Vstm3 is a member of the CD28 family and an important modulator of T-cell function - PubMed (original) (raw)

doi: 10.1002/eji.201041136. Epub 2011 Mar 18.

David W Taft, Cameron S Brandt, Christoph Bucher, Edward D Howard, Eric M Chadwick, Janet Johnston, Angela Hammond, Kristen Bontadelli, Daniel Ardourel, LuAnn Hebb, Anitra Wolf, Thomas R Bukowski, Mark W Rixon, Joseph L Kuijper, Craig D Ostrander, James W West, Janine Bilsborough, Brian Fox, Zeren Gao, Wenfeng Xu, Fred Ramsdell, Bruce R Blazar, Katherine E Lewis

Affiliations

Vstm3 is a member of the CD28 family and an important modulator of T-cell function

Steven D Levin et al. Eur J Immunol. 2011 Apr.

Abstract

Members of the CD28 family play important roles in regulating T-cell functions and share a common gene structure profile. We have identified VSTM3 as a protein whose gene structure matches that of the other CD28 family members. This protein (also known as TIGIT and WUCAM) has been previously shown to affect immune responses and is expressed on NK cells, activated and memory T cells, and Tregs. The nectin-family proteins CD155 and CD112 serve as counter-structures for VSTM3, and CD155 and CD112 also bind to the activating receptor CD226 on T cells and NK cells. Hence, this group of interacting proteins forms a network of molecules similar to the well-characterized CD28-CTLA-4-CD80-CD86 network. In the same way that soluble CTLA-4 can be used to block T-cell responses, we show that soluble Vstm3 attenuates T-cell responses in vitro and in vivo. Moreover, animals deficient in Vstm3 are more sensitive to autoimmune challenges indicating that this new member of the CD28 family is an important regulator of T-cell responses.

Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

PubMed Disclaimer

Figures

Figure 1

Figure 1

CD155 expression is sufficient to convey Vstm3 binding on activated DC and transfected cells. (A and B) DC were generated from mouse BM cells by culturing total BM at 106 cells/mL in 100 ng/mL Flt3-L for 7 days. Resultant DC were activated for 24 h with 10 ng/mL IFN-γ and 1 μg/mL CD40-L and then stained with (A) 2 μg/mL Alexa-647 conjugated Vstm3-Fc or (B) Alexa-647 labeled Vstm3-tetramer alone (solid lines) or in the presence of a tenfold excess of unlabeled Vstm3-Fc or Vstm3 tetramer respectively (dotted lines). Control Fc-fusion protein or an irrelevant tetrameric protein (shaded histograms) served as controls. (C) P815 cells transfected with mouse CD155 were stained with 2 μg/mL of an Alexa-647 conjugated Vstm3 tetramer (solid line) alone or in the presence of a tenfold excess unlabeled Vstm3 protein (dotted line). Staining with an irrelevant tetramer (shaded histogram) served as a negative control. (D) HEK-293 cells transfected with mouse CD155 were stained with 2 μg/mL Alexa-647 conjugated Vstm3-tetramer (solid line) or 2 μg/mL Alexa-647 labeled soluble CD226 (dashed line). Untransfected HEK-293 cells stained with either Vstm3-tetramer or Vstm-Fc served as negative controls. All results are representatives of at least two experiments.

Figure 2

Figure 2

Vstm3 and CD226 cross-compete for binding to CD155 and anti-mouse Vstm3 mAbs inhibit Vstm3 binding to CD155. (A) P815 cells expressing human CD155 were left unstained or stained with 1 μg/mL Alexa-647 labeled human VSTM3 (left graph) or CD226 (right graph) in absence (no comp) or presence of a 100-fold excess of CD226-Fc, VSTM3-Fc or an irrelevant (control) Fc protein. MFI was determined and the results are representatives of at least two experiments. (B) P815-CD155 cells were cultured in medium alone (No mAb) or medium plus Alexa-647 conjugated Vstm3-tetramer (2 μg/mL) and either an irrelevant isotype control (Control mAb), or three independent anti-mouse Vstm3 mAb clones (each at 10 μg/mL). Samples were run in triplicate and bars show mean±SEM. Results are representatives of at least two experiments.

Figure 3

Figure 3

Schematic comparing the CD226-Vstm3 network of costimulation/inhibition between APC and T/NK cells with the CD28-CTLA-4 network. Vstm3 is a putative inhibitory receptor like CTLA-4 whereas CD226 is an activating receptor like CD28. Both sets of molecules are engaged by two sets of counter-structures, Vstm3 and CD226 by CD155 (PVR) and CD112 (nectin-2) and CTLA-4 and CD28 by CD80 (B7.1) and CD86 (B7.2). Activation is indicated by +; inhibition by −; arrows indicate protein–protein interactions.

Figure 4

Figure 4

CD155 delivers a costimulatory signal to T cells, but Vstm3 is an inhibitory receptor. (A) BHK cells stably transfected with I-Ad and the indicated co-receptors, and untransfected BHK cells, were loaded with 1 μg/mL DO11.10-specific OVA peptide and assessed for their ability to stimulate proliferation of DO11.10 TCR TG T cells. (B) Peptide-loaded BHK expressing I-Ad alone or together with CD155 were tested for their effects on DO11.10 T-cell proliferation in the presence or absence of 5 μg/mL soluble Vstm3-tetramer or blocking anti-Vstm3 antibody. (A and B) Graphs show the mean percentage of proliferating T cells±SEM (triplicate cultures), are derived from separate experiments and are representatives of at least five experiments. The dashed line indicates the mean values for BHK cells expressing only I-Ad; * indicates values significantly different from this value (p<0.05, Student’s _t_-test) while ** indicates values differing both from this value and from the value for cells expressing I-Ad plus CD155 (p<0.05, Student’s _t_-test).

Figure 5

Figure 5

Soluble Vstm3 inhibits disease in a CIA model whereas a blocking anti-Vstm3 mAb accelerates disease development. (A and B) Mice were treated starting one day before the second collagen injection with PBS, Vstm3-Fc, Vstm3-Tet or anti-Vstm3 mAb and the average paw score (±SEM) was measured on the initial day of treatment and daily thereafter; Graphs show average paw score versus time (in days) after the second collagen injection *p<0.05 by two-way ANOVA. Results shown are representatives of at least three experiments. Serum (C) IL-6 or (D) IL-10 levels were determined by Luminex assay on serum samples harvested on day 12 after CIA induction. Each dot represents the mean of triplicate determinations from a single mouse and the horizontal lines are the mean values from all animals in a treatment group; _p_-values are calculated relative to the PBS-treated mice (one-way ANOVA). UI, untreated, non-CIA mice.

Figure 6

Figure 6

Soluble Vstm3 attenuates disease in CIA by inhibiting T-cell activation. On day 12 after CIA induction, cells from the DLN were isolated, acutely restimulated in the presence of Golgi-blockers and surface and the percentage of cells staining for (A) IL-17A, (B) TNF-α, (C) CD4+ cells with a memory (CD44high) phenotype, (E) IFN-γ+ cells, and (F) IL-10+ cells, as well as (D) the number of lymphocytes harvested from the paws of animals, was determined by flow cytometry. Each dot is the mean of triplicate cultures of cells from one animal and the horizontal lines are the mean values from all animals in a treatment group; _p_-values are results from Student’s _t_-test comparing PBS-treated mice to those treated with Vstm3-Tet and only those values showing significant differences from controls have _p_-values associated with them. Results shown are representatives of at least three experiments.

Figure 7

Figure 7

Vstm3-deficient (KO) mice are more sensitive to autoimmune challenge whereas TG mice are protected. (A) Total splenocytes from Vstm3 KO (left panel, dashed line), TG mice (right panel, dotted line) or control WT mice (solid lines) were stained with anti-mouse Vstm3 mAb and and MFI measured. Plots show Vstm3 expression gated on total splenocytes as indicated in the Supporting Information Fig. 5B. (B) Graph shows average disease score (±SEM) versus time after pertussis toxin administration in the MOG EAE model in WT, Vstm3-KO and Vstm3-Tg mice as described in the materials and methods section. * indicates p<0.05 by two-way ANOVA KO/TG versus the respective WT. Results shown are representatives of at least three experiments. (C) T-cell-depleted BM was transferred (day 0) into lethally irradiated B10.BR mice without (BM only) or with purified T cells from Vstm3 KO (KO) or control mice (WT) in the absence/presence of anti-Vatm3 mAb and cell survival was measured over time; p<0.0002 for WT versus KO and WT versus WT + Anti-vstm3 (log-rank test). Results shown are representatives of two experiments.

Figure 8

Figure 8

Agonistic activity of an anti-VSTM3 mAb inhibits T-cell responses. mAb were coupled to beads (anti-CD3 at the indicated percentage of bead capacity and anti-VSTM3 mAb or an isotype-matched control antibody each at 80%) and the beads incubated at a bead: cell ration of 1:1 with purified T cells. Proliferation was judged by CFSE dilution on T cells (gated as indicated in the Supporting Information Fig. 5C) for anti-CD3 plus isotype control (left hand panels) or anti-CD3 plus anti-VSTM3 (center panels). Upregulation of CD25 was also assessed by FACS (right panels) using the beads coated with anti-CD3 plus the isotype control (dark line) or anti-CD3 plus anti-VSTM3 (grey line). Profiles shown are gated on CD4+ T cells (Supporting Information Fig. 5B), but similar results were obtained examining the same parameters on CD8+ cells in the same cultures. Results are representatives of at least three experiments.

Similar articles

Cited by

References

    1. Greenwald RJ, Freeman GJ, Sharpe AH. The B7 family revisited. Annu.Rev.Immunol. 2005;23:515–548. - PubMed
    1. Keir ME, Sharpe AH. The B7/CD28 costimulatory family in autoimmunity. Immunol.Rev. 2005;204:128–143. - PubMed
    1. Sharpe AH, Freeman GJ. The B7-CD28 superfamily. Nat.Rev.Immunol. 2002;2:116–126. - PubMed
    1. Sedy JR, Gavrieli M, Potter KG, Hurchla MA, Lindsley RC, Hildner K, Scheu S, Pfeffer K, Ware CF, Murphy TL, Murphy KM. B and T lymphocyte attenuator regulates T cell activation through interaction with herpesvirus entry mediator. Nat.Immunol. 2005;6:90–98. - PubMed
    1. Parry RV, Riley JL, Ward SG. Signalling to suit function: tailoring phosphoinositide 3-kinase during T-cell activation. Trends Immunol. 2007;28:161–168. - PubMed

MeSH terms

Substances

Grants and funding

LinkOut - more resources