As we enter the season of giving (and receiving), I thought it would be a great time to consider how antigens are presented to the immune system. A robust adaptive immune response is driven by antigen stimulation of both B cells and T cells. While both cellular populations are activated by the same antigen(s), they engage the antigen quite differently. In past articles I have spent a great deal of time discussing B cell receptor (BCR)/ antibody development and maturation, diversity, and their ability to develop specificity to antigens. However, I have yet to describe how T cells recognize and interact with antigens. In this article we will explore the mechanisms of antigen processing and how this contributes to T cell recognition of antigen and the regulation of the immune response.
II. Antigen Receptors
There are three primary types of immune receptors that have the specific purpose of binding protein antigens (Figure 1): B cell receptors (and antibodies), T cell receptors, and Major Histocompatibility Complex (MHC). Each of these have a specific role in responding to antigens and regulating the immune response. The details of antibody structure and function has already been adequately covered (here) so I will not spend time in this article repeating the specifics of B cell derived antigen receptors. I do wish to emphasize, however, that both BCR and antibody binding to epitopes on the native antigen is dependent on the epitope being structurally appropriate and available.
T cells have a T cell receptor (TCR) which is structurally related to a BCR, but unlike BCRs, TCRs do not interact with native antigens. Rather TCRs have antigens presented by MHC as a peptide fragment (TCR epitope) and specifically recognize both the MHC and the peptide. The MHC displays the peptide fragment in the context that is recognizable for the TCR to engage with the epitope. There are two major MHC types: class I (MHC I) and class II (MHC II). While there are some structural differences, which I will not highlight here, the functional distinction is the source of the antigens and the type of T cell that binds to the specific MHC (see below).
III. Antigen Processing
As indicated above, T cells only recognize antigen when presented by MHC, and they only recognize peptide fragments and not the native protein structure. Therefore, the protein antigens must be digested, processed, and bound by MHC to be presented to T cells. MHC I will present intracellular antigens and MHC II will present extracellular antigens. In both cases, the antigens are processed and bound by the MHC and the peptide antigen-MHC complex is displayed on the surface of the cell to allow for T cell surveillance. All nucleated cells have the ability to process intracellular proteins (antigens) and present the peptides through MHC I on the surface of the cell. A defined population of cells, called antigen presenting cells (APCs) have the ability of importing extracellular antigens, processing them into peptides and presenting them via MHC II molecules on their cell surface.
In order for the MHC I or II to present the peptides, the native protein antigens must be digested and processed such that they can be bound by the MHC and transported to the cell surface for presentation (Figure 2). Normal turnover and degradation of intracellular proteins is predominantly driven by the proteosome complex. The proteosome generates peptide fragments, which are transported into the endoplasmic reticulum by the Transporter associated with Antigen Processing (TAP). MHC I is synthesized as a transmembrane complex into the endoplasmic reticulum (ER) membrane with the peptide binding cleft on the luminal side (inside) where it can interact with the transported peptides in its binding cleft. The MHC I peptide binding cleft has a restricted or closed cleft that restricts the peptide size to be 8-9 amino acids in length. The MHC I with the bound peptide can then be transported to the cell surface by vesicle transport, which results in the peptide displayed on the outside of the cell.
MHC II presents protein antigens that are brought into the cell from the extracellular environment. Macrophages and dendritic cells phagocytose proteins from the extracellular space and engulf these proteins into endosomes inside the cell. B cells interact with specific extracellular proteins by BCR recognition. Once the protein (antigen) is bound the BCR and antigen will be endocytosed and engulfed into an endosome. All of the APCs are capturing proteins (specifically or generally) and encapsulating them inside the cell in the endosome. The proteins are degraded into peptides in the endosome. The MHC II is synthesized into the ER membrane (similarly to the MHC I) but its peptide binding cleft is blocked by the Invariant chain to prevent MHC I peptides from binding. The MHC II is transported to the endosome by vesicle transport, where the Invariant chain is removed by CLIP and peptides from the endosome are loaded into the MHC II binding cleft by HLA-DM. The MHC II binding cleft is more open than the MHC I cleft and can handle peptides that are 14-20 amino acids in length. The MHC II (with bound peptide) is then transported to the plasma membrane and the peptide is displayed on the outside of the APC.
Individuals inherit multiple MHC I and MHC II genes from each parent and all are expressed. Each individual MHC molecule has preference for different populations of peptides and therefore the composition of peptides displayed by the MHC are defined by the combination of MHC genes that are inherited maternally and paternally.
IV. Antigen Presentation and Effector Function
TCRs activation requires the recognition of both the MHC directly and through associated proteins (CD4 or CD8) as well as the peptide. Similarly to B cell development, TCRs thatrecognize “self” antigens during development are removed. The cellular proteins presented by MHC I should not be recognized by T cells (specifically the TCR). However, proteins from a virus or bacteria inside the cell will be processed and presented similarly to the normal cellular proteins. These peptides were not tolerized during development and therefore TCRs will be able to recognize these peptides as foreign and identify cells that are invaded by potentially harmful pathogens. MHC I peptides are recognized by CD8 T cells and the activation of the T cell receptor will result in the CD8 T cell killing the target cell (Figure 3).
As APCs capture proteins in the extracellular environment, recognition of foreign peptides in MHC II by T cells does not result in cytotoxic killing of the target cell, because the pathogen would not be present in the APC. CD4 T cells will interact specifically with MHC II, which will result in the stimulation of the T cell to produce cytokines and activate effector cells (macrophages, neutrophils, etc) to increase phagocytic activities and other regulatory processes to capture, kill, and remove extracellular pathogens from the area (Figure 3). In addition, B cells, as APCs will be activated to produce antibodies to assist in the specific recognition of the pathogen for efficient capture and destruction.
The type of effector function generated will be driven by how the antigen (peptide) is presented and the context of the TCR activation (CD4 vs CD8). Co-stimulatory molecules are also critical to defining the type of response and to insure the antigen is presented appropriately. If co-stimulation does not occur (or inhibitory receptors are activated) then the T cell is suppressed as mechanism to insure that T cells get activated only when danger is present. CD4 T cell activation can result in several types of cellular responses and this will also result in driving isotype switching and drive specific types of antibody responses (IgG1, IgG2, IgA, IgE, etc).
V. Concluding Remarks
T cells are incapable of directly interacting with proteins and therefore must have them presented in order to respond. Most MHC molecules are presenting peptides that will not be recognized and stimulate TCRs, but allow for the immune surveillance to determine when a threat is present. The MHC gift wraps the peptide for the TCR to identify those that are foreign and activate an appropriate response.
In the context of hybridoma development, we must consider the activation of T cells to support appropriate antibody responses. Therefore antigen presentation to both T cells and B cells must be considered. If protein antigens are highly homologous to native mouse proteins, the likelihood of developing a robust immune response resulting in antibody production is reduced. However, this may be overcome by conjugating a homologous target to a foreign carrier protein to assist in driving T cell responses. Also, small molecules and peptides are typically considered haptens, meaning they can be recognized by antibodies but not drive T cell responses. This will not yield good B cell activation, isotype switching, or affinity maturation (all require T cells), which means hybridoma development and monoclonal antibody production is nearly impossible. Again, if these are appropriately conjugated to carrier proteins, then T cell antigens/epitopes will be provided by the carrier and support B cell activation and antibody production. Diversity can also be impacted by T cell responses and as different strains of mice have different MHC haplotypes they will respond to the immunogen differently, therefore consideration of mouse strains, immunization strategy, and antigen design can improve the likelihood of generating a monoclonal antibody with the desired characteristics.