Learning goals for “Acquired in Rheumatologic Disease”

(Something for everyone)

Following this presentation Fellows should be able to:

1. Describe the molecular basis for Macfarlane Burnet’s “ theory” and how this explains immunologic memory. 2. Discuss HLA association of disease and why diseases with seropositive inflammatory arthritis are associated with MHC II while those with seronegative inflammatory arthritis are associated with MHC I. 3. Discuss problems with central tolerance including why patients with rheumatoid arthritis and the HLA-DR alleles containing the “shared ” develop against citrullinated peptides. 4. Describe why against associated with DNA or RNA are so common in rheumatic diseases. 5. Describe the major CD4 effector populations and why TH2 cells might be protective in erosive arthritis. 6. Discuss the multiple effectors of and which immunoglobulin isotypes are most likely to be associated with neonatal lupus. 7. Discuss differentiation and how tissue resident and/or bystander T cells might play a role in tissue . 8. Discuss the challenge of long lived plasma cells.

Answers (and additional information)

1. Describe the molecular basis for Macfarlane Burnet’s “clonal selection theory” and how this explains immunologic memory.

• The adaptive learns from prior exposure to an infectious agent, protecting the organism for subsequent exposure to the same infectious agent. B cells and T lymphocytes (aka B cells and T cells) are the lymphocytes that form the . Antigens are molecules recognized by or T cells. B cells recognize native . • Precursors of B cells and T cells undergo gene rearrangements to form the variable regions of the B cell antigen receptor and the variable region of the antigen receptor. Thus, these antigen receptors are not present in the germline and the number of different antigen receptors can be on the order of 1010. • The progeny of T cells and B cells that have undergone rearrangement form clones with unique antigen receptors. The interaction between these receptors and antigen triggers activation, proliferation, and induction of a host of cellular changes that result in a robust protective response with subsequent exposure, i.e. immunologic memory.

Additional notes:

• B cell antigen receptor – surface immunoglobulin consists of dimers of heavy and light chain with variable regions binding antigen and constant regions; surface immunoglobulin contain a transmembrane domain that interacts non- covalently with other membrane proteins to form the B cell antigen receptor complex; crosslinking surface immunoglobulin results in juxtaposition of immune tyrosine activating motifs (ITAMs) and recruitment and activation of tyrosine kinases; alternative mRNA translation results in secreted immunoglobulin that lacks transmembrane domain, the Fc region of secreted immunoglobulin can bind Fc receptors and activate complement • T cell antigen receptor – alpha and beta chains or gamma and delta chains containing variable regions and constant regions; non-covalent interaction with membrane molecules (CD3 molecules) to form the T cell antigen receptor complex; crosslinking surface immunoglobulin results in juxtaposition of immune tyrosine activating motifs (ITAMs) and recruitment and activation of tyrosine kinases • Rearrangement of the VDJ gene of heavy chains and the VJ genes of the light chains generates the variable region of surface immunoglobulin. These gene rearrangements are imprecise with nucleotides added or subtracted at each step. Thus, between the pairing of heavy and light chains and the recombination of heavy and light chain variable region genes, the number of unique surface immunoglobulins in on the order of 1010. The alpha and beta or gamma and delta chains of the T cell antigen receptors are similarly generated by gene rearrangement. Rearrangement of the variable regions of immunoglobulins occurs in the ; rearrangement of the T cell receptor variable regions occurs in the .

2. Discuss HLA association of disease and why diseases with seropositive inflammatory arthritis are associated with MHC II while those with seronegative inflammatory arthritis are associated with MHC I.

• CD4 T cells recognized peptides bound to MHC II and CD8 T cells recognize peptides presented by MHC I. Antigens that are phagocytosed or endocytosed are bound by MHC II. B cells are an important antigen presenting cells that are able to endocytose antigens recognized by their specific cell surface immunoglobulin and then present these antigens in the context of MHCII to CD4 T cells which then provide help for B cell activation. Thus, antigens that induce autoantibodies are going to recruit CD4 T cell help and diseases with seropositive inflammatory arthritis are most likely to be associated with the MHC II alleles that present these antigens. Antigens that form in the cytoplasm of cells are digested by the proteasome are transferred into the endoplasmic reticulum and bound by MHC I. Antigens recognized and endocytosed by surface immunoglobulin are not presented by MHC I.

Additional notes:

• Bacteria can be phagocytosed by or dendritic cells. Phagocytosis of particulate antigens such as bacteria can be presented by MHC II but with phagocytosis antigen can be transferred from the phagocytic vesicle to the cytoplasm. Thus, antigens from phagocytosed bacterial can be presented via MHC I. Some microbes on mucosal surfaces can be invaded epithelial cells, cells that generally express MHC I but not MHC II. Some of these invasive microbes escape from phagocytic vesicles and are released into the cytoplasm of macrophages. Thus, control of infections in epithelial cells or in macrophages where the organism enters into the cytoplasm often requires lysis of cells by CD8 T cells specific for MHC I + microbial peptide.

3. Discuss problems with central tolerance including why patients with rheumatoid arthritis and the HLA-DR alleles containing the “shared epitope” develop antibodies against citrullinated peptides.

• For B cells central tolerance involves deletion of cells recognizing self-antigens in bone marrow. For T cells central tolerance involves deletion of cell recognizing self-antigen with a high affinity. (T cells have to be selected to have some affinity for MHC molecules. Otherwise they would not be able to recognized self-MHC + peptide.) Thus, T cells and B cells specific for self-antigens that are not expressed in the thymus or bone marrow escape central tolerance. Some of these self-antigens are in privileged sites such as the eye, testes, ovaries, or brain. Release of these antigens can trigger an autoimmune response. In addition, inflammation and other processes can modify antigens or change how antigens are processed into peptides by antigen presenting cells. If these modified antigens or alternatively processed peptides induce immune responses they are called neoantigens. In addition to self-antigens and neoantigens, there are a large number of non-self-antigens (food, pollen, animal dander, commensal organisms, etc.) that are innocuous and/or beneficial; the immune response to these antigens must be carefully controlled to prevent reactions. Although T cells coming out of the thymus cannot further modify their antigen receptors, B cells can. The immunoglobulin genes in antigen-specific, germinal center B cells can develop point- mutations. Selection of the B cells based on how well these mutations bind antigen leads to the development of high affinity antibodies (). However, this process means that some of these mutated immunoglobulin genes in germinal centers will be self-reactive. • The HLA-DR molecules with the shared epitope have positively charged amino acids in the peptide binding grove. Thus, self-peptides with arginine which is also positively charged may be excluded from the binding grove. Peptide arginine deaminase (PAD) modified arginine to citrulline, a non-charged amino acid allowing these citrullinated peptides to bind to HLA-DR with the shared epitope and creating a neoantigen for T cells. PAD4 is induced by inflammation. In this inflammatory environment antigen presenting cells taking up these citrullinated peptides will also be activated to express co-stimulatory molecules and pro-inflammatory leading to activation of naïve citrullinated-peptide specific T cells that can then help activated B cells making anti-citrullinated-peptide autoantibodies.

4. Describe why autoantibodies against antigens associated with DNA or RNA are so common in rheumatic diseases.

• B cells antigens include proteins, carbohydrates, lipids, nucleic acids, and a whole variety of chemicals. In contrast T cells recognize only peptides that are generated in antigen presenting cells and bound to MHC. Although B cells and T cells often recognize the same antigen they do not necessarily recognized the same part of an antigen. Thus, B cells may recognize either the protein or the nucleic acid part of the complex while T cells will only recognize the protein. • Many self-proteins bind tightly to nucleic acids, e.g. histones-DNA, Sm-RNA. Surface immunoglobulin with affinity for these complexes can induce endocytosis into B cells. Under the right circumstances the toll-like receptors within endocytic vesicles can bind and be activated by the nucleic acid portion of these complexes (TLR9 for DNA complexes and TLR7 or 8 for RNA complexes). Stimulation of these TLR serves as a second signal for B cell activation. Moreover, since B cells do not process antigen exactly like myeloid cells there is the opportunity for generation of T cell neoantigens. The net effect is activation of both autoantigen specific B cells and T cells with induction of high affinity, class-switched autoantibodies. • Immune complexes formed by autoantibodies against antigens associated with DNA and RNA and their antigens are extremely effective at inducing the production of α interferon by plasamcytoid dendritic cells and other cells leading to a pro-inflammatory/pro- feedback loop.

Additional notes:

• Many of the proteins that bind DNA and RNA have homology in the microbiome. Therefore, microbial infections have an opportunity to induce antibodies that cross react with autoantigens. • The autoantigens associated with DNA and RNA are generally intracellular. When cells die by , apoptotic bodies are very rapidly endocytosed and very little extracellular “leak” of these self-antigens. When there is a defect in apoptosis or when there is necrosis, the level of these autoantigens in extracellular space can spike, markedly increasing the opportunity to induce an autoimmune response.

5. Describe the major CD4 effector populations and why TH2 cells might be protective in erosive arthritis.

• TH-1, induced by IL-12, secretes IFNƳ, in host defenses protects from intracellular organisms such and TB and viruses. • TH-2, induced by IL-4, secretes IL-4/IL-13/IL-5, in host defenses important for helminth infections • TH-17, induced by IL-23/IL-6/IL-1β/TGFβ, secretes IL-17/IL-22, in host defenses important for mucosal and dermal infection with extracellular organisms • Tfh, induced by IL-6 and IL-21, secretes IL-21/IL-4 and express CD40L and ICOS, in host defenses important for production • iTreg, induced by TGBβ and IL-2, secretes TGFβ/IL-10, important for tolerance and production of IgA • TH17 cells promote erosions by activating synovial fibroblast like cells to secrete RANKL and activate osteoclasts. TH2 cells down regulate the production and activation of TH17 cells. (TH1 cell production of IFNƳ can also block osteoclast activation.)

Note, proteins in bold can be targeted by currently approved biologics.

6. Discuss the multiple effectors of humoral immunity and which immunoglobulin isotypes are most likely to be associated with neonatal lupus.

• IgM – complement activation, primarily in the intravascular space, important in cryoglobulinemia and cold agglutinin disease • IgG – complement activation, engagement of IgG FcRs, intravascular and extravascular space, important for most autoimmune diseases • IgA – poor at activating complement and engaging FcRs, monomeric intravascular and extravascular space, dimeric transported across epithelium via the polymeric immunoglobulin receptor, important for Henoch-Schonelein and IgA nephropathy • IgE – triggers mast cells and via high affinity IgE receptor • IgG1 and IgG3 are the most important isotypes for neonatal lupus. Placental transfer IgG1 and IgG3 >> IgG2 > IgG4. IgM, IgA and IgE do not cross the placenta. IgG1 and IgG3 are also particularly effective for complement activation and for activation of myeloid cells via IgG FcRs.

7. Discuss lymphocyte differentiation and how bystander T cells might play a role in tissue inflammation.

• Naïve T and B lymphocytes recirculate between secondary lymphoid tissue and the blood. CD61L and CCR7 are important for entry into lymph nodes via high endothelial venules. The marker for naïve T cells is CD45RA (naïve). • Upon activation T cells become effector T cells and memory T cells. The marker for memory T cells is CD45RO (memory). Central memory T cells continue to recirculate between the secondary lymph nodes and blood and continue to express CD61L and CCR7. Activated T cells can also become effector memory T cells that do not express CD61L or CCR7. These effector memory T cells recirculate first to peripheral tissues, then migrate to lymph nodes via efferent lymphatics and eventually back to the blood. • A fraction of effector memory T cells reside in tissue for long periods of time as tissue resident memory T cells. These T cells remain in a state of readiness and can be rapidly activated by antigen, cytokines, or PAMPs/DAMPs. Recurrence of rashes in specific locations, arthritis in specific joints, and enthesitis may be explained, in part, by tissue resident T cells • Different effector T cells subsets express different receptors. Thus TH2 and TH17 cell activating in cutaneous lymph nodes can selectively be recruited to the rash of atopic dermatitis or psoriatic arthritis. • If effector memory T cells are strongly activated by exposure to antigen the can become terminally differentiated effector memory T cells that re-express the markers for naïve T cells. These cells are described as terminal effectors (TE) and express CD45RA. After activation these cells die rather than deactivate to become memory T cells. • Memory T cells that have been activated in lymph nodes draining skin express CLA (cutaneous lymphocyte antigen), a homing receptor for migration to skin. Memory T cells that have been activated in lymph nodes draining the GI tract express the α4β7 integrin, a homing receptor for the small bowel and colon. • In target tissue antigen specific T cells represent a small fraction of the total number of T cells. Nevertheless, they are preferentially activated early in the immune response and can be considered the pioneers. The rest of the T cells can be described as bystanders. They are recruited into the site but are not able to find their cognate antigen. These bystander T cells are predominantly effector memory T cells. Although they are not activated by their cognate antigen they can be activated by cytokines and PAMPs/DAMPs and thus contribute to host defenses and/or tissue inflammation and destruction.

8. Discuss the challenge of long lived plasma cells.

• Some naïve and memory B cells activated in secondary lymphoid tissue do not go to germinal centers to potentially become memory B cells or long lived plasma cells. Instead they rapidly differentiate into plasma cells in the extrafollicular space providing a rapid response to infectious agents but also in some cases autoimmune plasma cells that are short lived. • A subset of B cells activated in germinal centers have the potential to become long lived plasma cells. Some of the precursors for long lived plasma cells home from secondary lymphoid tissues to the bone marrow where they occupy a special niche that allows them to survive in some case for the lifetime of the individual. Other precursors home to inflamed tissues like the kidney. These plasma cells will continue to survive at those sites until the inflammation is brought under control and their niche disappears • If pathogenic autoantibodies are produced by long lived plasma cells in the bone marrow, therapies directed at inflammation or dividing cells will not be effective. New strategies will have to be developed that specifically target these cells. Most of the therapies are being developed to treat myeloma or macroglobulinemia. Approved agents include proteasome inhibitors, anti-CD38, anti-SLAMF7. CAR T cells targeting BCMA are in development.