Leaders in Hematology Review Series: T-cell and natural killer cell therapies for hematologic malignancies after hematopoietic stem cell transplantation: enhancing the graft-versus-leukemia effect

Hematopoietic stem cell transplantation has revolutionized the treatment of hematologic malignancies, but infection, graft-versus-host disease and relapse are still important problems. Calcineurin inhibitors, T-cell depletion strategies, and immunomodulators have helped to prevent graft-versus-host disease, but have a negative impact on the graft-versus-leukemia effect. T cells and natural killer cells are both thought to be important in the graft-versus-leukemia effect, and both cell types are amenable to ex vivo manipulation and clinical manufacture, making them versatile immunotherapeutics. We provide an overview of these immunotherapeutic strategies following hematopoietic stem cell transplantation, with discussions centered on natural killer and T-cell biology. We discuss the contributions of each cell type to graft-versus-leukemia effects, as well as the current research directions in the field as related to adoptive cell therapy after hematopoietic stem cell transplantation.     

Favorable impact of natural killer cell reconstitution on chronic graft-versus-host disease and cytomegalovirus reactivation after allogeneic hematopoietic stem cell transplantation

Natural killer cells are the first lymphocyte subset to reconstitute, and play a major role in early immunity after allogeneic hematopoietic stem cell transplantation. Cells expressing the activating receptor NKG2C seem crucial in the resolution of cytomegalovirus episodes, even in the absence of T cells. We prospectively investigated natural killer-cell reconstitution in a cohort of 439 adult recipients who underwent non-T-cell-depleted allogeneic hematopoietic stem cell transplantation between 2005 and 2012. Freshly collected blood samples were analyzed 3, 6, 12 and 24 months after transplantation. Data were studied with respect to conditioning regimen, source of stem cells, underlying disease, occurrence of graft-versus-host disease, and profiles of cytomegalovirus reactivation. In multivariate analysis we found that the absolute numbers of CD56^bright natural killer cells at month 3 were significantly higher after myeloablative conditioning than after reduced intensity conditioning. Acute graft-versus-host disease impaired reconstitution of total and CD56^dim natural killer cells at month 3. In contrast, high natural killer cell count at month 3 was associated with a lower incidence of chronic graft-versus-host disease, independently of a previous episode of acute graft-versus-host disease and stem cell source. NKG2C⁺CD56^dim and total natural killer cell counts at month 3 were lower in patients with reactivation of cytomegalovirus between month 0 and month 3, but expanded greatly afterwards. These cells were also less numerous in patients who experienced later cytomegalovirus reactivation between month 3 and month 6. Our results advocate a direct role of NKG2C-expressing natural killer cells in the early control of cytomegalovirus reactivation after allogeneic hematopoietic stem cell transplantation.     

Expression of a dominant T-cell receptor can reduce toxicity and enhance tumor protection of allogeneic T-cell therapy

Due to the lack of specificity for tumor antigens, allogeneic T-cell therapy is associated with graft-versus-host disease. Enhancing the anti-tumor specificity while reducing the graft-versus-host disease risk of allogeneic T cells has remained a research focus. In this study, we demonstrate that the introduction of ‘dominant’ T-cell receptors into primary murine T cells can suppress the expression of endogenous T-cell receptors in a large proportion of the gene-modified T cells. Adoptive transfer of allogeneic T cells expressing a ‘dominant’ T-cell receptor significantly reduced the graft-versus-host toxicity in recipient mice. Using two bone marrow transplant models, enhanced anti-tumor activity was observed in the presence of reduced graft-versus-host disease. However, although transfer of T-cell receptor gene-modified allogeneic T cells resulted in the elimination of antigen-positive tumor cells and improved the survival of treated mice, it was associated with accumulation of T cells expressing endogenous T-cell receptors and the development of delayed graft-versus-host disease. The in vivo deletion of the engineered T cells, mediated by endogenous mouse mammary tumor virus MTV8 and MTV9, abolished graft-versus-host disease while retaining significant anti-tumor activity of adoptively transferred T cells. Together, this study shows that the in vitro selection of allogeneic T cells expressing high levels of a ‘dominant’ T-cell receptor can lower acute graft-versus-host disease and enhance anti-tumor activity of adoptive cell therapy, while the in vivo outgrowth of T cells expressing endogenous T-cell receptors remains a risk factor for the delayed onset of graft-versus-host disease.     

Genome editing of donor-derived T cells to generate allogeneic chimeric antigen receptor-modified T cells: optimizing αβ T-cell-depleted haploidentical hematopoietic stem cell transplantation

Allogeneic hematopoietic stem cell transplantation is an effective therapy for high-risk leukemias. In children, graft manipulation based on the selective removal of αβT cells and B cells has been shown to reduce the risk of acute and chronic graft-versus-host disease, thus allowing the use of haploidentical donors which expands the population of recipients in whom allogeneic hematopoietic stem cell transplantation can be used. Leukemic relapse, however, remains a challenge. T cells expressing chimeric antigen receptors can potently eliminate leukemia, including those in the central nervous system. We hypothesized that by engineering the donor αβT cells that are removed from the graft by genome editing to express a CD19-specific chimeric antigen receptor, while simultaneously inactivating the T-cell receptor, we could create a therapy that enhances the anti-leukemic efficacy of the stem cell transplant without increasing the risk of graft-versus-host disease. Using genome editing with Cas9 ribonucleoprotein and adeno-associated virus serotype 6, we integrated a CD19-specific chimeric antigen receptor inframe into the TRAC locus. More than 90% of cells lost T-cell receptor expression, while >75% expressed the chimeric antigen receptor. The initial product was further purified with less than 0.05% T-cell receptorpositive cells remaining. In vitro, the chimeric antigen receptor T cells efficiently eliminated target cells and produced high cytokine levels when challenged with CD19⁺ leukemia cells. In vivo, the gene-modified T cells eliminated leukemia without causing graft-versus-host disease in a xenograft model. Gene editing was highly specific with no evidence of off-target effects. These data support the concept that the addition of αβ T-cell-derived, genome-edited T cells expressing CD19-specific chimeric antigen receptors could enhance the anti-leukemic efficacy of αβT-celldepleted haploidentical hematopoietic stem cell transplantation without increasing the risk of graft-versus-host disease.     

Rabbit antithymocyte globulin (Thymoglobulin?) impairs the thymic output of both conventional and regulatory CD4+ T cells after allogeneic hematopoietic stem cell transplantation in adult patients

Rabbit antithymocyte globulin-Genzyme™ is used to prevent graft-versus-host disease after allogeneic hematopoietic stem cell transplantation. Common disadvantages of treatment are infectious complications. The effects of rabbit antithymocyte globulin-Genzyme™ on thymic function have not been well-studied. Multicolor flow cytometry was used to analyze the kinetics of conventional and regulatory T cells in adult patients treated (n=12) or not treated (n=8) with rabbit antithymocyte globulin-Genzyme™ during the first 6 months after allogeneic hematopoietic stem cell transplantation. Patients treated with rabbit antithymocyte globulin-Genzyme™ had almost undetectable levels of recent thymic emigrants (CD45RA⁺CD31⁺) of both conventional and regulatory CD4T cells throughout the 6 months after allogeneic hematopoietic stem cell transplantation whereas CD4⁺CD45RA-memory T cells were less affected, but their levels were also significantly lower than in patients not treated with rabbit antithymocyte globulin-Genzyme™. In vitro, rabbit antithymocyte globulin-Genzyme™ induced apoptosis and cytolysis of human thymocytes, and its cytotoxic effects were greater than those of rabbit antithymocyte globulin-Fresenius™. Rabbit antithymocyte globulin-Genzyme™ in combination with a conditioning regimen strongly impairs thymic recovery of both conventional and regulatory CD4⁺ T cells. The sustained depletion of conventional and regulatory CD4⁺T cells carries a high risk of both infections and graft-versus-host disease. Our data indicate that patients treated with rabbit antithymocyte globulin-Genzyme™ could benefit from thymus-protective therapies and that trials comparing this product with other rabbit antithymocyte globulin preparations or lymphocyte-depleting compounds would be informative.     

A Good Manufacturing Practice procedure to engineer donor virus-specific T cells into potent anti-leukemic effector cells

A sequential, two-step procedure in which T-cell-depleted allogeneic stem cell transplantation is followed by treatment with donor lymphocyte infusion at 6 months can significantly reduce the risk and severity of graft-versus-host disease, with postponed induction of the beneficial graft-versus-leukemia effect. However, patients with high-risk leukemia have a substantial risk of relapse early after transplantation, at a time when administration of donor lymphocytes has a high likelihood of resulting in graft-versus-host disease, disturbing a favorable balance between the graft-versus-leukemia effect and graft-versus-host disease. New therapeutic modalities are, therefore, required to allow early administration of T cells capable of exerting a graft-versus-leukemia effect without causing graft-versus-host disease. Here we describe the isolation of virus-specific T cells using Streptamer-based isolation technology and subsequent transfer of the minor histocompatibility antigen HA-1-specific T-cell receptor using retroviral vectors. Isolation of virus-specific T cells and subsequent transduction with HA-1-T-cell receptor resulted in rapid in vitro generation of highly pure, dual-specific T cells with potent anti-leukemic reactivity. Due to the short production procedure of only 10–14 days and the defined specificity of the T cells, administration of virus-specific T cells transduced with the HA-1-T-cell receptor as early as 8 weeks after allogeneic stem cell transplantation is feasible. (This clinical trial is registered at www.clinicaltrialsregister.eu as EudraCT number 2010-024625-20).     

Mesenchymal stromal cells transiently alter the inflammatory milieu post-transplant to delay graft-versus-host disease

Background

Multipotent mesenchymal stromal cells suppress T-cell function in vitro, a property that has underpinned their use in treating clinical steroid-refractory graft-versus-host disease after allogeneic hematopoietic stem cell transplantation. However the potential of mesenchymal stromal cells to resolve graft-versus-host disease is confounded by a paucity of pre-clinical data delineating their immunomodulatory effects in vivo.

Design and Methods

We examined the influence of timing and dose of donor-derived mesenchymal stromal cells on the kinetics of graft-versus-host disease in two murine models of graft-versus-host disease (major histocompatibility complex-mismatched: UBI-GFP/BL6 [H-2^b]→BALB/c [H-2^d] and the sibling transplant mimic, UBI-GFP/BL6 [H-2^b]→BALB.B [H-2^b]) using clinically relevant conditioning regimens. We also examined the effect of mesenchymal stromal cell infusion on bone marrow and spleen cellular composition and cytokine secretion in transplant recipients.

Results

Despite T-cell suppression in vitro, mesenchymal stromal cells delayed but did not prevent graft-versus-host disease in the major histocompatibility complex-mismatched model. In the sibling transplant model, however, 30% of mesenchymal stromal cell-treated mice did not develop graft-versus-host disease. The timing of administration and dose of the mesenchymal stromal cells influenced their effectiveness in attenuating graft-versus-host disease, such that a low dose of mesenchymal stromal cells administered early was more effective than a high dose of mesenchymal stromal cells given late. Compared to control-treated mice, mesenchymal stromal cell-treated mice had significant reductions in serum and splenic interferon-γ, an important mediator of graft-versus-host disease.

Conclusions

Mesenchymal stromal cells appear to delay death from graft-versus-host disease by transiently altering the inflammatory milieu and reducing levels of interferon-γ. Our data suggest that both the timing of infusion and the dose of mesenchymal stromal cells likely influence these cells’ effectiveness in attenuating graft-versus-host disease.     

Loss of endothelial thrombomodulin predicts response to steroid therapy and survival in acute intestinal graft-versus-host disease

Steroid-refractory graft-versus-host disease causes significant morbidity and mortality after allogeneic stem cell transplantation. The pathomechanism of steroid resistance is currently not understood, but it has been suggested that endothelial cell dysfunction plays a role. Endothelial thrombomodulin was quantified along with histological markers of epithelial damage and cytotoxic T cells in colon biopsies from 51 allografted patients, and retrospectively correlated with response to steroids and survival. Loss of endothelial thrombomodulin was the strongest predictor of response to steroids (P=0.02) and nonrelapse mortality (P=0.01) in multivariate analyses adjusting for T-cell infiltrates, histological grading, vessel density, disease status, donor type, and conditioning therapy. Our data provide evidence that at disease onset, loss of endothelial thrombomodulin expression rather than excessive T-cell infiltration associates with steroid-refractory graft-versus-host disease and mortality. Prospective histological investigations are now warranted to improve diagnosis and prognostication of this core complication of stem cell transplantation.Key words: allogeneic stem cell transplantation, acute GvHD, thrombomodulin, endothelial cells, steroid-resistance, cytotoxic T cells, TIA-1     

Interleukin-10 spot-forming cells as a novel biomarker of chronic graft-versus-host disease

Although there are National Institutes of Health consensus criteria for the global assessment of chronic graft-versus-host disease, no validated biomarkers have been established for this disease. Furthermore, whereas the role of T cells, B cells, and dendritic cells in chronic graft-versus-host disease has been established, the contribution of monocytes has not been clearly addressed. Using an enzyme-linked immunospot assay, we measured the spot-forming cells for interferon-γ, interleukin-4, interleukin-10, and interleukin-17 in unstimulated peripheral blood of patients following allogeneic hematopoietic stem cell transplantation. Other immunological examinations, including skin biopsy, were also done. Fifty-seven patients were enrolled. Interleukin-10 spot-forming cells were evaluable for therapeutic monitoring in 16 patients with chronic graft-versus-host disease. The number of interleukin-10 spot-forming cells in patients with active chronic graft-versus-host disease was significantly higher than the number in those with no or inactive chronic graft-versus-host disease. Interleukin-10 was predominantly produced by monocytes. CD29 expression on monocytes in patients with active chronic graft-versus-host disease was elevated. The level of plasma fibronectin, a ligand of CD29, correlated with the number of interleukin-10 spot-forming cells. Immunohistochemical analysis of the skin in active chronic graft-versus-host disease showed that infiltrating CD29⁺ monocytes might produce interleukin-10. A novel biomarker, interleukin-10 spot-forming cells, shows promise as both a diagnostic and prognostic indicator for chronic graft-versus-host disease, and may allow for early intervention prior to the onset of the disease. Measurement of interleukin-10 spot-forming cells would be helpful in clinical trials and in patients'' management.     

Persistence of recipient-type endothelium after allogeneic hematopoietic stem cell transplantation

Background

The possibility that allogeneic hematopoietic stem cell transplantation performed across the ABO blood group-barrier is associated with an increase of graft-versus-host disease, in particular endothelial damage, has not been elucidated so far. For this reason, we investigated the level of endothelial cell chimerism after allogeneic hematopoietic stem cell transplantation in order to delineate the role of hematopoietic stem cells in endothelial replacement.

Design and Methods

The frequency of donor-derived endothelial cells was analyzed in 52 hematopoietic stem cell transplant recipients, in 22 normal skin biopsies, in 12 skin samples affected by graft-versus-host disease, various tissues from five autopsies and four secondary solid tumors by ABH immunohistochemistry, XY fluorescence in situ hybridization and short tandem repeat analysis of laser captured endothelial cells.

Results

Skin biopsies from two patients transplanted with minor ABO-incompatible grafts (i.e. O in A) showed 3.3% and 0.9% H antigen-positive donor-derived endothelial cells by ABH immunohistochemistry. Tumor biopsies from two recipients showed 1.2% and 2.5% donor-derived endothelial cells by combined immunohistochemistry/ fluorescence in situ hybridization. All other skin samples, heart, liver, bone-marrow, and tumor tissues failed to reveal donor-type endothelial cells up to several years after ABO-incompatible hematopoietic stem cell transplantation.

Conclusions

Endothelial cell replacement by bone marrow-derived donor cells after allogeneic hematopoietic stem cell transplantation is a rare event. It does not seem to represent a major mechanism of physiological in vivo blood vessel formation, tumor neoangiogenesis, vascular repair after graft-versus-host disease episodes or acceptance of ABO-incompatible grafts.     

A randomized phase II study to evaluate tacrolimus in combination with sirolimus or methotrexate after allogeneic hematopoietic cell transplantation

Background

There is evidence suggesting that sirolimus, in combination with tacrolimus, is active in the prevention of graft-versus-host disease. Sirolimus-based immune suppression may suppress alloreactive T cells, while sparing the survival and function of regulatory T cells.

Design and Methods

We conducted a randomized trial to compare the impact of sirolimus/tacrolimus against that of methotrexate/tacrolimus on the prevention of graft-versus-host disease and regulatory T-cell reconstitution.

Results

Seventy-four patients were randomized 1:1 to sirolimus/tacrolimus or methotrexate/tacrolimus, stratified for type of donor (sibling or unrelated) and the patients'' age. The rate of grade II-IV acute graft-versus-host disease at 100 days was 43% (95% CI: 27-59%) in the sirolimus/tacrolimus group and 89% (95% CI: 72-96%) in the methotrexate/tacrolimus group (P<0.001). The rate of moderate/severe chronic graft-versus-host disease was 24% (95% CI: 7-47%) in the sirolimus/tacrolimus group and 64% (95% CI: 41-79%) in the methotrexate/tacrolimus group (P=0.008). Overall survival and patient-reported quality of life did not differ between the two groups. On days 30 and 90 post-transplant, sirolimus-treated patients had a significantly greater proportion of regulatory T cells among the CD4⁺ cells in the peripheral blood, and isolated regulatory T cells were functional.

Conclusions

These data demonstrate that sirolimus/tacrolimus prevents grade II-IV acute graft-versus-host disease and moderate-severe chronic graft-versus-host disease more effectively than does methotrexate/tacrolimus, and supports regulatory T-cell reconstitution following allogeneic hematopoietic cell transplantation. Trial registration: ({"type":"clinical-trial","attrs":{"text":"NCT00803010","term_id":"NCT00803010"}}NCT00803010)Key words: tacrolimus, sirolimus, methotrexate, combination therapy, GVHD prophylaxis     

Recipient and donor cells in the graft-versus-solid tumor effect: It takes two to tango

Allogeneic hematopoietic stem cell transplantation (alloHSCT) produces –similar to the long-established graft-versus-leukemia effect– graft-versus-solid-tumor effects. Clinical trials reported response rates of up to 53%, occurring mostly but not invariably in association with full donor chimerism and/or graft-versus-host disease. Although donor-derived T cells are considered the principal effectors of anti-tumor immunity after alloHSCT or donor leukocyte infusion (DLI), growing evidence indicate that recipient-derived immune cells may also contribute. Whereas the role of recipient-derived antigen-presenting cells in eliciting graft-versus-host reactions and priming donor T cells following DLI is well known, resulting inflammatory responses may also break tolerance of recipient effector cells towards the tumor. Additionally, mouse studies indicated that post-transplant recipient leukocyte infusion produces anti-leukemia and anti-solid-tumor effects that were exclusively mediated by recipient-type effector cells, without graft-versus-host disease. Here, we review current preclinical and clinical evidence on graft-versus-solid-tumor effects and growing evidence on the effector role of recipient-derived immune cells in the anti-tumor effect of alloHSCT.     

Identification of 4 novel HLA-B*40:01 restricted minor histocompatibility antigens and their potential as targets for graft-versus-leukemia reactivity

Background

Patients with hematologic malignancies can be successfully treated with donor lymphocyte infusion after HLA-matched allogeneic hematopoietic stem cell transplantation. The effect of donor lymphocyte infusion is mediated by donor T cells recognizing minor histocompatibility antigens. T cells recognizing hematopoietic restricted minor histocompatibility antigens may induce selective graft-versus-leukemia reactivity, whereas broadly-expressed antigens may be targeted in graft-versus-host disease.

Design and Methods

We analyzed in detail CD8⁺ T-cell immunity in a patient with relapsed chronic myelogenous leukemia who responded to donor lymphocyte infusion with minimal graft-versus-host disease of the skin. CD8⁺ T-cell clones specific for 4 HLA-B*40:01 restricted minor histocompatibility antigens were isolated which were identified by screening a plasmid cDNA library and whole genome association scanning. Detailed T-cell reactivity and monitoring experiments were performed to estimate the clinical and therapeutic relevance of the novel antigens.

Results

Three antigens were demonstrated to be expressed on primary leukemic cells of various origins as well as subtypes of non-malignant hematopoietic cells, whereas one antigen was selectively recognized on malignant hematopoietic cells with antigen presenting cell phenotype. Skin derived fibroblasts were only recognized after pre-treatment with IFN-γ by two T-cell clones.

Conclusions

Our data show evidence for different roles of the HLA-B*40:01 restricted minor histocompatibility antigens in the onset and execution of the anti-tumor response. All antigens may have contributed to a graft-versus-leukemia effect, and one minor histocompatibility antigen (LB-SWAP70-1Q) has specific therapeutic value based on its in vivo immunodominance and strong presentation on leukemic cells of various origins, but absence of expression on cytokine-treated fibroblasts.     

Depletion of T regulatory cells through selection of CD127-positive cells results in a population enriched in memory T cells: implications for anti-tumor cell therapy

Background

Donor lymphocyte infusions can induce remissions in patients with relapse after allogeneic hematopoietic stem cell transplantation. Nevertheless, some grafted patients never display any signs of alloreactivity, either following allogeneic hematopoietic stem cell transplantation or after donor lymphocyte infusions. Consequently, they do not develop graft-versus-host disease and frequently do not respond to donor lymphocyte infusions. In a recently published clinical trial, we observed that elimination of CD4⁺CD25⁺Foxp3⁺ natural regulatory T cells from the donor lymphocyte product could improve alloreactivity and the associated anti-tumor effect in a small proportion of patients with relapsed hematologic malignancies. Here, we aimed to improve the effect of donor lymphocyte infusion by modifying the procedure for depletion of T regulatory cells.

Design and Methods

We directly compared depletion of regulatory T cells from human peripheral blood mononuclear cells achieved by selection of CD127-positive cells or by selection of CD25-negative cells. We tested the manipulated products (i) in vitro in mixed lymphocyte reactions and against pathogen-derived recall antigens and (ii) in vivo in experimental graft-versus-host disease.

Results

In vitro, we found that depletion of regulatory T cells through CD127 positive selection improved both alloreactive and pathogen-specific immune responses. In vivo, we observed accelerated donor T-cell division and enhanced graft-versus-host disease due to efficient regulatory T-cell depletion accompanied by enrichment in memory T cells.

Conclusions

Our results show that the strategy of CD127 positive selection is an efficient way of eliminating regulatory T cells from donor lymphocyte infusions and improves alloreactivity. This supports the investigation of CD127 positive selection in place of elimination of CD25-positive cells for clinical applications.Key words: CD127 positive selection, Treg, alloreactivity, donor lymphocyte infusion     

The global severity of chronic graft-versus-host disease, determined by National Institutes of Health consensus criteria, is associated with overall survival and non-relapse mortality

Background

The 2005 National Institutes of Health Consensus Development Conference on chronic graft-versus-host disease proposed major changes in the classification and grading of severity of chronic graft-versus-host disease.

Design and Methods

We aimed to study the association of the proposed chronic graft-versus-host disease classification and global severity with transplantation outcomes among a consecutive series of patients who received pharmacokinetically-targeted doses of intravenous busulfan and fludarabine conditioning followed by transplantation of allogeneic peripheral blood stem cells.

Results

From a total cohort (n = 242) of patients surviving more than 100 days after hematopoietic stem cell transplantation, 181 (75% of those at risk) had some manifestations of graft-versus-host disease after day 100. Of these, at onset 13 (7%) had late acute graft-versus-host disease, 62 (34%) had classic chronic graft-versus-host disease, and 106 (59%) had the overlap subtype of chronic graft-versus-host disease. The global severity of the chronic graft-versus-host disease was mild in 25% of cases, moderate in 46%, and severe in 29%. Multivariable modeling demonstrated the independent association of global severity of chronic graft-versus-host disease with overall survival (moderate/severe versus mild; HR 2.9, 95% CI 1.8–4.7, P<0.0001) and non-relapse mortality (moderate versus mild; HR 3.86, 95% CI 1.17–12.73, P=0.03, and severe versus mild (HR 10.06, 95% CI 3.07–32.97, P<0.001). The type of onset of progressive chronic graft-versus-host disease and the platelet count at the time of diagnosis of the disease were significantly associated with overall survival. The occurrence and severity of chronic graft-versus-host disease was also significantly associated with primary disease relapse.

Conclusions

Patients with moderate to severe chronic graft-versus-host disease, as determined by National Institutes of Health Consensus criteria, have an inferior overall survival and worse non-relapse mortality. Clinical and research advances are needed to improve the outcomes of affected patients.     

High pre-transplant serum nitrate levels predict risk of acute steroid-refractory graft-versus-host disease in the absence of statin therapy

Steroid-refractory graft-versus-host disease is a life-threatening complication after allogeneic stem cell transplantation. Evidence is accumulating that steroid-refractory graft-versus-host disease is associated with endothelial distress. Endothelial cell homeostasis is regulated by nitric oxide, and serum nitrates are derived from nitric oxide synthase activity or dietary sources. In this retrospective study based on 417 patients allografted at our institution we investigated whether quantification of serum nitrates could predict steroid-refractory graft-versus-host disease. Elevated pre-transplant levels of serum nitrates (>26.5 μM) predicted steroid-refractory graft-versus-host disease (P=0.026) and non-relapse mortality (P=0.028), particularly in combination with high pre-transplant angiopoietin-2 levels (P=0.0007 and P=0.021, respectively). Multivariate analyses confirmed serum nitrates as independent predictors of steroid-refractory graft-versus-host disease and non-relapse mortality. Differences in serum nitrate levels did not correlate with serum levels of tumor necrosis factor or C-reactive protein or expression of inducible nitric oxide synthase in blood cells. Patients with high pre-transplant nitrate levels had significantly reduced rates of refractory graft-versus-host disease (P=0.031) when pravastatin was taken. In summary, patients at high risk of developing steroid-refractory graft-versus-host disease could be identified prior to transplantation by serum markers linked to endothelial cell function. Retrospectively, statin medication was associated with a reduced incidence of refractory graft-versus-host disease in this endothelial high-risk cohort.     

T-cell reconstitution after allogeneic stem cell transplantation: assessment by measurement of the sjTREC/βTREC ratio and thymic na?ve T cells

The immune reconstitution after allogeneic hematopoietic stem cell transplantation comprises thymus-dependent and thymus-independent pathways. We wanted to improve the understanding of this complex process using two different measurements at definite checkpoints of T-cell neogenesis. We therefore assessed the thymus-dependent pathway by combining measurements of single joint T-cell receptor excision circles (sjTREC) and β T-cell receptor excision circles (βTREC) in an improved quantitative light-cycler hybridization polymerase chain reaction assay. In a subgroup of patients, we additionally assessed the proliferation kinetics of the CD31⁺ thymic naïve cell population, which corresponds to recent thymic emigrants by six-color immunostaining. After the establishment of normal values in 22 healthy volunteers, we applied our polymerase chain reaction to 66 patients undergoing allogeneic hematopoietic stem cell transplantation at a median age of 44 years. It took more than 2 years after transplant to restore the pre-transplant thymic proliferation capacity. Only one third of the patients in our longitudinal study reached age-adjusted normal values for both sjTREC and βTREC at a median follow-up of 558 days, with acute graft-versus-host disease being the most prominent negative factor by univariate analysis. We observed several patterns of sjTREC and βTREC recovery suggesting different mechanisms of thymic damage in individual patients. In a comparison of CD31⁺ thymic naïve cells between volunteers and patients after transplant we found a significantly higher peak proliferation rate within the latter population in the first year after transplantation. The combination of measurements of sjTREC and βTREC by our simplified polymerase chain reaction assay provides insight about the stage of T-cell development affected by different types of damage and may help to choose the correct therapeutic intervention. Besides the sole thymic T-cell neogenesis, proliferation within the CD31⁺ thymic naïve cell compartment contributed to the replenishment of the naïve T-cell pool after transplantation.     

Nuclear factor-�� B inducing kinase is required for graft-versus-host disease

Background

Donor T lymphocytes are directly responsible for graft-versus-host disease. Molecules important in T-cell function may, therefore, be appropriate targets for graft-versus-host disease therapy and/or prophylaxis. Here we analyzed whether nuclear factor-κ B inducing kinase might have a role in graft-versus-host disease.

Design and Methods

We studied the expression of nuclear factor-κ B inducing kinase in human samples from patients with graft-versus-host disease. We also explored the effect of nuclear factor-κ B inducing kinase in a murine model of graft-versus-host disease using donor cells from aly/aly mice (deficient in nuclear factor-κ B inducing kinase) and C57BL/6 mice (control).

Results

We detected expression of nuclear factor-κ B inducing kinase in T-lymphocytes in the pathological lesions of patients with acute graft-versus-host disease. Mice transplanted with aly/aly T lymphocytes did not develop graft-versus-host disease at all, while mice receiving C57BL/6 cells died of a lethal form of the disease. Deficiency of nuclear factor-κ B inducing kinase did not affect the engrafting ability of donor T cells, but severely impaired their expansion capacity early after transplantation, and aly/aly T cells showed a higher proportion of apoptosis than did C57BL/6 T cells. Effector T lymphocytes were the T-cell subset most affected by nuclear factor-κ B inducing kinase deficiency. We also detected lower amounts of inflammatory cytokines in the serum of mice receiving aly/aly T cells than in the serum of mice receiving C57BL/6 T cells.

Conclusions

Our results show that nuclear factor-κ B inducing kinase has a role in graft-versus-host disease by maintaining the viability of activated alloreactive T lymphocytes.     

Cytomegalovirus shapes long-term immune reconstitution after allogeneic stem cell transplantation

Immune reconstitution after allogeneic stem cell transplantation is a dynamic and complex process depending on the recipient and donor characteristics, on the modalities of transplantation, and on the occurrence of graft-versus-host disease. Multivariate methods widely used for gene expression profiling can simultaneously analyze the patterns of a great number of biological variables on a heterogeneous set of patients. Here we use these methods on flow cytometry assessment of up to 25 lymphocyte populations to analyze the global pattern of long-term immune reconstitution after transplantation. Immune patterns were most distinct from healthy controls at six months, and had not yet fully recovered as long as two years after transplant. The two principal determinants of variability were linked to the balance of B and CD8⁺ T cells and of natural killer and B cells, respectively. Recipient’s cytomegalovirus serostatus, cytomegalovirus replication, and chronic graft-versus-host disease were the main factors shaping the immune pattern one year after transplant. We identified a complex signature of under- and over-representation of immune populations dictated by recipient’s cytomegalovirus seropositivity. Finally, we identified dimensions of variance in immune patterns as significant predictors of long-term non-relapse mortality, independently of chronic graft-versus-host disease.     

Cyclosporine and methotrexate-related pharmacogenomic predictors of acute graft-versus-host disease

Effective immunosuppression is mandatory to prevent graft-versus-host disease and to achieve a successful clinical outcome of hematopoietic stem cell transplantation. Here we tested whether germline single nucleotide polymorphisms in 20 candidate genes related to methotrexate and cyclosporine metabolism and activity influence the incidence of graft-versus-host disease in patients who undergo stem cell transplantation for hematologic disorders. Recipient genetic status of the adenosine triphosphate-binding cassette sub-family C1 and adenosine triphosphate-binding cassette sub-family C2 transporters, 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase/ inosine monophosphate cyclohydrolase within the methotrexate pathway, and nuclear factor of activated T cells (cytoplasmic 1) loci exhibit a remarkable influence on severe acute graft-versus-host disease prevalence. Indeed, an increased risk of acute graft-versus-host disease was observed in association with single nucleotide polymorphisms located in 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase/inosine monophosphate cyclohydrolase (hazard ratio=3.04; P=0.002), nuclear factor of activated T cells (cytoplasmic 1) (hazard ratio=2.69; P=0.004), adenosine triphosphate-binding cassette sub-family C2 (hazard ratio=3.53; P=0.0018) and adenosine triphosphate-binding cassette sub-family C1 (hazard ratio=3.67; P=0.0005). While donor single nucleotide polymorphisms of dihydrofolate reductase and solute carrier family 19 (member 1) genes are associated with a reduced risk of acute graft-versus-host disease (hazard ratio=0.32–0.41; P=0.0009–0.008), those of nuclear factor of activated T cells (cytoplasmic 2) are found to increase such risk (hazard ratio=3.85; P=0.0004). None of the tested single nucleotide polymorphisms was associated with the occurrence of chronic graft-versus-host disease. In conclusion, by targeting drug-related biologically relevant genes, this work emphasizes the potential role of germline biomarkers in predicting acute graft-versus-host disease. Further investigations are warranted to improve our understanding of these relationships to personalize immunosuppressive therapy and optimize outcomes.