THE BASICS OF IMMUNE RESPONSE AGAINST ALLOANTIGENS

Transplantation of organs or tissues (allografts) is a life-saving medical procedure, but it is fundamentally challenged by the recipient’s immune system recognizing the allograft as foreign. This immune response is a complex interplay of cellular and humoral mechanisms, orchestrated primarily against molecules known as alloantigens. Understanding these components is crucial for managing transplant rejection and improving long-term graft survival.

Here is a detailed breakdown of key elements in the immune response to allografts:

Definition of Alloantigen

An alloantigen is an antigen that is present in some members of a species but not in others. In the context of transplantation, alloantigens are molecules expressed by the donor graft that are recognized as foreign by the recipient’s immune system, triggering an immune response aimed at rejecting the graft. These antigens are primarily responsible for initiating transplant rejection.

The most significant alloantigens are the major histocompatibility complex (MHC) molecules. In humans, MHC molecules are known as human leukocyte antigens (HLA). These cell surface proteins are highly polymorphic, meaning they vary significantly between individuals. HLA molecules are classified into two main types:

• MHC Class I (HLA-A, -B, -C): Expressed on the surface of almost all nucleated cells. They typically present peptides derived from proteins synthesized within the cell (e.g., viral proteins).
• MHC Class II (HLA-DR, -DQ, -DP): Primarily expressed on the surface of antigen-presenting cells (APCs) such as dendritic cells, macrophages, and B cells. They typically present peptides derived from extracellular proteins taken up by the cell.

Differences in HLA alleles between the donor and recipient are the primary drivers of the alloimmune response. The recipient’s T lymphocytes, specifically T helper cells (CD4+) and cytotoxic T lymphocytes (CD8+), recognize these foreign HLA molecules, either directly (recognizing intact donor MHC) or indirectly (recognizing donor MHC peptides presented by recipient MHC), initiating the rejection process.

While MHC/HLA molecules are the strongest alloantigens, other non-MHC antigens, known as minor histocompatibility antigens (mHAs), can also contribute to rejection, particularly in HLA-matched transplants. These are polymorphic proteins that differ between individuals, and peptides derived from these proteins can be presented by MHC molecules.

The presence and degree of disparity in these alloantigens between donor and recipient are critical determinants of the strength and speed of the immune response, directly impacting whether the allograft is accepted, acutely rejected, or chronically rejected.

Relative Importance of CD4+, CD8+, and Delayed-Type Hypersensitivity (DTH) T Cells in Response to Allografts

T cells are central players in cellular immunity and are critically involved in allograft rejection. Different subsets of T cells contribute distinct functions to this complex process.

• CD4+ T Helper Cells: CD4+ T cells are often considered the “master regulators” of the adaptive immune response to allografts. They are crucial for orchestrating both cellular and humoral immunity. CD4+ T cells recognize alloantigens primarily presented by MHC Class II molecules. This recognition can occur via two main pathways:
  • Direct Pathway: Recipient CD4+ T cells recognize intact donor MHC Class II molecules expressed on donor APCs within the graft. This pathway is most important in acute rejection.
  • Indirect Pathway: Recipient CD4+ T cells recognize processed peptides derived from donor alloantigens (e.g., donor MHC molecules) that are presented by recipient APCs on recipient MHC Class II molecules. This pathway is thought to be more important in chronic rejection and for activating B cells to produce alloantibodies.

  Upon activation, CD4+ T cells differentiate into various effector subsets, each with distinct roles:

  • Th1 cells: Produce pro-inflammatory cytokines like interferon-gamma (IFN-γ) and TNF-alpha, promoting cellular immunity, activating macrophages, and contributing to inflammation. Th1 responses are strongly associated with cellular rejection.
  • Th2 cells: Produce cytokines like IL-4, IL-5, IL-6, and IL-13, primarily supporting humoral immunity and B cell activation, which is important for antibody-mediated rejection.
  • Th17 cells: Produce IL-17 and other cytokines, contributing to inflammation and neutrophil recruitment, and have been implicated in both acute and chronic rejection.
  • Regulatory T cells (Tregs): A subset of CD4+ T cells (often FOXP3+) that suppress immune responses. Naturally occurring Tregs play a role in maintaining self-tolerance, and induced Tregs can potentially promote tolerance to the allograft. Their function is critical in modulating the intensity of the alloimmune response.

  CD4+ T cells are essential for providing “help” to CD8+ T cells and B cells. They secrete cytokines that are necessary for the activation, proliferation, and differentiation of cytotoxic T lymphocytes (CTLs) and for efficient antibody production by B cells. Without adequate CD4+ T cell help, optimal CD8+ T cell and B cell responses against the graft are significantly impaired.

• CD8+ Cytotoxic T Lymphocytes (CTLs): CD8+ T cells are the primary effector cells responsible for directly killing graft cells during cellular rejection. They recognize alloantigens, primarily presented by MHC Class I molecules, predominantly via the direct pathway, where they recognize intact donor MHC Class I on the surface of graft cells.Upon activation by donor APCs (often with concurrent help from CD4+ T cells), CD8+ T cells proliferate and differentiate into effector CTLs. These CTLs migrate to the graft and directly engage target cells (graft cells expressing foreign MHC Class I). CTLs induce apoptosis (programmed cell death) in target cells through several mechanisms, including the release of cytotoxic molecules like perforin and granzymes (discussed in section 3) and engagement of death receptors (e.g., FasL binding to Fas).CD8+ CTLs are the principal mediators of acute cellular rejection, causing widespread damage to the graft endothelium and parenchyma by lysing donor cells.
• Delayed-Type Hypersensitivity (DTH) T Cells: Delayed-type hypersensitivity (DTH) is a subset of cell-mediated immune response characterized by tissue inflammation that peaks 24-72 hours after antigen exposure. In the context of transplantation, DTH is largely mediated by activated CD4+ Th1 cells and macrophages.Recipient CD4+ T cells, particularly Th1 cells, recognize alloantigens (typically via the indirect pathway) within the graft and secrete cytokines like IFN-γ. IFN-γ activates macrophages, which then migrate to the graft site and release pro-inflammatory mediators (cytokines, chemokines, proteases, reactive oxygen species). These inflammatory mediators contribute to tissue damage, vascular injury, and fibrosis within the allograft.While CD8+ CTLs are crucial for direct cell killing in acute rejection, DTH mediated by CD4+ T cells and macrophages contributes significantly to the inflammatory milieu and tissue pathology observed in both acute and chronic rejection. The recruitment and activation of macrophages play a substantial role in the chronic changes that lead to graft dysfunction over time.

Relative Importance Summary: All three T cell types are important, often acting synergistically.

• CD4+ T cells are arguably the most centrally important as they are required for optimal activation of both CD8+ T cells and B cells (for antibody production) and orchestrate the inflammatory DTH response. They are critical for both acute and chronic rejection pathways.
• CD8+ T cells are the primary executioners in acute cellular rejection, directly killing graft cells.
• DTH, mediated by CD4+ T cells and macrophages, contributes significantly to inflammation, tissue damage, and chronic changes in the graft.

Effective immunosuppressive therapy typically targets the activation, proliferation, and function of both CD4+ and CD8+ T cells.

Role and Source of Perforin and Granzyme

Perforin and granzymes are critical cytotoxic molecules used by certain immune effector cells to induce apoptosis (programmed cell death) in target cells.

• • Source: The primary source of perforin and granzymes are:
    • Cytotoxic T Lymphocytes (CTLs): Activated CD8+ T cells are a major source, using these molecules to kill target cells expressing foreign antigens (like those in allografts).
    • Natural Killer (NK) Cells: NK cells are innate lymphoid cells that can recognize and kill abnormal cells (e.g., infected cells, tumor cells, or potentially allogeneic cells) without prior sensitization. They also utilize perforin and granzymes for this cytotoxic activity.
    • NKT Cells and Gamma Delta T Cells: Other less common lymphocyte subsets also employ this cytotoxic mechanism.
  • Role and Mechanism: Perforin and granzymes are stored in lytic granules within the cytoplasm of effector cells (CTLs and NK cells). When the effector cell encounters and engages a target cell (e.g., a graft cell expressing foreign MHC Class I for CTLs, or a stressed/antibody-coated graft cell for NK cells), the lytic granules migrate to the point of contact (immunological synapse) and release their contents into the narrow space between the two cells.The mechanism proceeds as follows:
    1. Perforin: Perforin monomers are released and polymerize in the presence of calcium ions to form channels or pores in the membrane of the target cell.
    2. Granzymes: Granzymes, which are serine proteases (most notably Granzyme B), enter the target cell cytoplasm through the perforin pores.
    3. Activation of Apoptosis Pathways: Once inside the target cell, granzymes (especially Granzyme B) initiate a cascade of intracellular events leading to apoptosis. The primary mechanism involves cleaving and activating caspase enzymes, which are the executioners of apoptosis. Granzyme B can directly activate key caspases (e.g., caspase-3) or cleave Bid, a pro-apoptotic protein, which then triggers the mitochondrial pathway of apoptosis. Granzymes can also degrade inhibitory proteins or activate DNases to fragment the target cell’s DNA.

    This perforin-granzyme pathway is a highly efficient mechanism for effector cells to induce programmed cell death in target cells while often minimizing damage to surrounding tissues, unlike lytic mechanisms that cause membrane rupture. In the context of allograft rejection, this pathway is a major way CD8+ CTLs and NK cells destroy donor cells.

The Role of Donor Specific Antibody (DSA) in Relation to Allograft Survival

Donor specific antibodies (DSAs) are antibodies produced by the recipient’s immune system that are directed against alloantigens on the donor graft. These antibodies, particularly those targeting donor HLA molecules, play a critical role in various forms of allograft rejection and significantly impact long-term graft survival.

The role of DSAs in allograft survival can be broadly categorized:

• Hyperacute Rejection: This is a rare but devastating form of rejection that occurs minutes to hours after reperfusion of the graft. It is mediated by pre-existing DSAs in the recipient’s circulation (often resulting from prior blood transfusions, pregnancies, or previous transplants). These antibodies bind to antigens (primarily HLA Class I and ABO blood group antigens) on the donor graft endothelium, activating the complement system and leading to rapid microvascular thrombosis, ischemia, and graft necrosis. Effective crossmatching procedures (testing recipient serum against donor lymphocytes) are used to detect pre-existing DSAs and prevent hyperacute rejection.
• Acute Antibody-Mediated Rejection (AMR), also known as Acute Humoral Rejection: This occurs days, weeks, months, or even years after transplantation and is mediated by newly formed DSAs. The recipient’s immune system, particularly B cells (often with help from CD4+ T cells), are sensitized to donor alloantigens and produce DSAs. These antibodies bind to the graft endothelium and parenchyma, leading to injury through several mechanisms:
  • Complement Activation: DSA binding can activate the classical complement pathway, leading to the formation of membrane attack complexes (MAC) that damage cell membranes, recruit inflammatory cells, and cause vascular injury (capillaritis, glomerulitis).
  • Antibody-Dependent Cell-Mediated Cytotoxicity (ADCC): DSAs bound to graft cells (especially endothelium) can be recognized by Fc receptors on effector cells like NK cells, macrophages, and neutrophils, triggering these cells to release cytotoxic molecules and kill the target cells (discussed in section 5).
  • Non-Complement Mediated Injury: DSAs can also cause injury independent of complement, for instance, by directly activating endothelial cells, leading to pro-thrombotic or pro-inflammatory states, or by inducing proliferation and thickening of the vessel wall.
• Chronic Antibody-Mediated Rejection (Chronic AMR): DSAs are strongly implicated in chronic rejection, a slower process leading to progressive graft dysfunction over months to years. While the exact mechanisms are debated and likely multifactorial, DSAs contribute to chronic vascular changes (transplant vasculopathy), glomerular injury (transplant glomerulopathy), and interstitial fibrosis/tubular atrophy. Ongoing DSA binding and complement activation can lead to repetitive injury, inflammation, and remodeling of the graft tissue.
• Impact on Graft Survival: The presence of DSAs, particularly those against HLA Class I and Class II, is a significant risk factor for both acute and chronic rejection and is strongly associated with poorer long-term allograft survival across various organ types. Monitoring for the development of de novo DSAs after transplantation is now a standard practice to identify patients at high risk for rejection and intervene with appropriate therapy.

In summary, DSAs are powerful mediators of graft injury and are detrimental to allograft survival, playing a causative role in hyperacute, acute, and chronic rejection pathways.

Description of Antibody-Dependent Cell-Mediated Cytotoxicity (ADCC)

Antibody-dependent cell-mediated cytotoxicity (ADCC) is a crucial immune mechanism employed by certain effector cells to kill target cells that have been coated with antibodies. It is a key pathway whereby the humoral immune response (antibodies) can direct cellular effectors (like NK cells) to eliminate specific target cells.

The process of ADCC involves the following steps:

1. Antibody Binding: Antibodies, typically IgG antibodies, bind to specific antigens present on the surface of a target cell. In the context of allograft rejection, these would be DSAs (e.g., anti-HLA antibodies) binding to HLA molecules expressed on donor graft cells, particularly the endothelium or parenchymal cells.
2. Effector Cell Engagement: Effector cells, which include Natural Killer (NK) cells, macrophages, neutrophils, and eosinophils, express receptors on their surface that specifically bind to the Fc region (the constant tail) of the antibody molecule. The most important of these is the Fc-gamma receptor IIIa (FcγRIIIA), also known as CD16, which is highly expressed on NK cells.
3. Fc Receptor Ligation and Activation: When multiple antibody molecules are bound to the target cell surface, their Fc regions are available for binding to Fc receptors on the approaching effector cell. The cross-linking or ligation of multiple Fc receptors on the effector cell surface triggers intracellular signaling pathways.
4. Effector Cell Activation and Cytotoxicity: Signaling through Fc receptors activates the effector cell, leading to the release of cytotoxic molecules and/or apoptotic signals.
   • NK Cells: Activated NK cells release the contents of their lytic granules, containing perforin and granzymes, into the space between the NK cell and the target cell (similar to the mechanism used by CTLs, described in section 3). Perforin forms pores in the target cell membrane, allowing granzymes to enter and induce apoptosis. NK cells are considered the primary mediators of ADCC due to their high expression of FcγRIIIA (CD16) and efficient cytotoxic machinery.
   • Macrophages and Neutrophils: These phagocytic cells can also mediate ADCC, though perhaps less efficiently than NK cells in some contexts. They may kill target cells by releasing cytotoxic mediators (like reactive oxygen species, nitric oxide, or TNF-alpha) or, in addition to phagocytosis, potentially via perforin/granzyme.
5. Target Cell Death: The release of cytotoxic molecules or activation of death pathways leads to the killing of the antibody-coated target cell, typically through apoptosis or sometimes lysis.

In the context of allograft rejection, ADCC mediated by NK cells is a significant mechanism by which DSAs contribute to graft injury, particularly endothelial damage in antibody-mediated rejection. DSAs bind to endothelial cells, making them targets for destruction by recipient NK cells. This contributes to vascular inflammation, microvascular injury, and the eventual development of transplant vasculopathy and graft loss. Understanding ADCC highlights the intertwined nature of humoral (antibody) and cellular (NK cell, macrophage) immunity in mediating allograft rejection.

This overview provides a foundation for understanding the key cellular and humoral immune components involved in the response to allografts, highlighting the central roles of alloantigens, different T cell subsets, cytotoxic molecules like perforin and granzyme, donor-specific antibodies, and the ADCC mechanism in determining transplant success or failure.