Steve Cobbold

T cell depletion with CAMPATH-1 in allogeneic bone marrow transplantation.

A total of 282 patients with leukemia have been treated by transplantation from HLA-matched siblings using marrow depleted of T cells with CAMPATH-1 and autologous complement. The incidence of graft-versushost disease (GVHD) of grades 2–4 was only 12% but the maximum incidence of graft failure was 15%. A significant increase in relapse cannot yet be detected in acute leukemia but relapse in chronic granulocytic leukemia (CGL) was substantially above that reported before T cell depletion. The most important predictive factor for relapse in CGL appeared to be slow engraftment. This finding suggests an alternative explanation for the graft-versus-leukemia effect other than a direct attack on leukemia cells. This is that donor T cells may affect the balance of competition between donor and recipient haemopoesis by preventing a rejection reaction to donor stem cells. Recipient leukemic cells would benefit (i.e. relapse) if recipient hemopoiesis gained an advantage. If this explanation were true we would expect extra immunosuppressive preconditioning of recipients to reduce the incidence of relapse, as well as preventing graft rejection.

Skin allograft rejection by L3/T4+ and Lyt-2+ T cell subsets.

The L3/T4+ and Lyt-2+ T-cell subsets can be depleted from mice, using selected monoclonal antibodies in vivo, at different times during rejection of, or priming to, allogeneic skin grafts. Although L3/T4+ cells are sufficient to reject skin grafts in naive Lyt-2-depleted mice, we show that Lyt-2+ cells can become involved, after an initial delay, in intact mice. Furthermore, these Lyt-2+ cells are primed to dominate the accelerated rejection of a normal secondary response. Mice depleted of L3/T4+ cells cannot be primed in this way, suggesting that priming of Lyt-2+ cells is dependent on help from L3/T4+ cells. However, in mice depleted of Lyt-2+ cells, priming for rapid rejection can be achieved, presumably via the L3/T4+ population. This suggests that the rejection of skin allografts in a given situation reflects different contributions of multiple effector mechanisms.

The Depletion of T Cell Subsets in Vitro and in Vivo

One of the major complications of allogeneic bone marrow transplantation is graft-versus-host disease. This can be avoided by removing the mature T cells from the marrow, most conveniently by the use of monoclonal antibodies. However, T cell purging results in an increased tendency for the recipient to reject the donor marrow. We have developed monoclonal antibodies to L3/T4 and Lyt-2 that specifically deplete functional T cell subsets in mice. We demonstrate that such reagents can be used to control both graft-versus-host disease and marrow rejection in mouse models of bone marrow transplantation across one-haplotype or two-haplotype major histocompatibility differences. Such strategies to abrogate host resistance, by administration of anti-T-cell monoclonal antibodies to the recipient, may complement marrow T cell purging for human allogeneic bone marrow transplantation.

Resistance to experimental autoimmune thyroiditis: L3T4+ cells as mediators of both thyroglobulin-activated and TSH-induced suppression.

Mechanisms suppressive to induction of murine experimental autoimmune thyroiditis (EAT) can be activated by pretreatment with tolerogenic doses of mouse thyroglobulin (MTg) or prior TSH infusion to raise circulatory MTg levels. MTg-activated suppressor T cells (Ts), shown earlier to be Thy-1+ and probably I-J+, were further characterized by in vivo administration of paired rat monoclonal antibodies to distinct epitopes on the L3T4 or Lyt-2 molecule, either on the day of, or subsequent to, initiation of the tolerogenic regimes. The cells required at the time of MTg pretreatment were L3T4+, Lyt-2- and low anti-L3T4 doses had no effect on their activation. The cells that mediated the strong MTg-induced resistance following pretreatment were also L3T4+; their suppressor function could only be abrogated by depletion of L3T4+, but not Lyt-2+, cells. Injection of cyclophosphamide (20-100 mg/kg) either prior to EAT induction or after Ts activation did not affect the severity of disease. Similarly, the suppressor state evoked by TSH infusion could only be abrogated by anti-L3T4 treatment. These findings indicate that both MTg-activated and TSH-induced suppression are mediated by L3T4+ cells. We hypothesize that MTg-specific Ts are present in normal, EAT-susceptible mice in low numbers to contribute to the maintenance of self-tolerance and that they are stimulated by increased levels of circulatory MTg to expand/differentiate and mediate the marked resistance to EAT induction.

Depletion of L3T4+ and Lyt-2+ cells by rat monoclonal antibodies alters the development of adoptively transferred experimental autoimmune thyroiditis

To delineate the contribution of L3T4+ and Lyt-2+ cells in the pathogenesis of experimental autoimmune thyroiditis (EAT), synergistic pairs of monoclonal antibodies (mAb) to the T cell subsets were used in conjunction with the adoptive transfer of mouse thyroglobulin (MTg)-activated cells from immunized mice. Initial experiments verified the important role of L3T4+ cells in the transfer of EAT. Subsequent experiments pointed to the relative contribution of both L3T4+ and Lyt-2+ cells, depending on the stage and extent of disease development. Treatment during disease with L3T4, but not Lyt-2, mAb alone significantly reduced thyroiditis. However, in situ analysis of the cellular infiltrate in thyroid sections revealed that, after treatment with mAb, the appropriate subset was eliminated without altering the amount of the other subset in the remaining lesion. In addition, treatment during severe thyroiditis following the transfer of MTg-activated lymph node cells showed that Lyt-2 mAb alone also reduced thyroid infiltration. When the recipients were pretreated with either pair of mAb before transfer, disease development was only moderately affected. We conclude that (i) donor L3T4+ cells are the primary cells responsible for the initial transfer and development of thyroiditis; and (ii) previous in vitro cytotoxicity data, plus current monoclonal antibody treatment of disease and in situ analysis, further implicate a role for Lyt-2+ cells in EAT pathogenesis.

Immunohistological screening in the selection of monoclonal antibodies: the use of isotype-specific antiglobulins

This paper describes the use of the immunoperoxidase technique for the screening of rat hybridoma culture supernatants on tissue sections. By combining the avidin-biotin system with mouse monoclonal antibodies specific to different rat immunoglobulin isotypes, it is possible to resolve the specificity patterns of complex mixtures of monoclonal antibodies from uncloned culture wells. This strategy is particularly useful in the derivation of monoclonal antibodies to cell surface antigens.

A Theoretical Framework for Self-tolerance and its Relevance to Therapy of Autoimmune Disease

Abstract Therapeutic intervention in autoimmune diseases should be based on a knowledge of how the normal immune system maintains unresponsiveness to ‘self’ and how this state of unresponsiveness may be broken. We have proposed that ‘self’ from the viewpoint of T cells may represent only a small fraction of the peptides that are available in the body. These would be the peptides that successfully access MHC molecules on a limited number of antigen presenting cells. As the number of self peptides is far greater than that of useful MHC molecules, then the set that are privileged to access MHC on presenting cells will compete or buffer out the others. In other words the peptides which are immunologically visible establish tolerance to themselves whilst ensuring that many others remain cryptic. On this model, organ-specific autoimmunity is not a breakdown of tolerance but rather a failure to keep certain peptides from associating with MHC molecules on cells involved in antigen presentation. This could be at either the inductive side of the response or on the target side if mimicry by foreign antigens has primed the effector arm of the immune response. Monoclonal antibodies (MoAbs) have proved to be useful immuno-suppressive agents. MoAbs to certain T-cell adhesion molecules may also permit tolerance to occur to antigens administered simultaneously with them. The possibility of establishing tolerance to exposed peptides in autoimmunity is discussed. We propose that T cells whatever their stage of maturation can be tolerized as long as they see antigen in the absence of helpful stimuli from other cells.

Rat monoclonal antibodies for bone marrow transplantation--the CAMPATH series.

Successful allogeneic marrow grafting requires that T-cell mediated responses of both donor and recipient be prevented (Korngold and Sprent, 1978; van Bekkum, 1984). Marrow transplantation is unique in the sense that there is an interplay between Graft versus Host (GvHD) and Host versus Graft (HvG) processes. Subclinical GvHD probably contributes to the immunosuppression which guarantees stem cell engraftment. The corollary of this is that success at purging T cells from donor marrow risks the prospect of marrow rejection by the recipient (see van Bekkum, 1984; Vallera et al, 1982; Vriesendorp et al, 198lab Miller et al, 1983, Wagemaker et al, 1981).

CD 4 and CD 8 monoclonal antibody therapy in canine renal allografts

Abstract Therapy with CD 4 and CD 8 monoclonal antibodies was evaluated in dogs which received double‐haplotype MHC‐mismatched renal allografts. Neither CD4 nor CD8 monoclonal antibodies given alone prolonged allografts survival (creatinine ≥ 300 μol/l) beyond 7 days. However, combined therapy with CD 4 and CD 8 antibodies given up to day 10 did prolong allograft survival to a median of 14 days. A longer (21 day) course of CD4 and CD 8 antibodies did not extend allograft survival further. The effect of prolonged antibody therapy was restricted by the occurrence of both an antiglobulin response and an anaphylactoid reaction to the monoclonal antibody preparation. When the CD 4 and CD 8 antibodies were combined with a pan‐T‐cell‐depleting Thy‐1 antibody, the survival of double‐haplotype mismatched allografts was further prolonged (median 16 days). The median survival of single‐haplotype mismatched renal allografts on this triple therapy was 21 days, with one surviving to day 36.