Immunology and Xenotransplant

Question 1

The pig has a large population of what were initially considered null cells. Describe these?

Answer 1

Lack expression of CD2, CD4, and CD8 = Null
Express CD3, classifying them as T cells.

Question 2

Describe gamma delta T cells in pigs.

Answer 2

Largely comprises lymphoid population, found in large number especially in mucosal sites such as uterus. Also highly prominent in newborn. Similar to cells described in ruminants.

Question 3

Describe CD4 and DC8 cell expression.

Answer 3

Swine, like monkeys and humans, have a unique lymphocyte subset that expresses both CD4 and CD8 markers. Not mutually exclusive as in other species. May represent a type of memory cell or lineage that differentiates from DC4+CD8- to CD4+CD8+.

Question 4

What molecules/cells does colostrum contain?

Answer 4

Neutrophils and T cells, IgG antibodies

Question 5

What antibody do pigs lack?

Answer 5

Pig lacks IgD, which is precursor immunoglobulin in the differentiation pathway to IgM.

Question 6

Describe IgG, IgA, and IgE in pig.

Answer 6

IgG: Large number of subclasses
IgA: Dimer in blood and tissues, monomer in mucosal secretions
IgE: Serum and mucosal tissues

Question 7

What are swine leukocyte antigens? Where are they found?

Answer 7

Equivalent of human major histocompatibility complex. Coded for on chromosome 7.

Question 8

What are the differences between MHC-I, MHC-II, and MHC-III (SLA) in pigs?

Answer 8

MHC-I: Expressed by all nucleated cells, function to restrict CD8+ T-cell activation, particularly antiviral immune responses.
MHC-II: Restricted to professional antigen presenting cells such as B cells, macrophages, and dendritic cells. Similar to humans (unlike mice) also expressed in lymphocytes and vascular endothelium. Upregulation occurs during inflammation.
MHC-III: Function in complement system. Antigen-antibody elimination occurs via lung in pigs, as compared to liver and spleen in humans.

Question 9

Describe antigen classification of swine RBC. Why is this important for transplantation work?

Answer 9

Complex. 16 genetic systems consisting of 78 blood factors. Disparities between donor and recipient can induce antibody-mediated hyperacute rejection.

Question 10

Combining skin or solid organ transplant with what tissue may contribute to tolerance in allotransplantation?

Answer 10

Bone marrow.

Question 11

How have immunodeficient swine been developed?

Answer 11

Acquired: Thymectomy, splenectomy, and strong pan-immunosuppressants
Management/Spontaneous: Inadequate colostrum, stress, or poor nutrition
Autoimmune: Poorly documented. Hemolytic disease related to postnatal absorption of maternal IgG (erythroblastosis fetalis) and two forms of glomerulonephritis (Norwegian Yorkshire inherited, Japanese slaughter IgA nephropathy)

Question 12

What agents are of particular risk for xenotransplantation? How common are they in swine?

Answer 12

Retroviruses. Swine have fewer than other vertebrates and infections have not been documented.

Question 13

Cross-breeding of outbred miniature swine from the Andes and Rockies developed three lines of miniature swine homozygous for what? What are these animals used for?

Answer 13

Homozygous for different SLA alleles: SLA aa, SLA cc, and SLA dd. Lines are fixed at SLA loci and heterozygous at minor histocompatibility loci. MHCs matched with minor or full mismatch transplants available to emulate clinical paradigms.
MHC within a line - Transplants between MHC identical human siblings
Between lines - Full MHC mismatch
Between F1 hybrids - Haplo-identical transplants or parent-to-offspring transplants

Question 14

It is easier to develop tolerance to what type of MHC mismatch?

Answer 14

Full MHC-I instead of MHC-II. MHC-II though to permit optimal function of regulatory T cells, which are dependent on MHC-II matching.

Question 15

What is the transplant hierarchy of tolerance?

Answer 15

Greatest to least: Liver > Kidney > Heart > Lungs > Skin

Question 16

What are the two drivers of xenograft rejection?

Answer 16

Humoral and cellular

Question 17

Describe immediate graft loss. What is the cause? What enzyme is the causative agent produced by? How can this be avoided?

Answer 17

Hyperacute rejection occurring in seconds to minutes. Humoral, driven by natural antibodies that recognize the sugar moiety Gala1-3GalB1-4GlcNAc (a1,3Gal) present in porcine epithelium. Produced by enzyme a-1,3-glactosyltransferase, present in most mammals but not humans and OWM. Antibody-mediated recognition of this epitope triggers complement cascade. Absorb natural anti-Gal antibodies or use grafts deficient of Gal antigen.

Question 18

What causes acute humoral xenograft rejection? How long does it take for it to occur?

Answer 18

Non-Gal xenoreactive antibodies. Graft lost over several days t weeks by complement activation.

Question 19

Are complement-mediated mechanisms the core of all graft losses?

Answer 19

No

Question 20

Describe delayed rejection of xenografts. How long does the process take? What cells are involved?

Answer 20

Through cell-mediated responses over 3-4 days, involve activation of endothelial cells of the graft. Activation leads to loss of thrombomodulin and ATP, leads to prothrombosis, proinflammatory gene activation increasing expression of adhesion molecules, prothrombotic factors, and cytokines. Involves T-cells, NK cells, macrophages, and neutrophils.

Question 21

Why has T-cell mediated rejection not as well characterized as humoral responses? How does the Gal-KO pig play a role?

Answer 21

Difficulty avoiding HAR and AHXR. Permitted studies in large animals of non-humoral xenograft rejection mechanisms.

Question 22

How does control of CD4+ T-helper and CD8+ T cytotoxic cells play a role in xenotransplant success?

Answer 22

CD4+ important for T-cell mediated response, but also control of AHXR.
CD8+ cytolysis continues to be a potent method of xenograft rejection.

Question 23

Activation of what innate cells by pathogen-associated molecular patterns plays a role in xenograft rejection? How are NK cells inhibited? How does this result in rejection? How could this be overcame?

Answer 23

NK and macrophages. NK cells function by sensing inhibitory signals through MHC ligation. Xenogeneic MHC cannot provide the necessary inhibitory signals (compared to allogeneic responses) so NK is more potent. Overcome by addition of human MHC expression on Gal-KO pigs.

Question 24

What is another mechanisms of NK-cell mediated kill mechanism?

Answer 24

Antibody-dependent cell cytotoxicity (ADCC). Both natural and IgG-specific antibody responses can induce. Macrophages also involved, as they phagocytose target tissues if they do not receive a negative signal through interaction of CD172 on macrophage and CD47 on cell surface (‘do not eat me’ signal). Interspecies incompatibilities induce macrophage-mediated xenograft rejection.

Question 25

What is immunoabsorption? Is this effective? What are the two methods?

Answer 25

Removal of natural anti-Gal xenoantibodies. Efficient, but incomplete, and xenoreactive natural antibodies rebound relatively quickly. In vitro, using a-Ga immunoaffinity columns, or in vivo via extracorporeal perfusion of a donor organ (often liver as it is large and vascular).

Question 26

How were alpha-Gal KO pigs developed?

Answer 26

Sachs and colleagues knocked out gene in the most inbred of the MGH miniature swine (‘D’ haplotype) creating a pig devoid of alpha-Ga from SLA dd pigs.

Question 27

What are additional approaches to eliminating xenoantibodies?

Answer 27

Anti-CD20 monoclonal antibodies to kill B cells, but there are B cells that do not express CD20.
Cobra venom for preventing complement activation.
Use of pigs expressing human complement inhibitory molecules.

Question 28

What is theorized to be the most effective method to circumvent xenograft rejection? What does this method address? What methods have been attempted?

Answer 28

Development of immunological tolerance. Addresses humoral and T-cell-mediated responses. Bone marrow transplantation to create hematological chimeras in allograft model, difficult in xenograft model.

Question 29

How well does pig marrow/hematopoiesis occur in primate marrow?

Answer 29

Not well

Question 30

Can transfer and expression of xenogenic (swine-origin) class II transgenes be achieved in baboons? Why is this important?

Answer 30

Yes. This therapy may prevent late T-cell dependent responses to porcine xenografts, including non-a-Gal IgG antibody responses.

Question 31

What occurs following thymic xenograft transplant?

Answer 31

In immunodeficient mice, supported normal development of polyclonal, functional T cells specifically tolerant to SLA antigens of porcine thymus donor.

Question 32

What xenograft tolerance approach has provided the longest survival and donor-specific tolerance?

Answer 32

Bone marrow chimerism