CD28 (Antigen CD28) have been characterized as a co-receptor for the TCR-CD3 (T-Cell Receptor-CD3 Antigen) complex and is responsible for providing the co-stimulatory signal required for T-cell activation. CD28 also act as a receptor independent of the TCR and can initiate signaling events without concomitant TCR ligation. Priming of naive T-cells in lymphoid organs depends on the interaction between CD28, which is constitutively expressed in T-cells, and both CD80 (CD80 Antigen) and CD86 (CD86 Antigen) and induces subsequent IL-2 (Interleukin-2) production and clonal expansion for effective immune response. CD28 is a major positive co-stimulatory molecule required for T-cell activation and functional differentiation, and that CTLA4 (Cytotoxic T-Lymphocyte Antigen-4) upon ligation with CD80 and CD86 provides a negative co-stimulatory signal for the termination of activation and cellular function of T-cells (Ref.1 & 2). T-cells help to control and eliminate microbial infections by detecting the causative agent and initiation of this process requires a “go-between” represented by sentinel cells called DCs (Dendritic Cells), a special class of antigen presenting cells. DCs detect and “process” microbes in the invaded tissue, storing information later delivered to T-cells by directly contacting them in the appropriate anatomical site (for example, a draining lymph node). The message transferred through the DCs is essentially composed of two signals. One signal is captured by the TCR upon detection of non-self structures, such as foreign peptides bound to MHC (Major Histocompatibility Complex) proteins (peptide-MHC complexes) on the DC. The other signal, called co-stimulatory, is relayed by CD28, which is a T-cell specific homodimeric receptor that serves to amplify the signal triggered by TCR ligation. The T-Helper cells recognize antigen when processed by antigen presenting cells/DCs containing MHC-Class II and CD4 (CD4 Antigen) (Ref.3).
In naive T-cells, CD28 co-stimulation enhances cell cycle entry, potently stimulates expression of the mitogenic lymphokine IL-2 and also facilitates the activation of an anti-apoptotic program. At the same time, CD28 co-stimulation enhances the acquisition of T-Helper cell-1 and 2 phenotypes, as well as the induction of CD8 (CD8 Antigen) effector responses. Once ligated by CD80, CD28 provides the T-cell with an initial adhesion capable of approximating the T-cell and antigen presenting cell membranes. Many other transmembrane receptors are known to modulate specific elements of TCR and CD28 signaling. One of such important receptor is the CD45 (CD45 Antigen) that occurs as a component of a complex of proteins associated with the antigen receptor, and may regulate signal transduction by modulating the phosphorylation state of the tyrosine kinases like Lck (Lymphocyte-Specific Protein-Tyrosine Kinase). This phosphorylation state of Lck is reversed by CSK (c-Src Tyrosine Kinase). Lck and Fyn (Fyn Oncogene Related to Src,FGR, YES) remains attached to the cytoplasmic domain of either CD4 or CD8. Concomitantly, activation of Lck and Fyn phosphorylates ZAP70 (Zeta-Chain-Associated Protein Kinase), SYK (Spleen Tyrosine Kinase) and Vav1 (Oncogene Vav1) (Ref.4). Activated Lck in turn activates CD28 and induce activation of LAT (Linker for Activation of T-Cells). This is critical for coupling the pre-TCR surface complexes to intracellular signaling pathways in the developmental response. LAT is a palmitoylated integral membrane adaptor protein that resides in lipid membrane rafts and binds to the adaptor GADS (Growth Factor Receptor-Bound Protein-2-Related Adaptor Protein-2), SLP76 (SH2 Domain-Containing Leukocyte Protein-76), ITK (IL-2 (Interleukin-2) inducible T-cell Kinase), Vav1 and Tec (Tec Protein Tyrosine Kinase); this interaction activates PLC-Gamma (Phospholipase-C-Gamma), RLK (Resting Lymphocyte Kinase), CARMA1 (CARD-MAGUK Protein-1), BCL10 (B-Cell Cell/Lymphoma-10), CDC42 (Cell Division Cycle-42) and Rac(Ras-Related C3 Botulinum Toxin Substrate) thereby facilitating the recruitment of key signal transduction components to drive T-cell activation (Ref.5).
Further binding of CD28 to Class-I regulatory PI3K (Phosphatidylinositde-3 Kinase) recruits the Class-I catalytic PI3K to the membrane that generates PIP3 (Phosphatidylinositol 3,4,5-trisphosphate) and anchors proteins that contain a pleckstrin-homology domain to the plasma membrane including the Class-III catalytic PI3K, PIK3C3 (Phosphoinositide-3-Kinase-Class-3). The catalytic PI3Ks help to convert PIP2 (Phosphatidylinositol 4,5-bisphosphate) to PIP3. Excess PIP3 is removed by PTEN (Phosphatase and Tensin Homolog). PI3K is required for activation of Akt
(v-Akt Murine Thymoma Viral Oncogene Homolog), which in turn regulate many downstream targets via Akt Signaling that are involved in diverse functions such as protein translation, cellular metabolism, inhibition of apoptosis; thus leading to cell survival. Despite the importance of the CD28-PI3K-Akt pathway, binding of PI3K alone is insufficient to account for the full effects of CD28 (Ref.6). In addition to NFAT
(Nuclear Factor of Activated T-Cells) and NF-KappaB
(Nuclear Factor-KappaB) has a crucial role in the regulation of gene transcription of the IL-2 promoter and anti-apoptotic factors. For this PLC-Gamma utilizes PIP2 as a substrate to generate IP3 (Inositol 1,4,5-trisphosphate) and DAG (Diacylglycerol). IP3 elicit release of Ca2+ (Calcium Ions) via IP3R (IP3 Receptor), whereas DAG activates PKC-Theta (Protein Kinase-C-Theta). Under the influence of RLK, PLC-Gamma, and Ca2+; PKC-Theta, along with Calm
(Calmodulin) and CalmK2 (Calcium/Calmodulin-Dependent Protein Kinase-2) regulates the phosphorylation state of IKK
(Inhibitor of Kappa Light Polypeptide Gene Enhancer in B-Cells Kinase) complex through direct as well as indirect interactions. This activation ultimately results in translocation of NF-KappaB
(Nuclear Factor-KappaB) to the nucleus, followed by degradation of I-KappaBs (Inhibitor of Kappa Light Chain Gene Enhancer in B-Cells). Moreover activation of CARMA1 (CARD-MAGUK Protein-1) phosphorylates BCL10 (B-Cell Leukemia-10) and dimerizes MALT1 (Mucosa Associated Lymphoid Tissue Lymphoma Translocation Gene-1), an event that is sufficient for the activation of IKKs
(Ref.3 & 7).
The two CD28-responsive elements in the IL-2 promoter have NF-KappaB
binding sites. NF-KappaB
dimers are normally retained in cytoplasm by binding to inhibitory I-KappaBs. Phosphorylation of I-KappaBs initiates its ubiquitination and degradation, thereby freeing dimers to translocate to the nucleus. Likewise translocation of NFAT
to the nucleus as a result of Calm-Caln
(Calcineurin) interaction effectively enhances IL-2 gene expression. CD28 interfaces with Actin-mediated cytoskeletal reorganization by activating LAT and Vav1. Activation of Vav1 by TCR-CD28-PI3K signaling, connects CD28 with the activation of Rac
and CDC42 and this enhances TCR-CD3-CD28 mediated cytoskeletal re-organization. Rac regulates the polymerization to drive lamellipodial protrusion and membrane ruffling, whereas CDC42 generates polarity and induces formation of filopodia and microspikes. These GTPases, CDC42 and Rac, function sequentially to activate downstream effectors like WASP (Wiskott-Aldrich Syndrome Protein) and PAK1 (p21/CDC42/Rac1-Activated Kinase-1) to induce activation of ARPs (Actin-Related Proteins) resulting in Actin alterations in the cytoskeleton (Ref.3 & 5). CD28 impinges on the Rac/PAK1-mediated IL-2 gene transcription through subsequent activation of MEKK1 (MAP/ERK Kinase Kinase-1), MKKs
(Mitogen-Activated Protein Kinase Kinases) and JNKs
(c-Jun Kinases). JNKs
phosphorylate and activate c-Jun (Jun Oncogene) and c-Fos (Cellular Oncogene Fos)-a part of the Activator Protein-1 complex essential for transcription of IL-2. Signaling through CD28 promotes cytokine IL-2 mRNA production and entry into the cell cycle, T-cell survival, T-Helper cell differentiation and Ig (Immunoglobulin) isotype switching and because of the strong biological effects of co-stimulatory signals mediated by CD28 family molecules on various immune responses, this pathway remains an attractive target for new therapeutic strategies for the treatment of tumors, autoimmune diseases and graft rejection (Ref.8 & 9).