Intravenous antigen administration as a therapy for autoimmune demyelinating disease.

Experimental allergic encephalomyelitis is a prototypic autoimmune disease characterized by central nervous system inflammation and demyelination. Previously, we demonstrated that intravenous administration of high doses of myelin basic protein abrogated the clinical and pathological signs of experimental allergic encephalomyelitis by causing the deletion of encephalitogenic, CD4+, myelin basic protein-specific T cells through antigen-induced programmed cell death. In the present study, we further characterized the ability of intravenous antigen administration to attenuate an immune response by myelin basic protein-reactive encephalitogenic T cells. We demonstrated that multiple injections of myelin basic protein are required to achieve a therapeutic response, and that this form of therapy is effective even after prolonged chronic disease. These studies showed that although interleukin-2-stimulated cell cycling is an important factor leading to T-cell death, the administration of exogenous interleukin-2 with antigen can result in the aggravation of clinical disease compared to administration of antigen alone. More importantly, administration of myelin basic protein alone without interleukin-2 was sufficient to reduce autoreactive T cells and clinical disease in experimental autoimmune encephalomyelitis. Our experiments support the rationale for antigen-specific therapy aimed at inducing the programmed death of autoreactive T cells in autoimmune diseases, potentially including the human demyelinating disease multiple sclerosis.

Antigen-induced programmed T cell death as a new approach to immune therapy.

We describe recent advances in understanding a mechanism of peripheral tolerance that operates through antigen-induced programmed cell death of mature T lymphocytes. A three-phase model of this process, termed propriocidal regulation, involves: (i) activation of T cells to express growth lymphokines and their receptors, (ii) lymphokine-stimulated cell-cycle progression, and (iii) T cell receptor reengagement leading to programmed cell death. Based on this model, antigen was used to treat experimental allergic encephalomyelitis and caused profound deletion of autoreactive, encephalitogenic T cells as well as dramatic clinical and pathological improvement of the disease. The potential strengths and weaknesses of this approach to the clinical treatment of T-cell-mediated diseases are discussed.

Parameters controlling the programmed death of mature mouse T lymphocytes in high-dose suppression.

We have characterized parameters of both T cells and antigen-presenting cells (APCs) that influence high-dose suppression due to apoptosis. Blockade of interleukin-2 (IL-2) utilization is shown to inhibit both proliferation and the ensuing death. An analysis of sublines of a mature T cell clone demonstrates a correlation between IL-2 receptor alpha chain (IL-2R) induction, increased proliferation, and greater suppression at high antigen doses. Profound loss of cells at high antigen dose was found to require at least 48 to 72 hr to develop. Antigen add-back experiments showed that strong T cell receptor reengagement of activated, cycling cells was essential for proliferative suppression and cell loss. Increasing the ratio of APC:T lymphocytes to 50:1 augmented cell death. For antigen-induced death of lymph node T cells, fresh T-depleted splenocytes were more effective than splenocytes that had been irradiated or treated with mitomycin C. Thus, T lymphocyte apoptosis at high antigen doses is a function of the activation response of the T lymphocyte as well as the efficiency of antigen presentation by the APC. These results strengthen the theory that apoptosis takes part in a feedback regulatory mechanism that has been called propriocidal regulation, which limits T cell expansion at high antigen doses.

Amelioration of autoimmune reactions by antigen-induced apoptosis of T cells.

We have shown that T cells vigorously cycling in response to growth lymphokines are driven into apoptosis by potent TCR restimulation. This process, termed propriocidal regulation, appears to be a normal feedback inhibitory mechanism to prevent excessive T cell proliferation and lymphokine production. Exposure of T cells to repeated high dose antigen treatments creates the conditions just described by activating T cells, and stimulating the production of growth lymphokines and their receptors. High growth lymphokine levels induced by the large amount of antigen present, stimulate vigorous cycling. The continued presence of high antigen levels subjects the cycling T cells to strong TCR restimulation as they enter the vulnerable S phase, inducing apoptosis in T cells responsive to the administered antigen. Thus, simple, repetitive, intravenous administration of high dose antigen may be used to delete potentially destructive clones of T cells, resulting in a state of peripheral tolerance. This has obvious therapeutic potential in disorders where the elimination of pathogenic T cell clones could be beneficial. We have described in EAE, an animal model for MS, that high dose MBP therapy is effective in preventing CNS pathology and the onset of disease as well as reducing the severity of the clinical symptoms of established EAE. We are currently involved in expanding this approach to other animal models of autoimmunity and graft rejection, as well as refining the immunotherapy in the EAE model with the objective of developing a clinical therapy for human demyelinating disease.

T cell deletion in high antigen dose therapy of autoimmune encephalomyelitis.

Encounters with antigen can stimulate T cells to become activated and proliferate, become nonresponsive to antigen, or to die. T cell death was shown to be a physiological response to interleukin-2-stimulated cell cycling and T cell receptor reengagement at high antigen doses. This feedback regulatory mechanism attenuates the immune response by deleting a portion of newly dividing, antigen-reactive T cells. This mechanism deleted autoreactive T cells and abrogated the clinical and pathological signs of autoimmune encephalomyelitis in mice after repetitive administration of myelin basic protein.

Stereochemical course of the reaction catalyzed by the cyclic GMP phosphodiesterase from retinal rod outer segments.

The stereochemical course of hydrolysis catalyzed by the cyclic GMP phosphodiesterase from bovine retinal rod outer segments was determined. The Sp diastereomer of guanosine 3',5'-cyclic monophosphorothioate was hydrolyzed by cyclic GMP phosphodiesterase in H2(18)O to give [16O,18O]guanosine 5'-monophosphorothioate. This isotopomer was reacted with diphenyl phosphorochloridate to form the two diastereomers of P1-(5'-guanosyl) P2-(diphenyl) 1-thiodiphosphate. The 31P NMR spectrum of this mixture of diastereomers was identical to that obtained from [16O,18O]guanosine 5'-monophosphorothioate resulting from the hydrolysis of the Rp diastereomer of guanosine 5'-p-nitrophenyl phosphorothioate by snake venom phosphodiesterase. This finding indicates that the 18O is bridging in the Rp diastereomer of the P1-(5'-guanosyl) P2-(diphenyl) 1-thiodiphosphate and nonbridging in the Sp diastereomer. As the snake venom phosphodiesterase reaction is known to proceed with retention of configuration, it follows that hydrolysis by retinal rod cyclic GMP phosphodiesterase proceeds with inversion of configuration at the phosphorus atom.