responses. IL-2, through its pivitol role in activation-induced cell death (AICD), is involved in peripheral tolerance through the elimination of self-reactive T cells. In contrast, in general IL-15 manifests antiapoptotic actions and inhibits IL-2-mediated AICD. IL-15 stimulates persistence of memory phenotype CD8+ T cells, whereas IL-2 inhibits their expression. Humanized monoclonal antibodies that recognize IL-2Ra, the private receptor for IL-2, are being employed to inhibit allograft rejection and to treat T-cell leukemia/lymphoma. Therapies directed toward inhibiting the actions of the inflammatory cytokine, IL-15, are proposed for an array of autoimmune disorders as well as diseases associated with the retrovirus human T-cell lymphotrophic virus 1.

Meeting abstracts IL-15 activates and induces the proliferation of CD8+ T cells and NK cells. ALT-803 (Altor Bioscience, Miramar, FL) is a superagonist of IL-15 consisting of 2 molecules of IL-15 N-to-D substituted at position 72, bound to 2 molecules of the "sushi" domain of IL-15 alpha receptor

IL-15 is a 14-15 kDa member of the four α-helix bundle of cytokines that acts through a heterotrimeric receptor involving IL-2/IL-15R β, γc and the IL-15 specific receptor subunit IL-15R α. IL-15 stimulates the proliferation of T, B and NK cells, and induces stem, central and effector memory CD8 T cells. In rhesus macaques, continuous infusion of recombinant human IL-15 at 20 μg/kg/day was associated with approximately a 10-fold increase in the numbers of circulating NK, γ/δ cells and monocytes, and an 80- to 100-fold increase in the numbers of effector memory CD8 T cells. IL-15 has shown efficacy in murine models of malignancy. Clinical trials involving recombinant human IL-15 given by bolus infusions have been completed and by subcutaneous and continuous intravenous infusions are underway in patients with metastatic malignancy. Furthermore, clinical trials are being initiated that employ the combination of IL-15 with IL-15R α(+/-) IgFc.

IL-2 and IL-15 have overlapping functions since they share the IL-2Rbetagamma receptor complex. However, each cytokine has a private alpha receptor namely IL-2Ralpha for IL-2 and IL-15Ralpha for IL-15. As a consequence the effects of the two cytokines may differ. We describe the differential effects of the two cytokines regarding the induction of cell surface expression of the IL-2Ralpha subunit on YT-l cells. Both cytokines induced transcription of the IL-2Ralpha gene. Furthermore translation of IL-2Ralpha leading to intracellular expression of the receptor was observed following either IL-2 or IL-15 addition. However, only IL-15 was associated with the induction of cell surface expression of IL-2Ralpha. With IL-2 there appears to be an impediment to the translocation of IL-2Ralpha to the cell membrane. Since surface expression of IL-2Ralpha is a key element in the formation of the high affinity IL-2 receptor, translocation of IL-2Ralpha to the membrane represents another level of control of the immune response in addition to regulation of IL-2Ralpha transcription and translation.

Natural killer (NK) cells are cytotoxic innate lymphoid cells, which play a major role in tumor surveillance. We have tested the safety and efficacy of allogeneic NK cell adoptive transfer from heathy haploidentical donors and demonstrate that in vivo expansion and persistence of the adoptively transferred NK cells at Day 14 after infusion correlates with 30-50% remission in patients with refractory AML. However, the factors that influence successful persistence of donor-derived NK cells are unclear. We hypothesized that recipient T cells play a role in the rejection of allogeneic NK cells and a correlation could exist between persistence of donor-derived NK cells and exhaustion in recipient T cells. T cell exhaustion, a well-established state of T-cell dysfunction occurring in response to chronic and continuous antigen stimulation, is well-documented in human cancer, and characterized by progressive and hierarchical loss of effector functions including sustained up-regulation and co-expression of multiple inhibitory receptors such as PD-1 and Tim-3 and altered expression of key transcription factors including the gain of Eomes and T-bet. We used samples from a phase I/II trial of CD3/CD19 depleted, IL-15-activated, haploidentical donor NK cells delivered following conditioning with cyclophosphamide (50mg/kg) and fludarabine (35 mg/m2 x 3days) in adults with chemotherapy refractory AML. Patients received donor NK cells on Day 0 followed by 10 doses of recombinant human (rh) IL-15 (2 mcg/kg/day) manufactured by the NCI and delivered SQ on Days 1-5 and 8-12. A significant proportion of patients experienced donor NK cell expansion at Day 14 (expanders), but there were some that did not (non-expanders). Therapeutic benefit has only been noted among the expanders. We examined samples from a total of 10 patients with refractory AML, 5 expanders and 5 non-expanders, along with their 10 respective donors. Cryopreserved patient PBMCs were thawed and rested overnight in RPMI-1640 with 2% FBS. The cells were stained for viability, for surface markers using antibodies against CD3, CD8, CD56, PD-1, and Tim-3, intracellularly stained for Eomes and T-bet. We evaluated CD8+ T cell expression of PD-1 and Tim-3, in expanders and non-expanders, prior to chemotherapy and at Day 14. Paired donor T cells from the non-mobilized apheresis products served as controls. Prior to chemotherapy, both patient groups had equivalently elevated expression of both PD1 and Tim-3 on CD8+ T cells. However, at Day 14, the expanders had persistence of PD-1 and Tim-3 while expression on non-expander CD8+ T cells fell to donor level (Figure 1A). Furthermore, expanders had a significantly higher proportion of CD8+ T cells that either co-expressed PD-1 and Tim-3 (p=0.017) or had a PD-1high phenotype (p=0.032) at Day 14, both of which are suggestive of an exhausted state, as opposed to an activated one (Figure 1B,C). Next, we examined Eomes and T-bet expression in recipient T cells. While generally low among healthy T cell populations, as T cells become exhausted, they gain expression of these transcription factors. We looked specifically at the expression of these transcription factors in the recipient CD8+ T cell populations with the highest likelihood of being exhausted, i.e. those co-expressing PD-1 and Tim-3 or those with the PD-1high phenotype. Eomes expression in recipient PD-1high CD8+ T cells and in PD-1+Tim-3+ CD8+ T cells at Day 14 was significantly higher (p=0.01 and p=0.04, respectively) among expanders compared to non-expanders (Figure 2A,B). Likewise, T-bet expression was greater (p=0.004) among expanders in the PD-1high population (Figure 2A). There was no difference in the T-bet expression in PD-1+Tim-3+ CD8+ T cells between groups (Figure 2B). While all patients with refractory AML receiving NK cell adoptive transfer had an elevated percentage of CD8+ T cells with an exhausted phenotype prior to therapy, only patients with donor-derived NK cell expansion had persistence of the exhausted T cell phenotype at Day 14. Thus, T cell mediated rejection is a major obstacle to overcome for successful adoptive NK cell transfer which could in part be aided by a link between recipient T cell exhaustion and expansion of NK cells. This might further suggest that IL-15 reverses T cell exhaustion among those who failed to achieve donor-derived NK cell expansion. Disclosures Miller: Fate Therapeutics: Consultancy, Research Funding; Oxis Biotech: Consultancy, Other: SAB.

A transgenic (Tg) mouse expressing human IL-15 was generated to define the role of IL-15 in the normal immune response. Overexpression of IL-15 resulted in an increase of NK, CD44 hi CD8 memory T cells, and γδ T cells. Additionally, we observed the emergence of a novel type of NK-T cells with CD8αα′ expression. Due to the expansion and activation of NK cells, the IL-15Tg mouse showed enhanced innate immunity. In adaptive T cell immunity, the roles of IL-15 contrasted with those of IL-2. IL-15 inhibited IL-2-induced T cell death, which plays a role in the maintenance of peripheral self-tolerance. IL-15 thus seems to contribute to enhanced immune memory by selectively propagating memory T cells and by blocking T cell death mediated by IL-2.

HER2/neu is a receptor protein whose overexpression strongly correlates with poor prognosis in breast carcinomas. It is used increasingly as a therapeutic target for breast carcinomas in clinical human trials. In particular, monoclonal antibodies (mAbs) that target HER2/neu have been investigated for therapeutic applications. Anti-Her2 mAbs linked to radionuclides have been used in pre-clinical models and in patients for radioimmunolocalization of tumors using external scintigraphy and intraoperative handheld gamma detecting probes. Initial efforts of systemic radioimmunotherapy employed radio-iodinated mAbs. In a murine human breast cancer xenograft model, the radiolabeled mAb was 20-fold more effective than the unarmed mAb but did not yield permanent eradication of the tumor. Due to the limitations of radio-iodine ((131)I), metallic radionuclides (e.g, the beta(-)-emmiter (90)Y or the alpha-emitters (213)Bi, (212)Bi, (212)Pb) linked to mAb's are being evaluated in murine models. Complete elimination of beast cancer xenografts was possible in an adjuvant setting using (212)Pb linked to an anti-HER2/neu mAb. Finally, major efforts are underway to increase the access of the radiolabel to the tumor cell in large solid tumors. One such approach involved a pre-targeting strategy with an initial administration of streptavidin linked to mAb that, after a clearing step, was followed by biotin armed with the radionuclide. Although major challenges must be addressed, HER2/neu represents a very attractive target for systemic radioimmunotherapy of breast cancer.

The stoichiometry of the p16INK4A and p15INK4B proteins bound to the cyclin D-CDK4/6 complex regulates the entry of cells into the G1 phase of the cell cycle. Thus, their level of expression is essential in maintaining regulated cell growth. In several tumors, deletion of these genes has been reported and, more recently, promoter methylation has been suggested as an alternative mechanism to decrease the expression of these cell cycle inhibitor proteins. Here, we studied the methylation status and the integrity of the p16INK4A and p15INK4B genes in 8 chronically HTLV-I-infected T cell lines and in ex vivo cells from 14 ATLL patients. Deletion of the locus carrying both genes was not found in the HTLV-I-infected T cell lines but was found in seven of eight acute ATLL cases and in none of the PBMCs from the chronic cases or the affected lymph nodes of the lymphoma type. In contrast, partial or complete methylation of one or both genes was found only in chronically HTLV-I T cells. Thus, HTLV-I infection targets the p16INK4A and p15INK4B loci both in vitro and in vivo, although the mechanisms may differ.

PURPOSE: To evaluate the toxicity, pharmacokinetics, immunogenicity, and antitumor activity of anti-Tac(Fv)-PE38 (LMB-2), an anti-CD25 recombinant immunotoxin that contains an antibody Fv fragment fused to truncated Pseudomonas exotoxin. PATIENTS AND METHODS: Patients with CD25+ hematologic malignancies for whom standard and salvage therapies failed were treated with LMB-2 at dose levels that ranged from 2 to 63 μg/kg administered intravenously over 30 minutes on alternate days for three doses (QOD × 3). RESULTS: LMB-2 was administered to 35 patients for a total of 59 cycles. Dose-limiting toxicity at the 63 μg/kg level was reversible and included transaminase elevations in one patient and diarrhea and cardiomyopathy in another. LMB-2 was well tolerated in nine patients at the maximum-tolerated dose (40 μg/kg QOD × 3); toxicity was transient and most commonly included transaminase elevations (eight patients) and fever (seven patients). Only six of 35 patients developed significant neutralizing antibodies after the first cycle. The median half-life was 4 hours. One hairy cell leukemia (HCL) patient achieved a complete remission, which is ongoing at 20 months. Seven partial responses were observed in cutaneous T-cell lymphoma (one patient), HCL (three patients), chronic lymphocytic leukemia (one patient), Hodgkin’s disease (one patient), and adult T-cell leukemia (one patient). Responding patients had 2 to 5 log reductions of circulating malignant cells, improvement in skin lesions, and regression of lymphomatous masses and splenomegaly. All four patients with HCL responded to treatment. CONCLUSION: LMB-2 has clinical activity in CD25+ hematologic malignancies and is relatively nonimmunogenic. It is the first recombinant immunotoxin to induce major responses in cancer. LMB-2 and similar agents that target other cancer antigens merit further clinical development.

The preparation and characterization of a new tetramercaptocalix[4]arene and its 211At complex is described. The in vivo stability of the complex has been evaluated in nude mice for the purpose of α-radioimmunotherapy of cancer. The study shows that while this complex lacks adequate stability in vivo, 211At may behave as a heavy metal ion and may be successfully sequestered for biomedical applications. Copyright © 2000 John Wiley & Sons, Ltd.

This article focuses on the recent dramatic advances in the applications of monoclonal antibody therapy to hematopoietic and neoplastic disease. The increase in the understanding of the role of growth factors and their receptors in the pathogenesis of malignancy and other undesirable hematological events taken in conjunction with the ability to produce humanized chimeric monoclonal antibodies to these targets is providing a new perspective for the treatment of leukemia, lymphoma and breast cancer, autoimmune disease and for prevention of ischemic complications. Dr. Waldmann describes approaches targeting the Her2/neu and the II-2/IL-15 receptor systems. The Her2/neu receptor is overexpressed in select breast, ovarian, gastric and pancreatic neoplasms. The use of trastuzumab (Herceptin) in the treatment of patients with breast cancer whose tumors overexpress this receptor are reviewed. The IL-2 receptor (Tac) is expressed on select malignant cells (adult T cell leukemia, hairy cell leukemia) and activated T cells involved in autoimmune disease and organ rejection. Humanized anti-Tac alone (daclizumab, Zenapax) or armed with toxins or radionuclides have been used successfully in the treatment of leukemia. Dr. Levy updates the experience with rituximab targeting CD20 on B cell lymphomas and reviews the antibodies to CD3, CD22, CD33, CD52, HLA-DR β chain and HLA-D currently in or proposed for clinical trials, including radiolabelled antibodies. In the last section, Dr. Coller reviews the therapeutic results achieved with abciximab (ReoPro), an antagonist of platelet receptor GPIIbIIIa for the prevention of restenosis in percutaneous coronary interventions and the treatment of unstable angina. The mechanism of action, pharmacology and safety and efficacy of abciximab are reviewed.

The authors have previously reported that the soluble serum form of the alpha subunit of the IL-2 receptor (sIL-2Ralpha), whose natural half-life is approximately 40 min, survived much longer in the circulation when bound by a specific antibody. In the present study, the authors evaluated the extent to which sIL-2Ralpha protected IL-2 in freshly collected serum using biochemical analyses, and a functional CTLL-2 assay. In particular, sIL-2Ralpha protected IL-2 from forming complexes with alpha(2)-macroglobulin and from inactivation in vitro. In addition, the authors demonstrated that the anti-IL-2Ralpha monoclonal antibody 7G7/B6, which does not inhibit the binding of IL-2 to its binding site on sIL-2Ralpha, protected IL-2 from degradation and inactivation in vivo in the presence of sIL-2Ralpha. Both(125)I-labelled and unlabelled IL-2 were injected into mice preinjected with humanized anti-Tac (hTac) or 7G7/B6 and sIL-2Ralpha, or sIL-2Ralpha alone. Using size-exclusion HPLC, ELISA, and CTLL-2 cell proliferation assays, we observed that the presence of 7G7/B6 led to formation of complexes with sIL-2Ralpha and increased the serum levels of IL-2 more than 3- to 40-fold those of groups receiving IL-2 alone, sIL-2Ralpha, or hTac. Taken as a whole, these results suggest that the complex of 7G7/B6 and sIL-2Ralpha not only prolongs the survival of IL-2 in vivo, but also maintains the bioactivity of IL-2. The use of antibodies against endogenous soluble receptors could increase the in vivo survival of cytokines, protect their bioactivity and thereby facilitate their clinical use in the treatment of various malignancies and AIDS.

Human T lymphotropic virus type I (HTLV-I) is the causative agent of an inflammatory neurological disease termed HTLV-I-associated myelopathy/tropical spastic paraparesis (HAM/TSP). An ongoing lymphocyte activation exists in patients with HAM/TSP, which was demonstrated by the spontaneous proliferation of their PBMC ex vivo. It was shown that spontaneous proliferation present in HAM/TSP is due, in part, to an IL-2/IL-2R autocrine loop. However, addition of Abs against IL-2 or IL-2Rα only partially inhibited the spontaneous proliferation. Since IL-15 is a cytokine with similar functional characteristics to those of IL-2, we reasoned that IL-15 might be an additional growth factor that contributes to the spontaneous proliferation observed in HAM/TSP. In this study, we demonstrated that IL-15 mRNA expression was elevated in PBMC obtained from HAM/TSP patients when compared with those of the normal donors. Furthermore, we showed that the addition of blocking Abs against IL-15 or its receptor inhibited the spontaneous proliferation of HAM/TSP PBMC. Addition of Abs directed toward both IL-15 and IL-2, or their receptors, inhibited the proliferation almost completely. These data suggest the existence of two autocrine loops involving IL-15/IL-15R and IL-2/IL-2R, both contributing to the spontaneous proliferation of HAM/TSP PBMC.

To evaluate the safety and potential therapeutic activity of humanized anti-IL-2 receptor mAb (Daclizumab) therapy in the treatment of patients with severe, sight-threatening, intermediate and posterior noninfectious uveitis, a nonrandomized, open-label, pilot study was performed. Patients with uveitis were treated with a minimum of 20 mg of prednisone, cyclosporine, antimetabolites, or any combination of these agents were eligible. Patients were weaned off their systemic immunosuppressive agents according to a standardized schedule, while ultimately receiving Daclizumab infusions every 4 weeks. Anti-IL-2 receptor antibody therapy, given intravenously with intervals of up to 4 weeks in lieu of standard immunosuppressive therapy, appeared to prevent the expression of severe sight-threatening intraocular inflammatory disease in 8 of 10 patients treated over a 12-month period, with noted improvements in visual acuity. One patient met a primary endpoint with a loss of vision of 10 letters or more from baseline in one eye and another patient discontinued therapy because of evidence of increased ocular inflammation. All patients were able to tolerate the study medications without the need for dose reduction. We report effective long-term use of anti-IL-2 therapy for an autoimmune indication. These initial findings would suggest that anti-IL-2 receptor therapy may be an effective therapeutic approach for uveitis and, by implication, other disorders with a predominant Th1 profile.

IL-15 is a T cell growth factor that shares many biological activities with IL-2 and uses the same β/γ polypeptides of the IL-2R complex for signal transduction. Accumulating evidence implicates an important role for this cytokine in the inflammatory response of the host. Consistent with such a role, IL-15 has been shown to be a chemoattractant for T lymphocytes, NK cells, and neutrophils. Extending these observations, we now show that IL-15 is a potent inducer of CC-, CXC-, and C-type chemokines in T lymphocytes. In addition, we demonstrate that IL-15 induces CC chemokine receptors, but not CXC chemokine receptors, in a dose-dependent manner. Thus, our findings suggest that the proinflammatory effects of IL-15 at least in part may be due to the induction of chemokines and their receptors in T cells. Furthermore, we demonstrate that IL-15 promotes entry and replication of macrophage-tropic HIV in T lymphocytes and suggest a plausible mechanism by which IL-15, a cytokine that is elevated in HIV-infected individuals, may promote the transition of HIV displaying the M-tropic phenotype primarily associated with the initial transmission into the T cell-tropic phenotype that predominates as the disease progresses.

Human T-lymphotropic virus type I (HTLV-I)-associated myelopathy/tropical spastic paraparesis (HAM/TSP) is a neurological disease that results from an interaction of retroviral infection and immune activation. In this study, five doses (1 mg/kg) of humanized anti-Tac antibody were administered to 9 HAM/TSP patients at weeks 0, 2, 6, 10, and 14. Preliminary immunological studies on HAM/TSP patients treated with humanized anti-Tac indicate that there is a selective down-regulation of activated T cells and a decrease in the HTLV-I viral load in peripheral blood lymphocytes, most likely through the selective removal of HTLV-I-infected, activated CD4+ lymphocytes.

Interleukin-15, that activates antitumor T and natural killer cells, has potential applications in cancer immunotherapy. IL-15 administered at 20 μg/kg/day by continuous intravenous (CIV) infusion for 10 days to nonhuman primates yielded an 80–100-fold expansion of circulating CD8+ effector memory T-cells, a 15-fold increase in the number of monocytes and an 8-fold increase in NK cells. Based on these observations we performed a first-in-human phase 1 clinical trial of rhIL-15 administered by bolus infusions and a trial of IL-15 by CIV in patients with metastatic malignancy. When administered by CIV at 2 μg/kg per day IL-15 led to a 30-fold increase in the number of NK cells and over a 200-fold increase in the number of CD56+ CD16− NK cells. It was clear from these clinical trials that to be effective IL-15 would have to be administered in combination with agents with specificity directed toward the tumor. Given the capacity of IL-15 to increase NK cells an attractive strategy was to use it in conjunction with an antitumor antibody to augment ADCC. When IL-15 was administered with rituximab it markedly increased the ADCC effectiveness of this antibody in a syngeneic mouse model of human B-cell lymphoma involving EL4 leukemia cells transfected with human CD20. Activity of this combination was lost in Fc alpha-R-1- mice. Furthermore IL-15 augmented the ADCC of alemtuzumab in a xenograft model of human adult T-cell leukemia (ATL). These studies support our clinical trial involving IL-15 with alemtuzumab in patients with acute ATL.

IL-2Rα augments IL-2 signaling. Although this is generally believed to occur only when the three known components of IL-2R are associated within a single cell membrane, we demonstrate here an intercellular interaction. Cocultivation of cells individually expressing chimerae incorporating the extracellular domain of IL-2Rα alone with cells expressing chimerae of IL-2Rβ alone permitted IL-2 dose-dependent oligomerization of the chimerae. Likewise, native IL-2Rα-bearing cells augmented the IL-2 proliferative response of ex vivo large granular lymphocytic leukemia cells expressing IL-2Rβ/γc but lacking IL-2Rα. In both cases, the response was inhibitable by an Ab to IL-2Rα. Intercellular augmentation of cytokine effects, acting in trans, has important implications for biology and medicine.

We previously reported that the AUG-burdened 5′ untranslated region (UTR) of IL-15 mRNA impedes its translation. Here we demonstrate that the nucleotide or protein sequences of the IL-15 signal peptide and carboxyl terminus also contribute to the poor translation of IL-15 transcripts. In particular, the exchange of the IL-15 signal peptide coding sequence with that of IL-2 increased cellular and secreted levels of IL-15 protein 15- to 20-fold in COS cells, while IL-2 transcripts with the IL-15 signal peptide generated 30- to 50-fold less IL-2 protein than wild-type IL-2. Furthermore, the addition of an artificial epitope tag to the 3′ coding sequence of IL-15 increased its protein production 5- to 10-fold. Combining these two IL-15 message modifications, in addition to removing the 5′ UTR, increased IL-15 synthesis 250-fold compared with a wild-type construct with an intact 5′ UTR. These data suggest that IL-15 mRNA, unlike IL-2 mRNA, may exist in translationally inactive pools. By storing translationally quiescent IL-15 mRNA, cells might respond to intracellular infections or other stimuli by rapidly transforming IL-15 message into one that can be efficiently translated.

Prior in vivo studies using the 125I-labeled anti-Tac disulfide-stabilized variable region fragment (125I-anti-Tac dsFv) of monoclonal antibody in the presence of the circulating soluble alpha subunit of the interleukin-2 receptor (sIL-2R alpha) have shown formation of complexes which interfere with biodistribution. In this study we evaluated the effects of preinjecting HuTac and 7G7/B6, two immunoglobulin Gs (IgGs) that recognize different epitopes of sIL-2R alpha, on the biodistribution of 125I-anti-Tac dsFv in mice bearing SP2/Tac tumor xenografts, which produce sIL-2R alpha, or on nude mice injected with 500 ng of sIL-2R alpha. We also evaluated the biodistribution in mice of 125I-labeled sIL-2R alpha injected alone or with HuTac and 7G7/B6. Injection of either HuTac or 7G7/B6 resulted in complexes with the sIL-2R alpha in serum. Injection of HuTac before 125I-anti-Tac dsFv, in SP2/Tac tumor-bearing mice, resulted in faster clearance of the dsFv from the blood (7.6% ID/g at 30 min), compared to 23.2% ID/g for the no-antibody control; preinjection of 7G7/B6 prolonged the retention of 125I-anti-Tac dsFv to 35.3% ID/g, with more complexes in serum. In mice pre-injected with 7G7/B6 the concentration of 125I-anti-Tac dsFv in tumor was lower (5.2 +/- 0.3% ID/g) than in mice preinjected with HuTac (7.9 +/- 1.2% ID/g) or in the control group (5.6 +/- 0.7% ID/g). In conclusion, while both IgGs formed complexes with sIL-2R alpha and prolonged its retention, preinjection of 7G7/B6 was detrimental, because the increased circulating sIL-2R alpha still had the epitope recognized by the dsFv available for binding and neutralized the anti-Tac dsFv upon injection, whereas preinjection of HuTac blocked the epitope.

Interleukin 15 (IL-15) mRNA is expressed in a wide variety of tissue types. However, with the exception of some T cell lines, IL-15 transcript expression has not been described in T cells. Herein we demonstrate that IL-15 mRNA can be detected in freshly isolated normal T cells and T cell lines. Furthermore, its expression is 3- to 4-fold higher in human T cell lymphotropic virus type I (HTLV-I)-infected T cells. By using reporter constructs bearing the 5′ regulatory region of the IL-15 gene, we observed a positive correlation between HTLV-I Tax protein expression and IL-15 promoter activity in HTLV-I-infected T cells. Additionally, by using a Jurkat T cell transfectant that expresses Tax under an inducible promoter, we demonstrated that the expression of IL-15 mRNA increased 3-fold as Tax was expressed, suggesting that the Tax protein activates IL-15 transcription. An NF-κB consensus sequence is located at the −75 and −65 region of the IL-15 5′ regulatory region. Mutations in the NF-κB motif or deletion of this sequence abrogated the promoter activity in both HTLV-I-positive and Jurkat Tax-transfectant cells. These data represent evidence for trans-activation of the IL-15 gene by the HTLV-I Tax protein through an NF-κB motif and suggest a potential role for IL-15 in HTLV-I-associated diseases such as adult T cell leukemia and HTLV-I-associated myopathy/tropical spastic paraparesis.

Natural killer (NK) cells are critical for both innate and adaptive immunity. The development of NK cells requires interactions between their progenitors and the bone-marrow microenvironment; however, little is known about the molecular nature of such interactions. Mice that do not express the transcription factor interferon-regulatory factor-1 (IRF-1; such mice are IRF-1(-/-) mice) have been shown to exhibit a severe NK-cell deficiency. Here we demonstrate that the lack of IRF-1 affects the radiation-resistant cells that constitute the microenvironment required for NK-cell development, but not the NK-cell progenitors themselves. We also show that IRF-1(-/-) bone-marrow cells can generate functional NK cells when cultured with the cytokine interleukin-15 and that the interleukin-15 gene is transcriptionally regulated by IRF-1. These results reveal, for the first time, a molecular mechanism by which the bone-marrow microenvironment supports NK-cell development.

These studies describe the production and purification of 211At as well as the procedure for labeling humanized anti-Tac, the antibody to the alpha-chain of the IL-2 receptor (IL-2R alpha), which has been shown to be a useful target for immunotherapy. The optimized protocol combines the advantages of the two-stage dry distillation procedure with the astatination of trialkylstannyl substances as labeling compounds for proteins. The 211At was produced by bombarding either an external or a recently developed disposable internal bismuth target with alpha-particles from a Cyclotron Corporation CS-30 cyclotron. The 211At was found to contain less than 0.01% 210At. The production rate for the external target was 0.15 mCi +/- 0.056 microA(-1) h(-1) (n = 9) (5.55 MBq mcroA[-1] h[-1]). The production rate for the internal target was 0.44 +/- 0.14 mCi microA(-1) h(-1) (n = 16) (16.28 MBq mcroA[-1] h[-1]).

Two isoforms of human interleukin 15 (IL-15) exist. One isoform has a shorter putative signal peptide (21 amino acids) and its transcript shows a tissue distribution pattern that is distinct from that of the alternative IL-15 isoform with a 48-aa signal peptide. The 21-aa signal isoform is preferentially expressed in tissues such as testis and thymus. Experiments using different combinations of signal peptides and mature proteins (IL-2, IL-15, and green fluorescent protein) showed that the short signal peptide regulates the fate of the mature protein by controlling the intracellular trafficking to nonendoplasmic reticulum sites, whereas the long signal peptide both regulates the rate of protein translation and functions as a secretory signal peptide. As a consequence, the IL-15 associated with the short signal peptide is not secreted, but rather is stored intracellularly, appearing in the nucleus and cytoplasmic components. Such production of an intracellular lymphokine is not typical of other soluble interleukin systems, suggesting a biological function for IL-15 as an intracellular molecule.

Human T cell leukemia/lymphotropic virus type I (HTLV-I) induces adult T cell leukemia/lymphoma (ATLL). The mechanism of HTLV-I oncogenesis in T cells remains partly elusive. In vitro , HTLV-I induces ligand-independent transformation of human CD4 + T cells, an event that correlates with acquisition of constitutive phosphorylation of Janus kinases (JAK) and signal transducers and activators of transcription (STAT) proteins. However, it is unclear whether the in vitro model of HTLV-I transformation has relevance to viral leukemogenesis in vivo . Here we tested the status of JAK/STAT phosphorylation and DNA-binding activity of STAT proteins in cell extracts of uncultured leukemic cells from 12 patients with ATLL by either DNA-binding assays, using DNA oligonucleotides specific for STAT-1 and STAT-3, STAT-5 and STAT-6 or, more directly, by immunoprecipitation and immunoblotting with anti-phosphotyrosine antibody for JAK and STAT proteins. Leukemic cells from 8 of 12 patients studied displayed constitutive DNA-binding activity of one or more STAT proteins, and the constitutive activation of the JAK/STAT pathway was found to persist over time in the 2 patients followed longitudinally. Furthermore, an association between JAK3 and STAT-1, STAT-3, and STAT-5 activation and cell-cycle progression was demonstrated by both propidium iodide staining and bromodeoxyuridine incorporation in cells of four patients tested. These results imply that JAK/STAT activation is associated with replication of leukemic cells and that therapeutic approaches aimed at JAK/STAT inhibition may be considered to halt neoplastic growth.

Geldanamycin belongs to the family of benzoquinoid ansamycin tyrosine-kinase inhibitors. We have examined its effects on Her-2/neu kinase activity, protein expression level, and proliferation of Her-2+ malignant cells. In SK-BR-3 breast-cancer cells, short-time treatment with geldanamycin completely abrogated gp30-ligand-induced activation of Her-2 without a change of receptor-expression level. Longer treatment of intact cells with geldanamycin induced decreased steady-state Her-2 autophosphorylation activity, which correlated with reduction of Her-2 protein expression and phosphotyrosine content of several proteins. The decrease was time- and dose-dependent, starting after 1 hr at 100 nM concentration and reaching completion by 24 hr. The reduction of the Her-2 protein level probably resulted from increased degradation, since the Her-2 mRNA level remained constant. Geldanamycin effects were not specific for Her-2, since the non-receptor tyrosine-kinase fyn was inhibited equally. In contrast to these results, protein-kinase-C activity was not affected. In 3 other malignant cell lines expressing different amounts of Her-2 (SK-BR-3 > SK-OV-3 > OVCAR3 > MCF7), geldanamycin also effectively reduced Her-2-kinase activity proportionally to the decrease of protein expression. In contrast, in a [3H]-thymidine-uptake assay, cell growth was meaningfully inhibited by geldanamycin at nanomolar concentrations only in SK-BR-3 (IC50 2 nM) and MCF7 (IC50 20 nM), while OVCAR3 was only moderately sensitive (IC50 2 microM) and SK-OV-3 was clearly resistant to geldanamycin. In direct comparison with herbimycin A, another benzoquinoid ansamycin that has been more thoroughly characterized, the biologic effects of geldanamycin were more pronounced.

A number of antitumor vaccines have recently shown promise in upregulating immune responses against tumor antigens and improving patient survival. In this study, we examine the effectiveness of vaccination using interleukin (IL)-15-expressing tumor cells and also examine their ability to upregulate immune responses to tumor antigens. We demonstrated that the coexpression of IL-15 with its receptor, IL-15Rα, increased the cell-surface expression and secretion of IL-15. We show that a gene transfer approach using recombinant adenovirus to express IL-15 and IL-15Rα in murine TRAMP-C2 prostate or TS/A breast tumors induced antitumor immune responses. From this, we developed a vaccine platform, consisting of TRAMP-C2 prostate cancer cells or TS/A breast cancer cells coexpressing IL-15 and IL-15Rα that inhibited tumor formation when mice were challenged with tumor. Inhibition of tumor growth led to improved survival when compared with animals receiving cells expressing IL-15 alone or unmodified tumor cells. Animals vaccinated with tumor cells coexpressing IL-15 and IL-15Rα showed greater tumor infiltration with CD8(+) T and natural killer (NK) cells, as well as increased antitumor CD8(+) T-cell responses. Vaccination with IL-15/IL-15Rα-modified TS/A breast cancer cells provided a survival advantage to mice challenged with unrelated murine TUBO breast cancer cells, indicating the potential for allogeneic IL-15/IL-15Rα-expressing vaccines.

An intense effort has been launched to develop improved anthrax vaccines that confer rapid, long lasting protection preferably with an extended stability profile amenable for stockpiling. Protective antigen (PA)-based vaccines are most favored as immune responses directed against PA are singularly protective, although the actual protective mechanism remains to be unraveled. Herein we show that contrary to the prevailing view, an efficacious PA-based vaccine confers protection against inhalation anthrax by preventing the establishment of a toxin-releasing systemic infection. Equally importantly, antibodies measured by the in vitro lethal toxin neutralization activity assay (TNA) that is considered as a reliable correlate of protection, especially for PA protein-based vaccines adjuvanted with aluminum salts appear to be not absolutely essential for this protective immune response.

Adult T-cell leukemia (ATL) is an aggressive malignancy of CD4(+)CD25(+) lymphocytes caused by human T-cell lymphotropic virus type 1. Currently, there is no accepted curative therapy for ATL. In gene expression profiling, the antiapoptotic protein survivin (BIRC5) demonstrated a striking increase in ATL, and its expression was increased in patient ATL cells resistant to the anti-CD52 monoclonal antibody alemtuzumab (Campath-1H). In this study, we investigated the antitumor activity of a small-molecule survivin suppressant YM155 alone and in combination with alemtuzumab in a murine model of human ATL (MET-1). Both YM155 alone and its combination with alemtuzumab demonstrated therapeutic efficacy by lowering serum soluble IL-2Rα (sIL-2Rα) levels (P.001) and prolonged the survival of tumor-bearing mice (P.0001). Moreover, the combination of YM155 with alemtuzumab demonstrated markedly additive antitumor activity by significantly lowering serum sIL-2Rα levels and improving the survival of leukemia-bearing mice compared with monotherapy with either YM155 (P.001) or alemtuzumab (P.05). More significantly, all mice that received the combination therapy survived and were tumor free6 months after treatment. Our data support a clinical trial of the combination of YM155 with alemtuzumab in ATL. This trial was registered at www.clinicaltrials.gov as #NCT00061048.

Long-lived memory T cells are able to persist in the host in the absence of antigen; however, the mechanism by which they are maintained is not well understood. Recently, a subset of human T cells, stem cell memory T cells (TSCM cells), was shown to be self-renewing and multipotent, thereby providing a potential reservoir for T cell memory throughout life. However, their in vivo dynamics and homeostasis still remain to be defined due to the lack of suitable animal models. We identified T cells with a TSCM phenotype and stem cell–like properties in nonhuman primates. These cells were the least-differentiated memory subset, were functionally distinct from conventional memory cells, and served as precursors of central memory. Antigen-specific TSCM cells preferentially localized to LNs and were virtually absent from mucosal surfaces. They were generated in the acute phase of viral infection, preferentially survived in comparison with all other memory cells following elimination of antigen, and stably persisted for the long term. Thus, one mechanism for maintenance of long-term T cell memory derives from the unique homeostatic properties of TSCM cells. Vaccination strategies designed to elicit durable cellular immunity should target the generation of TSCM cells.

We reported previously that the human T-cell lymphotrophic virus type I (HTLV-I)-associated adult T-cell leukemia line HuT-102 produces a cytokine designated interleukin (IL) T that requires interleukin (IL) 2 receptor beta-subunit expression for its action. Using anti-cytokine antibodies, we demonstrated that IL-T is identical to the simultaneously described IL-15. When compared to activated monocytes, IL-15 mRNA expression was 6- to 10-fold greater in HuT-102 cells. The predominant IL-15 message from HuT-102 is a chimeric mRNA joining a segment of the R region of the long terminal repeat of HTLV-I and the 5'-untranslated region (UTR) of IL-15. Normally, by alternative splicing, this 118-nucleotide R element represents the most 5' region of several HTLV-I transcripts including tax, rex, and env. The introduction of the R element eliminated over 200 nucleotides of the IL-15 5'-UTR, including 8 of 10 upstream AUGs that are present in normal IL-15 messages. On analysis of the 5'-UTR of normal IL-15, we demonstrated that the presence of these 10 upstream AUGs interferes with IL-15 mRNA translation. Thus, IL-15 synthesis by the adult T-cell leukemia line HuT- 102 involves an increase in IL-15 mRNA transcription and translation secondary to the production of an HTLV-I R element fusion message that lacks many upstream AUGs.