Transplant Immunopathology

What is the probability that a sibling will be a perfect match?

1:4 or 25%. Perfect match is when recipient and donor are matched for HLA-A*, B*, C*, DRB1*, and DQB1* at the allele level.

Describe differences between acute and chronic GVHD.

Acute: onset typically less than 100 days. Maculopapular skin rash (skin), nausea and/or anorexia (plus positive histology; Upper GI), watery diarrhea +/- severe abdominal pain +/- bloody diarrhea (exclude others; lower GI), cholestatic hyperbilirubinemia (liver) Chronic: Onset typically after 100 days post transplant. Skin: Dypsipigmentation, new onset alopecia, poikiloderma, lichen planus-like eruptions or sclerotic features Nails: Nail dystrophy or loss Mouth: Ulcers, restrictions from sclerosis, xerostomia, lichen-type features Eyes: dry eyes, sicca, ciatrical conjunctivitis Muscles: fascitis, mycostitis, joint stiffness Female Genitalia: vaginal sclerosis, ulceraions GI tract: anorexia, weight loss esophageal web or strictures Liver: jaundice, transaminitis Lungs: Restrictive or obstructive defects, bronchiolitis obliterans, pleural effusions Kidneys: nephrotic syndrome Heart: pericarditis Marrow: thrombocytopenia, anemia, neutropenia

Review this slide that shows C4d positivity

Antibody mediated-Humoral Rejection

Besides donor, what is another way to do hematopoietic stem cell transplantation?

Autologous stem cell transplantation. Freeze patient's cell, use very high doses of chemo and/or radiation to "wipe out" immune system, and replace with own stem cells. No GVHD, but potentially cancer cells reinjected with frozen stem cells

What is rejection? How does it occur?

Body "rejects" transplanted tissue. Two different pathways are responsible for allograft rejection: *Direct pathway*: APCs (eg. dendritic cells) of donor origin, transferred along with the allograft (passenger leukocytes), are encountered within the allograft or draining lymph nodes by the recipient's cytotoxic (CD8+) T cells that recognize disparities in HLA class I and helper (CD4+) T cells that recognize disparities in HLA class II. Acute cellular rejection is *primarily mediated by activated cytotoxic T cells* that directly damage allograft tissue. In addition, activated helper T cells that produce cytokines (eg. IFNy) activate macrophages that may also mediate allograft tissue injury. *Indirect pathway*: recipient's antigen presenting cells process foreign allograft tissue antigens and present them along with HLA class II to helper (CD4+) T cells. These helper T cells may stimulate antibody-mediated rejection by interacting with B cells or acute cellular rejection by *activating helper T cells that produce cytokines* (eg. IFNy) that activate macrophages.

Describe hyperacute rejection.

Briefly: antibody mediated, occurs in minutes to hours Occurs almost immediately (within minutes to hours) after blood flow has been established to the allograft and is due to the *presence of preformed donor specific HLA antibodies in the recipient*. Antibodies are usually directed against HLA class I antigens formed in response to prior pregnancies, transplants, or blood transfusions. Hyperacute rejection can also occur when an ABO blood group incompatibility is present. Antibodies enter the graft and attach to endothelial cells. *Complement is activated* which causes direct damage to endothelial cells and also attracts neutrophils into the area. These inflammatory cells secrete cytokines that result in further endothelial cell damage. Platelets adhere to denuded areas within blood vessels forming clots that occlude the blood vessels and result in *tissue ischemia and allograft loss*. (Note: Hyperacute rejection is rare due to the use of the pre-transplant crossmatch and virtual crossmatching)

Describe chronic rejection

Briefly: occurs in months to years, multifactorial Occurs several months to years after a transplant. This type of rejection is responsible for the inability to maintain long-term allograft function in most patients. Cause is most likely multifactorial but appears to be due to continuous immunological insult (cellular and/or antibody mediated) against the allograft. I.e in kidney: endothelial cell injury results in duplicated glomerular basement membranes (transplant glomerulopathy) and peritubular capillary basement membrane multilayering. Over time, chronic damage to arteries causes intimal fibrosis and vascular narrowing resulting in chronic tissue ischemia. In lung: arterial changes are seen, most affected structure in the lung is the bronchioles where fibrosis narrows or obliterates air passages resulting in obstructive lung disease. This process is called obliterative bronchiolitis. In liver: arterial narrowing and chronic tissue ischemia affects the bile ducts, main blood supply for the biliary system is the hepatic arteries. As a result, bile ducts begin to disappear and cholestasis develops which over time produces hepatic fibrosis. Only treatment, once well established, is re-transplantation. Primary focus is prevention: minimizing acute rejection episodes. Some centers perform allograft biopsies at defined intervals to help diagnose potentially clinically silent acute rejection.

Describe acute rejection.

Briefly: weeks to few months. 2 types: cellular and antibody-mediated Encountered in solid organ transplantation usually within a few weeks to a few months after the transplant, can occur anytime especially when a patient becomes *noncompliant* with their immunosuppressive medications. Two types, *cellular and antibody mediated (humoral)*. Cellular rejection is primarily mediated by cytotoxic T cells, which infiltrate the graft and cause tissue damage. Usually successfully treated with an increase in immunosuppression (e.g. high dose steroids). Antibody-mediated rejection occurs when the recipient synthesizes antibodies directed against mismatched donor HLA class I and/or II antigens that are present in the allograft. These attach to endothelial cells in the allograft and cause tissue injury (usually not as severe as what is seen in hyperacute rejection). Can be identified by reactive endothelial cell changes and damage identified in light microscopy; antibody-mediated rejection may be also identified by detecting *C4d deposition* within interstitial capillaries. Presence in capillaries indicates complement activation. C4d is used instead of other complement cleavage components because it is degraded more slowly. Much more difficult to control with conventional immunosuppressive agents due to their inability to effectively shut down antibody production by B lymphocytes and plasma cells. Currently, the 2 main treatments are plasmapheresis (used to remove the detrimental antibody), and intravenous immunoglobulin (IVIG), (mech. unknown). Rituximab (monoclonal antibody against the B cell marker CD20), Bortezomib (reversible 26S proteasome inhibitor monoclonal antibody that depletes plasma cells), and Eculizumab (complement C5 inhibitor monoclonal antibody) increasingly used.

Slide of acute GVHD in liver

Can see bile duct. Cells w/ the "halo" effect are donor T cells

Review this slide of chronic rejection in the lung

Can see oliberated bronchioles/narrowing of bronchii. Hardly any lumen left.

Review this slide of HLA typing.

DN1 not a match, DN2 was a high-resolution match

True or false: HLA antibody screening is only performed pre-transplant.

False, is also routinely performed in post-transplant period to look for the presence of donor specific antibodies. It is the mismatched HLA antigens that the recipient's immune system may recognize as foreign and produce antibodies against. If an antibody or antibodies are identified in the recipient, they are compared with the list of mismatched HLA antigens to determine if they are indeed donor specific and how strong they are. If they are donor specific, this information is interpreted in the context of how the allograft is functioning since the presence of donor specific HLA antibodies is not always associated with allograft rejection.

Review these slides of hyperacute infection in kidney and heart.

Glomerulus deflated due to clots --> lack of perfusion Myocytes in heart severely damaged with edema

What is GVHD?

Graft vs host disease - largely limited to patients that receive an allogeneic hematopoietic stem cell transplant. Transplant process begins conditioning regimen w/ chemo and radiation to prepare marrow space. This causes significant tissue injury --> release of tissue antigens and cytokines that activate APC. Then, donor stem cell prepared (with some donor T cells) and infused into recipient. Donor T cells become activated by recipient's APCs and proliferate into activated cytotoxic and helper T cells. Donor activated cytotoxic T cells are capable of causing GVHD with helper T cells aiding in process. Damage, sometimes severe, occurs to skin, GI, and liver. Immunosuppressive therapy is used to control but can sometimes be life threatening or become chronic. Chronic GVHD may cause fibrosis in skin, cholestatic jaundice in liver, and strictures in GI tract. In addition, depletion of host immune system cause pt to be susceptible to serious infections.

What is one of the most important molecules involved in transplant immunopathology? Describe them.

HLA antigens, play a major role in graft recognition and subsequent rejection. Encoded for on chromosome 6, except for B2-microgobulin (component of Class I) on chromosome 15. All nucleated cells express HLA Class I (lymphoid probably express the most); Class II on APC cells (B cells, dendritic cells, monocytes, etc.) Class I peptides present *endogenous antigens* to CD8 cells. Class II peptides present *exogenous antigens* to CD4 cells

Review this slide of chronic rejection in the heart

Have narrow lumen due to plaque (differen from what is found in atherosclerosis) from Circumferential intimal fibrosis. Results in myochardial ischemia, can cause CHF or MI

What is a virtual cross match?

Helps to filter out non-"perfect" matches/incompatible transplants. Detection of HLA antibodies in brecipient directed against donor antigens (mismatch) may cause graft rejection/failure to engraft. If these antibodies are detected, this is incompatible and organ not transplanted. Hopefully, there is an organ found where the donor HLA typing and the recipient HLA typing are compatible (no antibodies directed against donor) Note: this is all done virtually

Name the four types of rejection discussed in class (detailed cards for each later)

Hyperacute rejection, acute rejection, chronic rejection, Graft-vs-host disease (GVHD)

Explain why HLA matching is important in transplants, and when it might not be as important

Important because the closer the match, the higher likelihood the body's immune system will "accept" the transplant/not reject it. Renal transplants can sometimes have a mismatched transplant and do fine w/ immunosuppressive drugs. Also, in thoracic transplants (lungs, heart); sometimes cannot wait for the perfect match. In these cases, ABO blood group compatibility and organ size become most important factors. Extremely important in hematopoietic stem cell transplantation to lower risk of significant graft vs host disease in the recipient.

Review these slides of acute cellular rejection

In kidney, see endothelialitis --> higher rejection In liver, can see portal vein with lymphocytes damaging it In lung, can see mononuclear cells surrounding the vessels with some bleeding

Review this slide of chronic rejection in the liver

Loss of bile duct

Explain HLA tissue typing.

Performed on DNA typically isolated from leukocytes using one or more molecular assays to identify genetic sequences (alleles) that give rise to HLA antigens. Molecular assays allow for more specific characterization of HLA class I and II antigens than serological techniques (lymphocyte cytotoxicity), especially when identifying the subtypes needed for hematopoietic stem cell transplants. DNA sequencing by Sanger or Next Generation are used for high resolution HLA typing. Each person has two of each type of HLA antigens, with one (HLA-A, B, C, DR, DQ, and DP) haplotype being contributed by each parent.

What are some downsides to immunosuppressive therpy?

Predispose patient to infections and neoplasms, especially if used in high doses. Infections - opportunistic infections (viral, bacterial, fungal, protozoan). Most common in first 6 months post-transplant Neoplasms - skin cancer (basal cell and squamous cell carcinoma); viral infection related: squamous cell carcinoma, cervix (HPV), Post-transplant lymphoproliferative disorder (EBV); Kaposi's sarcoma (HHV 8)

What are some populations that might have "excess" antibody formation?

Pregnancy: 30-50% of women with 3+ pregnancies Blood transfusions: 50% of patients who receive multiple transfusions develop antibodies, leukocyte reduce blood decreases risk Previous transplant: 90% of patients develop antibodies within 2 weeks of allograft failure. Greater likelihood of HLA-DPB1* antibodies.

If donor T cells cause root of issue for GVHD, why not transplant stem cells w/o donor T cells?

Removal of some T cells from collected donor hematopoietic stem cells prior to infusion may help; however, if too many T cells are depleted, the hematopoietic stem cells may not engraft in the recipient. Indicates that T cells play an important role in the engraftment process. In addition, depletion of T cells results in a higher relapse rate in patients who receive a hematopoietic stem cell transplant for cancers like acute leukemias because of a decrease in the *"graft vs. leukemia"* effect that appears to be mediated by the T cells. Combination of removing some donor T cells and getting "perfect" match seems to mitigate risk of GVHD.

Describe the typical initial antibody screen in potential transplant recipients.

Screening test to look for preformed HLA antibodies (Class I and Class II) in potential transplant recipients (antibodies to HLA-A, B, etc.; may be detected) Patient's serum is tested against many HLA antigens coupled to beads. HLA antibody specificity determined and listed, %cPRa calculated--indicates risk of a positive crossmatch Note: a patient having preformed HLA antibodies is said to be sensitized. If test is rerun, new HLA antibodies detected are listed as "Newly Identified Unacceptables"

Review this slide of acute GVHD

See rash, separation in layers of epidermis that caused blistering

Describe the two ways Crossmatch is performed.

Serological: mix donor's lymphocytes, (separated into T and B cells) with the recipient's serum followed by the addition of AHG (anti-human globulin), rabbit complement and a vital dye that increases the sensitivity of the test (binds to human Ig), and in turn enhances rabbit complement activation and cell death. Upon completion of the assay, the numbers of cells killed, if any, are scored. If minimal to no cells have died, the crossmatch is considered compatible, transplant may take place with minimal risk of hyperacute rejection since no pre-existing anti-donor antibody(s) are detected. Flow cytometry: more sensitive and easier. Performed by incubating recipient serum with donor lymphocytes. Fluorescent tagged antibodies to CD3 (T cells) and CD19 (B cells) are added. Fluorescent tagged anti-human IgG is added to detect any antibody that may have bound to the T cells and/or B cells. If positive reaction occurs with both T cells and B cells, an antibody to HLA class I only or a combination of HLA class I and class II is present. If a positive reaction occurs with only B cells, an antibody to HLA class II only is present.

Another slide of acute GVHD

Tissue is hemorrhagic. Epithelial cells were damaged

What is Crossmatch?

Usually the last, most critical laboratory test performed before the transplant (no longer virtual, in the lab) Detects preformed donor specific HLA antibodies in the potential recipient. Test utilizes donor lymphocytes and recipient serum. Positive test (incompatible) = do not transplant Negative test (compatible) = transplant Minimizes risk of hyperacute rejection Might be impractical--test takes time, some organs do not do well outside of body. Also, if urgent, don't necessarily have time to do testing.

Briefly describe the immunosuppressive regimen seen in transplant patients.

Utilized to prevent graft rejection in the case of solid organ transplants and to prevent primarily GVHD in the case of hematopoietic stem cell transplants. Typically two or three different drugs, each with slightly different mechanisms of action, are used in combination with each other so that lower doses of each drug may be used. Important because the majority of drugs used have side effects that are more significant at higher doses. Currently, the most common combination of immunosuppressive drugs used for solid organ transplantation is one of the *calcineurin inhibitors (Cyclosporine or Tacrolimus), Mycophenolate mofetil, and corticosteroids*. Treatment should not be viewed to just prevent the immune system from attacking the graft, but to also prevent acute allograft rejection from destroying the transplanted organ until the transplant recipient achieves some degree of tolerance. This theory is supported by the fact that as the time from the transplant increases, the majority of patients will be able to undergo a decrease in the dosage of their immunosuppressive drugs.