Harnessing regulatory T cells for clinical use in transplantation: the end of the beginning.

Owing to the adverse effects of immunosuppression and an inability to prevent chronic rejection, there is a pressing need for alternative strategies to control alloimmunity. In three decades, regulatory T cells (Tregs) have evolved from a hypothetical mediator of adoptively transferred tolerance to a well-defined population that can be expanded ex vivo and returned safely to patients in clinical trials. Herein, we review the historical developments that have permitted these advances and the current status of clinical trials examining Tregs as a cellular therapy in transplantation. We conclude by discussing the critical unanswered questions that face this field in the coming years.

GITR ligation blocks allograft protection by induced CD25+CD4+ regulatory T cells without enhancing effector T-cell function.

The induction of operational tolerance prior to transplant could provide a solution to the complications of current immunosuppression in transplantation. In rodents, operational tolerance frequently correlates with the presence of CD25(+)CD4(+) regulatory T cells (Tregs) but their function is usually demonstrated by adoptive transfer into lymphopenic hosts leading some to question their relevance to normal immunocompetent recipients. The role of these cells in primary transplant recipients has been explored using anti-CD25 antibody but specific targeting of Treg is not possible since CD25 is also up-regulated on activated effector T cells. To overcome this limitation we targeted the Treg associated molecule GITR in tolerized primary transplant recipients. This reverses regulation resulting in acute allograft rejection. This is not due to co-stimulation of effector cells since rejection mediated by isolated populations of CD4(+)CD25(-) or CD8(+)CD25(-) T cells transferred into Rag(-/-) mice was not enhanced by anti-GITR antibody. Furthermore, GITR cross-linking does not provide co-stimulation for in vitro proliferation of the same CD4(+)CD25(-) or CD8(+)CD25(-) T-cell populations in response donor-strain APC. Thus, CD4(+)CD25(+)GITR(+) Treg play an essential role in early graft protection in primary transplant recipients following tolerance induction providing further support for protocols that might generate similar populations in clinical transplantation.

Critical role for IL-4 in the development of transplant arteriosclerosis in the absence of CD40-CD154 costimulation.

Blockade of the CD40-CD154 pathway can inhibit CD4(+) T cell activation but is unable to prevent immune responses mediated by CD8(+) T cells. However, even in the absence of CD8(+) T cells, inhibition of the CD40-CD154 pathway is insufficient to prevent the development of transplant arteriosclerosis. This study investigated the mechanisms of transplant arteriosclerosis in the absence of the CD40 pathway. C57BL/6 CD40(-/-) (H2(b)) recipients were transplanted with MHC-mismatched BALB/c (H2(d)) aortas. Transplant arteriosclerosis was evident in both CD40(-/-) and CD40(+/-) mice (intimal proliferation was 59 +/- 5% for CD40(-/-) mice vs 58 +/- 4% for CD40(+/-) mice) in the presence or absence of CD8(+) T cells (intimal proliferation was 46 +/- 7% for CD40(-/-) anti-CD8-treated mice vs 50 +/- 10% for CD40(+/-) anti-CD8-treated mice), confirming that CD8(+) T cells are not essential effector cells for the development of this disease. In CD40(-/-) recipients depleted of CD8(+) T cells, the number of eosinophils infiltrating the graft was markedly increased (109 +/- 24 eosinophils/grid for CD40(-/-) anti-CD8-treated mice vs 28 +/- 7 for CD40(+/-) anti-CD8-treated mice). The increased presence of eosinophils correlated with augmented intragraft production of IL-4. To test the hypothesis that IL-4 was responsible for the intimal proliferation, CD8 T cell-depleted CD40(-/-) recipients were treated with anti-IL-4 mAb. This resulted in significantly reduced eosinophil infiltration into the graft (12 +/- 5 eosinophils/grid for CD40(-/-) anti-CD8(+), anti-IL-4-treated mice vs 109 +/- 24 for CD40(-/-) anti-CD8-treated mice), intragraft eotaxin, CCR3 mRNA production, and the level of intimal proliferation (18 +/- 5% for CD40(-/-) anti-CD8(+)-, anti-IL-4-treated mice vs 46 +/- 7% for CD40(-/-) anti-CD8-treated mice). In conclusion, elevated intragraft IL-4 production results in an eosinophil infiltrate and is an important mechanism for CD8(+) T cell-independent transplant arteriosclerosis in the absence of CD40-CD154 costimulation.

Paper alert. Immunology.

A selection of interesting papers that were published in the two months before our press date in major journals most likely to report significant results in immunology.

Immunoregulation by CD4 T cells in the induction of specific immunological unresponsiveness to alloantigens in vivo: evidence for a reduction in the frequency of alloantigen-specific cytotoxic T cells in vitro.

Donor-specific unresponsiveness to allogeneic cardiac allografts in mice can be induced by the combined pretreatment with donor alloantigen and anti-CD4 antibody (anti-CD4+DST). We have investigated whether the induction of unresponsiveness in this model is due to the presence of T cells that regulate immune responsiveness towards the allograft. First, we analysed the functional characteristics of splenocytes from pretreated mice at the time of transplantation. A significant reduction in the frequency of donor specific cytotoxic precursor was found only after the anti-CD4+DST treatment. Next, we designed an in vitro assay to identify the phenotype of the splenocyte population responsible. CD4+ and CD4- fractions were purified from mice treated with anti-CD4+DST or anti-CD4 alone (controls) by cell sorting. Interestingly, only the addition of CD4+ cells from anti-CD4+DST treated mice resulted in a selective reduction and a bimodal distribution in the donor specific CTLp response, indicating the presence of a regulatory population. CD4+ cells from controls did not have this effect. These in vitro findings were substantiated by adoptive transfer experiments in vivo. These data demonstrate that CD4+ cells with the ability to regulate immune responsiveness to a cardiac allograft are present at the time of transplantation following pretreatment with donor alloantigen in combination with anti-CD4.

Ischemia-reperfusion-induced muscle damage. Protective effect of corticosteroids and antioxidants in rabbits.

We examined the potential protective effect of pretreatment with corticosteroids or antioxidants (ascorbic acid or allopurinol) in rabbits with reperfusion-induced damage to skeletal muscle after ischemia. 4 hours of limb ischemia induced by a pneumatic tourniquet, followed by reperfusion for 1 hour, caused a considerable amount of ultrastructural damage to the anterior tibialis muscles accompanied by a rise in circulating creatine kinase activity. Pretreatment of animals with depomedrone by a single 8 mg bolus injection led to a preservation of the anterior tibialis structure on both light and electron microscopy. High-dose continuous intravenous infusion with ascorbic acid (80 mg/hr) throughout the period of ischemia and reperfusion also preserved skeletal muscle structure, although allopurinol in various doses had no protective effect. These data are fully compatible with a mechanism of ischemia/reperfusion-induced injury to skeletal muscle, involving generation of oxygen radicals and neutrophil sequestration and activation. They also indicate that damage to human skeletal muscle caused by prolonged use of a tourniquet is likely to be reduced by simple pharmacological interventions.

Donor-recipient microchimerism is not required for tolerance induction following recipient pretreatment with donor-specific transfusion and anti-CD4 antibody. Evidence of a clear role for short-term antigen persistence.

There is considerable current interest in the possibility that long-term graft acceptance in clinical solid-organ transplantation might be dependent upon the development of a microchimeric state between the donor and recipient. This possibility has been prompted by the observation that in some transplant patients cells of donor origin can be detected in peripheral sites such as the skin, and it has been proposed that these cells play an essential role in maintaining graft survival. The hypothesis that peripheral microchimerism is an absolute requirement for the long-term survival of solid-organ allografts was tested in a well-characterized model of transplantation tolerance in which adult recipient mice are pretreated 28 days before transplant with a single donor-specific transfusion under the cover of a depleting anti-CD4 antibody. Mice pretreated with this protocol accept donor-specific cardiac allografts (MST > 100 days) but reject those of a third party (MST 16.5 days). The protocol leads to operational tolerance in the long term in that donor-specific skin grafts show prolonged survival while those from a third party strain are rejected acutely. Since peripheral blood contains haematopoietic stem cells we speculated that the success of the anti-CD4/DST protocol might be dependent on the development of microchimerism. To address this possibility the DST was irradiated before administration under anti-CD4 antibody cover in order to prevent donor stem cells in the transfusion from establishing a microchimeric state in the recipient animals. Mice in this group rejected their grafts acutely (MST 12 days), suggesting indeed that stem cells might be very important in the success of this model. However, when the protocol was modified by giving three additional doses of irradiated whole blood to increase the possibility that recipient T cells would be engaged during antibody-induced immunocompromise, graft prolongation was restored (MST > 100 days). These results demonstrate that persistence of donor antigen at the time of anti-CD4 antibody treatment is critical for the induction of unresponsiveness in this model and show that microchimerism is not an absolute requirement for long-term graft survival.

The progressive appearance of multiple urinary Bence-Jones proteins and serum paraproteins in a child with immune deficiency.

Multiple urinary Bence-Jones proteins and serum paraproteins were found in a child with type I dysgammaglobulinaemia (Seligmann et al., 1968). These showed a continually evolving pattern over a period of 4 months in relation to systemic infections and with no evidence of underlying malignancy.

A histopathologic, histobacteriologic, and radiographic study of periapical endodontic surgical specimens.

Data available on 230 periapical endodontic surgical specimens were studied. It was found that bacteria occurred in the periapical tissue very infrequently. In addition, there was no correlation between the presence of acute inflammatory cells and the presence or absence of pain. Granulomas with epithelium occurred in 61 out of 230 cases, while 14 of these were cysts. Of the 110 cases with radiographic follow-up data, 67 were classified as successful, 40 were uncertain, and 3 were unsuccessful according to a modification of Strindberg's criteria, whereas 107 would have been successes according to the criteria of Bender and Seltzer and their associates. No valid biologic or clinical basis for endodontic therapy as suggested by Bhaskar was found in this material.