Retrospective study of a TTR FAP cohort to modify NIS+7 for therapeutic trials.

Protein stabilization and oligonucleotide therapies are being tested in transthyretin amyloid polyneuropathy (TTR FAP) trials. From retrospective analysis of 97 untreated TTR FAP patients, we test the adequacy of Neuropathy Impairment Score+7 tests (NIS+7) and modifications to comprehensively score impairments for use in such therapeutic trials. Our data confirms that TTR FAP usually is a sensorimotor polyneuropathy with autonomic features which usually is symmetric, length dependent, lower limb predominant and progressive. NIS+7 adequately assesses weakness and muscle stretch reflexes without ceiling effects but not sensation loss, autonomic dysfunction or nerve conduction abnormalities. Three modifications of NIS+7 are suggested: 1) use of Smart Somatotopic Quantitative Sensation Testing (S ST QSTing); 2) choice of new autonomic assessments, e.g., sudomotor testing of distributed anatomical sites; and 3) use of only compound muscle action potential amplitudes (of ulnar, peroneal and tibial nerves) and sensory nerve action potentials of ulnar and sural nerve - than the previously recommended attributes suggested for the sensitive detection of diabetic sensorimotor polyneuropathy. These modifications of NIS+7 if used in therapeutic trials should improve characterization and quantification of sensation and autonomic impairment in TTR FAP and provide better nerve conduction tests.

Long-term remission in a patient with metastatic collecting duct carcinoma treated with taxol/carboplatin and surgery.

Collecting duct carcinoma of the kidney is a rare and aggressive neoplasm of the distal collecting tubules for which there is no established therapy. We describe a young woman with metastatic collecting duct carcinoma who responded to Taxol/carboplatin chemotherapy with an 80% reduction in her tumor burden, including complete regression of lymph node metastases and significant shrinkage of a renal mass. She was rendered free of disease through nephrectomy and has been without a recurrence for 20 months. This suggests that Taxol/carboplatin chemotherapy and surgery should be considered for the treatment of metastatic collecting duct carcinoma.

Agranulocytosis and hemolytic anemia in patients with renal cell cancer treated with interleukin-12.

Interleukin-12 (IL-12) is a cytokine with effects on immune function and hematopoiesis. In this article, the authors describe two patients with renal cell cancer in whom grade 4 neutropenia and grade 3 hemolytic anemia developed, respectively, during treatment with twice-weekly intravenous recombinant human interleukin-12 (rhIL-12) during a phase 1 trial. The severe neutropenia was associated with bone marrow agranulocytosis and a preponderance of large granular lymphocytes in the peripheral blood, whereas the hemolytic anemia was negative for the Coombs test and associated with splenomegaly. The agranulocytosis and hemolytic anemia persisted after the rhIL-12 was stopped, but both subsequently responded to treatment with cyclophosphamide. steroids, or both. These findings indicate that rhIL-12 can induce unique hematologic toxic effects that can be reversed with immunosuppressive drugs.

CD2 stimulation leads to the delayed and prolonged activation of STAT1 in T cells but not NK cells.

OBJECTIVE: T lymphocytes can be activated by soluble factors such as cytokines or through direct cell-cell interactions. Although cytokine receptors are known to signal through STAT family transcription factors, the mechanisms by which other cell-surface molecules, such as CD2, transduce signals is unclear. The goal of this study was to determine whether stimulation of T cells through CD2 recapitulates aspects of cytokine-induced T-cell activation by use of STAT transcription factors. MATERIALS AND METHODS: T cells were treated with anti-CD2 antibodies or cells bearing the natural CD2 ligand CD58, after which signaling through STAT transcription factors was assessed. RESULTS: Stimulation of CD2 on primary T lymphocytes leads to the tyrosine phosphorylation, nuclear translocation, and DNA binding of STAT1. In contrast to stimulation by cytokines, the activation of STAT1 in response to CD2 ligation is delayed and does not involve Jak kinases. Furthermore, while STAT phosphorylation induced by cytokines is generally transient, STAT1 phosphorylation following CD2 stimulation persists for a period of days. Transcription of key target genes such as IRF1 and c-fos proceeds with delayed kinetics following CD2 stimulation, suggesting that this unique pattern of STAT activation may lead to a distinct cellular response following CD2 ligation. This pathway appears to be restricted to T cells, as stimulation of CD2 on NK cells does not lead to STAT1 activation. CONCLUSION: Stimulation of T cells through cell-surface molecules such as CD2 involves activation of STAT transcription factors, thus recapitulating elements of cytokine signaling.

Impairment of STAT activation by IL-12 in a patient with atypical mycobacterial and staphylococcal infections.

IL-12 plays a pivotal role in the stimulation of immune responses against intracellular infections. This role is manifested in the increased susceptibility to atypical mycobacterial and salmonella infections among individuals whose lymphocytes lack expression of IL-12Rbeta1. Here, we report on a patient with Mycobacterium avium infection, recurrent Staphylococcus aureus sinusitis, and multiple adverse drug reactions whose T cells were unable to produce IFN-gamma or proliferate in response to IL-12 despite the expression of wild-type IL-12Rbeta1 and IL-12Rbeta2. The defect in these functional responses to IL-12 was selective, as cytolytic activity induced by IL-12 was intact, and lymphocytes were responsive to stimulation by IL-2. An examination of cytokine signaling revealed that STAT4 and extracellular regulated kinase 1 (ERK1) activation by IL-12 was intact, whereas the activation of STAT1, -3, and -5 by IL-12 was lost. This impairment of STAT activation was specific for IL-12, as STAT activation by IL-2, IL-15, and IFN-gamma was unaffected. These findings demonstrate that the activation of STAT4 alone is not sufficient for IL-12-induced IFN-gamma production and proliferation and suggest that other STATs play a role in these responses to IL-12. While the etiology of the impaired IL-12 signaling in this patient has not yet been elucidated, the absence of mutations in IL-12Rbeta1 or IL-12Rbeta2 and the preservation of STAT4 activation raise the possibility that there may be a mutation in an as yet undiscovered component of the IL-12 signaling complex that is normally required for the recruitment and activation of STAT1, -3, and -5.

Phase I trial of twice-weekly intravenous interleukin 12 in patients with metastatic renal cell cancer or malignant melanoma: ability to maintain IFN-gamma induction is associated with clinical response.

The aim of this study was to examine the tolerability, antitumor activity, and biological effects of a new schedule of i.v. recombinant human interleukin 12 (rhIL-12). Twenty-eight patients were enrolled in a Phase I trial in which rhIL-12 was administered twice weekly as an i.v. bolus for 6 weeks. Stable or responding patients were eligible to receive additional 6-week cycles until there was no evidence of disease or until tumor progression. Patient cohorts were treated with escalating doses of rhIL-12 (30-700 ng/kg). The maximum tolerated dose (MTD) was 500 ng/kg, with dose-limiting toxicities consisting of elevated hepatic transaminases and cytopenias. At the MTD (n = 14), there was one partial response occurring after 6 cycles of rhIL-12 in a patient with renal cell cancer. Two additional renal cell cancer patients treated at the MTD had prolonged disease stabilization, with one of these exhibiting tumor regression after 8 cycles of rhIL-12. IFN-gamma, IL-15, and IL-18 were induced in patients treated with rhIL-12. Whereas IFN-gamma and IL-15 induction were attenuated midway through the first cycle in patients with disease progression, those patients with tumor regression or prolonged disease stabilization were able to maintain IFN-gamma, IL-15, and IL-18 induction. The down-modulation of IFN-gamma induction during rhIL-12 treatment did not relate to IL-10 production or alterations in rhIL-12 bioavailability but was associated with an acquired defect in lymphocyte IFN-gamma production in response to IL-12, IL-2, or IL-15. This defect could be partially overcome in vitro through combined stimulation with IL-12 plus IL-2. These findings show that the chronic administration of twice-weekly i.v. rhIL-12 is well-tolerated, stimulates the production of IL-12 costimulatory cytokines and IFN-gamma, and can induce delayed tumor regression. Strategies aimed at maintaining IFN-gamma induction, such as the addition of IL-2, may further augment the response rate to this schedule of rhIL-12.

The functional synergy between IL-12 and IL-2 involves p38 mitogen-activated protein kinase and is associated with the augmentation of STAT serine phosphorylation.

IL-12 and IL-2 can stimulate mitogen- or CD3-activated T cells to proliferate, produce IFN-gamma, and kill tumor cells. The magnitude of these functional responses is greatly augmented when T cells are activated by the combination of IL-12 and IL-2. Although peripheral blood T cells are largely unresponsive to these cytokines without prior activation, a small subset of CD8+ T cells (CD8+CD18bright) is strongly activated by the combination of IL-12 and IL-2. In this report we show that the functional synergy between IL-12 and IL-2 in CD8+CD18bright T cells correlates with the activation of the stress kinases, p38 mitogen-activated protein (MAP) kinase and stress-activated protein kinase (SAPK)/Jun N-terminal kinase, but not with the activation of the extracellular signal-regulated kinases. The functional synergy between IL-2 and IL-12 is also associated with a prominent increase in STAT1 and STAT3 serine phosphorylation over that observed with IL-12 or IL-2 alone. By contrast, STAT tyrosine phosphorylation is not augmented over that seen with either cytokine alone. A specific inhibitor of p38 MAP kinase completely inhibits the serine phosphorylation of STAT1 and STAT3 induced by IL-12 and IL-2 and abrogates the functional synergy between IL-12 and IL-2 without affecting STAT tyrosine phosphorylation. This suggests that p38 MAP kinase may play an important role in regulating STAT serine phosphorylation in response to the combination of IL-12 and IL-2. Furthermore, these findings indicate that the optimal activation of T cells by IL-12 and IL-2 may depend on an interaction between the p38 MAP kinase and Janus kinase/STAT signaling pathways.

Characterization of a novel subset of CD8(+) T cells that expands in patients receiving interleukin-12.

IL-12 has significant antitumor activity in mice that may be mediated by CD8(+) T cells. We show in this report that repeated subcutaneous injections of IL-12 in patients with cancer resulted in the selective expansion of a subset of peripheral blood CD8(+) T cells. This T cell subset expressed high levels of CD18 and upregulated IL-12 receptor expression after IL-12 treatment in vivo. In normal subjects, these CD3(+)CD8(+)CD18(bright) T cells expressed IL-12 and IL-2 receptors and adhesion/costimulatory molecules to a greater degree than other CD8(+) and CD4(+) T cells. They appeared morphologically as large granular lymphocytes, although they did not express NK cell markers such as CD56. In addition, CD8(+)CD18(bright) T cells were almost exclusively T cell receptor (TCR) alphabeta+, and exhibited a TCR Vbeta repertoire that was strikingly oligoclonal, whereas the Vbeta repertoire of CD18(dim) T cells was polyclonal. Although CD8+CD18(bright) T cells demonstrated little functional responsiveness to IL-12 or IL-2 alone in vitro, they responded to the combination of IL-12+IL-2 with strong IFN-gamma production and proliferation and enhanced non-MHC-restricted cytolytic activity. In contrast, CD18(dim) T cells were not activated by IL-12 or IL-2, alone or in combination. These findings demonstrate that CD8+CD18(bright) T cells are a unique population of peripheral blood lymphocytes with features of both memory and effector cells that are capable of TCR-independent activation through combined stimulation with IL-12+IL-2. As this activation results in IFN-gamma production and enhanced cytolytic activity, these T cells may play a role in innate as well as acquired immunity to tumors and infectious pathogens. Additional studies will be necessary to determine whether CD8+CD18(bright) T cells mediate the antitumor effect of IL-12 or IL-2 administered to cancer patients, and if so, whether maximal activation of these T cells with the combination of IL-12+IL-2 in vivo can augment the clinical effectiveness of these cytokines.

Impairment of mycobacterial immunity in human interleukin-12 receptor deficiency.

In humans, interferon gamma (IFN-gamma) receptor deficiency leads to a predisposition to mycobacterial infections and impairs the formation of mature granulomas. Interleukin-12 (IL-12) receptor deficiency was found in otherwise healthy individuals with mycobacterial infections. Mature granulomas were seen, surrounded by T cells and centered with epithelioid and multinucleated giant cells, yet reduced IFN-gamma concentrations were found to be secreted by activated natural killer and T cells. Thus, IL-12-dependent IFN-gamma secretion in humans seems essential in the control of mycobacterial infections, despite the formation of mature granulomas due to IL-12-independent IFN-gamma secretion.

Altered interleukin-12 responsiveness in Th1 and Th2 cells is associated with the differential activation of STAT5 and STAT1.

T-cell activation in response to interleukin-12 (IL-12) is mediated through signaling events that include the tyrosine phosphorylation of STAT4. IL-12 responsiveness and the ability of IL-12 to activate STAT4 is different in T cells induced to differentiate into a Th1 or Th2 phenotype. In this report, we show that STAT5, STAT1alpha, and STAT1beta, in addition to STAT4, are tyrosine phosphorylated in response to IL-12 in phytohemagglutinin (PHA)-activated human T cells. To understand how the activation of these STATs contributes to T-cell IL-12 responsiveness, we analyzed the IL-12-induced activation of STAT5 and STAT1 in T cells stimulated to undergo Th1 or Th2 differentiation. The IL-12-induced tyrosine phosphorylation of STAT5 and STAT1, but not STAT4, is augmented in T cells activated into Th1 cells with PHA + interferon-gamma (IFN-gamma) compared with T cells activated with PHA alone. STAT5 DNA binding induced by IL-12 is also augmented in T cells activated with PHA + IFN-gamma compared with T cells activated with PHA alone, whereas STAT4 DNA binding is not increased. In contrast, the IL-12-induced activation of these STATs is inhibited in T cells activated into Th2 cells with PHA + IL-4. The enhancement of IL-12 signaling by IFN-gamma is not a direct effect of IFN-gamma on T cells, but rather is mediated by IL-12 that is produced by antigen-presenting cells in response to IFN-gamma. This positive autoregulatory effect of IL-12 on the activation of select STATs correlates with an increase in T-cell IFN-gamma production in response to IL-12. These findings suggest that the activation of STAT5 and STAT1 may augment select STAT4-dependent functional responses to IL-12 in Th1 cells.

Interferon-gamma and interleukin-4 regulate T cell interleukin-12 responsiveness through the differential modulation of high-affinity interleukin-12 receptor expression.

Interferon-gamma (IFN-gamma) and interleukin-4 (IL-4) are mutually antagonistic cytokines that stimulate CD4+ T cells to develop into either Th1 or Th2 cells. One feature of Th2 differentiation in mice is the loss of IL-12-induced Jak2 and Stat4 activation, which is accompanied by the inability to produce IFN-gamma in response to IL-12. In this report, we show that freshly isolated human T cells activated with phytohemagglutinin (PHA) in the presence of IL-4 exhibit a greatly diminished response to IL-12, whereas the IL-12 response of T cells activated with PHA plus IFN-gamma is enhanced. Radiolabeled IL-12 binding studies demonstrate that the impairment of T cell IL-12 responsiveness by IL-4 is associated with the down-regulation of high-affinity IL-12 receptor expression. In contrast, the enhancement of IL-12 responsiveness by IFN-gamma is associated with the upregulation of high-affinity IL-12 receptor expression. Through the use of a newly synthesized neutralizing antibody to the low-affinity IL-12 receptor beta subunit (IL-12Rbeta), we show that neither IL-4 nor IFN-gamma affect the expression of IL-12Rbeta, which we determine to be one of at least two low-affinity subunits required for high-affinity IL-12 binding. These findings suggest that IL-4 and IFN-gamma exert opposite effects on T cell IL-12 responsiveness by differentially modulating the expression of low-affinity IL-12 receptor subunits that are distinct from IL-12Rbeta and required, together with IL-12Rbeta, for high-affinity IL-12 binding and IL-12 responsiveness. This provides a basis for understanding the interplay between different cytokines at the level of cytokine receptor expression, and offers insight into one of the mechanisms governing Th1 and Th2 development.

CD2-CD58 interaction and the control of T-cell interleukin-12 responsiveness. Adhesion molecules link innate and acquired immunity.

Interleukin-12 is an antigen-presenting cell (APC)-derived cytokine that stimulates T helper cell type 1 (Th1) differentiation as well as proliferation, cytolytic activity, and IFN-gamma production among T and natural killer (NK) cells. By immunizing mice with activated T cells and screening for antibodies that could inhibit IL-12-induced proliferation, CD2 was identified as a regulator of T-cell IL-12 responsiveness. Antibodies specific for the adhesion domain of either CD2 or its primary ligand, CD58, inhibited the major functional responses of T cells to IL-12 without affecting the response to IL-2. This inhibition did not involve any alteration in IL-12 binding to T cells. The CD2-CD58 interaction between activated T cells and monocytes was found to be a critical factor in the modulation of T-cell IL-12 responsiveness, and experiments using CD58-transfected Chinese hamster ovary (CHO) cells, and stimulatory pairs of CD2 antibodies confirmed the key role of CD2 ligation in optimizing T-cell IL-12 signaling. These findings illustrate how APCs, through the differential expression of a specific adhesion molecule, can control the T-cell response to a cytokine independent of cytokine receptor expression. This has important implications for an understanding of Th1 development and the conditions required to link innate with acquired immune responses.

Molecular interaction between CD58 and CD2 counter-receptors mediates the ability of monocytes to augment T cell activation by IL-12.

IL-12 stimulates both T and NK cells and is pivotal in the development of the Th1 immune response. In this work, we show that an interaction between CD2 and CD58 on activated T cells and monocytes, respectively, regulates the T cell response to IL-12. B cells provide little IL-12-specific costimulation, and this correlates with the low level of CD58 on B cells relative to monocytes and the lack of significant up-regulation in response to IFN-gamma or PHA activation. CHO cell transfectants expressing CD58 at a level comparable with that found on monocytes restore IL-12 responsiveness to APC-depleted T cells. This effect is not observed with CHO cells expressing CD48, a second CD2 ligand with a low avidity for CD2 relative to CD58. Thus, in addition to augmenting adhesion between T cells and their cognate APCs and facilitating TCR-triggered activation, the CD2-CD58 interaction uniquely optimizes the T cell response to IL-12.

CD2 regulates responsiveness of activated T cells to interleukin 12.

Interleukin (IL) 12 is a 70-kD heterodimeric cytokine produced by antigen-presenting cells (APCs) such as macrophages in response to infectious pathogens and interferon (IFN) gamma. The varied immunomodulatory effects of IL-12 include the stimulation of proliferation and IFN-gamma production by T cells, and it also has a central role in the development of the T helper cell type 1 immune phenotype. We undertook the production of antibodies capable of modulating the response of T cells to IL-12, and in the process we discovered two antibodies that inhibited the ability of IL-12 to stimulate T cell proliferation. In this report, we demonstrate that these anti-bodies recognize CD2, and we show how antibodies directed toward either the adhesion domain of CD2 or its ligand, CD58, specifically inhibit IL-12 induced proliferation and IFN-gamma production by phytohemagglutinin-activated T cells, leaving the response to IL-12 unaffected. A three-to fourfold reduction in proliferation and IFN-gamma production was observed at IL-12 concentrations as high as 1 nM, with complete inhibition occurring at < or = 1 pM. This novel effect is not directly mediated at the level of the IL-12 receptor, as shown by the inability of these antibodies to block IL-12 binding to activated T cells. Furthermore, by using activating pairs of CD2 antibodies, we show that CD2 stimulation strongly synergizes with IL-12, even at 0.1 pM, in inducing both T cell proliferation and IFN-gamma production. Cytolytic T lymphocyte-associated antigen 4-immunoglobulin-mediated inhibition of the B7/CD28 interaction did not affect the T cell response to either IL-12 or IL-2, but the removal of APCs selectively diminished the proliferative response to IL-12. Based on this data, we hypothesize that CD2 has a central role in an IL-12/IFN-gamma positive feedback loop between T cell and APC, providing the key functional link via a CD2/CD58 interaction that controls T cell responsiveness to IL-12. This model provides a basis for future investigations aimed at defining the signaling mechanisms that mediate this cytokine-specific regulatory effect of CD2, and it offers insight into how a cytokine receptor and distinct adhesion molecule can interact to modulate responsiveness to that cytokine. In addition, it underscores the possibility that the clinical potential of an immunomodulatory drug like IL-12 may be governed by the presence or absence of specific costimulation.