Christine C. B. Soderling

Bone marrow transplantation across major histocompatibility barriers in mice. Effect of elimination of t cells from donor grafts by treatment with monoclonal thy-1.2 Plus complement or antibody alone.

SUMMARY The current studies were designed to evaluate optimal conditions for reduction of graft-versus-host disease (GVHD) by removal of donor T cells from bone marrow inoculum. A model was used in which the addition of spleen cells to donor marrow heavily favored the development of lethal GVHD. Treatment of donor bone marrow plus spleen cells with monoclonal anti-Thy-1.2 antibody plus complement protected lethally irradiated recipients from GVHD across major histocompatibility barriers better than donor cells treated with the same dilution of antibody alone. Engraftment was demonstrated by the presence of high percentages of donor cells in the peripheral blood of these animals and the long-term survival of donor skin grafts. These results may be important in light of the development of new antihuman T cell monoclonal antibodies which may be used in the treatment of donor marrow in clinical transplantation.

Monoclonal antibody-toxin conjugates reactive against human T lymphocytes: A comparison of antibody linked to intact ricin toxin with antibody linked to ricin A chain

A non-complement-binding monoclonal antibody, TAIL, recognizing determinants on human T lymphocytes, was linked to the plant seed toxin ricin, either the intact molecule or purified ricin A chain. Peripheral blood lymphocytes were pretreated with conjugate for 2 hr, washed, and then measured in vitro for T cell proliferation. Studies showed that antibody-intact ricin conjugates were up to 39-fold more inhibitory than antibody-A-chain conjugates. Killing was selective because an unreactive control antibody linked to toxin had minimal inhibitory effect. Dose response curves obtained in human studies were nearly identical to curves obtained in an animal model in which a monoclonal anti-murine T cell antibody (anti-Thy 1.1) was linked to ricin and ricin A chain. In the human system, longer exposures of peripheral blood cells to conjugates did not alter our findings. TA-1 -ricin conjugates were tested against human ALL cell lines. KOPN-1, a cell line bearing the determinant reactive with TA-1 was selectively eliminated within 2 days after pretreatment with 500 ng/ml. Even 10-fold greater concentrations of TA-1-A chain were not adequate for leukemic cell destruction. These findings (1) show for the first time in a human model that monoclonal antibodies, directed against certain differentiation antigens when linked to ricin A chain are not as effective in normal or malignant cell killing as when linked to intact ricin; (2) contribute to the growing body of evidence showing that monoclonal antibody A chain conjugates do not permit the acquisition of levels of toxin sufficient to destroy target cells; and (3) are important relative to increasing interest in use of antibody-toxin conjugates for graft-versus-host disease prophylaxis in allogeneic bone marrow transplantation.

Absence of a facilitory role for NK 1.1-positive donor cells in engraftment across a major histocompatibility barrier in mice.

Ex vivo T cell depletion of donor marrow grafts in humans and mice has virtually eliminated severe graft-versus-host disease (GVHD). However, as a consequencl of T cell depletion, sustained donor cell engraftment is likely compromised. Since the majority of T cell depletion techniques also deplete natural killer (NK) cells, we investigated the role of donor NK cells in engraftment and GVHD in a murine model. Using a monoclonal antibody directed against an NK-specific epitope, we have selectively depleted NK cells while preserving donor marrow T cells. In an established model of engraftment, selective NK depletion demonstrated that removal of donor NK cells did not impair the engraftment process under conditions in which donors and recipients are major histocompatibility complex–disparate. In contrast, recipients of anti-Thy 1.2 plus complement (entreated marrow grafts had a significantly higher incidence of either partial engraftment or graft rejection as compared with recipients of selective NK-depleted donor grafts or control grafts. In addition, we have observed that NK-specific depletion of donor marrow/ splenocyte inocula does not alter the incidence of GVHD. Recipients of NK-depleted donor grafts developed lethal acute GVHD, whereas recipients of anti-Thy 1.2–depleted donor grafts did not (P < 0.0001). Interestingly, NK 1.1–depleted donor graft recipients had a significantly increased mortality in comparison with control groups receiving C‘-treated grafts (P = 0.04) or anti-Thy 1.2 plus C’-treated grafts (P < 0.05). Thus, NK depletion may reduce immunosurveillance, thereby increasing the risk of posttransplant infection. We conclude from these results that donor NK cells play an insignificant role in engraftment as well as in the afferent phase of GVHD, but may be important in immunosurveillance when murine bone marrow is transplanted across the major histocompatibility barrier.

The effect of T subset depletion on the incidence of lethal graft-versus-host disease in a murine major-histocompatibility-complex−mismatched transplantation system

Experiences sur souris. Lors de greffes de moelle+cellules spleniques incompatibles, la maladie GVH est evitee si les cellules Lyt2 + et L 3 T 4 + ont ete eliminees prealablement

Bone marrow transplantation across major histocompatibility barriers in mice: IV. Graft-versus-host disease in TLI-conditioned mice

We studied the effect of transplanting lymphohematopoietic grafts across major histocompatibility barriers into mice conditioned with either single dose 900 rad total body irradiation (TBI), fractionated 17 × 200 rad total lymphoid irradiation (TLI, or single dose 900 rad TLI plus cyclophosphamide (TLI + CY). In all three conditioning regimens, survival following BALBc → C57BL6 grafting was inversely related to the number of immunocompetent T cells in the donor graft. These findings show that regardless of conditioning protocol (TBI or TLI), it was not possible to protect mice from lethal GvHD when the number of T cells in donor grafts approximated those found in patient bone marrow aspirates. When T cells were eliminated from the donor graft by pretreatment with monoclonal anti-Thy 1.2 plus complement prior to injection into TBI-conditioned recipients, long-term protection from lethal GvHD was indeed possible.

Assessment of immunocompetence by limiting dilution analysis in long-term T cell depletion chimeras transplanted across the MHC barrier

Chimeras were generated in a system in which donor C57BL/6 bone marrow plus spleen cells were T-cell-depleted prior to transplantation into lethally irradiated DBA/2 recipients. This protocol permits donor lymphohematopoietic engraftment and protects transplanted mice from development of lethal GVHD. The frequencies of alloantigen-specific cytotoxic T cells (CTL) and/or CTL precursors (CTL-P) in the chimera spleens were determined by limiting dilution analysis. This identified a small population of host-reactive CTL-P. The presence of host-reactive CTL-P in the absence of detectable anti-host immune response raises questions concerning the maintenance of the tolerant state in chimeras. Using mixtures of chimera and normal C57BL/6 splenocytes we found no evidence by limiting-dilution analysis for regulatory cells capable of dampening antihost immune reactivity in chimera spleens. We next measured the frequency of third-party-reactive CTL-P in chimeras. Chimeras displayed low CTL-P frequency by the 30th day posttransplant, which increased 15–21-fold over a five-month interval. Interestingly, both chimeric and irradiated syngeneic reconstituted control mice recovered anti-third-party CTL-P at a similar rate, but CTL-P levels never reached those measured in normal unirradiated control mice, suggesting that the radiation regimen has a long-lasting influence on host immunocompetence. In concomitant experiments we measured third-party CTL generation in MLC. Our findings suggest that measurement of CTL generation in MLC may be a less sensitive assessment of immunocompetence than LDA analysis. Our data also suggest that irradiated T-cell-depleted chimeras may suffer prolonged immunologic deficiencies based on reduced frequencies of alloreactive CTL-P.