Encyclopedia of Signaling Molecules

2018 Edition
| Editors: Sangdun Choi


Reference work entry
DOI: https://doi.org/10.1007/978-3-319-67199-4_101843


Historical Background

CD28 was identified in early 1980s by Martin and colleagues by monoclonal antibodies recognizing a 44 kDa protein on the surface of human T lymphocytes (Martin et al. 1980). Further experiments from Gmunder and Lessener evidenced the crucial role of this molecule in costimulating T cell responses by synergizing with PHA in inducing T cell proliferation (Gmunder and Lesslauer 1984). Later in 1987, Aruffo and Seed cloned the cDNA of human CD28 and showed that it encodes for a 23 kDa glycosylated type I transmembrane protein expressed as disulfide-linked homodimer on the surface of 80% of human CD4+ T cells and 50% of human CD8+ T cells (Aruffo and Seed 1987). In mouse, CD28 was cloned in 1990 by Gross and colleagues and found to be expressed 100% on both CD4+ and CD8+ T cells (Gross et al. 1990). In the same year, the natural ligands of CD28 were also identified. In 1989, Freeman and colleagues identified B7.1, also known as CD80, on the surface of activated B lymphocytes (Freeman et al. 1989), and further studies from Azuma and colleagues demonstrated that B7.1 binds to CD28 (Azuma et al. 1993). Later, the same group also cloned and identified B7.2 or CD86 as a CD28 binding partner (Freeman et al. 1993).

Since its discovery, it has become clear that CD28 was the most prominent costimulatory molecule able to deliver the signal two necessary for full T lymphocyte activation. The two signal model of T lymphocyte activation predicts that optimal T cell response to antigen is achieved following the recognition of peptide-major histocompatibility complex (MHC) by TCR (signal one) together with a subset of costimuli (signal two), generally provided by counterreceptors expressed on the surface of APCs. Extensive in vitro and animal model studies demonstrated that CD28 delivers signals that complement TCR in both qualitative and quantitative manners, thus promoting/enhancing cell proliferation, high levels of cytokines, cell survival, and T cell differentiation. More recent studies also evidenced the ability of CD28 to function in a TCR-independent manner (Porciello and Tuosto 2016).


CD28 monomers consist of an extracellular immunoglobulin (Ig)-V-like domain, a transmembrane region, and a short cytoplasmic tail (Fig. 1). The extracellular IgV-like domain binds to B7-1 (CD80) and B7-2 (CD86), expressed on the surface of activated APCs (i.e., macrophages, dendritic cells, B lymphocytes), through a MYPPPY motif. The measurement of the relative affinity of CD28 binding to CD80 and CD86 revealed that CD80 is a stronger ligand (KD = 4 μM) compared to CD86 (KD = 20 μM).
CD28, Fig. 1

Structure of human CD28 and the main signalling molecules recruited to its cytoplasmic motifs. The extracellular IgV-like domain of CD28 binds to B7-1/CD80 and B7-2/CD86 through a MYPPPY motif. The sequence of SH3 and SH2 binding motifs within human cytoplasmic tails of CD28 is shown. The YMNM motif binds the SH2 of p85 subunit of class 1A PI3K. The N-terminal PRRP motif binds the SH3 domain of Itk. The C-terminal PYAP motif binds the SH3 domain of Lck, filamin-A (FLNa), and Vav1

The short cytoplasmic tail of CD28 (41 aa in human and 38 aa in mouse) is highly conserved and has no enzymatic activity. However, it contains several tyrosine and proline-based motifs, which bind the Src homology (SH)2 and SH3 domain of intracellular signalling molecules. It contains an N-terminal YMNM motif that following phosphorylation binds the SH2 domain of the p85 subunit of class 1A PI3K and the adaptor proteins Grb2 and Grb2-related adaptor downstream of Shc (GADS). The YMNM motif is followed by two proline-rich regions, the N-terminal PRRP that binds the SH3 domain of the IL-2 inducible kinase (Itk) and a C-terminal motif PYAP that binds important signalling molecules, including Lck, filamin-A, and Vav1 (Porciello and Tuosto 2016) (Fig. 1).

CD28 and the Regulation of IS Formation

CD28 engagement by either agonistic antibodies or its natural ligands CD80/CD86 lowers the T cell activation threshold and leads to the augmentation of TCR signalling events necessary for efficient cytokine production (via augmented transcriptional activity and messenger RNA stabilization), cell cycle progression, survival, and T cell differentiation. Activation of T cells by APCs bearing the appropriate peptide-MHC complexes requires rapid cytoskeletal reorganization events leading to the polarization of membrane receptors and signalling molecules within the contact site between T cell and APC, a process that is referred as the immunological synapse (IS). TCR CD28 and coreceptors segregate into central supramolecular activation clusters (cSMACs), whereas LFA-1 adhesion molecule and CD45 are enriched into the peripheral SMACs. CD28 plays a critical role in immunological synapse (IS). CD28 mediates the rearrangement of membrane lipid rafts, thus generating a dynamic platform at the IS where many signalling proteins are concentrated and protected from phosphatases (Viola et al. 2010). CD28 also enhances the close contact between T cells and APCs and triggers the actin cytoskeleton rearrangement events, which are necessary for the recruitment and the organization of molecular signalling complexes (Acuto and Cantrell 2000). CD28 capability to promote cytoskeleton rearrangement events relies on its ability to recruit Vav1 and filamin-A (Tavano et al. 2006; Muscolini et al. 2015).

Vav1 is a guanine nucleotide exchange factor of small Rho GTPases, Rac1, and Cdc42 and is required for CD28-dependent signals and actin nucleation (Acuto and Michel 2003; Rudd and Schneider 2003). CD28 stimulation promotes Vav1 tyrosine phosphorylation and activation and Vav1 binding through C-terminal PYAP motif (Muscolini et al. 2015), thus bringing to the membrane the N-Wiskott Aldrich syndrome protein (WASp)/actin related protein (Arp)2/3 complex and filamin-A. All these proteins in turn cooperate in inducing cortical actin polymerization.

CD28-dependent regulation of actin dynamic and cytoskeleton reorganization is fundamental for its role of general amplifier of TCR signalling functions (Boomer and Green 2010).

CD28 Contribution to TCR-Signalling Functions

One of the earliest events initiated by TCR recognition of peptide-MHC complexes is the activation of Src family tyrosine kinases p56lck and p59fyn that phosphorylate tyrosine residues of the immunoreceptor tyrosine-based activation motifs (ITAMs) of CD3 and ζ chains. Tyrosine phosphorylated ITAMs bind the Syk family tyrosine kinase Zap-70 that following activation by p56lck and/or p59fyn phosphorylates the linker for activation of T cells (LAT). Tyrosine phosphorylated LAT binds and recruits to the membrane PLC-γ1 and the growth-factor receptor-bound protein (Grb2). PLCγ1 Hydrolyzes phosphatidylinositol 4,5-biphosphate (PIP2) into inositol-1,4,5-trisphosphate (IP3) and diacylglycerol (DAG). Soluble IP3 triggers intracellular Ca2+ mobilization, thus leading to the activation of calcineurin and nuclear factor of activated T cells (NF-AT). TCR ligation in absence of CD28 costimulation results in a strong reduction of both PLCγ1 phosphorylation and release of endoplasmic reticulum Ca2+ into the cytoplasm.

The membrane lipid DAG activates the protein kinase C (PKC)θ and nuclear factor-κB (NF-κB), and CD28 stimulation is essential to trigger the activation of the NF-ĸB pathway by favoring the recruitment of PKCθ to the IS.

Grb2 recruitment to LAT leads to the activation of the mitogen-activated protein kinase (MAPK) cascades. Indeed, Grb2 recruits Son of Seveless (SoS), a guanine nucleotide exchange factor for p21Ras, that in turn activates the extracellular signal regulated kinases (ERK) pathway. Moreover, Grb2 also favors the recruitment of Vav1 and the activation of Jun N terminal kinases (JNK). JNK and ERK cooperate to induce the transcriptional activation of AP-1. CD28 engagement by B7 cooperates with TCR for achieving the optimal activation of both ERK and JNK (Fig. 2).
CD28, Fig. 2

CD28 amplifies early and downstream signalling pathways triggered by TCR. Upon TCR recognition of peptide-MHC complexes on the surface of APCs, Lck and Fyn phosporylate ITAMs within CD3 and ζ chains, which bind ZAP-70. ZAP-70 phosphorylates LAT that in turn binds PLC-γ1. PLC-γ1 hydrolyze phosphatidylinositol 4,5-biphosphate (PIP2) in IP3, which induces the activation of Ca2+/Calcineurin (CN) and NF-AT, and DAG that participates in activating PKCθ/CARMA1/Bcl10/MALT1 and NF-κB; Grb2, by bringing SoS and Vav1, activates Ras/ERK and Rac-1/JNK, respectively, thus inducing AP-1; Grb2/Vav1 also activates Cdc42/WASP/Arp2/3 and actin cytoskeleton rearrangements necessary for the mobilization of membrane lipid rafts. NF-AT, NF-kB, and AP-1 cooperate to transactivate the IL-2 gene promoter

The first readout of CD28 costimulatory signals in TCR-mediated T cell activation is the enhancement of IL-2 expression and secretion at both transcriptional and posttranscriptional levels. The IL-2 promoter is, indeed, specifically regulated by NF-AT, NF-κB, and AP-1 transcription factors that, as described above, are all amplified by CD28. Moreover, the IL-2 promoter contains a specific enhancer region, known as CD28-responsive element (CD28RE), which is composed of NF-κB binding sites with adjacent NF-IL-2B AP-1 sites (Verweij et al. 1991; Shapiro et al. 1997). This regulative unit controls CD28 responsiveness in the IL-2 promoter and results in a site for signal integration and thus mutations of RE/AP strongly impair the transcriptional increase of IL-2 induced by CD28 costimulation.

CD28 and NF-κB Activation

Despite the relevance of CD28 in enhancing all TCR-mediated signalling pathways, NF-κB may be considered the most relevant CD28 biochemical target (Tuosto 2011) (Fig. 3).
CD28, Fig. 3

Schematic model of CD28-mediated activation of both canonical and noncanonical NF-κB pathways. Following engagement of human CD28 by B7, tyrosine phosphorylated YMNM binds the SH2 domain of p85 regulatory subunit of class 1A PI3K and C-terminal PYAP binds Vav1, filamin-a (FLNa) and associated NIK. Class 1A PI3K phosphorylates PIP2 and generates PIP3 that favors the recruitment and activation of PDK1 that in turn cooperates with TCR in activating PKCθ/CARMA1/Bcl10/MALT1 and the tripartite IKKγ/α/β complex. The IKK complex leads to the activation of canonical NF-κB1 pathway. PDK1 also activates Akt that cooperates with NIK to activate IKKα and noncanonical NF-κB2 pathway

NF-κB family consists of transcriptionally active heterodimers containing RelA, c-Rel, or RelB in association with p50 (NF-κB1) and/or p52 (NF-κB2). In most unstimulated cells, inhibitory proteins belonging to the inhibitor of NF-κB (IκB) family, which include IκBα, IκBβ, and IκBε, bind and sequester the active heterodimers in the cytosol. A protein kinase complex containing two serine kinases, IκB kinase (IKK)α and IKKβ, and a third subunit, IKKγ/NEMO, with regulatory functions, phosphorylates IκBs, thus leading to their proteolytic degradation and release of NF-κB into the nucleus. TCR stimulation induces the activation of the canonical RelA/p50 or c-Rel/p50 heterodimers by recruiting PKCθ and the ternary complex caspase recruitment domain membrane associated guanylate kinase protein 1 (CARMA1), Bcl10, and mucosa-associated lymphoid tissue lymphoma translocation protein 1 (MALT1) that links TCR to the IKK complex.

CD28 contributes to the activation of the canonical NF-κB1 pathway by binding the SH2 domain of p85 subunit of class 1A PI3K through the tyrosine phosphorylated YMNM motif. Class 1A PI3K generates the phosphatydilinositol 3,4,5 triphosphates (PIP3) lipids that bind the pleckstrin homology (PH) domains of phosphoinositide-dependent protein kinase 1 (PDK1). PDK1 leads to the activation and membrane recruitment of PKCθ that cosegregates with CD28 to a spatially unique subregion within the IS, thus favoring the activation of CARMA1/Bcl10/MALT1 complex and IKKs. Moreover, PDK1 also contributes to the phosphorylation and activation of Akt that both cooperates with PKCθ in stimulating the canonical NF-κB1 pathway and synergize with CD28 to activate noncanonical NF-κB2 pathway (Tuosto 2011).

The noncanonical NF-κB2 pathway is generally activated by the IKKα activator NF-κB-inducing kinase (NIK) that, by phosphorylating IKKα homodimers, leads to the processing of NF-κB2 and the release of p52-containing heterodimers. CD28 stimulation is able to recruit and activate NIK and IKKα, thus leading to the nuclear translocation and activation of noncanonical NF-κB2 dimers. This CD28 unique capability to activate NF-κB converges to the selective regulation of the expression of several genes, including antiapoptotic and proapoptotic genes of the Bcl-2 family and cytokine/chemokine genes (Tuosto 2011; Porciello and Tuosto 2016).


Since its discovery and on the basis of the high homology between rodent (mouse and rat) and human CD28, for several years, in vivo mouse models have been used for understanding the mechanisms of T lymphocyte activation and differentiation and the role of CD28 costimulation in health and immune diseases. When Thomas Hunig’s group discovered that CD28 superagonistic antibodies (CD28SAbs) were able to preferentially activate and expand immunosuppressive regulatory T cells (Lin and Hunig 2003), an enormous amount of preclinical experiments have been performed to evaluate the potential use of these CD28SAbs to ameliorate the onset, progression, and clinical course of human autoimmune diseases. However, on March 2006, the phase I clinical trial of a humanized CD28SAb (TGN1412) turned in a catastrophe, because this antibody induced a rapid and massive cytokine production (i.e., IFN-γ, IL-1, IL-6, TNF-α), thus causing a severe systemic inflammatory response syndrome in all healthy volunteers (Suntharalingam et al. 2006). These data evidence that despite the enormous progresses made in identifying the mechanisms and molecules involved in CD28 signalling, much remains to be elucidated, especially in the light of the differences in CD28 signalling capabilities between human and mouse.


  1. Acuto O, Cantrell D. T cell activation and the cytoskeleton. Annu Rev Immunol. 2000;18:165–84. doi: 10.1146/annurev.immunol.18.1.165.CrossRefPubMedGoogle Scholar
  2. Acuto O, Michel F. CD28-mediated co-stimulation: a quantitative support for TCR signalling. Nat Rev Immunol. 2003;3:939–51. doi: 10.1038/nri1248.CrossRefPubMedGoogle Scholar
  3. Aruffo A, Seed B. Molecular cloning of a CD28 cDNA by a high-efficiency COS cell expression system. Proc Natl Acad Sci USA. 1987;84:8573–7.CrossRefPubMedPubMedCentralGoogle Scholar
  4. Azuma M, Ito D, Yagita H, Okumura K, Phillips JH, Lanier LL, et al. B70 antigen is a second ligand for CTLA-4 and CD28. Nature. 1993;366:76–9. doi: 10.1038/366076a0.CrossRefPubMedGoogle Scholar
  5. Boomer JS, Green JM. An enigmatic tail of CD28 signaling. Cold Spring Harb Perspect Biol. 2010;2:a002436. doi: 10.1101/cshperspect.a002436.CrossRefPubMedPubMedCentralGoogle Scholar
  6. Freeman GJ, Freedman AS, Segil JM, Lee G, Whitman JF, Nadler LM. B7, a new member of the Ig superfamily with unique expression on activated and neoplastic B cells. J Immunol. 1989;143:2714–22.PubMedGoogle Scholar
  7. Freeman GJ, Gribben JG, Boussiotis VA, Ng JW, Restivo Jr VA, Lombard LA, et al. Cloning of B7-2: a CTLA-4 counter-receptor that costimulates human T cell proliferation. Science. 1993;262:909–11. doi: 10.1126/science.7694363.CrossRefPubMedGoogle Scholar
  8. Gmunder H, Lesslauer W. A 45-kDa human T-cell membrane glycoprotein functions in the regulation of cell proliferative responses. Eur J Biochem. 1984;142:153–60. doi: 10.1111/j.1432-1033.1984.tb08263.x.CrossRefPubMedGoogle Scholar
  9. Gross JA, St John T, Allison JP. The murine homologue of the T lymphocyte antigen CD28. Molecular cloning and cell surface expression. J Immunol. 1990;144:3201–10.PubMedGoogle Scholar
  10. Lin CH, Hunig T. Efficient expansion of regulatory T cells in vitro and in vivo with a CD28 superagonist. Eur J Immunol. 2003;33:626–38. doi: 10.1002/eji.200323570.CrossRefPubMedGoogle Scholar
  11. Martin PJ, Hansen JA, Nowinski RC, Brown MA. A new human T-cell differentiation antigen: unexpected expression on chronic lymphocytic leukemia cells. Immunogenetics. 1980;11:429–39.CrossRefPubMedGoogle Scholar
  12. Muscolini M, Camperio C, Porciello N, Caristi S, Capuano C, Viola A, et al. Phosphatidylinositol 4-phosphate 5-kinase alpha and Vav1 mutual cooperation in CD28-mediated actin remodeling and signaling functions. J Immunol. 2015;194:1323–33. doi: 10.4049/jimmunol.1401643.CrossRefPubMedGoogle Scholar
  13. Porciello N, Tuosto L. CD28 costimulatory signals in T lymphocyte activation: emerging functions beyond a qualitative and quantitative support to TCR signalling. Cytokine Growth Factor Rev. 2016;28:11–9. doi: 10.1016/j.cytogfr.2016.02.004.CrossRefPubMedGoogle Scholar
  14. Rudd CE, Schneider H. Unifying concepts in CD28, ICOS and CTLA4 co-receptor signalling. Nat Rev Immunol. 2003;3:544–56. doi: 10.1038/nri1131.CrossRefPubMedGoogle Scholar
  15. Shapiro VS, Truitt KE, Imboden JB, Weiss A. CD28 mediates transcriptional upregulation of the interleukin-2 (IL-2) promoter through a composite element containing the CD28RE and NF-IL-2B AP-1 sites. Mol Cell Biol. 1997;17:4051–8.CrossRefPubMedPubMedCentralGoogle Scholar
  16. Suntharalingam G, Perry MR, Ward S, Brett SJ, Castello-Cortes A, Brunner MD, et al. Cytokine storm in a phase 1 trial of the anti-CD28 monoclonal antibody TGN1412. N Engl J Med. 2006;355:1018–28. doi: 10.1056/NEJMoa063842.CrossRefPubMedGoogle Scholar
  17. Tavano R, Contento RL, Baranda SJ, Soligo M, Tuosto L, Manes S, et al. CD28 interaction with filamin-A controls lipid raft accumulation at the T-cell immunological synapse. Nat Cell Biol. 2006;8:1270–6. doi: 10.1038/ncb1492.CrossRefPubMedGoogle Scholar
  18. Tuosto L. NF-kappaB family of transcription factors: biochemical players of CD28 co-stimulation. Immunol Lett. 2011;135:1–9. doi: 10.1016/j.imlet.2010.09.005.CrossRefPubMedGoogle Scholar
  19. Verweij CL, Geerts M, Aarden LA. Activation of interleukin-2 gene transcription via the T-cell surface molecule CD28 is mediated through an NF-kB-like response element. J Biol Chem. 1991;266:14179–82.PubMedGoogle Scholar
  20. Viola A, Contento RL, Molon B. Signaling amplification at the immunological synapse. Curr Top Microbiol Immunol. 2010;340:109–22. doi: 10.1007/978-3-642-03858-7_6.PubMedGoogle Scholar

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© Springer International Publishing AG 2018

Authors and Affiliations

  1. 1.Istituto Pasteur-Fondazione Cenci Bolognetti, Department of Biology and Biotechnology Charles DarwinSapienza University of RomeRomeItaly