Permanent, lowered HLA class I expression using lentivirus vectors with shRNA constructs: Averting cytotoxicity by alloreactive T lymphocytes.

Transplantation of many tissues requires histocompatibility matching of human leukocyte antigens (HLA) to prevent graft rejection, to reduce the level of immunosuppression needed to maintain graft survival, and to minimize the risk of graft-versus-host disease, particularly in the case of bone marrow transplantation. However, recent advances in fields of gene delivery and genetic regulation technologies have opened the possibility of engineering grafts that display reduced levels of HLA expression. Suppression of HLA expression could help to overcome the limitations imposed by extensive HLA polymorphisms that restrict the availability of suitable donors, necessitate the maintenance of large donor registries, and complicate the logistics of procuring and delivering matched tissues and organs to the recipient. Accordingly, we investigated whether knockdown of HLA by RNA interference (RNAi), a ubiquitous regulatory system that can efficiently and selectively inhibit the expression of specific gene products, would enable allogeneic cells to evade immune recognition. For efficient and stable delivery of short hairpin-type RNAi constructs (shRNA), we employed lentivirus-based gene transfer vectors, which provide a delivery system that can achieve integration into genomic DNA, thereby permanently modifying transduced graft cells. Our results show that lentivirus-mediated delivery of shRNA targeting pan-Class I and allele-specific HLA can achieve efficient and dose-dependent reduction in surface expression of HLA in human cells, associated with enhanced resistance to alloreactive T lymphocyte-mediated cytotoxicity, while avoiding MHC-non-restricted killing. We hypothesize that RNAi-induced silencing of HLA expression has the potential to create histocompatibility-enhanced, and, eventually, perhaps "universally" compatible cellular grafts.

Hyperacute and acute kidney graft rejection due to antibodies against B cells.

Because of the perception of its uncertain clinical significance, the B cell crossmatch is not universally performed before renal transplantation. Even though sporadic cases of hyperacute rejection associated with B cell antibodies have been reported, doubts remain in light of other studies suggesting no effect on graft survival. This report describes 4 cases of graft rejection (3 hyperacute and 1 acute) that occurred in patients with anti-B-cell antibodies specific against donor HLA-DR or DQ antigens. Absence of anti-donor class I antibodies was confirmed in all cases by 2-color flow cytometry. Strong evidence for an antibody-mediated mechanism was found in one patient with anti-class I and anti-class II antibodies in serum transplanted with a class II mismatched kidney. In this case, only anti-class II antibodies were recovered in the eluate of the nephrectomy specimen. These four cases were compiled from three different institutions over a four-year period, which confirms the infrequent occurrence of these events. While anti-class II antibodies may not always be detrimental for graft survival, these results also confirm that they have the potential to cause hyperacute or acute graft loss. We conclude that the information provided by the B cell crossmatch should be available at the time that a decision to proceed with a renal transplant is made.

A cytotoxic monoclonal antibody to colon adenocarcinoma.

We have produced a monoclonal antibody CCOL1 that is cytotoxic at a titer of 1:20,000 using human sera as a complement source. CCOL1 reacts to the immunizing colon carcinoma line as well as to one other colon carcinoma line. However, it does not react with another colon adenocarcinoma line or with 28 other cultured tumor cells. It is noncytotoxic to lymphocytes, monocytes, granulocytes, RBC, and platelets. When tested by enzyme-linked immunosorbent assay against the normal cell membranes, it did not react to colon, lung, kidney cortex, heart, pancreas, liver, stomach, intestine, or kidney medulla but was strongly positive to the cell membrane of the colon adenocarcinoma line. The antibody reacted with 4 colon cancer tissue sections by immunoperoxidase. Control tissue sections of skin, spleen, kidney, brain, and uterus were negative. CCOL1 showed slight staining of epithelial cells in the tissue sections of normal lung, esophagus, colon, pancreas, and stomach. From preliminary studies, the antigen appears to be a glycoprotein, since it was highly sensitive to Pronase and sodium periodate.

HLA-DR3 associated with improved kidney transplant survival.

Using the National Kidney Recipient Pool and selecting all patients who were HLA-DR typed, immunized by either transfusion or kidney allograft, and had recorded cytotoxic antibody against a panel of lymphocytes, we found significantly lower levels of cytotoxic antibody in patients with HLA-DR3 (p less than 0.05). Moreover, when we examined patients with only HLA-DR3 (presumed homozygous) we found that the low response effect was even stronger and was significant at 30%, 67%, or 90% cytotoxic antibody cutoffs. One of the immediate predictions of postulating that homozygous HLA-DR3 patients are low responders is that these patients should have better kidney transplant survival. Indeed, when we examined transplant survival in HLA-DR3 homozygous transplant patients and non-HLA-DR3 patients, the 1-year survival was 74% +/- 9% vs. 49 +/- 4%, respectively. When one stratifies the data for transfusion effect, the 0-4 transfusion category shows 43% +/- 5% survival for non-HLA-DR3 recipients vs. 79% +/- 10% for HLA-DR3 only recipients. These data strongly suggest HLA-DR3 individuals have a low responsiveness to histocompatibility antigens.

Antibodies to human immunoglobulin detected by hemolysis of human immunoglobulin-coated red blood cells.

A hemolysis assay was developed to detect alloantibodies to human immunoglobulin. A total of 1035 serum samples was tested. Anti-IgM antibodies were found in 8% of 59 normal persons and in 13% of 439 multiparous women, with the highest incidence of 67% in 341 dialysis patients. Although the anti-IgM antibodies were inhibited by both IgM and IgG, it appeared that they were also inhibited by F(ab')2 bu not by Fc. Anti-IgG antibodies were more strongly inhibited by Fc than F(ab')2. These results suggest that anti-IgM antibodies might be analogous to antiidiotypic antibodies directed to F(ab')2, whereas anti-IgG antibodies tend to have greater reactivity to Fc.

Preliminary evidence of dual-marked lymphocytes in thoracic duct lymph fluid.

Thoracic duct lymphocytes from patients receiving thoracic duct drainage as a pretransplant therapy were examined for cell surface markers. Patients followed over the drainage time period showed a variable but decreasing percentage of E-rosette-positive cells in the lymph fluid. A substantial percentage of these E-rosette-positive cells also had C3 receptors on their cell surface. Reactions of the whole lymphocytes with a heteroantisera to human B-lymphocyte antigens reflected the increasing proportion of B cells in the samples, but also indicated that a fraction of the T cells have Ia-like antigens on their surface marker characteristics. Significance of these cells with respect to graft survival is discussed.

Human IgM anti-IgM cytotoxin for B lymphocytes.

B lymphocytes were shown previously to be killed at 5 degrees C by some autologous and allogeneic human sera. We show here that such cold cytotoxins are directed against immunoglobulins on the surface of B lymphocytes. The activity is partially removed by passing serum through IgG-coupled Sepharose and usually completely removed by passing serum through IgM-coupled Sepharose. Activity is regained from the columns by acid elution and IgM inhibits the cytotoxicity of these eluates. 125I-labeled eluates bind to IgG- and IgM-coupled Sepharose beads and binding is inhibited by IgM and to a lesser degree by IgG thus showing a primarily more avid binding to IgM as compared to IgG. Furthermore, the 125I-labeled cytotoxic eluates recognize the same determinant(s) on IgM and IgG. We suggest tht IgM anti-IgM antiimmunoglobulin may regulate immune reactivity by binding to B-lymphocyte surfaces.

IgM anti-IgM cold lymphocytotoxins to B cells.

Cytotoxic antibodies to B lymphocytes have been shown to be IgM. They are not absorbed by red blood cells and therefore not absorbed by red blood cells and therefore not directed against the I antigen of red cells. They are also not inhibited by mannose, as are certain natural cytotoxins against lymphocytes. Methods for producing purified eluates of IgM anti-IgM antibodies are given. These antibodies are postulated to be immunoregulative by acting on B lymphocytes.

A simple, rapid micro-latex fixation test.

A micro-latex fixation test (LFT) for the determination of rheumatoid factor (RF) is presented. Its advantages compared to similar tests are greater precision, simplicity, increased sensitivity, lower cost, reproducibility and adaptibility to large-scale testing. Micro-LFT titres are presented from a wide range of sample populations. The majority of normal samples show measureable titres whereas rheumatoid patients show high titres. A large sampling of pre- and post-transplant sera from kidney patients was studied and the micro-LFT titres were in the range of normal persons. The transplant sera were tested for lymphocytotoxic antibodies and no correlation was observed with the micro-LFT titres.

Thymectomy in the adult mouse: a new approach.

Adult mice were thymectomized using a retrosternal approach. The sternothyroid muscle was divided, the sternum was elevated, and the lobes of the thymus were removed by aspiration. The technique was used to thymectomize about 300 mice of several strains. The incidence of thymic remnants was less than 1% and losses from surgery were less than 10%.