**7. Cure of autoimmune diseases by cord blood transplantation**

Although many patients with autoimmune diseases (AD) can have a relatively normal life expectancy with treatment, some patients with severe, progressive and therapy-refractory autoimmunity require more than medication. Patients with systemic sclerosis (SSc), for example, have shown disappointing results in prospective randomized trials of almost all therapeutic agents reported [102]; therefore, HSCT, both autologous and allogeneic, has been proposed as an effective potential treatment for such patients.

The basis for autologous HSCT for AD is the immune system reset by the generation of fresh self-tolerant lymphocytes after chemotherapy-induced elimination of self or auto-reactive lymphocytes. Elimination of auto-reactive T-effector cells, long-living plasma cells and antigen-presenting cells as well as increased T-regulatory cells, restoration of thymic function, normalization of T-receptor repertoire, reduced auto-antibodies and long-lasting lymphopenia are the intended effects of autologous HSCT for AD [103]. Autologous HSCT carries the risk that if the basic defect of the AD is in the stem cell, the autoimmunity will probably recur after autologous HSCT; however, if the primary defect is an aberrant immune response to an acquired, for example, viral antigen, or self-antigen, there is a theoretical possibility that tolerance may be acquired in the newly reconstituted autologous immune system—assuming ablation of the offending memory cells.

Autologous HSCT has been applied to treat severe autoimmune diseases (SAD) since 1996 [104], when some of the first autologous transplants specifically for AD were performed by Tyndall and colleagues [105]. Autologous HSCT for AD is based on the elimination of autoreactive effectors through potent immunosuppressive conditioning followed by subsequent regeneration of self-tolerated lymphocytes capable of "resetting" the immune system. This approach has been recognized to induce remission for some patients with various AD, including SSc, systemic lupus erythematosus (SLE), multiple sclerosis (MS), rheumatoid arthritis (RA), adjuvant arthritis and severe Crohn's disease [104–112]. Multiple sclerosis has been the main indication for autologous HSCT, along with SLE, therapy-refractory Crohn's, vasculitis, autoimmune cytopenia, diabetes mellitus type 1, polyarthritis, adults with rheumatoid arthritis and children with juvenile idiopathic arthritis; however, RA relapse is frequent [103]. According to the data from European Group for Blood and Marrow Transplantation (EBMT) registry from 1996 to 2007, the 5-year OS was 85% among the 900 patients who underwent autologous HSCT for AD; however, the 5-year progression-free survival (PFS) was only 43% [104]. Even lower values, 33% for 5-year PFS (78% for 5-year OS), were reported by the British Society of Blood and Marrow Transplantation (BSBMT) data registry for 1997– 2009 [113]. Although these studies proved the relative safety of autologous HSCT, the fact that more than half of the patients suffered from disease relapse is unsatisfactory. This was especially problematic for patients with RA, with a 3-year PFS of only 23% despite its high 3 year OS of 98% [104]. This tendency to relapse was also confirmed in the report from Snowden et al. [111]. With data from both EBMT and Autologous Blood and Marrow Transplant Registry (ABMTR), Snowden suggested that the majority of RA patients experienced a reactivation of the disease eventually and required the re-introduction of immunosuppressive drugs. Though somewhat better than RA, autologous HSCT for other ADs is also hampered by disease progression or relapse, showing unimpressive 3-year PFS ranging from 34 to 63% [104]. Illei and colleagues suggested that the reactivation of lupus is a major contributing factor to the deaths of SLE patients after autologous HSCT treatment [114]. Similar association between autologous HSCT and relapse was also identified with SSc patients [115]. T cell depletion has been proposed to be a relapse-preventing strategy in several clinical trials; however, no significant improvement in either OS or PFS has been observed [104]. The unavoidable high recurrence rate, together with the elevated risks of stem collection procedures on AD patients themselves, lead to increased adverse events or even mortality of patients.

Compared to autologous HSCT, which only produces sustained responses in 30–40% patients, allogeneic HSCT can achieve sustained response in 60–70% patients [113]. Allogeneic HSCT, which does not need stem cell collection from the patient and is less prone to disease relapse, is therefore considered higher GvHD and TRM risk, but may produce more favorable longterm results with AD relapse. In fact, evidence for the potential of HSCT to cure or ameliorate AD comes from many cases when patients undergo allogeneic HSCT for another indication with coincident AD, such as RA, psoriasis, psoriatic arthritis and ulcerative colitis, with the AD often in remission post-transplant. Moreover, the converse observation has also been reported, that is, passive transfer of AD from the donor graft through allogeneic HSCT, including myasthenia gravis, Graves' disease and autoimmune diabetes mellitus.

antigen-presenting cells as well as increased T-regulatory cells, restoration of thymic function, normalization of T-receptor repertoire, reduced auto-antibodies and long-lasting lymphopenia are the intended effects of autologous HSCT for AD [103]. Autologous HSCT carries the risk that if the basic defect of the AD is in the stem cell, the autoimmunity will probably recur after autologous HSCT; however, if the primary defect is an aberrant immune response to an acquired, for example, viral antigen, or self-antigen, there is a theoretical possibility that tolerance may be acquired in the newly reconstituted autologous immune system—assuming

Autologous HSCT has been applied to treat severe autoimmune diseases (SAD) since 1996 [104], when some of the first autologous transplants specifically for AD were performed by Tyndall and colleagues [105]. Autologous HSCT for AD is based on the elimination of autoreactive effectors through potent immunosuppressive conditioning followed by subsequent regeneration of self-tolerated lymphocytes capable of "resetting" the immune system. This approach has been recognized to induce remission for some patients with various AD, including SSc, systemic lupus erythematosus (SLE), multiple sclerosis (MS), rheumatoid arthritis (RA), adjuvant arthritis and severe Crohn's disease [104–112]. Multiple sclerosis has been the main indication for autologous HSCT, along with SLE, therapy-refractory Crohn's, vasculitis, autoimmune cytopenia, diabetes mellitus type 1, polyarthritis, adults with rheumatoid arthritis and children with juvenile idiopathic arthritis; however, RA relapse is frequent [103]. According to the data from European Group for Blood and Marrow Transplantation (EBMT) registry from 1996 to 2007, the 5-year OS was 85% among the 900 patients who underwent autologous HSCT for AD; however, the 5-year progression-free survival (PFS) was only 43% [104]. Even lower values, 33% for 5-year PFS (78% for 5-year OS), were reported by the British Society of Blood and Marrow Transplantation (BSBMT) data registry for 1997– 2009 [113]. Although these studies proved the relative safety of autologous HSCT, the fact that more than half of the patients suffered from disease relapse is unsatisfactory. This was especially problematic for patients with RA, with a 3-year PFS of only 23% despite its high 3 year OS of 98% [104]. This tendency to relapse was also confirmed in the report from Snowden et al. [111]. With data from both EBMT and Autologous Blood and Marrow Transplant Registry (ABMTR), Snowden suggested that the majority of RA patients experienced a reactivation of the disease eventually and required the re-introduction of immunosuppressive drugs. Though somewhat better than RA, autologous HSCT for other ADs is also hampered by disease progression or relapse, showing unimpressive 3-year PFS ranging from 34 to 63% [104]. Illei and colleagues suggested that the reactivation of lupus is a major contributing factor to the deaths of SLE patients after autologous HSCT treatment [114]. Similar association between autologous HSCT and relapse was also identified with SSc patients [115]. T cell depletion has been proposed to be a relapse-preventing strategy in several clinical trials; however, no significant improvement in either OS or PFS has been observed [104]. The unavoidable high recurrence rate, together with the elevated risks of stem collection procedures on AD patients

themselves, lead to increased adverse events or even mortality of patients.

Compared to autologous HSCT, which only produces sustained responses in 30–40% patients, allogeneic HSCT can achieve sustained response in 60–70% patients [113]. Allogeneic HSCT,

ablation of the offending memory cells.

204 Umbilical Cord Blood Banking for Clinical Application and Regenerative Medicine

In all but one of the cases reporting the use of allogeneic HSCT for concomitant AD, the donor was an HLA-identical sibling. Therefore, one possible explanation for recurrence of the AD may be the presence of shared genetic factors between the related donors and recipients. Another possible explanation is the persistence of host immune cells resulting in recurrence of disease activity. These case reports, although small in number, were important in establishing our understanding of the potential role of allogeneic HSCT for treatment of patients with severe autoimmune diseases.

The failure of autologous HSCT to cure spontaneous-onset AD and to maintain long-term regression could be due to the incomplete elimination of self-responsive memory T cells and B cells. While the conditioning regimes through chemotherapy or irradiation are unable to eradicate every single memory lymphocyte, complete immune ablation can be achieved by allogeneic HSCT via combined effects of immune system replacement and graft-versusautoimmunity (GVA) effect [116]. In allogeneic HSCT, the host auto-reactive immune system is replaced by the donors' non-auto-reactive, but potentially alloreactive cells. Therefore, alloreactive donor lymphocytes can undergo a GVA process similar to the graft-versusleukemia (GVL) effect or GvHD and attack the residual self-reactive host effectors [116, 117]. Thus, the biggest problem with allogeneic adult donor HSCT for AD is GvHD. In fact, several cases of complete donor chimerism after adult donor HSCT cured the patients' AD, but were accompanied by severe acute or chronic GvHD post-transplant [118, 119]. In contrast, cases of mixed chimerism, which sometimes occurs with reduced intensity or non-myeloablative conditioning regimens, often exhibit mild and sometimes no GvHD [120–122]. Despite the differences in the conditioning regimen used by the different groups, many patients treated with allogeneic HSCT have shown sustained remission to AD and amelioration of symptoms, and among patients with severe treatment-refractory ADs, the response rate was higher than 75% [123]. With reduced intensity conditioning, the adverse effects and treatment toxicity observed in myeloablative autologous HSCT could be reduced [107]. However, relatively high transplant-related mortality (22.1% at 2 years and 33.7% at 5 years) and GvHD remain as the biggest challenges to allogeneic HSCT for AD [123]; yet, the 5-year OS for allogeneic HSCT is not significantly different than the 78% 5-year OS for autologous HSCT reported by the BSBMT [113], but appears to be lower than the 85% OS for autologous HSCT reported by the EMBT [104].

To minimize the risks of GvHD with allogeneic HSCT for AD, there are emerging interests in using CB as the alternative source of stem cells to the traditionally used (or simply "traditional") adult stem cell sources of bone marrow (BM) and peripheral blood (PB). While tolerating far greater degrees of HLA mismatch, CBT is still associated with lower incidences and severity of acute and chronic GvHD than HSCT from adult donors [124]. According to the recent study of 143 patients in 2010, the rate of grade II or higher GvHD is only 9% among HLA-matched (n = 60) and 50% among HLA-mismatched (n = 18) patients [125]. Being less mature than stem cells from adult grafts, CB stem cells show lower alloreactivity and immunogenicity without increasing long-term relapse incidences [126]. Moreover, CB has other advantages including low contamination rate, easy storage and immediate accessibility via cryopreservation [125].

Together with its relatively lenient requirement for HLA matching, CB has been used commonly as an alternative source for transplantation. Indeed, one of the first cases to treat AD with CBT was performed due to lack of HLA-matched, appropriate adult stem cell source [127]. Raetz and colleagues performed myeloablative conditioning HSCT on a 5-year-old boy with severe Evans syndrome, which consists of immune thrombocytopenia and Coombspositive hemolytic anemia. The graft was a CB from an HLA-matched sibling with 3.85 × 107 nucleated cells/kg patient and 0.96 × 105 CD34+ cells/kg patient infused, leading to complete remission. Acute GvHD prophylaxis regimen was with cyclosporine, with G-CSF initiated on day +1. The patient engrafted with an absolute neutrophil count (ANC) greater than 0.5 × 109 / l on day +16. The following day, he developed symptoms of acute GvHD, with temperatures of up to 40°C, skin rash that on biopsy was consistent with GvHD and severe pulmonary insufficiency. He was intubated for 2 days and treated with high-dose steroids, with rapid resolution of symptoms. Platelet engraftment was delayed, with sustained platelets greater than 30 × 109 /l by day +170. He was platelet independent from day +240 and RBC independent from day +210. Reevaluation of his RBC antibody status revealed a DAT that was only microscopically positive by day +20 and negative on day +286. Anti-platelet antibodies were negative on day +115 and day +176. He had no evidence of response with cyclosporine pretransplant and no evidence of disease recurrence 2 months after discontinuation of cyclosporine and 4 months after discontinuation of prednisone. At the time of death, the patient was off all immunosuppression and had complete resolution of his autoimmune disease. However, the patient died unexpectedly from acute hepatic failure 9 months after transplantation [127].

Itamura et al. reported another case for a 48-year-old female patient with RA [128], who was subsequently diagnosed as having autoimmune hemolytic anemia (AIHA). Four months after the diagnosis of AIHA, she suddenly developed hemophagocytic syndrome (HPS) with disseminated intravascular coagulation (DIC) and *Staphylococcus aureus* bacteremia. Her HPS transiently improved after treatment, but relapsed 3 weeks later. Without an immediately available related adult donor, an unrelated donor CB graft was used for HSCT. The patient had HLA antibodies against multiple HLA class I antigens; however, the CB unit did not express HLA antigens reactive to her HLA antibodies and contained sufficient cell dose. HLA type of the CB graft was A\*02:06/A\*11:01, B\*40:06/B\*67:01, DRB1\*09:01/DRB1\*16:02 and that of the recipient was A\*11:01/A\*26:02, B\*40:06/B\*67:01, DRB1\*09:01/DRB1\*16:02. Four months after the onset of HPS, she received a CBT following a conditioning regimen of melphalan (40  mg/m2 /day 2 days), fludarabine (25 mg/m2 /day 5 days) and total-body irradiation. Tacrolimus was used for the prophylaxis of GvHD. Engraftment of neutrophils ANC500 and platelets 20K occurred on days 14 and 32, respectively. Bone marrow examination on day 60 showed complete donor-type chimerism by short tandem repeat-polymerase chain reaction and no evidence of HPS. EBV-DNA load was under the detectable limit. Post-transplant, the patient was not only cured for HPS, but also showed marked amelioration of preexisting RA and AIHA [128].

Others have performed unrelated donor CBT for AD, including at least two pediatric cases of scleroderma and systemic sclerosis where MaxCell CB products were provided by the StemCyte CB bank founded by the corresponding author. Both cases were performed at the same transplant center; however, the attending transplant physician relocated to a different hospital subsequently and both cases were lost to follow-up [R. Chow, unpublished data]. These CBT cases suggest that allogeneic CBT can potentially be a powerful tool for curing AD without significant GvHD provided that the cell dose and HLA match is adequate.

In summary, by optimizing transplant conditions and maximizing cell dose through the use of products manufactured by the MaxCell CB processing technologies, use of double CBT and direct infusion without post-thaw wash, unrelated CBT has been shown in the largest patient series of its kind to be capable of producing outstanding clinical outcome for thalassemia major, with excellent long-term TRM, OS and DFS that rivals or approaches that of related CBT. Such strategies may be employed to optimize unrelated donor CBT for other nonmalignant conditions, such as HIV infection or AD. For HIV infection, optimization of cell dose may make the difference between a HIV-resistant CB graft being eligible for HSCT, especially if in combination with other grafts. Similarly, cell dose maximization will minimize graft failure and TRM for CBT of AD. After the establishment of large racially and ethnically diverse CB banks with MaxCell CB products, further CBT studies of more unrelated patients with thalassemia, HIV infection and AD will be needed to see whether these hypotheses can be validated and the strategies can be widely applicable.
