I12 - Transplantation and Tumor Immunology

Busulfan

(DNA alkylating agent) Preconditioning (myeloablative) for bone marrow transplant

Cyclophosphamide

(DNA alkylating agent) Preconditioning (myeloablative) for bone marrow transplant

Azathioprine

(Inhibits adenine & guanine synthesis) Used extensively in '80s for immunosuppression Now induction agent

Mycophenolate mofetil

(Inhibits guanine synthesis) Induction agent GVHD treatment

Methotrexate

(Inhibits thymidine synthesis) GVHD prophylaxis

Effects of Cyclosporin A and Tacrolumus on T Cells, B Cells, and Granulocytes

*T Lymphocytes*: reduced cytokine expression, reduced cell division, reduced Ca-dependent exocytosis of cytoxic granules, inhibition of antigen-driven apoptosis *B lymphocytes*: inhibition of cell division, inhibition of antigen-driven cell division, induction of apoptosis after B-Cell activation *Granulocytes*: Reduced Ca-dependent exocytosis of granules

Transfusion Reaction

*Type II Hypersensitivity* (fixed, IgG) Prevent by typing and cross-matching (see pervious lecture on ABO Antigens)

Transplantaion Key Points

- 4 type of transplant rejection based on timing of response which is due to immune mechanism in action as a result of ABO and/or MHC mismatch - MHC mismatch gives rise to an alloreaction - For bone marrow transplants must of near identity for MHC allotypes - 3 categories of immunosuppressive drugs: corticosteroids, T cell activation inhibitors, cytotoxic

Categories of Immunosupressive Drugs

- Corticosteroids - T cell activation inhibitors - Cytotoxic drugs Given in combination to decrease side effects

T-Cell Inhibitors

- Cyclosporin A - Tacrolimus - Sirolimus - Antibodies against cytokines, cytokine receptors, e.g. IL2Rα (anti-CD25) - Antibodies against T cell markers, e.g. CD3 (anti-CD3); panspecific ATG/ALG

Chronic GVHD in Marrow Transplants

- Occurs in 25-45% of marrow transplant patients who live longer than 6 months post-op - Course of disease resembles autoimmune disease and eventually produces severe immunodeficiency leading to life-threatening recurrent infections.

Minor Histocompatibility Antigens: Difference between sexes

- X and Y analogous genes produce slightly different peptides. In a male receiving a marrow donation from a female sibling, the sister's marrow T-Cells may reject the Y chromosome derived proteins in the recipient's body

NK-cell alloreaciton occur when...

..recipient's HLA class I allotypes provide ligands for fewer types of inhibitory KIR than the donor's class I allotypes. - Patients with *acute myelogenous leukema (AML)* benefit from NK-cell alloreaction - Patients with acute lymphocytic leukemia (ALL) do not

Cytotoxic Drugs

1.) *Azathioprine* 2.) *Mycophenolate mofetil* 3.) Methotrexate 4.) Cyclophosphamide 5.) Busulfan

Acute Rejection - Direct Pathway of Allorecognition

1.) Donor dendritic cells in the graft carry complexes of donor HLA:donor peptides on their surfaces. The dendrites are carried to a draining secondary lymphoid organ (e.g. spleen), where they move to T-Cell areas 2.) Recipient's T-lymphocytes with receptors that can bind specifically to the complexes of allogenic donor HLA (class I and II) in combo with donor peptides 3.) After activation, effector T Cells travel via blood to implanted organ and attack cells containing that donor HLA:peptide

Consequence of Complete HLA Mismatch

1.) Donor derived thymocytes are positively selected on recipient HLA allotypes 2.) Circulating T-Cells are restricted by recipient, but not donor HLA allotypes 3.) APCs in tissues present antigens on donor HLA allotypes 4.) Upon infection no T-Cells can respond to pathogen-derived antigens presented by APCs 5.) No adaptive immune response is made and infection persists ==> *Severe Combined Immunodeficiency*

Consequence of Some Shared HLA

1.) Donor derived thymocytes positively selected on recipient and some donor HLA allotypes 2.) Circulating T-Cells are restricted by recipient and some donor HLA allotypes 3.) APCs in tissue present antigens on some recipient HLA allotypes 4.) Upon infection some T-Cells do respond to pathogen-derived antigens 5.) An adaptive immune response is made that terminates the infection

3 Types of Blood Transfusion

1.) Red cells, most common 2.) Platelets and clotting factors 3.) Plasma

Two Complimentary Approaches to avoid MHC Mismatch

1.) Select a donor who is as similar as possible in HLA I and II types to the recipient - this reduces alloreactive T-Cells that can be triggered 2.) Use battery of immunosuppresive drugs to prevent and interfere with activation/proliferation of T-Cells

Normal Antigen-Induced Activation of T-Cells

1.) Signals from T-Cell Receptor (TCR) lead to hydrolysis of membrane lipids to produce IP3 2.) IP3 ---> Ca release from ER 3.) [Ca2+] increase activates cytoplasmic Serine/Threonine Phosphatase *Calcineurin* 4.) Calcineurin dephosphorylates and activates transcription factor *NFAT* 5.) NFAT enters nucleus and binds transcription factor AP-1 to form a transcriptional regulatory complex that turns on transcription of IL-2

Hyperacute Rejection: ABO Mismatch or Anti-Class I Abs

ABO antigens are expressed on the endothelial cells of blood vessels. If there is an ABO mismatch between donor and recipient, the recipient's circulating antibodies will bind to blood vessels throughout the graft. By fixing complement throughout the graft they produce very rapid graft rejection - even before the patient leaves the operating room. - Vessel occlusion also occurs in the graft.

Cross-Matching Test

ABO compatibility Test the recipient's antibodies against the donor blood, not the other way around - amount of donor antibodies in the transfusion is not enough to produce significant hemolysis, it is the recipient's antibodies that are the real issue

Transplant Rejection

After transplantation of solid organs alloreactions developed by recipient's immune system are directed at the cells of the graft and can kill them

Histocompatibility in Transfusions

Alloreactions may occur between Rh types Rhesus D is the main alloantigen. When a transfused patient has preformed antibodies that bind the Rh, hemolysis occurs. Serological typing between RBC alloantigens must be performed - Although there are many different RBC antigens, important ones are *A,B,O, and RhD*

Alloantigens

Antigen that differs between members of the same species, e.g. HLA antigens and blood group antigens - *determined by alleles of polymorphic genes*

Tumor Associated Antigens

Antigens expressed on tumor cells but also found on certain normal cells in smaller amounts - Reactivation of embryonic genes not normally expressed in differentiated cell - Overexpresion of normal self-protein by tumor cell changes the density of self-peptide, allowing recognition by T-Cells

Tumor Specific Antigens

Antigens present on tumor cells but not on normal cells (bottom left) - have structures not present in normal cells and can derive from viral proteins, mutated parts of mutant cell proteins, aa sequences spanning tumor-specific recombination site between genes.

Source of Transplant

Autograft = self, autologous Isograft = same MHC, syngenic Allograft = different MHC, allogenic Xenograft = different species, xenogenic

HLA Class Divisions

Based on first serologic typing, then by alleles via DNA sequencing. e.g. HLA-A2 antigen comprises >130 alleles that differ by only a few nucleotide substitutions

ABO Compatibility

Blood transfusions can causes Type II Hypersensitivity reaction due to circulating antibodies that bind to the surface of transfused RBCs. - Major barrier to RBC transfusion is structural polymorphisms in the carb portion of glycolipids on the RBC surface. (ABO System) - A and B blood group antigens are similar to some bacterial structures and if a patient is not tolerant of them, they will react (e.g. a person Type O will attach A or B blood)

Immune Priveledged Sites

Brain Eye Testis Fetus Placenta

Type AB Compatibility

Can receive any blood: O, A, B, or AB

Two Types of Tumor Antigens

Cancer cells (viral or point mutation) have genomic differences that allow them to be recognized by the adaptive immune response. The antigens to which they respond = tumor antigens. 1.) Tumor Specific 2.) Tumor Associated - Embryonic - Over-Expression

Intestinal and Liver Reaction in GVHD

Causes cramps and diarrhea Inflammation of the bile ducts in liver cause hyperbilirubinemia and a rise in the level of liver enzymes in bloood

Skin Reaction in GVHD

Characteristic skin rash of GCHD tends to develop with the kinetics of a primary immune response during the 10-28 days following transplantation. - Fine, diffuse erythematous rash begins on palms of hands, soles of feet, and head and then spreads to trunk - Eventual blistering and desquamation in Grade IV GVHD

Tumor Evasion of Immune System: Loss of MHC Class I

Cytotoxic CD8 T-Cells are the best effector cells for cancer killing. Tumors avoid cytotoxic cells by stopping the expression of MHC I that presents the tumor antigen to the T-Cell - 1/3-1/2 of human tumors have defective expression of HLA class I, indicating that patients have made a CD8 T-Cell response to the tumor but cells lacking the MHC I have evaded the T-cells to continue growing

Mixed Lymphocyte Reaction (MLR)

Determines extend to which recipient's T-Cells will respond to transplanted tissue (similar to cross-match test in blood transfusions). 1.) Peripheral blood cells from recipient are mixed with lethally irradiated (prevents replication) cells from potential donor 2.) Proliferation of T-Cells and effector function of donor-specific Ctyotoxic T-Cells are both measured

Allograft

Different MHC, allogenic

Xenograft

Different species, xenogenic

For Reconstitution to Occur...

Donor and Recipient must share at least one HLA class I allotype and one HLA class II allotype

T-Cell Repertoire Depletion by too many MHC Isoforms

Effect of negative selection increases disproportionately as the number of different HLA molecules expressed by an individual increases.

Organ Transplant Data

First organ transplant 1954, kidney 122,325 people are waiting today, 78,286 people are active 18 people will die today waiting January 2014: 2,401 transplants from 1,210 donors

Blood Transfusion

First transplant, 1812 1 in 4 people Transfusion reaction type II hypersensitivity Prevent by typing and cross-matching

Peptide Splicing

Generation of novel peptide from self-proteins within tumor cells via the splicing of tumor-cell glycoproteins into novel sequences. e.g. In melanocyte Glycoprotein gp100, 2 peptides are cut out and pliced together by a proteosome. The spliced gp100 peptide is presented by *HLA-A32* and recognized as non-self by CD8 T-Cells

MHC Mismatches Lead to...

Graft Failure

Acute Rejection: CD4 T Cells respond to

HLA class 2 differences

Acute Rejection: CD8 T Cells respond to

HLA class I differences

AB Rh+

Have all 3 antigens (A,B, and RhD) and are "Universal Recipients" who can get blood from anyone, but can only donate to other AB Rh+ donors

Maximum time from organ recovery to transplantation

Heart or lung: 6 hours, blood type match only Liver: 24 hours, blood type match only Kidney and pancreas: long enough to do HLA typing

What is an Alloreaction

Histocompatibility antigen = transplant antigen = alloantigen - Immune response against differences between donor & recipient MHC (Major Histocompatibility Complex) - Think about what cells express what classes of MHC, what immune cells recognize MHC, and what effector mechanisms those cells use

Types of Rejection

Hyperacute (minutes) Acute (up to a year) Chronic (years 2-?) Graft versus host

Bone Marrow Transplants

In Bone Marrow transplants the recipient's immune system is destroyed and eventually replaced with one reconstituted from the hematopoietic stem cells in the bone marrow graft

The "Real Version"

In hypothetical version where recipient shared no MHC allotypes with donor, the recipient would end up with Severe Combined Immunodficiency, even if the transplant was successful. - due to the fact that reconstituting T-Cells selected on the recipient's HLA molecules in thymus would be unable to recognize peptide antigens produced by donor HLA and other APCs in the periphery. - Extent to which mature T-Cells respond to antigens presented by professional APCs of donor HLA type directly correlates to numer of HLA class I and II allotypes donor and recipient share.

Indirect Pathway of Allorecognition

In indirect pathway some of donor-derived dendritic cells migrate to draining lymphoid tissue and die by apoptosis. Membrane fragments containing HLA are phagocytosed by recipient dendritic cells and processed for presentation by HLA II allotypes of recipient. If the peptides presented are different in a.a. sequence from those produced by degradation of the host's own dendritic cells a CD4 T-Cell Alloreaction occurs.

"Fit as a Fiddle" Version

In theory marrow transplantation works by killing all of a patient's bone marrow and replacing it from a healthy donor graft which then reconstitutes the entire hematopoietic system. - Success is directly correlated to the degree of HLA matching between patient and donor

Indirect Pathway of Allorecognition (Chronic Rejection)

Indirect pathway responsible for stimulating production of anti-HLA antibodies that cause Chronic Rejection. Processing and presentation of allogenic HLA Class I by a Dendritic Cell of recipient activates Helper CD4 T-Cells which in turn activate B-Cells that have bound and internalized donor HLA molecules --> antibody production Anti-HLA Class II antibodies can be produced similarly. Because activated endothelium expresses both HLA I and II, antibodies agaisnt both classes of HLA can contribute to chronic rejection.

Chronic Rejection

Initiated by interaction of anti-HLA class I alloantibodis with blood vessels of graft. Antibodies bind to endothelial cells (E) to recruit Fc receptor bearing monocytes and neutrophils. - Accumulating damage leads to Internal Elastic Lamina (EL) thickening and infiltration of underlying intimas with smooch muscle cells (SMC), Macrophages (M) and Granulocytes (G), Alloreactive T-Cells (T) and Antibodies. Net Effect: Narrowing of lumen of vessel and chronic inflammation that intensifies tissue remodeling. - Eventual vessel obstruction, ishchemia, and fibrosis

Cyclosporin A (T-Cell Inhibitor)

Interferes with Calcineurin activity, shutting down T-Cell activation, proliferation, and differentiation at an early stage 1.) Diffuses across T-Cell membrane and binds Cyclophilins (peptidyl-prolyl isomerase enzyme) 2.) Cyclophilin:Cyclosporin complexes bind Calcineurin, inhibiting phosphatase activity and preventing activation of NFAT and subsequent production of IL-2

Sirolimus (T-Cell Inhibitor)

Interferes with T-Cell activation at a later stage Binds to FK-binding proteins FK-BP:Sirolimus prevents phosphorylation of p70S6 Kinase --? blocks cytokine receptor signaling

IκBα and NFkB

IκBα is an inhibitor of NFκB* (Transcription factor important for cytokine production and cellular activation) - Normally NFkB is held in cytoplasm via association with IκBα, upon activation IκBα is phosphorylated, releasing NFkB so it can enter the nucleus and act as a transcription factor - Suppression of IκBα suppresses cytokines --> reduces inflammation

Current Solid Organ Transplants

Kidney, Liver, Pancreas, Lung, Intestine, Heart - All can be living donor except for heart, but almost all are cadaveric

ORhD-

Lack all 3 antigens (A, B, and RhD) and are "Universal Donors" who can provide blood for anyone, but can only receive blood from other O RhD- donors

Haploidentical Transplant

Leukemia patients waiting for donors - Family members often share one HLA halotype with them, but differ in the second - to prevent GVHD, graft is stringently depleted of T-Cells and recipient is infused with anti-T-Cell antibodies - Graft Rejection is prevented via combo of intense condistioning regimen and larger than normal dose of hematopoietic stem cells

Negative Selection in the Thymus limits the Number of Expressed MHC Isoforms: Part 2

MHC diversity is advantageous to an individual bc it broadens the repertoire of pathogen-derived peptides presented. Here we see how a similar expression in the number of self-peptides presented leads to an equivalent increase in the population of positively selected T-Cells in Thymus. However, with each additional type of MHC molecule the proportion of MHC cells that are negatively selected goes up, not in an arithmetic manner but in a geometric manner that increases with the square of the total number of MHC Molecules. Result is...bla bla

Negative Selection in the Thymus limits the Number of Expressed MHC Isoforms: Part 1

Magnitude of the alloreactive response to nonself MHC provides a measure of the T-cells that are negatively selected in an individual's Thymus. If MHC from another person activate a certain fraction of mature T-Cells, then a smiliar stimulation by self MHC during T-cell development should lead to deletion of a similar faction of the positively selected T-Cells.Pic shows effects of positive and negative selection of changing the number of different MHC molecules.

Malignant Diseases Treatable by Bone Marrow Transplantation

Marrow transplantation important treatment for cancer patients, esp those with tumors of immune-system cells. In chemo/radiation patients the advantages of killing off malignant cells must be balanced against the damage caused to normally proliferating vital tissues. Bone marrow is most susceptible to chemo/radiation and transplantation allows opportunity to increase anticancer treatment beyond the point of lethality, after which patient is rescued by an allogenic transplant from a healthy HLA-matched donor.

Allele Match

Means the donor and recipient have identical HLA-A, HLA-B HLA-C, and HLA-DR Loci - a single class I mismatch means recipient and donor differ by one HLA class I locus - Class I and Class II mismtach means they differ by one HLA I and one HLA II The fewer the Number of Mismatches the better survival and health and lower risk of GVHD

MHC Matching

Most important to match betwen *HL-A, HL-B, and HLA-DR* - most matchng is done via DNA analysis though serology can be used also

Acute Rejection

Most transplants occur across some HLA I/II difference CD4 and CD8 T-Cells respond to the HLA class differences and produce effector CD4 and CD8 T-Cells that attack and kill graft - takes days to develop - prevented by giving patient immunosuppressants prior to and forever after transplant and monitoring closely for signs of rejection (give more immunosuppressors OR Anti-T-Cell antibodies if they reject the organ)

Transplantation Antigens

Name given to MHCmolecules as they are the main antigen that provoke rejection of transplanted tissues. - Immune responses against transplanted tissues/organs caused by genetic differences between donor and recipient. - Most important difference = differences in highly polymorphic HLA class I and class II (transplant antigens) proteins coded for by MHC genes

HLA Matching for Marrow Transplants

Need to match at least 6 of 8, many transplant centers require 7 of 8 some screen all 10 8 HLA markers (allotypes): 2 each for HLA isotypes A, B, C DRB1 10 HLA markers: the above 8 plus 2 for HLA DRQ

Chronic Rejection Result

Occurs months or years post-op. Characterized by reactions in the vasculture of the graft that cause thickening of vessel walls and narrowing of the lumina. Blood supply becomes inadequate, causing ischemia and loss of function and graft eventually dies. - Causes failure of >50% kidney and heart transplants within 10 years of transplantation - Correlated with antibodies specific for *HLA class I* molecules on the graft

Negative Selection in the Thymus limits the Number of Expressed MHC Isoforms: Part 3

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Figure 15.9 Subtext

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Genetic Diseases for which Bone Marrow Transplantation is a Therapy

PIC Marrow transplants permanently replace an individual's entire hematopoietic system, including the immune system. Often used in patients who have *genetic diseases impairing the function of one or more types of hematopoietic cells*. (Also used in *genetically determined immunodeficiencies* such as *SCID*)

NK Mediated Graft versus Leukemia (GVL)

Patients given haploidentical transplant get no GVHDand require no further immunosupressive treatment after transplant. As their immune systems reconstitute, alloreactive NK cells can emerge. These provide a GVL effect that reduces the incidence of leukemia relapse. - NK-cell response waned and is undetectable 4 months post-op. With full reconstitution of the immune system, NK-cell population becomes tolerant to both recipient and donor cells

Minor Histocompatibility Antigens - Definition

Peptides of polymorphic cellular proteins that can lead to graft rejection when bound by MHC molecules and recognized by T-Cells

Conditioning Regimen

Prior to transplantation chemo and radiation are used to destroy rapidly multiplying marrow cells, attacking other rapidly multiplying tissues in the process - skin, intestines, liver. These tissues become inflamed (called "cytokine storm"), causing dendritic cells to activate and stimulate alloreactive T-Cells, as well as making the tissues more accessible to alloreactive effector T-Cells

Corticosteroids

Regulate many genes/pathways 1.) Induces expression of IκBα, inhibitor of NFκB 2.) Alter lymphocyte homin to prevent lymphocytes from entering secondary lymphoid tissues and being stimulated by alloantigens; preventing T-Cells from entering and attacking graft tissue Most effective as immunosupressive drugs right before transplantation so that cytokine gene expression is already altered at time of alloantigenic challenge. - Also used for acute immunosupression in rejection due to infections - have many adverse side-effects and continued use should be avoided whenever possible

Acute Graft-versus-Host Disease in Marrow Transplants

Rejection typically does not occur, but GVHD is a serious issue - major cause of mobidity and mortality after marrow transplantaion. Restricted to first few months after transplantation due to limited number of T-Cells in transplant. 1.) After transplantation, any mature CD4 and CD8 T-Cells in the graft that are specific to the the recipient's HLA allotypes become activated in secondary lymphoid tissues. 2.) Effector CD4 and CD8 T-Cells move into circulation and preferentially enter and *attack tissues that have been most damaged by the conditioning therapy of chemo/radiation - skin, intestines, and liver*

Isograft

Same MHC, syngenic

Autograft

Self, autologous

Minor Histocompatibility Antigens: Polymorphic Gene Encoded Proteins

Self-proteins are routinely digested by proteasomes in the cell's cytosol. Peptides from the proteins are delivered to the ER and bound to MHC I for presentation. If a polymorphic protein differs between a graft donor and recipient, it can give rise to an antigenic peptide that can be recognized by the recipient's T-Cells as non-self and elicit an immune response.

Tissue Reactions in GVHD

Severity of GVHD classified in 4 diagnostic stages Chart

HLA Matching Bone Marrow Donors

Success is directly correlated to the degree of HLA matching between patient and donor HLA matching: 1.) Reduces Graft-versus-Host Disease 2.) Ensures effective reconstitution of the adaptive immune system

Tacrolimus (FK506) (T-Cell Inhibitor)

Suppresses T-Cell activation w/ similar mechanism to Cyclosporin except the peptidyl-prolyl isomerase enzymes that it binds are *FK-binding proteins* instead of Cyclophilins FK-binding protein:Tacrolimus complex inhibits activation of NFAT

Reconstitution of the Immune System Post marrow Transplant

T-Cells developing from grafted stem cells migrate to Thymus for maturation under the influence of Thymic epithelial cells of the recipient and the HLA class I and II molecules they express. Developing T-Cells are positively selected fin the recipient's Thymus for interaction with recipient HLA allotypes. - to reconstitute the immune system, the new T-Cells MUST be able to respond to antigens presented by Professional APCs derived from bone marrow (which, after transplantation, will all be of donor type).

ABO Compatibility in Blood Transfusions

To prevent hemolysis and other transfusion reactions you must only give ABO compatible blood - Only 4 types to pick from: O, A, B, AB (much simpler than HLA) - Assessment of compatibility via Cross-Match Test

Alloreactions in Clinical Transplant

Transplant Rejection Graft-versus-Host Disease

Immune Privileged Antigens

Tumor associated antigens derived from normal cellular proteins to which the immune system isn't tolerant that become immunogenic when expressed by the tumor. Typically proteins expressed in immune-priveledged sites or proteins only expressed in very small amounts. e.g. Proteins expressed by immature sperm in testis, ot by early embryonic trophoblast - neither of which expresses HLA-A, HLA-B or HLA class II

Type A Compatibility

Type A can receive A or O, but not B or AB

Type O Compatibility

Type O lack A and B tolerance and will attach A, B or AB blood - can get O only

Graft-versus-Host Disease

When T-Cells created by implanted bone-marrow develop an alloreaction to and begin to attack the recipient's healthy tissues - effects almost all marrow recipients in varying degrees of severity

Graft-versus-Leukemia (GVL) Effect

When alloreactive T-Cells in the graft help to get rid of residual leukemia cells

Alloantigens Induce Tumor Rejection

When tumor cells are transplanted between MHC-incompatible patients, the tumor cells are rejected by the alloreactive immune response (CD8 T-Cells) to MHC differences. In MHC-compatible patients, the tumor cells are not rejected and the tumor is allowed to grow - if a tissue donor has cancer cells in the tissue these can then propagate in the recipient!!

Tissue Grafts Undergoing Chronic Rejection

are Infiltrated with CD4- espressing B-Cells and T-Helper cells expressing CD40 Ligand - consistent with antibodies being the cause of rejection

Type B Compatibility

can receive O or B, but not A or AB

Cancer/Testis (CT) Antigens

subset of tumor-associated antigens that are normally expressed only in immature sperm in testis. - Most genes (17/38) for CT antigens concentrated on X-Chromosomes - remainder expressed on autosomes, with no more than 3 CT genes each - Chart shows first 10 best characterized CT angitens

Type II Hypersensitivity Reaction

tissue-damaging immune reaction caused by the secondary response to small chemically active molecules that modify cell surface components and stimulate production of specific *IgG antibodies*

MLR T-Cell Activation

~*5% of the T-Cell population* can be activated by complexes of self-peptides and allogenic HLA class I and II encoded by a disparate HLA halotype. The strength of this response comparable to that induced by bacterial superantigens, emphasizes benefit of HLA matching and need for immunosuppressant drugs.