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.

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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.
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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.
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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.
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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.
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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.
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