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Targeting Trends Newsletter 10q2

Below are links to view the quarterly newsletter Targeting Trends. If you would like to be added to the mailing list, please complete the information on our contact page.

Newsletter Highlights

  • New Catalog Available (page 2)
  • Orexin-SAP (page 5)
  • Teaser Winners (page 4)
  • Fab-ZAP, Anti-6 His-ZAP, Anti-GFP-ZAP (page 7)
  • Complete and Submit the Targeting Teaser online!

Depletion of syndecan-4+ T lymphocytes by saporin-conjugated DC-HIL alleviates T cell-mediated imflammatory disease. – read article (continued on page 6)

Contributed by Kiyoshi Ariizumi, Hideo Akiyoshi, Jin-Sung Chung, Mizuki Tomiharu, Ponciano D. Cruz Jr.
Department of Dermatology, The University of Texas Southwestern Medical Center and Dermatology Section (Medical Service), Dallas Veterans Affairs Medical Center, Dallas, TX

Product information related to cover article: Streptavidin-ZAP (Cat. #IT-27)

New Product Catalog Now Available

Targeting Teaser Winners

Upcoming Events

  • American Association for Cancer Research • April 17-21, 2010 • Washington, DC • Booth #2011
  • American Association of Immunologists • May 7-11, 2010 • Baltimore, MD • Booth #208

Recent Scientific References (read online)

  • Akasaka E, Watanabe S, Himaki T, Ohtsuka M, Yoshida M, Miyoshi K, Sato M (2010) Enrichment of xenograft-competent genetically modified pig cells using a targeted toxin, isolectin BS-I-B4 conjugate. Xenotransplantation 17:81-89. (Cat. #IT-10)
  • Akiyoshi H, Chung JS, Tomihari M, Cruz PD, Jr., Ariizumi K (2010) Depleting Syndecan-4+ T Lymphocytes Using Toxin-Bearing Dendritic Cell-Associated Heparan Sulfate Proteoglycan-Dependent Integrin Ligand: A New Opportunity for Treating Activated T Cell-Driven Disease. J Immunol 184:3554-3561. (Cat. #IT-27)
  • Browning PG, Gaffan D, Croxson PL, Baxter MG (2010) Severe scene learning impairment, but intact recognition memory, after cholinergic depletion of inferotemporal cortex followed by fornix transection. Cereb Cortex 20:282-293. (Cat. #IT-15)
  • Carstens EE, Carstens MI, Simons CT, Jinks SL (2010) Dorsal horn neurons expressing NK-1 receptors mediate scratching in rats. Neuroreport 21:303-308. (Cat. #IT-11)
  • Conner JM, Kulczycki M, Tuszynski MH (2010) Unique Contributions of Distinct Cholinergic Projections to Motor Cortical Plasticity and Learning. Cereb Cortex [Epub Feb 24]. (Cat. #IT-01)
  • Dailey MJ, Bartness TJ (2010) Arcuate nucleus destruction does not block food deprivation-induced increases in food foraging and hoarding. Brain Res [Epub Feb 4]. (Cat. #IT-28; Cat. #IT-21)
  • Vincent BG, Young EF, Buntzman AS, Stevens R, Kepler TB, Tisch RM, Frelinger JA, Hess PR (2010) Toxin-Coupled MHC Class I Tetramers Can Specifically Ablate Autoreactive CD8+ T Cells and Delay Diabetes in Nonobese Diabetic Mice. J Immunol [Epub Mar 10]. (Cat. #IT-27)
  • Zipancic I, Calcagnotto ME, Piquer-Gil M, Mello LE, Alvarez-Dolado M (2010) Transplant of GABAergic precursors restores hippocampal inhibitory function in a mouse model of seizure susceptibility. Cell Transplant [Epub Feb 8]. (Cat. #IT-11)
  • Cao F, Chen SS, Yan XF, Xiao XP, Liu XJ, Yang SB, Xu AJ, Gao F, Yang H, Chen ZJ, Tian YK (2009) Evaluation of side effects through selective ablation of the mu opioid receptor expressing descending nociceptive facilitatory neurons in the rostral ventromedial medulla with dermorphin-saporin. Neurotoxicology 30:1096-1106. (Cat. #IT-12; Cat. #PR-01)
  • Harle P, Mobius D, Carr DJ, Scholmerich J, Straub RH (2005) An opposing time-dependent immune-modulating effect of the sympathetic nervous system conferred by altering the cytokine profile in the local lymph nodes and spleen of mice with type II collagen-induced arthritis. Arthritis Rheum 52:1305-1313. (Cat. #IT-03)
  • Harle P, Pongratz G, Albrecht J, Tarner IH, Straub RH (2008) An early sympathetic nervous system influence exacerbates collagen-induced arthritis via CD4+CD25+ cells. Arthritis Rheum 58:2347-2355. (Cat. #IT-03)
  • Itoi K, Sugimoto N (2010) The brainstem noradrenergic systems in stress, anxiety, and depression. J Neuroendocrinol [Epub Feb 20]. (Cat. #IT-03)
  • Lujan HL, Palani G, Zhang L, Dicarlo SE (2010) Targeted Ablation of Cardiac Sympathetic Neurons Reduces the Susceptibility to Ischemia-Induced Sustained Ventricular Tachycardia in Conscious Rats. Am J Physiol Heart Circ Physiol [Epub Feb 19]. (Cat. #IT-14)
  • Marques-Lopes J, Pinho D, Albino-Teixeira A, Tavares I (2010) The hyperalgesic effects induced by the injection of angiotensin II into the caudal ventrolateral medulla are mediated by the pontine A(5) noradrenergic cell group. Brain Res [Epub Feb 19]. (Cat. #IT-03)
  • Thomsen MS, Hay-Schmidt A, Hansen HH, Mikkelsen JD (2010) Distinct Neural Pathways Mediate {alpha}7 Nicotinic Acetylcholine Receptor-Dependent Activation of the Forebrain. Cereb Cortex [Epub Jan 4]. (Cat. #IT-01)
  • Vianna DM, Carrive P (2010) Cardiovascular and behavioural responses to conditioned fear and restraint are not affected by retrograde lesions of A5 and C1 bulbospinal neurons. Neuroscience 166:1210-1218.
    (Cat. #IT-03; Cat. #IT-18)

Targeting Talk (see text)

Targeting Tools: Featured Products

Gangsta Gallery

Targeting Technology Tutorial

Targeting Teaser (Jumble)

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Targeting Tools: Fab-ZAPs

New Secondary Conjugates – Fab-ZAPs – Use Monovalent Secondary Antibodies Linked to Saporin

For the past decade scientists have extensively used secondary conjugates (e.g. Mab-ZAP; Cat. #IT-04, and Hum-ZAP; Cat. #IT-22) to make their own targeted toxins for in vitro use. The ability to combine: 1) a primary antibody to a cell-surface marker with 2) a secondary conjugate, in order to eliminate cells is a valuable tool in determining antibody specificity and internalization.

In theory, there are some possible limitations to the original product line of secondary conjugates to serve as a singular diagnostic tool. These secondary conjugates are made with whole molecule IgG secondary antibodies that recognize both the heavy and light chain of primary antibodies. The bivalent nature of these antibodies offers the possibility that cross-linking could occur on the cell surface, which can contribute to a phenomenon known as “cap formation.” When molecules on the surface of a cell are cross-linked they are moved to one end of the cell to form a “cap,” the formation of which can induce some level of endocytosis that leads to false positives (due to inappropriate internalization) in a cytotoxicity assay.

Although we have no data or customer feedback that demonstrates that this has actually occurred in practice, we decided to be proactive and provide our customers with additional tools to meet any potential concerns. As a result, ATS is proud to release a new line of secondary conjugates (Fab-ZAP) produced with monovalent antibodies. These products eliminate the possibility of cap formation as cross-linking of the Fab-ZAP molecules cannot occur, while preserving all of the qualities that make an effective in vitro diagnostic tool. The Fab-ZAP products will still recognize the heavy and light chains of antibodies, and should be used in the same way and at the same molar concentrations as the original secondary conjugates. In fact, preliminary assays using Fab-ZAP-mouse have demonstrated an unexpected lower EC50 when directly compared to Mab-ZAP in a cytotoxicity assay (see Fig. 1).

Fab-ZAP (mouse) – Cat. #IT-48
25 μg / 100 μg / 250 μg
Fab-ZAP (mouse) uses your primary mouse monoclonal IgG antibody to target and eliminate cells that recognize your primary antibody.

Fab-ZAP (human) and Fab-ZAP (rabbit) – In Production

Fig. 1. PC12 cells were plated at 5000 cells/90 μl/well and incubated overnight. Saporin (PR-01) and OX7-SAP (IT-02) dilutions were made in cell media, and 10 μl was added to each well. OX7 antibody (AB-N08) was diluted in cell media containing, at a final concentration, either 100 ng/10 μl Mab-ZAP (IT-04)or 45 ng/10 μl Fab-ZAP (IT-48), and 10 μl was added to each well. The plates were incubated 72 hours. The medium was dumped off of the plate, and the cells were fixed with ice-cold 10% TCA for 1 hr at 4°C. The plate was washed three times with tap water and allowed to air dry. 50 μl of 0.4% sulfarhodamine B/1% acetic acid was added to each well and the plate was incubated for 30 min at room temperature. The plate was washed three times with 1% acetic acid and allowed to air dry. The dye was solubilized with 100 μl of 10 mM unbuffered tris base per well, with 5 min of gentle shaking. The plate was read at 564 nm, and data analysis was done with Prism software (GraphPad, San Diego).
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Targeting Tools: Anti-6 His-ZAP

Anti-6 His-ZAP (Cat. #IT-52)

Composed of an antibody to 6 His conjugated to saporin, this secondary conjugate should be used as a diagnostic tool for testing your primary, 6 His-tagged proteins for specific cell surface epitope binding and internalization. The 6 His tag is widely used because of its affinity to bind nickel or cobalt metal ions attached to sepharose, which can then be used to purify the protein in a native or denatured state.

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Targeting Tools: Anti-GFP-ZAP

Anti-GFP-ZAP (Cat. #IT-53)

The green fluorescent protein (GFP) gene can be introduced and maintained in the genome through breeding, injection with a viral vector, or cell transformation. Whether your cells express a surface protein with a GFP-coded region, or you have a protein that targets specific cells and contains a GFP tag, Anti-GFP-ZAP can be used to verify cell binding and epitope internalization to an extracellular GFP fused to a cell surface molecule.

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Targeting Talk: Testing Lots of Orexin-SAP

Our lab has been working with Orexin-SAP (Cat. #IT-20) and we need to order more to complete our experiments. Unfortunately, your website says the product is discontinued. Could you let us know why you have stopped selling Orexin-SAP? Is it possible for us to get more of it in the future?

Thank you for your inquiry regarding Orexin-SAP. We have been working for several months to try to produce a new lot of this targeted toxin. We discontinued the sale of this targeted toxin because we could not validate the product. We have a Quality Control (QC) assay, but the material we prepared did not perform to the level of the previous Orexin-SAP that we have been selling for several years.

Peptides made by three different suppliers have been conjugated; none performed as the previous lot of Orexin-SAP did. We don’t have an explanation for why the new material doesn’t work in our in vitroQC assay. We decided it was best to discontinue distribution of Orexin-SAP.

Since announcing the decision to discontinue Orexin-SAP, we have heard from several scientists, such as yourself, expressing the need for more of this targeted toxin. ATS is proud of our reputation for quality targeting reagents, and we are reluctant to promote a product for which we cannot provide QC data.

So, we came up with a solution. We do not currently have in vitro or in vivo data for any of the new lots of Orexin-SAP, and are entertaining proposals for a collaboration with experienced researchers who will test these lots and share their data with us. Collaborators will receive samples from 2-4 different lots of Orexin-SAP, and aliquots of Blank-SAP (control conjugate) will be provided at no charge.

Contact us if you are interested in this opportunity. We look forward to working with you.

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Targeting Topics 10q2

Enrichment of xenograft-competent genetically modified pig cells using a targeted toxin, isolectin BS-I-B4 conjugate

Akasaka E, Watanabe S, Himaki T, Ohtsuka M, Yoshida M, Miyoshi K, Sato M

Xenotransplantation 17(1):81-89, 2010.

Genetically-modified pigs lacking the gala1-3gal epitope may be suitable for production of organs that could be transplanted to humans. The ability to select for a homozygous population of donor somatic cells would accelerate the process of generating these animals, which would otherwise take approximately two years. The authors incubated a heterozygous population of 107 porcine embryonic fibroblasts with 1.6 µg of IB4-SAP (Cat. #IT-10). Even after six months the treated cells were negative for the agal epitope.

Severe scene learning impairment, but intact recognition memory, after cholinergic depletion of inferotemporal cortex followed by fornix transection

Browning PG, Gaffan D, Croxson PL, Baxter MG

Cereb Cortex 20(2):282-293, 2010.

In order to directly test depletion of cholinergic neurons in the inferotemporal cortex on learning and memory the authors lesioned the inferotemporal cortex, the rostral entorhinal cortex, and the perirhinal cortex of monkeys with ME20.4-SAP (Cat. #IT-15), 56-64 injections of 0.02 µg per injection). The data suggest that episodic memory is in part controlled by interactions between the fornix and cholinergic input to the inferotemporal cortex.

Unique Contributions of Distinct Cholinergic Projections to Motor Cortical Plasticity and Learning

Conner JM, Kulczycki M, Tuszynski MH

Cereb Cortex 20(11):2739-2748, 2010.

This work mapped the basal cholinergic forebrain system associations with skilled motor learning and motor function recovery after cortical injury. Rats were lesioned with 192-IgG-SAP (Cat. #IT-01). The animals received either two rostrocaudal injections of 75-112 ng; two 19-ng injections into the “prefrontal depletion site”; or two 19-ng injections into the “motor cortex depletion site.” Loss of motor cortex cholinergic systems disrupts map plasticity and skilled motor behavior, indicating that control of these systems rests within the motor cortex.

Depleting Syndecan-4+ T Lymphocytes Using Toxin-Bearing Dendritic Cell-Associated Heparan Sulfate Proteoglycan-Dependent Integrin Ligand: A New Opportunity for Treating Activated T Cell-Driven Disease

Akiyoshi H, Chung JS, Tomihari M, Cruz PD, Jr., Ariizumi K

J Immunol 184(7):3554-3561, 2010.

The dendritic cell-associated heparin sulfate proteoglycan-dependent integrin ligand (DC-HIL) exclusively associates with syndecan-4 (SD-4), which is expressed on some activated T-cells. The authors biotinylated DC-HIL and combined it with streptavidin-ZAP (Cat. #IT-27) and used in culture at a concentration of 10 µg/ml. Only activated T cells were bound and eliminated. (See cover article.)

Transplant of GABAergic precursors restores hippocampal inhibitory function in a mouse model of seizure susceptibility

Zipancic I, Calcagnotto ME, Piquer-Gil M, Mello LE, Alvarez-Dolado M

Cell Transplant 9(5):549-564, 2010.

Although medial ganglionic eminence-derived cells can be grafted into the neonatal brain and become functionally mature GABAergic neurons, it is not clear whether the grafted cells can rescue loss of function. The authors injected mice with 1.6-2.0 ng of SSP-SAP (Cat. #IT-11) into the anterior and posterior hippocampus to eliminate GABAergic interneurons. Neuron function in mice receiving the grafts returned to near normal.

Arcuate nucleus destruction does not block food deprivation-induced increases in food foraging and hoarding

Dailey MJ, Bartness TJ

Brain Res 1323:94-108, 2010.

While some aspects of food intake are understood, mechanisms that control hoarding of food have not been identified. This work investigates the role of NPY in the arcuate nucleus (Arc) in hoarding. Siberian hamsters received 48-ng injections of NPY-SAP (Cat. #IT-28) into the Arc; Blank-SAP (Cat. #IT-21) was used as a control. In lesioned animals food deprivation-induced hoarding was increased 100%, but baseline foraging and food hoarding were unchanged.

Dorsal horn neurons expressing NK-1 receptors mediate scratching in rats

Carstens EE, Carstens MI, Simons CT, Jinks SL

Neuroreport 21(4):303-308, 2010.

The itch signal is passed through the superficial dorsal horn. The authors investigated whether ablation of NK-1 receptor-expressing neurons in this area would affect itch-related scratching behavior. Rats received 20 µl of 2.27-µM SSP-SAP (Cat. #IT-11) as an intracisternal injection. The reduction in itch response to intradermal 5-hydroxytryptamine indicates that NK-1 receptor-expressing superficial dorsal horn neurons are important for spinal itch transmission.

Toxin-Coupled MHC Class I Tetramers Can Specifically Ablate Autoreactive CD8+ T Cells and Delay Diabetes in Nonobese Diabetic Mice

Vincent BG, Young EF, Buntzman AS, Stevens R, Kepler TB, Tisch RM, Frelinger JA, Hess PR

J Immunol 184(8):4196-4204, 2010.

MHC class I tetramers have been used to identify antigen-specific cells. In this work the authors used a biotinylated tetramer in conjunction with streptavidin-ZAP (Cat. #IT-27) to eliminate a specific subset of reactive T cells associated with islets in vivo. NOD mice received three 4.36-µg intravenous injections of the tetramer/saporin complex over 12 days. The onset of type I diabetes in the treated mice was significantly delayed.

An early sympathetic nervous system influence exacerbates collagen-induced arthritis via CD4+CD25+ cells

Harle P, Pongratz G, Albrecht J, Tarner IH, Straub RH

Arthritis Rheum 58(8):2347-2355, 2008.

The sympathetic nervous system can play conflicting roles in collagen-induced arthritis (CIA). CD4+CD25+ T cells can play an immunoregulatory effect in this system depending on the expression of the FoxP3 transcription factor. Mice received 5-µg ip injections of anti-DBH-SAP (Cat. #IT-03) to induce an early sympathectomy. The results indicate that the sympathetic nervous system increases disease severity in CIA by stimulating some of the proinflammatory aspects of CD4+CD25+ T cells.

An opposing time-dependent immune-modulating effect of the sympathetic nervous system conferred by altering the cytokine profile in the local lymph nodes and spleen of mice with type II collagen-induced arthritis

Harle P, Mobius D, Carr DJ, Scholmerich J, Straub RH

Arthritis Rheum 52(4):1305-1313, 2005.

In this work the authors examined the role of the sympathetic nervous system (SNS) in late stages of chronic arthritis. 5-µg intraperitoneal injections of anti-DBH-SAP (Cat. #IT-03) were given in mice. The results demonstrate that the SNS supports inflammation during the asymptomatic phase of arthritis, but inhibits inflammation during the chronic symptomatic phase.

The brainstem noradrenergic systems in stress, anxiety, and depression

Itoi K, Sugimoto N

J Neuroendocrinol 22(5):355-361, 2010.

In this review the authors examine the relationship between the central noradrenergic system, fear/anxiety states, and depression. The use of anti-DBH-SAP (Cat. #IT-03) to investigate the function of the noradrenergic system in these paradigms is described.

Evaluation of side effects through selective ablation of the mu opioid receptor expressing descending nociceptive facilitatory neurons in the rostral ventromedial medulla with dermorphin-saporin

Cao F, Chen SS, Yan XF, Xiao XP, Liu XJ, Yang SB, Xu AJ, Gao F, Yang H, Chen ZJ, Tian YK

Neurotoxicology 30(6):1096-1106, 2009.

It has been shown that injection of dermorphin-SAP (Cat. #IT-12) into the rostral ventromedial medulla (RVM) can abolish descending facilitation. In this work side effects produced by a 3-pmol injection of dermorphin-SAP into the RVM of rats were assessed (Saporin, Cat. #PR-01, was used as a control). Following select physiological functions over a three-month period post-lesion demonstrated that treatment with this targeted toxin produces no long-standing adverse toxicity.

Cardiovascular and behavioural responses to conditioned fear and restraint are not affected by retrograde lesions of A5 and C1 bulbospinal neurons

Vianna DM, Carrive P

Neuroscience 166(4):1210-1218, 2010.

To investigate the role of A5 neurons in some forms of psychological stress the authors injected 22 or 44 ng of anti-DBH-SAP (Cat. #IT-03) into the spinal cord of rats. Mouse IgG-SAP (Cat. #IT-18) was used as control. The data show that A5 presympathetic neurons are not essential for the expression of the tachycardic and pressor responses to conditioned fear and restraint.

Distinct Neural Pathways Mediate {alpha}7 Nicotinic Acetylcholine Receptor-Dependent Activation of the Forebrain

Thomsen MS, Hay-Schmidt A, Hansen HH, Mikkelsen JD

Cereb Cortex 20(9):2092-2102, 2010.

a7 nicotinic acetylcholine receptor (nAChR) agonists are potential treatments for some aspects of schizophrenia. The authors examine whether cholinergic neurons in the horizontal limb of the diagonal band of Broca (HDB) are a target for this treatment. Rats received 300-ng injections of 192-IgG-SAP (Cat. #IT-01) into the HDB. The results demonstrate that cholinergic neurons in the HDB are essential for a7 nAChR agonist activation of the medial prefrontal cortex.

Targeted Ablation of Cardiac Sympathetic Neurons Reduces the Susceptibility to Ischemia-Induced Sustained Ventricular Tachycardia in Conscious Rats

Lujan HL, Palani G, Zhang L, Dicarlo SE

Am J Physiol Heart Circ Physiol 298(5):H1330-9, 2010.

Reduction of cardiac sympathetic activity protects against ventricular tachy-arrhythmias, which are the leading cause of death in industrially-developed countries. Rats received 10-µg injections of CTB-SAP (Cat. #IT-14) into each stellate ganglion. Using comparison of ventricular tachycardia onset times after coronary artery occlusion it was found that lesioned rats were less susceptible to tachycardia events.

The hyperalgesic effects induced by the injection of angiotensin II into the caudal ventrolateral medulla are mediated by the pontine A(5) noradrenergic cell group

Marques-Lopes J, Pinho D, Albino-Teixeira A, Tavares I

Brain Res 1325:41-52, 2010.

Injection of angiotensin II into the caudal ventrolateral medulla (CVLM) has been shown to induce angiotensin type 1 receptor-mediated hyperalgesia. Here the authors lesioned the pontine A5 cell group with anti-DBH-SAP (Cat. #IT-03) to evaluate the role of these neurons in this model. Rats received a 1.1-µg injection of anti-DBH-SAP into the CVLM. Behavioral responses indicate that loss of noradrenergic neurons in the CVLM partially prevented angiotensin II-induced hyperalgesia.

Serotonin transport and metabolism in the mammary gland modulates secretory activation and involution

Marshall AM, Nommsen-Rivers LA, Hernandez LL, Dewey KG, Chantry CJ, Gregerson KA, Horseman ND

J Clin Endocrinol Metab 95(2):837-846, 2010.

This work begins to examine the role of the serotonin reuptake transporter (SERT) in the regulation of lactation homeostasis. The SERT monoclonal antibody (Cat. #AB-N09) was used for immunohistochemistry.

The cerebellum harbors a circadian oscillator involved in food anticipation

Mendoza J, Pevet P, Felder-Schmittbuhl MP, Bailly Y, Challet E

J Neurosci 30(5):1894-1904, 2010.

The authors report on a circadian oscillator in the cerebellum that is sensitive to feeding cues. Mice received icv injections of 0.12, 0.25, or 0.50 µg of OX7-SAP (Cat. #IT-02). Lesioned animals displayed attenuated food-anticipatory activity, and less locomotor activity after fasting.

Does Age Matter? Behavioral and Neuro-anatomical Effects of Neonatal and Adult Basal Forebrain Cholinergic Lesions

De Bartolo P, Cutuli D, Ricceri L, Gelfo F, Foti F, Laricchiuta D, Scattoni ML, Calamandrei G, Petrosini L

J Alzheimers Dis 20(1):207-227, 2010.

The authors characterized the differences caused by age on the effect of cholinergic lesions of the basal forebrain. Seven-day-old rats received 210-ng bilateral icv injections of 192-IgG-SAP (Cat. #IT-01). Eighty-day-old rats received 4-µg bilateral icv injections. Both experimental groups displayed similar behavior, indicating that development of a depleted cholinergic system yields similar results to cholinergic dysfunction in adulthood.

Recent Progress in Research on Ribosome Inactivating Proteins

Ng TB, Wong JH, Wang H

Curr Protein Pept Sci 11(1):37-53, 2009.

Brief descriptions of research done using 192-IgG-SAP (Cat. #IT-01), OX7-SAP (Cat. #IT-02), dermorphin-SAP (Cat. #IT-12), anti-SERT-SAP (Cat. #IT-23), SSP-SAP (Cat. #IT-11), anti-DBH-SAP (Cat. #IT-03), CTB-SAP (Cat. #IT-14), and other conjugates are provided.

Methylphenidate-induced impulsivity: pharmacological antagonism by beta-adrenoreceptor blockade

Milstein JA, Dalley JW, Robbins TW

J Psychopharmacol 24(3):309-321, 2010.

In this work bilateral 20-ng intracortical injections of anti-DBH-SAP (Cat. #IT-03) were used to examine the role of noradrenergic neurons in the control of psychostimulant-induced impulsivity. Although β-adrenoreceptor blockade abolished this impulsivity, lesioning noradrenergic neurons in the cortex had no effect.

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Cover Article: Depletion of syndecan-4+ T lymphocytes by saporin-conjugated DC-HIL alleviates T cell-mediated imflammatory disease

Contributed by Kiyoshi Ariizumi, Hideo Akiyoshi, Jin-Sung Chung, Mizuki Tomiharu, Ponciano D. Cruz Jr.
Department of Dermatology, The University of Texas Southwestern Medical Center and Dermatology Section (Medical Service),
Dallas Veterans Affairs Medical Center, Dallas, TX

T lymphocyte activation is regulated by stimulatory and inhibitory signals transduced by binding of T cell receptors to corresponding ligands on antigen-presenting cells (APC). Stimulatory receptors tend to be present constitutively even on resting T cells, whereas many inhibitory receptors require activation for expression. [1] Thus, inhibitory receptors may serve as a marker for the functional state of T cells.

We discovered a novel inhibitory pathway composed of the APC receptor DC-HIL and its exclusive T cell ligand, syndecan-4 (SD-4). DC-HIL specifically recognizes particular structures of heparan sulfate on SD-4 peculiar to T cells. SD-4 is expressed by activated (but not resting) T cells, including effector/memory CD4+ and CD8+ T cells. Infusion of soluble DC-HIL into mice inhibits the DC-HIL/SD-4 pathway, and results in enhanced immune responses. The current report addresses the hypothesis that depleting SD-4+ T lymphocytes using DC-HIL conjugated to a toxin will suppress elicitation of a T cell-mediated inflammatory response.

We biotinylated and conjugated soluble DC-HIL receptor or control Fc alone (IgG-SAP) to Streptavidin-ZAP (streptavidin conjugated to saporin; Cat. #IT-27), and showed that DC-HIL-SAP binds specifically to activated T cells, is internalized by these cells, and inhibits T cell proliferation in a SD-4- specific manner. These results document that DC-HIL-SAP selectively kills SD-4+ activated T cells.

We next examined the effect of DC-HIL- SAP on an ongoing contact hypersensitivity (CH) response, which is an established model of a delayed T cell-mediated response. Mice were sensitized to a contact allergen oxazolone (Ox) on abdominal skin (day 0), then challenged with Ox on ear skin (day 6). Mice were injected i.v. with DC-HIL-SAP, IgG-SAP (control conjugate), or PBS 3 h prior to challenge (Fig. 1). PBS-injected mice developed strong ear swelling, whereas DC-HIL-SAP- injected mice exhibited markedly reduced ear swelling by 80%. IgG-SAP had no effect. Our DC-HIL-SAP concentration was optimal since 20 nM caused 50% suppression, whereas 80 nM produced 80% reduction (similar dose of IgG-SAP causing increased toxicity). Histologic examination of Ox-painted ear skin in DC-HIL-SAP-injected mice revealed less thick ears and fewer infiltrating leukocytes (Fig. 2). Injection of DC-HIL-SAP following Ox challenge also reduced CH response. The unresponsive state to Ox lasted for 3 weeks (Fig. 3), even as these same mice were able to mount effective CH response against another contact allergen 2,4,6- trinitrochlorobenzene (TNCB) (Fig. 4).

These results indicate that a single infusion of DC-HIL- SAP efficiently blocks elicitation of an established immune response that lasts for 3 weeks and is restricted to the antigen introduced at the time of treatment.

We also examined the ability of DC-HIL-SAP to deplete SD-4+ T cells in immunized mice. Two days after challenging sensitized mice treated with DC-HIL-SAP or controls, SD-4+ T cells in Ox-painted ear skin or in draining lymph nodes (DLN) were counted by immunofluorescent staining (Fig. 5A) or by flow cytometry (Fig. 5B), respectively. There were none- to-very few T cells in untreated skin, but many CD4+ and CD8+ T cells in Ox-painted skin, almost all of which were SD-4+ (Fig. 5A). Numbers of CD4+ and CD8+ T cells in skin of mice injected with IgG-SAP were similar to those of mice treated with PBS, whereas both were reduced markedly following DC-HIL-SAP infusion. In DLN, infusion of DC- HIL-SAP depleted by 40% CD4+ and CD8+ T cells. These results indicate that a single infusion of DC-HIL-SAP depletes SD-4+ T cells in the inflamed skin and DLN.

Our studies in mice indicate that SD-4 can be targeted using toxin-bearing DC-HIL to alleviate a cutaneous inflammatory response that may find applications in many human disease states. The targeted nature (SD-4+ T cells) of this treatment may hold special advantage with respect to safety.

figure 1
Figure 1. Mice were injected i.v. with PBS, IgG-SAP, or DC-HIL-SAP (40 nM) 3 h prior to the challenge. Daily change in ear thickness was plotted for each panel.
figure 1
Figure 2. On day 2 after challenge with oxazolone (Ox), ear skin specimens of mouse representative in each group were stained with hematoxylin and eosin, and histologically examined under magnification of 10 X.
figure 1
Figure 3. These mice were kept for one week and then rechallenged with Ox weekly for second (2o), third (3o) and fourth challenges (4o). Ear thickness was measured one day following challenge.
*p<0.001 and **p=0.003: Student’s t test vs. ear thickness treated with IgG-SAP.
figure 1
Figure 4. BALB/c mice (n=4) were sensitized with Ox on day 0, i.v. injected with PBS or SAP conjugate 3 h prior to challenge (day 6). On the same day, mice were challenged with Ox and solvent alone on right (R-ear) and left ears (L-ear), respectively, and also sensitized to TNCB. On day 7, ear thickness was measured (1o Ox challenge). Day 12, all mice were challenged with Ox (2o Ox challenge) and TNCB on right and left ears, respectively. Ear thickness shown is measured on day 1 after every challenge. *p<0.05; as compared with ear thickness treated with IgG-SAP.
figure 1
Figure 5. Infusion of DC-HIL-SAP depletes SD-4+ T cells efficiently in Ox-painted skin and draining lymph nodes (DLN). BALB/c mice (n=3) were sensitized, injected with PBS or SAP conjugate, and challenged. Two days after challenge, ear skin (A) or DLN (B) were procured, doubly-stained with anti-CD4 or CD8 Ab and anti-SD-4 Ab. (A) Using confocal microscopy, SD-4+/CD4+ or SD-4+/CD8+ T cells were counted in three separate views, and the average with standard deviation is shown graphically. * p<0.01 as compared with % in skin of mice infused with IgG-SAP. (B) CD4+ or CD8+ T cells were purified from pooled DLN cells and counted by flow cytometry for SD-4+/CD4+ or SD-4+/CD8+ T cells per DLN.

References/Footnotes:     (back to top)

  1. T cell expression profiles of these receptors overlap but are disparate; cytotoxic T Lymphocyte antigen-4 (expressed by almost all recently activated T cells), programmed cell death-1 (restricted to effector T cells), B and T lymphocyte attenuator and T cell immunoglobulin mucin 3 (expressed preferentially by Th1 cells). Moreover, sustained high-level of programmed cell death-1 expression is a marker for T cells undergoing exhaustion in chronic viral infections and in cancer.
  2. Chung J-S, Sato K, Dougherty I, Cruz PD Jr, Ariizumi K. DC-HIL is a negative regulator of T cell activation. Blood 109:4320-4327, 2007.
  3. Akiyoshi H, Chung J-S, Tomihari M, Cruz PD Jr, Ariizumi K. Depleting syndecan-4+ T lymphocytes using toxin-bearing DC-HIL: A new
    opportunity for treating activated T cell-driven disease. J Immunol April 2010.