Regulatory T cells have been broadly implicated in immune homeostasis across a wide range of immune disorders including autoimmune disorders, infectious immunity and, most important, allogeneic organ transplantation. Thus, with the array of new drugs that are being developed and implemented in the transplant setting to block both acute and chronic graft rejection, it is essential that care is taken to ensure that these therapies do not interfere in Treg development and/or function.
In this study, we have focused on two such drugs, belatacept and basiliximab, a second-generation CTLA-4Ig and anti-CD25, respectively. Both drugs have been shown in animal models to compromise Treg activity and promote immunity by eliminating Tregs. Taking advantage of an ongoing phase II and phase III trials conducted by University of California San Francisco (UCSF) in conjunction with Bristol–Myers Squibb, we were able to study the short-and long-term effects of these drugs in patients treated with either an immunosuppressive regimen that included basiliximab, MMF and corticosteroids in conjunction with either a CNI or belatacept. The results show that belatacept treatment has no short- or long-term effects on circulating Treg numbers or function when compared to the CNI arm. In contrast, it appears that the anti-CD25 monoclonal antibody therapy led to a profound, but transient, reduction in CD4+CD25+FOXP3+ Tregs within 7 days of treatment. It remains possible that reduction of CD25+FOXP3+ cells was due to the generalized effects of organ transplantation and/or immunosuppression. However, as noted above, we observed that immunosuppressive treatment resulted in the selective loss of only the CD25+ subset of FOXP3+ cells. Previous studies have shown that basiliximab treatment leads to transient depletion of effector T cells.
Most significantly, an increased number of Treg cells were observed in rejecting allografts in patients treated with belatacept when compared to the CNI treated. Costimulation blockade has proven to be a very effective therapy in both small and large animal models of allograft transplantation. Thus, it was a surprise, and a concern, when some animal studies showed that CD28 was an essential costimulatory pathway for the development and survival of Tregs (17
). We have observed different results in our human studies—why? First, it is possible that human Tregs are not as sensitive to CD28 costimulation blockade as mouse Tregs. This might be due to a different balance of natural versus adaptive Tregs in the two settings (24
). For instance, our studies in the autoimmune nonobese diabetic mouse model have shown that adaptive Tregs can be induced in the CD28-deficient setting (25
). Thus, it is possible in humans that over time a larger number of adaptive Tregs may be created in response to the allotransplant and, thus, be more CD28 independent. Another possibility is that other costimulatory molecules may ‘substitute’ for the absence of CD28 blockade. In that regard, CD2 is not only highly expressed on human Tregs, but can also function as an extremely effective costimulatory pathway to promote Treg expansion and function (26
). Finally, we have noted that disruption of either of the two CD28 ligands, CD80 or CD86, in isolation had only minimal effects on Treg numbers in the periphery (data not shown). Moreover, although the treatment of mice with high concentrations of CTLA-4Ig led to a precipitous decline in the number of circulating Tregs, the Tregs returned rapidly reaching near-normal levels by 1 month (data not shown). Thus, it seemed possible that the long-term dosing schedule in kidney transplant recipients was subsaturating. The data presented here clearly showed that, although >95% of the CD86 was blocked immediately postdosing (due mostly to belatacept coating), only 80% of the CD86 molecule was blocked on the cell surface at the trough of the circulating drug. In fact, this level of ‘free’ CD86 is likely to be an underestimate of the percentage of free CD86 in the tissues, including the kidney allowing Tregs to get sufficient CD28 signals to maintain their survival.
The second surprising observation was the selective early loss of CD4+
Tregs during basiliximab therapy. Anti-CD25 mAbs have been approved for the treatment of kidney transplant recipients since the late 1990s. The antibody has been thought to target activated T cells and thus eliminate potential alloreactive cells. Thus, the finding that elimination of CD25+
T cells in animal models results in enhanced immunity and autoimmunity raised significant concern in the transplant setting. These concerns intensified when it became clear that CD25 deficiency resulted in loss of Tregs. However, to date there have been no reports of Treg deficiency in patients treated with these CD25-specific drugs early after induction therapy. In this study, we observed that anti-CD25 therapy resulted in a profound short-term depletion of CD25+
Tregs. This effect was not due to modulation of the CD25 molecule but due to elimination of the CD25+
T-cell subset as intracellular staining with a noncompeting antibody, as well as the use of other markers showed that the cells were indeed gone from the circulation (data not shown). However, all the FOXP3+
cells were not eliminated. In fact, the percentage of CD25−
Tregs was equal or increased over time. A combination of CD4 and CD127 was used to purify these cells and show that they retained their suppressive activity, although the functional suppressor assay of proliferation showed a somewhat compromised suppressor cell activity of the CD4+
cells presumably due to loss of CD25+
Treg subset. Finally, the cells returned by ~ 90 days posttreatment as did the suppressive function of the CD25+
Treg subset. The finding that the FOXP3+
returned to normal levels at 90 days is in contrast to the report of a protracted Treg decrease in basiliximab-and CNI-treated patients (27
), which may either reflect the antio-CD25 or the long-term CNI therapy. Importantly, we observed that the basiliximab therapy led to a dramatic decrease in CD25+
Tconv cells. This population presumably includes potential allograft-specific effector T cells. Thus, in spite of the transient Treg depletion following anti-CD25 therapy the net result was a shift toward regulation.
Another interesting observation was the finding of a significant increase of FOXP3+
cells in the rejecting allografts of patients treated with belatacept. In the study by Muthukumar et al., patients with increased expression of FOXP3 mRNA in the urine had better resolution of their rejection (13
). In our study, we had too few patients to evaluate the impact of FOXP3 cells on outcome, although in the overall phase II trial belatacept-treated patients had significantly better renal function and reduced incidence of chronic allograft nephropathy than the CNI-treated controls. The data suggest that belatacept may function to enhance Treg infiltration in renal allografts and facilitate recovery from rejection.
In summary, this study has shown that treatment with immunosuppressive drugs can have an inadvertent effect on Tregs. Whether the drug effects on Treg cells have an impact on the ultimate outcome of renal allografts remains unclear. However, in this study, we have demonstrated that belatacept therapy has no adverse impact on Treg cells and may in fact enhance their infiltration in the graft during rejection and counteract the effects from effector T cells.