Studies of the use of L-leucyl-L-leucine methyl ester in canine allogeneic marrow transplantation.

L-leucyl-L-leucine methyl ester (Leu-Leu-OMe) is a lysosomotropic agent that selectively kills cytotoxic T cells and their precursors, natural killer cells, and monocytes but not helper T cells or other cells of hematopoietic origin. In this study, the effects of treatment of bone marrow and peripheral blood buffy coat with Leu-Leu-OMe on the outcome of allogeneic marrow transplantation were studied in several canine models. Whereas incubation of autologous marrow with Leu-Leu-OMe had no adverse effects on subsequent engraftment, incubation of marrow from dog leukocyte antigen (DLA)-identical littermates resulted in a high rate of graft failure. Previous studies have demonstrated that the addition of peripheral blood buffy coat allows engraftment of unrelated DLA-nonidentical marrow, and in this study we found that incubation of buffy coat with Leu-Leu-OMe did not alter this graft promoting effect. In a final experiment it was demonstrated that incubation of both marrow and peripheral blood buffy coat did not prevent the development of graft-versus-host disease in recipients of marrow from DLA-haploidentical littermates. In considering the eventual application of Leu-Leu-OMe in the clinic, these results are less encouraging than those previously reported using murine models.

Monoclonal antibodies specific for canine CD4 and CD8 define functional T-lymphocyte subsets and high-density expression of CD4 by canine neutrophils.

The characteristics of canine homologues of CD4 and CD8, defined by murine monoclonal antibodies CA13.1E4 (IgG1) and CA9.JD3 (IgG2a) respectively, are described. These antibodies identify mutually exclusive subpopulations of non-B lymphocytes in peripheral lymphoid organs and blood. However, in thymus the antibodies defined populations of double-positive, double-negative and single-positive cells that showed a progressive maturation consistent with that described for CD4 and CD8 in other mammalian species. Furthermore, functional studies clearly associated cytotoxic effector cell function with the subpopulation reactive with CA9.JD3 (CD8). In contrast, proliferation stimulated by allogeneic cells and mitogens was more pronounced in the subpopulation reactive with CA13.1E4 (CD4). Cell and tissue distribution studies revealed that CA13.1E4 stained neutrophils with equivalent intensity to the staining of peripheral T cells. CA13.1E4 precipitated a 60 kD protein from the surface of T cells and highly purified neutrophils under both reducing and nonreducing conditions. CA9.JD3 precipitated a heterodimer of 32 kd and 36 kD under reducing conditions, and a 70 kD protein under non-reducing conditions. The expression of CD4 by canine neutrophils is without precedent in other mammalian species; the functional significance of neutrophil CD4 expression is puzzling in light of the current understanding of the functions of CD4 which include it's role as a receptor for nonpolymorphic regions of MHC class II molecules.

L-leucyl-L-leucine methyl ester treatment of canine marrow and peripheral blood cells. Inhibition of proliferative responses with maintenance of the capacity for autologous marrow engraftment.

Incubation of canine marrow and peripheral blood mononuclear cells with L-leucyl-L-leucine methyl ester resulted in the inhibition of mitogen- and alloantigen-induced blastogenesis, the elimination of allosensitized CTL and NK activity, and prevented the development of CTL from pCTL. The effects of these incubations were similar to those described in mice and humans. Additionally, in vitro CFU-GM growth from treated canine marrow was reduced, but could be regained when the Leu-Leu-OMe-treated marrow was cocultured with either untreated autologous peripheral blood mononuclear cells or monocyte-enriched PBMC but not with untreated monocyte-depleted PBMC. Six of seven dogs conditioned with 920 cGy total-body irradiation engrafted successfully after receiving autologous marrow that was incubated with Leu-Leu-OMe prior to infusion. These cumulative results indicate that incubation with Leu-Leu-OMe is a feasible method to deplete canine marrows of alloreactive and cytotoxic T cells prior to transplantation.

Comparative reactivity of anti-Ia monoclonal antibodies in man and laboratory animals.

Sixteen murine monoclonal antibodies (MAb), reactive with HLA-DR, DR + DP or DR + DQ, were tested, using indirect immunocytofluorescence, for their reactivity with peripheral blood mononuclear cells (PBMC) from the dog, cat, guinea pig, sheep, rabbit and rat. In addition, the MAb were evaluated for inhibitory activity in the canine mixed lymphocyte culture (MLC). Fourteen of 16 MAb reacted with canine PBMC. There was a greater tendency for DR + DP reactive MAb to inhibit canine MLC and subsequently react with PBMC of the guinea pig, sheep, and, cat. MAb failing to block the canine MLC were generally nonreactive with guinea pig PBMC (7 of 9 nonreactive) suggesting the guinea pig may be a useful model to study the functional relevance of specific Ia molecules. One MAb, H81.98.21 (reactive with HLA-DR) blocked canine MLC and reacted with PBMC from all species tested suggesting the determinant it recognized to be very well conserved in nature.

Selective inhibition of the canine mixed lymphocyte response by HLA-DR and DP-reactive monoclonal antibodies.

Twenty-three of 37 anti-Ia McAb reactive with human B cells, as determined by indirect immunocytofluorescence, were shown to be reactive with canine peripheral blood mononuclear cells (PBMC). Using a panel of human B cell lines that differ in their expression of HLA-DR, -DP, and -DQ molecules, it was shown that 15 of these antibodies identify HLA-DR and DP molecules (i.e., broadly reactive), while 22 identify only HLA-DR molecules. Fourteen of the 15 broadly reactive McAb were reactive with canine PBMC while only 9 of the 22 HLA-DR-specific McAb reacted with canine PBMC, suggesting that broadly reactive anti-Ia McAb are much more likely to react with canine cells than narrowly reactive McAb. Ten of the canine reactive McAb that were shown to identify typical Ia bimolecular structures on canine cells using immune precipitation analysis were tested for blocking activity in the canine mixed lymphocyte culture (MLC). All four of the broadly reactive McAb (B1F6, J-70, 9-49, and HB10a) plus two of the six narrowly reactive McAbs (H81.98.21 and H40.164.3) blocked the canine MLC when added to culture wells on day 0, suggesting that inhibition may be related to the specificity of the anti-Ia McAb employed. Since the MLC may reflect cellular interactions occurring during graft-versus-host disease, this assay may be useful for screening functionally relevant broadly reactive McAb in experimental canine bone marrow transplantation studies. These data suggest that the dog may be a useful model to study anti-Ia immunotherapy.

Two monoclonal antibodies recognizing subpopulations of canine T lymphocytes with or without suppressor/cytotoxic functions.

Two monoclonal antibodies (mAb) were studied in regards to their ability to differentiate functionally distinct subsets of canine lymphocytes. MAb DT-2 (IgG2a) reacts with 33-55% of canine peripheral blood mononuclear cells (PBMC), which are also surface immunoglobulin (SIg) negative. Cytolytic treatment with DT-2 eliminates the ability of PBMC to respond to allogeneic cells and mitogens but does not affect alloantigen-primed cytotoxic or suppressor cells. MAb E11 (IgG3) reacts with 15-40% of PBMC which are also SIg negative. Cytolytic treatment with E11 affects responses of PBMC to allogeneic cells or mitogens to a lesser extent than DT-2 but eliminates cytotoxic and suppressor cells among alloantigen-primed lymphocytes. Thus, canine helper cells appear to be Thy 1+, 7.2+, DT-2+, DLy 6+, DLy 1+, E11-, SIg-, and cytotoxic/suppressor cells Thy 1+, 7.2+, E11+, DT-2+, DLy 6-, DLy 1-, SIg-.

Specific tolerance and immunocompetence in haploidentical, but not in completely allogeneic, canine chimeras treated with methotrexate and cyclosporine.

Recipient dogs were conditioned with 9.2 Gy of total-body irradiation followed by the infusion of bone marrow and peripheral blood leukocytes from a DLA-haploidentical littermate (N = 10) or a completely allogeneic unrelated donor (n = 9). Graft-vs.-host disease (GVHD) prophylaxis consisted of methotrexate (MTX) and cyclosporine (CsA). Postgrafting all dogs were complete lymphohemopoietic chimeras. Lymphocytes of haploidentical chimeras without GVHD were unresponsive to stimulation by host lymphocytes cryopreserved pregrafting. Lymphocytes of haploidentical chimeras with GVHD proliferated in response to host cells, albeit less than donor cells pregrafting. In completely allogeneic chimeras, neither lymphocytes from dogs with GVHD, nor those from dogs without the disease showed responses to host lymphocytes. In addition, cells from haploidentical chimeras obtained early after transplantation nonspecifically suppressed donor cell proliferation. Later on, lymphocytes from dogs without GVHD showed specific suppression of donor cells, while lymphocytes from chimeras with GVHD continued to show nonspecific suppression. Cells from completely allogeneic chimeras both with and without GVHD never suppressed donor cells specifically. Both specific and nonspecific suppressor cells were enriched by nylon wool adherence, expressed T cell markers, and were not affected by the addition of indomethacin. Even after removing nylon wool-adherent cells, however, chimera cells were unresponsive to stimulation by host cells. By one year after transplant, chimera lymphocytes no longer showed suppression. In cell-mediated lympholysis assays, lymphocytes from all chimeras, regardless of GVHD, failed to generate cytotoxic cells against host cell targets. However, while haploidentical chimeras showed cytotoxicity against third-party targets, completely allogeneic chimeras did not. This deficiency was not overcome by the addition of mixed leukocyte culture supernatant or donor lymphocytes. All chimeras had basically normal antibody responses to keyhole limpet hemocyanin and phage X174. However, while haploidentical chimeras had normal responses to bacillus Calnette-Guerin (BCG) sensitization and rejected DLA-incompatible skin grafts within the normal time frame, completely allogeneic chimeras were not sensitized by BCG and showed delayed skin graft rejection. Histopathological studies revealed slow thymic reconstitution in all chimeras, particularly in the presence of GVHD. However, while healthy haploidentical chimeras eventually showed thymic histology normal for age, completely allogeneic chimeras did not.(ABSTRACT TRUNCATED AT 400 WORDS)

DLA-D-specific suppressor cells characterized by monoclonal antibodies.

Canine lymphocytes alloantigen-primed in mixed leukocyte culture (MLC) were tested for their ability to suppress MLC reactivity. These cells were added to fresh responder cells in MLC at ratios of 1:50 and 1:20 and suppressed reactivity 90 +/- 7% (mean +/- 1 SD) and 91 +/- 3% respectively. At lower ratios no suppression was observed. Monoclonal antibody E11 (IgG3) recognized a subset of canine Thy1+ T cells, and with complement treatment was capable of depleting suppressor cells from bulk MLC (11 +/- 6% of control suppression), but DT-2 (IgG2a), another canine T-cell antibody, had no such effect (84 +/- 11%) (P = 0.028). Antibody A5 (IgG2b), reactive with all T and most non-T cells, also eliminated suppression. Positively selected E11+ bulk-MLC cells suppressed (91%) and E11- bulk-MLC cells did not (4%). At the ratios described, suppression was specific for the DLA-D phenotype of the stimulator cells. In tests with the original stimulators, suppression was 91 +/- 7%; in tests with DLA-D identical stimulators it was 92 +/- 5%; and for DLA-D nonidentical stimulators it was 38 +/- 25% (P = 0.005). Nonspecific suppression increased with increasing numbers of suppressor cells. At ratios of 1:10 and 1:5 nonspecific suppression was 72 +/- 13% and 92 +/- 8%, respectively. These studies show that under appropriate conditions, Thy1+, E11+, A5+, DT-2- alloantigen-activated canine T lymphocytes function as DLA-D-specific suppressor cells and DT-2+ cells do not.