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Immune function is critical in health and disease. The control and regulation of immune reactions is an area of intense investigation that has important implications for allogeneic hematopoietic cell transplantation. Immune reactions are regulated in a number of important ways. Compartmentalization of immune responses and the production of both pro-inflammatory and anti-inflammatory cytokines play a major role. More recently several populations of T cells that regulate immune responses termed regulatory T cells have been identified. This manuscript will focus on CD4+CD25+FoxP3+ natural regulatory T cells (Treg) and αβ TCR+CD4 +NK1.1+ natural killer T (NK-T) cells which both suppress graft vs host disease but appear to function by distinct mechanisms.
Allogeneic hematopoietic cell transplantation (HCT) has proven to be an effective therapy for a broad range of hematologic malignancies and selected solid tumors as well as genetic abnormalities. The major benefits of allogeneic HCT is through the introduction of the hemtolymphoid system of the donor which can correct errors of hematopoiesis and generate and immune response against the underlying malignancy. However, allogeneic HCT is limited by graft vs host disease (GVHD) which is caused by alloreactive donor derived immune effector cells. GVHD is characterized by dysregulation and excessive immune activation resulting in tissue damage primarily to the skin, gastrointestinal tract and liver. Control of GVHD holds great promise not only for improving allogeneic HCT as it is currently practiced but also to allow for extension to other potential indications such as for the treatment of patients with severe autoimmune disease or for the induction of tolerance to organ transplantation. To date a number of different populations of cells capable of regulating immune responses have been isolated and characterized in murine and other model systems. Here I will focus on the role of CD4+CD25+FoxP3+ natural regulatory T cells (Treg) and αβTCR+CD4+DX5+ natural killer T (NK-T) cells.
Isolation and purification of regulatory T cell populations has been possible though magnetic cell separation and cell sorting such that highly purified (95–99% purity) populations of both Treg and NK-T cells can be readily obtained and utilized in experimental models to elucidate function. The discovery of Treg was facilitated by the development of the mixed lymphocyte reaction (MLR). In this reaction, irradiated stimulator cell populations are mixed with responding cells for several days and then pulsed with 3H-thymidine. An alternative approach involves the use of responding cells that are labeled with the dye CFSE that is diluted upon cell proliferation. Responses are quantified by the incorporation of 3H-thymidine or dilution of the CFSE during cell division. The addition of Treg resulted in a marked suppression of the proliferation of the responding cells in a dose dependent manner. Typically, a 1:1 ratio between Treg and conventional CD4 and CD8 cells (Tcon) is required for full suppression in the MLR. Interestingly, in vitro the Treg do not proliferate. A number of groups have demonstrated that the addition of Treg to Tcon in animal models of allogeneic hematopoietic cell transplantation, both across major and minor histocompatibility barriers, resulted in suppression of graft-versus-host disease (GVHD) and improved survival as compared to animals that received Tcon alone1–4. These studies have demonstrated the ability of Treg to have biological function in vivo.
Using bioluminescent imaging (BLI), we have demonstrated that tumor cells marked with luciferase will actively proliferate in these in vivo models when T cell depleted bone marrow is used alone disease progresses and animals die after ~6–8 weeks of progressive leukemia5. When CD4 and CD8+ Tcon alone are added, animals have control of disease, however, these animals dye very rapidly due to GVHD with 2–3 weeks. Importantly, when Treg are added to the Tcon, there is a marked suppression of GVHD yet GVT reactions are maintained. Maintenance of GVT was demonstrated in two different tumor model systems using both A20 and BCL1 tumor models that has been reproduced by others. In these studies when Treg are added to T cell depleted bone marrow (TCD-BM), there is no GVHD yet no GVT effects. The GVT reaction is dependent upon the Tcon that is maintained in the presence of Treg 5, 6. The understanding of this apparent separation of GVHD and GVT effects involved the dissection of Tcon function in the presence or absence of Treg. In these studies the most striking findings were that Treg suppress Tcon proliferation demonstrated by BLI and assessment of the number of donor-derived Tcon in different tissues following HCT5. These studies have demonstrated that in vivo Treg do not block Tcon activation, but block their proliferation. In the setting such as GVHD, Tcon proliferation is required for the full impact of GVHD mediated infiltration and destruction of target tissues whereas in these models proliferation of alloreactive or tumor reactive T cells is not required for GVT effects. These observations are likely dependent upon the precursor frequency of donor derived alloreactive T cells as well as the tumor burden. The opposite situation would be a setting where immunization is being performed for a very low frequency T cell population where significant T cell proliferation is required for example during immunization with a tumor vaccine. Here Treg would block the manifestation of this immune reaction through suppression of Tcon proliferation and depletion of Treg could be potentially beneficial.
An important question is what are the trafficking and survival characteristics of Treg compared to Tcon. We have address this question using BLI and created transgenic animals which constitutively express luciferase7. In these models the trafficking and survival of different populations of cells can be readily assessed by isolating cell populations such as Treg and Tcon from the luc+ transgenic mice and transplanting these cells along with TCD-BM into irradiated wild type recipient mice. When comparing Treg to Tcon both cell populations actively proliferate in allogeneic recipients, especially following total body irradiation. Both cell populations initially migrate to nodal sites such as the cervical lymph nodes, mesenteric lymph nodes, and the spleen, actively proliferate in these sites and then infiltrate GVHD target organs. Interestingly, Treg do not cause GVHD. Within the first 10 days following transplantation, Treg proliferate as actively as Tcon, yet Tcon continue to proliferate and progress resulting in GVHD whereas Treg proliferation wanes after ~4–6 weeks and do not cause significant pathology8. The reasons that Treg proliferation is limited remain active areas of on-going investigation.
These studies suggested to us that the addition of Treg prior to Tcon, which could be readily accomplished both experimentally and in the clinic, may allow for infusion of lower numbers of Treg for control of GVHD if these cells were allowed to traffic to nodal sites and begin to proliferate. If successful this could help solve one of the major problems in translating these concepts to the clinic namely the relatively low numbers of Treg available. In fact, the ratio of Treg to Tcon of 1:1 required when both cell populations are given the same day can be reduced to 1:3 or even 1:10 if the Treg are given 48 hours prior to the Tcon9. These studies demonstrated that the administration of Treg prior to Tcon was more effective and have influenced the design of the clinical trials extending these observations to the clinic.
One of the major concerns relating to the use of Treg clinically is the question of whether the Treg will result in a state of global immunosuppression increasing the risks of opportunistic infections. We addressed this concern by exploring the immune reconstitution following both Tcon and Tcon plus Treg infusions. One of the major sites of GVHD induced tissue destruction are nodal tissues where it could be demonstrated that in the presence of Tcon, both lymph nodes and the thymus were damaged resulting in smaller atrophied organs with disrupted architecture. In these animals, CD4 and CD8 T cell reconstitution was impaired. To assess the functional capabilities of these animals an infectious agent in this case, murine CMV (mCMV) was used to infect the recipient animals resulted in lethality when the mMCV was given at days 14, 30 and 63. When Tcon and Treg were both utilized, this resulted in preservation of thymic and nodal architecture, improved immune reconstitution and ability to better control this infectious challenge9. The Tcon plus Treg treated animals were capable of mounting an immune response demonstrated by ELISPOT analysis against mCMV. These studies demonstrate that the combined use of Tcon plus Treg results in improved immune recovery due to blocking the GVHD-induced damage to nodal structures which is required for immune reconstitution. These studies have important implications for clinical translation.
Another population of immune regulatory cells termed NK-T cells has also been implicated in the control of GVHD10. These cells express markers typically found on T cells (αβTCR and CD4), as well as NK cells (NK1.1 and DX5). The potential role of NK-T cells in the control of GVHD has been highlighted by the development of the total lymphoid irradiation and anti-thymocyte globulin (TLI/ATG) regimen which has been effective in both murine models and patients11. In these murine systems, as well as in clinical translational studies, following TLI and ATG there is an increase in the number of NK-T cells which produce the immunoregulatory cytokine IL-4 required for the suppression of GVHD. Animals prepared with TLI/ATG can tolerate up to 1,000 times the number of T cells as TBI prepared animals. We evaluated the adoptive transfer of NK-T cells from donor animals in the same model systems described above for the analysis of Treg. NK-T cells could also be highly purified by magnetic bead separation and FACS to >98% purity. The NK-T cell population is composed of both a TCR variant and an invariant population of cells both present at ~50% of the cells which can be identified by the reactivity to CD1d and can be separated using a CD1d tetramer. Using BLI, we demonstrated that NK-T cells also actively proliferate in these animal models, yet also do not cause GVHD. NK-T cells persist much longer than Treg, over 100 days, and traffic to many of the same sites as conventional T cells yet don’t infiltrate target tissues as readily and are retained more in nodal structures. Interestingly, when NK-T cells were added to the same models of GVHD across major histocompatibility barriers induced by donor derived Tcon as few as 10–20,000 NK-T cells were capable of significantly improving animal survival as compared to control animals who received Tcon alone12. Paradoxically, increasing numbers of NK-T cells were less effective in these animal models for reasons that remain to be elucidated. Both variant and invariant NK-T cells appeared to function in these models and IL-4 production was a critically important feature of the NK-T cells. As previously described, Treg appear to control GVHD by suppressing Tcon proliferation which can be demonstrated by BLI. When NK-T cells were evaluated in the BLI experiments these cells did not suppress luc+ Tcon proliferation which actively proliferated in the presence or absence of NK-T cells. This suggested that NK-T cells control GVHD through a different mechanism not related to suppression of T cell proliferation and related to IL-4 production. The mechanisms by which these different populations of regulatory cells suppress the GVHD reaction are areas of ongoing investigation (Table 1).
These studies, as well as many others from a variety of different laboratories, have suggested that these regulatory cell populations could be ideal populations for clinical translation. We felt that Treg hold the greatest promise and set out to translate these findings to the clinic. There are a number of challenges that must be overcome including that these are rare cell populations with a paucity of unique markers for their isolation. The MLR is a useful but limited functional assay since no information can be obtained about the ability of the Treg to traffic to nodal sites can be obtained. Further a number of regulatory requirements must be satisfied that pose significant challenges to clinical translation. We and others have pursued magnetic bead based cell separation technologies. Using CD25 separation is a very effective first step resulting in ~50–70% FoxP3 expressing populations of cells. In these studies, recovery of ~60–70% can be readily obtained on a clinical scale and these cells have been used for clinical translation with limited toxicity. This approach has been applied in haploidentical transplantation with exciting initial results by the group from the University of Perugia. In their studies where rigorous T cell depletion is required in the absence of immunosuppressive medications, very low numbers of T cells of >5×104/kg are unacceptable due to the risks of GVHD13. The concept of mega CD34 cell utilization has also been extremely important and results in successful transplantation across these histocompatible barriers with high rates of engrafment and limited GVHD14. A major issue has been that these individuals are immunosuppressed and at risk for opportunistic infections and disease relapse. The addition of Tcon has been impossible since even low numbers of cells result in GVHD. The setting of haploidentical transplantation is an ideal setting in which to test the concepts of Treg biology since the number of Tcon that is acceptable is known and low, no immunosuppressive medications are required that can have differential impact on Treg function and there is an unmet clinical need. The initial results from the University of Perugia are highly encouraging where up to 2×106 were infused following Treg with low levels of GVHD15. These patients appeared to develop more rapid immune recovery compared to their historical experience.
We sought to purify Treg to the same level of homogeneity as achieved in the animal models. A frequent criticism of animal models is that they don’t always translate to the clinic, however, it is also frequent that the clinical studies are not performed with the same rigor as the model systems for many technical, as well as reagent availability reasons. Nevertheless, we felt it was important to purify the Treg for these translational studies to determine how likely they were to result in GVHD, especially across major histocompatibility barriers. High speed cell sorting has been utilized on a clinical scale to isolate hematopoietic stem cells with high purity. A similar strategy was utilized for the isolation of Treg. The first approach utilized CD25 as the primary approach for Treg isolation, however, this proved to be problematic in that there was significant variability amongst different individuals in the setting of the gates. In some individuals, the top 20% brightest CD25+ cells resulted in uniformly FoxP3+ cells whereas in other individuals 40 or 50% of the CD25+ cell population was found to be FoxP3+. Further, a significant number of FoxP3+ cells were found within the negative cell population resulting in low yields. A key finding was that Treg typically do not express the IL-7 receptor (CD127)16. Isolation of CD4+CD25+CD127lo/− cell population resulted in a uniformly FoxP3+ population with high yields of the FoxP3+ cells. In these pre-clinical studies purity of 96% and a yield of ~1×108 cells per apheresis product can be readily obtained. These cells can be cryopreserved, suppress the MLR and are stable for over 2 years once cryopreserved. We have developed the technologies and clinical trial to test these concepts which is currently enrolling patients.
In conclusion, murine studies have demonstrated that both Treg and NK-T cells are capable of suppressing GVHD while maintaining GVT reactions. Both cell populations proliferate in vivo in allogeneic lymphopenic hosts. These studies set the stage for clinical translation that is on-going by a number of different groups. Initial studies are very encouraging and have demonstrated that Treg based clinical studies are feasible, do not result in toxicity and appear to suppress GVHD risk of Tcon while promoting enhanced immune reconstitution. These studies are not only important for allogeneic hematopoietic cell transplantation but could have major implications for the treatment of autoimmune disorders, as well as for induction of tolerance to solid organ transplantation.
Source of Funding: Studies discussed here were funded by NIH award P01 CA049605 and HL075462.
Conflict of Interest: There are no conflicts to report.
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