Early expression of CD94 and loss of CD96 on CD8+ T cells after allogeneic stem cell tranplantation is predictive of subsequent relapse and survival.

Allogeneic stem cell transplantation is used widely in the treatment of hematopoietic malignancy. However, relapse of malignant disease is the primary cause of treatment failure and reflects loss of immunological graft-versus-leukemia effect. We studied the transcriptional and phenotypic profile of CD8+ T cells in the first month following transplantation and related this to risk of subsequent relapse. Single cell transcriptional profiling identified five discrete CD8+ T-cell clusters. High levels of T-cell activation and acquisition of a regulatory transcriptome were apparent in patients who went on to suffer disease relapse. A relapse-associated gene signature of 47 genes was then assessed in a confirmation cohort of 34 patients. High expression of the inhibitory receptor CD94/NKG2A on CD8+ T cells within the first month was associated with 4.8 fold increased risk of relapse and 2.7 fold reduction in survival. Furthermore, reduced expression of the activatory molecule CD96 was associated with 2.2 fold increased risk of relapse and 1.9 fold reduction in survival. This work identifies CD94 and CD96 as potential targets for CD8-directed immunotherapy in the very early phase following allogeneic transplantation with the potential to reduce long term relapse rates and improve patient survival.

Early inflammatory markers as prognostic indicators following allogeneic stem cell transplantation.

Allogeneic stem cell transplantation is used widely in the treatment of hematopoietic malignancy although graft versus host disease and relapse remain major complications. We measured the serum protein expression of 92 inflammation-related markers from 49 patients at Day 0 (D0) and 154 patients at Day 14 (D14) following transplantation and related values to subsequent clinical outcomes. Low levels of 7 proteins at D0 were linked to GvHD whilst high levels of 7 proteins were associated with relapse. The concentration of 38 proteins increased over 14 days and higher inflammatory response at D14 was strongly correlated with patient age. A marked increment in protein concentration during this period associated with GvHD but reduced risk of disease relapse, indicating a link with alloreactive immunity. In contrast, patients who demonstrated low dynamic elevation of inflammatory markers during the first 14 days were at increased risk of subsequent disease relapse. Multivariate time-to-event analysis revealed that high CCL23 at D14 was associative of AGvHD, CXCL10 with reduced rate of relapse, and high PD-L1 with reduced overall survival. This work identifies a dynamic pattern of inflammatory biomarkers in the very early post-transplantation period and reveals early protein markers that may help to guide patient management.

Azacitidine for the treatment of steroid-refractory chronic graft-versus-host disease: the results of the phase II AZTEC clinical trial.

Chronic graft-versus-host disease (cGvHD) is a major cause of non-relapse morbidity and mortality following allogeneic stem cell transplant. Over half of patients with moderate or severe cGvHD fail to respond adequately to first-line treatment with systemic steroids, and although a range of second-line options have been employed, a lack of prospective evidence means there is no standard of care. The AZTEC trial is a prospective, single-arm, phase II study investigating the safety and activity of azacitidine for the treatment of cGvHD in patients who are resistant to, or intolerant of, systemic steroid therapy. The co-primary outcomes were treatment tolerability, and activity measured as objective response according to modified National Institutes of Health criteria. Fourteen patients were recruited to the first stage of the trial, of whom seven completed the planned six cycles of azacitidine 36 mg/m2 days 1-5 per 28-day cycle. Azacitidine was tolerated by 13/14 patients, and 7/14 showed an objective response. Clinical responses were mirrored by improvements in patient-reported cGvHD symptoms and quality of life. AZTEC demonstrates that azacitidine is a safe and promising option for the treatment of cGvHD, and continued evaluation in the second stage of this phase II efficacy study is supported.

DNA and modified vaccinia Ankara prime-boost vaccination generates strong CD8+ T cell responses against minor histocompatibility antigen HA-1.

Allogeneic immune responses underlie the graft-versus-leukaemia effect of stem cell transplantation, but disease relapse occurs in many patients. Minor histocompatibility antigen (mHAg) peptides mediate alloreactive T cell responses and induce graft-versus-leukaemia responses when expressed on patient haematopoietic tissue. We vaccinated nine HA-1-negative donors against HA-1 with a 'prime-boost' protocol of either two or three DNA 'priming' vaccinations prior to 'boost' with modified vaccinia Ankara (MVA). HA-1-specific CD8+ T cell responses were observed in seven donors with magnitude up to 1·5% of total CD8+ T cell repertoire. HA-1-specific responses peaked two weeks post-MVA challenge and were measurable in most donors after 12 months. HA-1-specific T cells demonstrated strong cytotoxic activity and lysed target cells with endogenous HA-1 protein expression. The pattern of T cell receptor (TCR) usage by HA-1-specific T cells revealed strong conservation of T cell receptor beta variable 7-9 (TRBV7-9) usage between donors. These findings describe one of the strongest primary peptide-specific CD8+ T cell responses yet recorded to a DNA-MVA prime-boost regimen and this may reflect the strong immunogenicity of mHAg peptides. Prime-boost vaccination in donors or patients may prove of substantial benefit in boosting graft-versus-leukaemia responses.

Very early lineage-specific chimerism after reduced intensity stem cell transplantation is highly predictive of clinical outcome for patients with myeloid disease.

BACKGROUND: The importance of chimerism status in the very early period after hematopoietic stem cell transplantation is unclear. We determined PBMC and T-cell donor chimerism 50 days after transplantation and related this to disease relapse and overall survival. METHODS: 144 sequential patients underwent transplantation of which 90 had AML/MDS and 54 had lymphoma. 'Full donor chimerism' was defined as ≥99% donor cells and three patient groups were defined: 40% with full donor chimerism (FC) in both PBMC and T-cells; 25% with mixed chimerism (MC) within both compartments and 35% with 'split' chimerism (SC) characterised by full donor chimerism within PBMC and mixed chimerism within T-cells. RESULTS: In patients with myeloid disease a pattern of mixed chimerism (MC) was associated with a one year relapse rate of 45% and a five year overall survival of 40% compared to values of 8% and 75%, and 17% and 60%, for those with SC or FC respectively. The pattern of chimerism had no impact on clinical outcome for lymphoma. CONCLUSION: The pattern of lineage-specific chimerism at 50 days after transplantation is highly predictive of clinical outcome for patients with myeloid malignancy and may help to guide subsequent clinical management.

Mixed chimerism established by hematopoietic stem cell transplantation is maintained by host and donor T regulatory cells.

Transplantation is an effective treatment of many clinical disorders, but the mechanisms that regulate immunological tolerance are uncertain and remain central to improving patient outcome. Hemopoietic stem cell transplantation (SCT) often establishes "mixed chimerism" in which immune cells from both the donor and patient coexist in vivo in a setting of immunological tolerance. We studied immune function in 69 patients within 2 months following SCT; 37 were fully donor and 32 displayed mixed chimerism. The proportion of T regulatory (Treg) cells was increased during mixed chimerism and comprised equal numbers of donor and host-derived regulatory cells. This was associated with a tolerogenic PD-L1+ profile on dendritic cells. Importantly, effector T cells from patients with mixed chimerism exhibited reduced cytotoxicity against host target cells in vitro, but this was restored following depletion of CD4+ Treg cells. These data show that Treg cells play a major role in sustaining immunological tolerance during mixed chimerism. These insights should help to guide novel interventions to improve clinical transplantation.

Unique features and clinical importance of acute alloreactive immune responses.

Allogeneic stem cell transplantation (allo-SCT) can cure some patients with hematopoietic malignancy, but this relies on the development of a donor T cell alloreactive immune response. T cell activity in the first 2 weeks after allo-SCT is crucial in determining outcome, despite the clinical effects of the early alloreactive immune response often not appearing until later. However, the effect of the allogeneic environment on T cells is difficult to study at this time point due to the effects of profound lymphopenia. We approached this problem by comparing T cells at week 2 after allograft to T cells from autograft patients. Allograft T cells were present in small numbers but displayed intense proliferation with spontaneous cytokine production. Oligoclonal expansions at week 2 came to represent a substantial fraction of the established T cell pool and were recruited into tissues affected by graft-versus-host disease. Transcriptional analysis uncovered a range of potential targets for immune manipulation, including OX40L, TWEAK, and CD70. These findings reveal that recognition of alloantigen drives naive T cells toward a unique phenotype. Moreover, they demonstrate that early clonal T cell responses are recruited to sites of subsequent tissue damage and provide a range of targets for potential therapeutic immunomodulation.

NK cells produce high levels of IL-10 early after allogeneic stem cell transplantation and suppress development of acute GVHD.

Natural killer (NK) cells rapidly reconstitute following allogeneic stem cell transplantation (allo-SCT), at the time when alloreactive T cell immunity is being established. We investigated very early NK cell reconstitution in 82 patients following T cell-depleted allo-SCT. NK cell number rapidly increased, exceeding T cell reconstitution such that the NK:T cell ratio was over 40 by day 14. NK cells at day 14 (NK-14) were donor-derived, intensely proliferating and expressed chemokine receptors targeted to lymphoid and peripheral tissue. Spontaneous production of the immunoregulatory cytokine IL-10 was observed in over 70% of cells and transcription of cytokines and growth factors was augmented. NK-14 cell number was inversely correlated with the incidence of grade II-IV acute graft versus host disease (GVHD). These findings reveal that robust reconstitution of immunoregulatory NK cells by day 14 after allo-SCT is an important determinant of the clinical outcome, suggesting that NK cells may suppress the development of the T cell-mediated alloreactive immune response through production of IL-10.

The number of CD56dim NK cells in the graft has a major impact on risk of disease relapse following allo-HSCT.

The graft-versus-leukemia (GVL) effect of allogeneic hemopoietic stem cell transplantation (allo-HSCT) is mediated by the donor immune system and acts to decrease the rate of disease relapse. Although studies of posttransplant immune reconstitution have identified correlates of clinical outcome, the number and profile of mature immune cells infused with the stem cell graft is also likely to be an important determinant and has been relatively poorly studied. We characterized immune cells within the stem cell graft of 107 patients who underwent T-cell-depleted allo-HSCT and related this to clinical outcome. The number of natural killer (NK) cells and T cells that were infused varied markedly between patients, but T-cell dose was not an important factor in subsequent outcome. In contrast, the number of NK cells was a powerful determinant of the risk of disease relapse. Patients who received an NK cell dose below the median level of 6.3 × 106 cells per kg had a relapse rate of 40% at 2 years posttransplant compared with only 6% for those whose stem cell graft contained a dose above this value. Analysis of NK subsets showed that this effect was mediated primarily by the CD56dim population of mature effector cells and that high-level expression of the activatory protein DNAM on donor NK cells was also strongly protective. These observations offer important insights into the mechanism of GVL and suggest that optimization studies of the number of NK cells within the stem cell graft should be considered as a means to reduce disease relapse.

Chemokine-mediated tissue recruitment of CXCR3+ CD4+ T cells plays a major role in the pathogenesis of chronic GVHD.

Chemokines regulate the migration of hemopoietic cells and play an important role in the pathogenesis of many immune-mediated diseases. Intradermal recruitment of CD8(+) T cells by CXCL10 is a central feature of the pathogenesis of cutaneous acute GVHD (aGVHD), but very little is known about the pathogenesis of chronic GVHD (cGVHD). Serum concentrations of the 3 CXCR3-binding chemokines, CXCL9, CXCL10, and CXCL11, were found to be markedly increased in patients with active cGVHD of the skin (n = 8). An 80% decrease in CD4(+) cells expressing CXCR3 was seen in the blood of these patients (n = 5), whereas CD4(+) cells were increased in tissue biopsies and were clustered around the central arterioles of the dermis. The well-documented increase in expression of CXCL10 in aGVHD therefore diversifies in cGVHD to include additional members of the CXCR3-binding family and leads to preferential recruitment of CD4(+) T cells. These observations reveal a central role for chemokine-mediated recruitment of CXCR3(+) T cells in cGVHD.

Azacitidine augments expansion of regulatory T cells after allogeneic stem cell transplantation in patients with acute myeloid leukemia (AML).

Strategies that augment a GVL effect without increasing the risk of GVHD are required to improve the outcome after allogeneic stem cell transplantation (SCT). Azacitidine (AZA) up-regulates the expression of tumor Ags on leukemic blasts in vitro and expands the numbers of immunomodulatory T regulatory cells (Tregs) in animal models. Reasoning that AZA might selectively augment a GVL effect, we studied the immunologic sequelae of AZA administration after allogeneic SCT. Twenty-seven patients who had undergone a reduced intensity allogeneic transplantation for acute myeloid leukemia were treated with monthly courses of AZA, and CD8(+) T-cell responses to candidate tumor Ags and circulating Tregs were measured. AZA after transplantation was well tolerated, and its administration was associated with a low incidence of GVHD. Administration of AZA increased the number of Tregs within the first 3 months after transplantation compared with a control population (P = .0127). AZA administration also induced a cytotoxic CD8(+) T-cell response to several tumor Ags, including melanoma-associated Ag 1, B melanoma antigen 1, and Wilm tumor Ag 1. These data support the further examination of AZA after transplantation as a mechanism of augmenting a GVL effect without a concomitant increase in GVHD.