Signals for T cell activation Costimulation and the B7:CD28 family Lecture outline Signals for T cell activation Costimulation and the B7:CD28 family Inhibitory receptors of T cells Responses of T cells Cytokines

The life history of T lymphocytes Abbas, Lichtman and Pillai. Cellular and Molecular Immunology, 7th edition, 2011 c Elsevier

Principles of lymphocyte activation Lymphocytes are normally in a resting state in lymphoid organs and circulation Rapid response to antigen (activation) --> proliferation, change to functionally active effector cells (differentiation) Migration to tissues, where they perform their function of eliminating infections Multiple possible steps for therapeutic targeting

Steps in the activation of T lymphocytes

Recognition of antigen by the TCR The TCR of CD4+ and CD8+ T cells recognizes MHC-bound peptide + portions of the MHC. Other T cells (gd T cells, NKT cells) recognize non-peptide antigens; these are small cell populations whose function is unclear.

Antigen recognition by T cells Each T cell sees a (self) MHC molecule and a bound peptide Dual recognition determines specificity and MHC restriction Multiple ligands on APCs and receptors on T cells, in addition to the TCR, participate in orchestrating responses to antigens Signaling: engagement of receptors --> activation of kinases (often via “adaptor proteins”) --> activation of transcription factors

Receptor-ligand pairs involved in T cell activation Different molecules involved in T cell responses to antigen serve distinct functions, seen even in this partial listing. Drugs that block these ligand-receptor pairs have been developed to treat immune-mediated inflammatory diseases, graft rejection.

Molecules involved in T cell activation Signal transduction CD4 and CD8 “co-receptors” recognize MHC molecules (class II or class I) at the same time as the TCR sees the peptide-MHC; CD4 and CD8 provide necessary activating signals for T cells CD28 is a receptor for “costimulators” expressed on APCs

Molecules involved in T cell activation Signal transduction CD4 and CD8 co-receptors recognize MHC molecules (class II or class I) at the same time as the TCR sees the peptide-MHC; CD4 and CD8 provide necessary activating signals for T cells CD28 is a receptor for “costimulators” expressed on APCs Strengthen adhesion with antigen-presenting cells Integrins function as adhesion molecules Affinity of integrins is increased by chemokines produced during inflammation, and by antigen recognition by TCRs Control routes of T cell migration Selectins and integrins control migration of naïve T cells through lymph nodes and of effector and memory T cells to sites of infection Therapeutic targets

Therapeutic targeting of molecules involved in T cell responses CD3: signaling molecule attached to the TCR on all T cells; anti-CD3 MAb to deplete T cells (transplants) Integrins (LFA-1, VLA-4, others): adhesion to APCs, endothelium; anti-integrin MAb’s to block leukocyte migration into tissues “Costimulators”: CD28, others; costimulatory blockade

The two-signal requirement for lymphocyte activation Costimulation: signal(s) in addition to antigen that are needed to initiate adaptive immune responses Best defined for CD4+ T cells Multiple pairs of ligands on APCs and receptors on T cells may serve this function; best defined are the B7-CD28 families of proteins Abbas, Lichtman and Pillai. Cellular and Molecular Immunology, 7th edition, 2011 c Elsevier

Role of costimulation in T cell activation

Two signal requirement for T cell activation Naïve lymphocytes need two signals to initiate their responses Signal 1: antigen recognition Ensures that the response is antigen-specific Signal 2: costimulators induced on APCs during infection (or upon recognition of necrotic cells) Ensures that the immune system responds best to microbes (or to necrotic cells, as in tumors) and not to harmless antigens Adjuvants stimulate expression of costimulators

The B7: CD28 family Different members of the B7-CD28 families serve different roles in stimulating and suppressing immune responses.

Major functions of selected B7-CD28 family members B7-CD28: initiation of immune responses ICOS-ICOS-L: T cell help in germinal center reactions (antibody responses) B7-CTLA-4: inhibits early T cell responses in lymphoid organs PD-1:PD-L1,2: inhibits effector T cell responses in peripheral tissues Activation Inhibition

Inhibitory receptors of the immune system One mechanism by which the system maintains a balance between activation and inhibition is to use different receptors for different outcomes Inhibitory receptors are present in NK cells, T cells and B cells; other immune cells? In many instances, activating receptors work by recruiting kinases (often via ITAMs) and inhibitory receptors activate phosphatases (via ITIMs)

The opposing functions of CD28 and CTLA-4 B7 CD28 APC B7-CD28 interaction Naïve T cell TCR Proliferation, differentiation CTLA-4 B7-CTLA-4 interaction Functional inactivation Inhibitory pathways function normally to prevent responses to self antigens: demonstrated by the finding that blocking or eliminating these inhibitors (CTLA-4, PD-1) causes autoimmune disease

Blocking CTLA-4 promotes tumor rejection Tumor recognition by T cells leads to engagement of CTLA-4 on the T cells and inhibition of immune responses. Blocking CTLA-4 increases anti-tumor response and leads to tumor rejection.

Inhibitory role of PD-1 in a chronic infection Virus-specific T cell response Residual virus In a chronic viral infection in mice, recognition of virus by specific T cells leads to PD-1 engagement, inhibition of T cell responses, and persistence of the virus. Blocking the PD-1 pathway releases the inhibition, enhances the T cell response, and leads to viral clearance.

Inhibitory receptors of T cells Prevent reactions against self antigens (their physiologic function) Suppress immune responses to some tumors, chronic infections (HCV, HIV) Similar roles are established for both CTLA-4 and PD-1

The balance between activation and inhibition How does a T cell choose to use CD28 to be activated or CTLA-4 to shut down? Both recognize B7 on APCs CTLA-4 (the inhibitory receptor) binds to B7 with much higher affinity than CD28 (the activating receptor)

The balance between activation and inhibition How does a T cell choose to use CD28 to be activated or CTLA-4 to shut down? Low B7 (e.g. when DC is displaying self antigen) --> engagement of high-affinity CTLA-4 High B7 (e.g. after microbe encounter) --> engagement of lower affinity CD28 Not well understood for the PD-1 pathway

Therapeutics based on the B7:CD28/CTLA-4 family 1. Costimulatory blockade CTLA-4.Ig is used for diseases caused by …?

Therapeutics based on the B7:CD28/CTLA-4 family 1. Costimulatory blockade CTLA-4.Ig is being tested in diseases caused by excessive T cell activation -- rheumatoid arthritis (FDA approved), psoriasis, graft rejection (approved)

Therapeutics based on the B7:CD28/CTLA-4 family 2. Removing the brakes on the immune response Anti-CTLA-4 antibody is used for ….?

Therapeutics based on the B7:CD28/CTLA-4 family 2. Removing the brakes on the immune response Anti-CTLA-4 antibody is approved for tumor immunotherapy (enhancing immune responses against tumors) Even more impressive results with anti-PD-1 in cancer patients

The emerging paradigm for cancer therapy Signaling (e.g. Kinase) inhibitor to block oncogenic pathways in tumor cells + Immune modulator Inhibiting endogenous regulators allows the host to mount an effective immune response (let the immune system do the work itself) Appears to be more effective than vaccination and other approaches for stimulating immunity Anti-PD1 may be more effective than anti-CTLA-4 (less toxic, greater effect on CTLs)

Costimulation Required for initiating T cell responses Many proteins on APCs and their receptors on T cells shown to “costimulate” (function with antigen to activate T cells); most important costimulators are B7:CD28 Ensures that T cells respond to microbes (the inducers of costimulators) and not to harmless antigens Source of costimulation in responses to tumors and transplants: products of dead cells? Therapeutic targets

T cell expansion and contraction (decline) Many aspects of T cell responses and functions are mediated by cytokines: initial activation -- IL-2; maintenance of memory cells -- IL-7; effector functions -- various

Cytokines Secreted proteins that mediate immune and inflammatory reactions, and communications among leukocytes and other cells Produced transiently in response to extrinsic stimuli Bind to high-affinity receptors on target cells Actions are most often autocrine and paracrine, rarely endocrine Cytokines are pleiotropic (one cytokine has multiple actions) and redundant (different cytokines have similar actions)

Role of IL-2 and IL-2 receptors in T cell proliferation Production of IL-2 and expression of high-affinity IL-2 receptors are both dependent on antigen recognition + costimulation

Clonal expansion (proliferation) of T cells Stimulated mainly by autocrine IL-2 T cell stimulation by antigen + costimulators induces secretion of IL-2 and expression of high-affinity IL-2 receptors Therefore, antigen-stimulated T cells are the ones that expand preferentially in any immune response, keeping pace with replicating microbes

Clonal expansion (proliferation) of T cells Stimulated mainly by autocrine IL-2 T cell stimulation by antigen + costimulators induces secretion of IL-2 and expression of high-affinity IL-2 receptors Therefore, antigen-stimulated T cells are the ones that expand preferentially in any immune response, keeping pace with replicating microbes CD8+ T cells may expand >50,000-fold within a week after an acute viral infection with minimal expansion of cells not specific for the virus (up to 10% of all CD8+ T cells in the blood may be specific for the pathogen) Some of the progeny of the expanded clone differentiate into effector and memory cells; the majority die by apoptosis

Naïve T cells differentiate into functional effector cells Effector T cells CD4+ helper T cells Cytokine secretion + antigen Differentiation + antigen APC Naïve T cell: Can recognize antigen but incapable of any functions Cell killing CD8+ CTLs

Memory T cells Long-lived, functionally silent More numerous than naïve cells specific for the antigen; respond more rapidly than do naïve cells -- explains why secondary response is “better” than primary response May consist of multiple subsets Some migrate to lymphoid organs (like naïve T cells), and proliferate and differentiate rapidly in response to antigen challenge (repeat infection) Others migrate to peripheral sites of infection, and rapidly perform effector functions upon encountering the antigen

The life history of T lymphocytes Precursors mature in the thymus Naïve CD4+ and CD8+ T cells enter the circulation Naïve T cells circulate through lymph nodes and find antigens Clonal expansion; differentiation into effector and memory cells Effector T cells migrate to sites of infection Eradication of infection