HLA-matched Allogeneic Stem Cell Transplantation Research Articles

Current Approaches for the Prevention and Treatment of Acute and Chronic GVHD.

Whereas aGVHD has strong inflammatory components, cGVHD displays autoimmune and fibrotic features; incidence and risk factors are similar but not identical; indeed, the aGVHD is the main risk factor for cGVHD. Calcineurin Inhibitors (CNI) with either Methotrexate (MTX) or Mycophenolate (MMF) still represent the standard prophylaxis in HLA-matched allogeneic stem cell transplantation (HSCT); other strategies focused on ATG, Post-Transplant Cyclophosphamide (PTCy), Abatacept and graft manipulation. Despite the high rate, first-line treatment for aGVHD is represented by corticosteroids, and Ruxolitinib is the standard second-line therapy; investigational approaches include Microbiota transplant and the infusion of Mesenchymal stem cells. GVHD is a pleiotropic disease involving any anatomical district; also, Ruxolitinib represents the standard for steroid-refractory cGVHD in this setting. It is a pleiotropic disease involving any anatomical district; also, Ruxolitinib represents the standard for steroid-refractory cGVHD in this setting. Extracorporeal Photopheresis (ECP) is still an option used for steroid refractoriness or to achieve a steroid-sparing. For Ruxolitinib-refractory cGVHD, Belumosudil and Axatilimab represent the most promising agents. Bronchiolitis obliterans syndrome (BOS) still represents a challenge; among the compounds targeting non-immune effectors, Alvelestat, a Neutrophil elastase inhibitor, seems promising in BOS. Finally, in both aGVHD and cGVHD, the association of biological markers with specific disease manifestations could help refine risk stratification and the availability of reliable biomarkers for specific treatments.

An Alternative Novel Therapy for the Treatment of Chronic Inflammatory Demyelinating Polyneuropathy: Adult Autologous Telomerase-Positive Stem Cells

Chronic inflammatory demyelinating polyneuropathy, e.g., chronic inflammatory demyelinating polyradiculoneuropathy (CIDP), is a rare autoimmune mediated peripheral neuropathy. CIDP is defined as symptomology of greater than two months duration and electro diagnostic evidence of peripheral nerve demyelination. The estimated overall prevalence of CIDP is 4.8 to 8.9 cases per 100,000 people. Symptomology includes motor, sensory, and autonomic involvement resulting in symmetrical proximal and distal muscle weakness, loss of strength, areflexia of greater than eight weeks duration, numbness, weakness, sensory ataxia, paresthesia, decreased peripheral temperature, and gait disorder. As CIDP progresses there is axonal loss within mixed peripheral nerves secondary to demyelination, which is associated with a poor prognosis. Autoantibodies identified for CIDP thus far include contactin-1 (CNTN1), contactin-associated protein-1 (Caspr1), contactin-2 (CNTN2), neurofascin-155 (Nfasc-155), neurofascin-140/186(Nfasc-140/186), LM1, gliomedin, and vinculin. Another marker of CIDP is sphingomyelin protein in the cerebral spinal fluid. Potential treatment options for CIDP are first-line therapies, such as corticosteroids, plasma exchange, and/or immunoglobulins. If patients are refractory to first-line treatments to halt progression of the disease, then second-line therapies, such as chemotherapeutic drugs, immunosuppressive drugs, and/or immunomodulatory drugs, are utilized. Lastly, if first- and second-line therapies fail, novel unconventional therapies have been utilized, such as high-dose cyclophosphamide to eradicate a defective immune system containing CIDP-associated autoantibodies to nodal and par anodal proteins. This is then followed with either autologous or HLA-matched allogeneic hematopoietic stem cell transplantation (HSCT) with the intent to replace the defective immune system with a normal immune system absent of CIDP-associated autoantibodies. Whatever therapeutic treatment regimen(s) is/are utilized, maintenance treatments are required for years to maintain stasis in individuals with CIDP. Unfortunately, while first-line, second-line, and/or HSCT treatments may halt the progression of the CIDP and maintain individuals in stasis, they do little to restore neurophysiological function to the individual. We proposed an alternative unconventional therapy to treat CIDP, the use of adult autologous adult telomerase positive stem cells to halt progression of the disease and restore (neuro-) physiological function to the tissues. This hypothesis was based on previous clinical studies utilizing telomerase positive stem cells with Parkinson disease, Alzheimer’s disease, age- related dry macular degeneration, traumatic blindness, traumatic spinal cord injury, myocardial infarction, chronic obstructive pulmonary disease, idiopathic pulmonary fibrosis, celiac disease, systemic lupus erythematosus, and osteoarthritis. Within this small cohort (n=3) clinical study, there were no adverse events reported for any participant treated. While there was no direct proof that the autologous telomerase positive stem cells contributed to the results seen in two of these participants, there was indirect proof for restoration of neurophysiological functions. This was demonstrated with respect to return of motor, sensory, and autonomic functions, e.g., increased strength, return of sensory input, return of reflexes, loss of numbness, increased blood flow, normal body temperature in extremities, and normal gait. Indirectly, this suggested that autologous telomerase positive stem cells are safe and demonstrate a 66% efficacy with respect to halting progression of chronic inflammatory demyelinating polyneuropathy and restoration of neurophysiological functions.

Transgenic lymphocytes targeting minor histocompatibility antigens for post-transplant immunotherapy

Background & Aim The relapse of acute leukemia occurs in one third of the patients after HLA-matched allogeneic stem cell transplantation (HSCT), resulting in high mortality. Disparities in minor histocompatibility antigens (MiHA, MHC-presented peptides derived from polymorphic self-proteins) can result in the development of allogeneic immune response. Several MiHA (HA-1, HA-2, and ACC-1Y) are encoded by the genes predominantly expressed in hematopoietic tissues. They represent promising targets for post-HSCT relapse therapy. The immune response towards them selectively targets patient hematopoietic cells sparing hematopoietic cells of donor origin and patients healthy tissues. Methods, Results & Conclusion Naïve CD8+ T cells from MiHA-negative donors were expanded in vitro using autologous dendritic cells, pulsed with MiHA peptides. Antigen-specific cells were sorted, and the sequences of T-cell receptor (TCR) alpha and beta chains were determined by NGS. 8, 10, and 1 TCRs specific for HA-1, HA-2, and ACC-1Y antigens, respectively, were cloned in the lentiviral vectors containing coreceptor CD8. T cell lines were generated for further analysis. To prevent off-target effects caused by the mispairing of endogenous and recombinant TCR chains, the endogenous TCR knockout was made using gRNA/Cas9 ribonucleoprotein complexes. Transgenic alpha and beta-chain constant domains were modified to be resistant to Cas9 cleavage and to form more stable TCR. Antigen-specificity was measured by binding to MHC-tetramers loaded with the MiHA peptide and by TCR-mediated activation of the Jurkat 76 triple parameter reporter cell line upon antigen stimulation. Most of the generated cell lines were specific to the desired antigen, and none were activated in response to irrelevant peptides presented in the same HLA. After analysis of cross-reactivity to allogeneic HLA on the panel of Epstein-Barr virus-transformed lymphoblastoid cell lines, the best receptors would be selected for further therapeutic application. Adoptive transfer of MiHA-specific genetically modified T cells holds great promise for the treatment of leukemia relapse after allo-HSCT. The work was supported by the Russian Foundation for Basic Research grant 19-29-04156. The relapse of acute leukemia occurs in one third of the patients after HLA-matched allogeneic stem cell transplantation (HSCT), resulting in high mortality. Disparities in minor histocompatibility antigens (MiHA, MHC-presented peptides derived from polymorphic self-proteins) can result in the development of allogeneic immune response. Several MiHA (HA-1, HA-2, and ACC-1Y) are encoded by the genes predominantly expressed in hematopoietic tissues. They represent promising targets for post-HSCT relapse therapy. The immune response towards them selectively targets patient hematopoietic cells sparing hematopoietic cells of donor origin and patients healthy tissues. Naïve CD8+ T cells from MiHA-negative donors were expanded in vitro using autologous dendritic cells, pulsed with MiHA peptides. Antigen-specific cells were sorted, and the sequences of T-cell receptor (TCR) alpha and beta chains were determined by NGS. 8, 10, and 1 TCRs specific for HA-1, HA-2, and ACC-1Y antigens, respectively, were cloned in the lentiviral vectors containing coreceptor CD8. T cell lines were generated for further analysis.

Optimized Whole Genome Association Scanning for Discovery of HLA Class I-Restricted Minor Histocompatibility Antigens

Patients undergoing allogeneic stem cell transplantation as treatment for hematological diseases face the risk of Graft-versus-Host Disease as well as relapse. Graft-versus-Host Disease and the favorable Graft-versus-Leukemia effect are mediated by donor T cells recognizing polymorphic peptides, which are presented on the cell surface by HLA molecules and result from single nucleotide polymorphism alleles that are disparate between patient and donor. Identification of polymorphic HLA-binding peptides, designated minor histocompatibility antigens, has been a laborious procedure, and the number and scope for broad clinical use of these antigens therefore remain limited. Here, we present an optimized whole genome association approach for discovery of HLA class I minor histocompatibility antigens. T cell clones isolated from patients who responded to donor lymphocyte infusions after HLA-matched allogeneic stem cell transplantation were tested against a panel of 191 EBV-transformed B cells, which have been sequenced by the 1000 Genomes Project and selected for expression of seven common HLA class I alleles (HLA-A∗01:01, A∗02:01, A∗03:01, B∗07:02, B∗08:01, C∗07:01, and C∗07:02). By including all polymorphisms with minor allele frequencies above 0.01, we demonstrated that the new approach allows direct discovery of minor histocompatibility antigens as exemplified by seven new antigens in eight different HLA class I alleles including one antigen in HLA-A∗24:02 and HLA-A∗23:01, for which the method has not been originally designed. Our new whole genome association strategy is expected to rapidly augment the repertoire of HLA class I-restricted minor histocompatibility antigens that will become available for donor selection and clinical use to predict, follow or manipulate Graft-versus-Leukemia effect and Graft-versus-Host Disease after allogeneic stem cell transplantation.

Results of a Phase III Double-Blind Study of the Addition of Tocilizumab Vs. Placebo to Cyclosporin/Methotrexate Gvhd Prophylaxis after HLA-Matched Allogeneic Stem Cell Transplantation

Results of a Phase III Double-Blind Study of the Addition of Tocilizumab Vs. Placebo to Cyclosporin/Methotrexate Gvhd Prophylaxis after HLA-Matched Allogeneic Stem Cell Transplantation

T-cell depletion effects of low-dose antithymocyte globulin for GVHD prophylaxis in HLA-matched allogeneic peripheral blood stem cell transplantation

T-cell depletion effects of low-dose antithymocyte globulin for GVHD prophylaxis in HLA-matched allogeneic peripheral blood stem cell transplantation

Integrated In Silico Prediction of Minor Histocompatibility Antigen Peptides in HLA-Matched Allogeneic Stem Cell Transplantation for Acute Myeloid Leukemia

Background and Rationale: T-cell responses to minor histocompatibility antigens (mHA) are important drivers of the beneficial graft versus leukemia (GvL) effect and harmful graft versus host disease (GvHD) pathology following HLA-matched allogeneic stem cell transplantation (alloSCT). Despite their importance, Genome Wide Association Studies (GWAS) have failed to elicit a prognostic set of individual mHA associated with the clinically observed GvL and GvH effects of alloSCT [Sato-Otsubo et al. Blood 2015]. This is likely due to a lack of public mHA shared across the global set of donor/recipient pairs (DRPs). Even an optimally frequent single nucleotide polymorphism (SNP) would result in a recipient restricted genetic variant in only 24% of DRPs with a matched unrelated donor (MUD) or 17% of DRPs with a matched related donor (MRD) [Armistead et al. PLoS One 2011]. Moreover, even when DRPs do share a recipient restricted genetic variant, any resulting peptide would have to bind a MHC molecule within the recipient for presentation to T-cells.Methods: To evaluate the role of mHA in alloSCT without requiring public mHA, we developed a bioinformatics pipeline to predict mHA peptides based on SNP differences taking into account peptide/MHC binding estimated by netMHCpan [Nielson et al. Genome Med 2016] and expected GvL vs GvH tissue expression derived from mRNA sequencing data from acute myeloid leukemia (AML) and normal hematopoietic, hepatobiliary, skin, and gastrointestinal tract tissues. In order to understand the distribution of mHA in HLA-matched alloSCT drawn from a diverse pool of DRPs, we applied this analysis to putative DRPs drawn from healthy individuals who underwent whole-exome sequencing as part of the 1000 Genomes Project (n = 1,916) [1000 Genomes Project Consortium et al. Nature 2015]. To evaluate the association of mHA with AML relapse and GvHD incidence following alloSCT, we predicted mHA from SNP data using clinical information from an earlier study [Armistead et al. PLoS One 2011].Results: To determine a baseline for the number of predicted mHA in an alloSCT, we considered every possible pair of samples in the 1000 Genomes data set as a theoretical alloSCT (n = 3,669,140). We determined each sample’s HLA type using PHLAT [Bai et al. BMC Genomics 2014] and then performed mHA predictions for all theoretical transplants with a 10 out of 10 HLA match (n = 10). Within each ethnicity represented in the 1000 Genomes data, the degree of HLA matching was greater than that of all ethnicities pooled, with the lowest HLA diversity in the Finnish, Chinese, and Japanese populations. The number of predicted mHA binding MHC with Kd < 500nM ranged from 6,217 to 13,545 in the 1000 Genomes theoretical HLA-matched DRPs, with mHA contained in genes selectively expressed in AML versus those selectively expressed in GvH target organs numbering 213 to 610 and 367 to 1,135, respectively. HLA-A*02:01 restricted mHA were predicted for 37 actual DRPs in the context of MUD alloSCT and 97 DRPs from MRD alloSCT [Armistead et al. PLoS One 2011]. There were significantly more predicted mHA in MUD transplants (Figure 1). Taking into account both predicted peptide/MHC binding and tissue expression, the number of predicted GvL mHA was significantly associated with remission and the aggregate number of predicted GvH mHA was significantly associated with grade 2-4 GvHD incidence in MUD transplants (Figure 2).Conclusions: Prediction of mHA based on whole-exome sequencing data is feasible and can be used to discover associations of mHA distribution features with clinical outcomes including AML remission and GvHD incidence. Future work in larger datasets will be required to validate these predicted associations and guide development of mHA-directed therapeutics. [Display omitted] [Display omitted] DisclosuresMolldrem:Astellas Pharma: Patents & Royalties.

The Value of Online Algorithms to Predict T-Cell Ligands Created by Genetic Variants

Allogeneic stem cell transplantation can be a curative treatment for hematological malignancies. After HLA-matched allogeneic stem cell transplantation, beneficial anti-tumor immunity as well as detrimental side-effects can develop due to donor-derived T-cells recognizing polymorphic peptides that are presented by HLA on patient cells. Polymorphic peptides on patient cells that are recognized by specific T-cells are called minor histocompatibility antigens (MiHA), while the respective peptides in donor cells are allelic variants. MiHA can be identified by reverse strategies in which large sets of peptides are screened for T-cell recognition. In these strategies, selection of peptides by prediction algorithms may be relevant to increase the efficiency of MiHA discovery. We investigated the value of online prediction algorithms for MiHA discovery and determined the in silico characteristics of 68 autosomal HLA class I-restricted MiHA that have been identified as natural ligands by forward strategies in which T-cells from in vivo immune responses after allogeneic stem cell transplantation are used to identify the antigen. Our analysis showed that HLA class I binding was accurately predicted for 87% of MiHA of which a relatively large proportion of peptides had strong binding affinity (56%). Weak binding affinity was also predicted for a considerable number of antigens (31%) and the remaining 13% of MiHA were not predicted as HLA class I binding peptides. Besides prediction for HLA class I binding, none of the other online algorithms significantly contributed to MiHA characterization. Furthermore, we demonstrated that the majority of MiHA do not differ from their allelic variants in in silico characteristics, suggesting that allelic variants can potentially be processed and presented on the cell surface. In conclusion, our analyses revealed the in silico characteristics of 68 HLA class I-restricted MiHA and explored the value of online algorithms to predict T-cell ligands that are created by genetic variants.

Cyclosporine (CsA) Pharmacogenetics Post HLA-Matched Sibling Allogeneic Hematopoietic Stem Cell Transplantation in Patients with Myeloid Leukemia: Value of CYP3A4 Gene Polymorphism

Cyclosporine (CsA) Pharmacogenetics Post HLA-Matched Sibling Allogeneic Hematopoietic Stem Cell Transplantation in Patients with Myeloid Leukemia: Value of CYP3A4 Gene Polymorphism

Impact of the GSTA1 Gene Polymorphism on Clinical Outcomes of HLA-Matched Sibling Allogeneic Hematopoietic Stem Cell Transplantation in Patients with Myeloid Leukemia

Impact of the GSTA1 Gene Polymorphism on Clinical Outcomes of HLA-Matched Sibling Allogeneic Hematopoietic Stem Cell Transplantation in Patients with Myeloid Leukemia

Integrated Whole Genome and Transcriptome Analysis Identified a Therapeutic Minor Histocompatibility Antigen in a Splice Variant of ITGB2.

In HLA-matched allogeneic hematopoietic stem cell transplantation (alloSCT), donor T cells recognizing minor histocompatibility antigens (MiHAs) can mediate desired antitumor immunity as well as undesired side effects. MiHAs with hematopoiesis-restricted expression are relevant targets to augment antitumor immunity after alloSCT without side effects. To identify therapeutic MiHAs, we analyzed the in vivo immune response in a patient with strong antitumor immunity after alloSCT. T-cell clones recognizing patient, but not donor, hematopoietic cells were selected for MiHA discovery by whole genome association scanning. RNA-sequence data from the GEUVADIS project were analyzed to investigate alternative transcripts, and expression patterns were determined by microarray analysis and qPCR. T-cell reactivity was measured by cytokine release and cytotoxicity. T-cell clones were isolated for two HLA-B*15:01-restricted MiHA. LB-GLE1-1V is encoded by a nonsynonymous SNP in exon 6 of GLE1 For the other MiHAs, an associating SNP in intron 3 of ITGB2 was found, but no SNP disparity was present in the normal gene transcript between patient and donor. RNA-sequence analysis identified an alternative ITGB2 transcript containing part of intron 3. qPCR demonstrated that this transcript is restricted to hematopoietic cells and SNP-positive individuals. In silico translation revealed LB-ITGB2-1 as HLA-B*15:01-binding peptide, which was validated as hematopoietic MiHA by T-cell experiments. Whole genome and transcriptome analysis identified LB-ITGB2-1 as MiHAs encoded by an alternative transcript. Our data support the therapeutic relevance of LB-ITGB2-1 and illustrate the value of RNA-sequence analysis for discovery of immune targets encoded by alternative transcripts. Clin Cancer Res; 22(16); 4185-96. ©2016 AACR.

Influence of genetic polymorphisms of cytokine genes in the outcome of HLA-matched allogeneic stem cell transplantation in a South East Asian population

Non-HLA gene polymorphisms have been shown to be associated with the risk of graft-versus-host disease (GVHD) and outcome of allogeneic haematopoietic stem cell transplantation (AHSCT). This study aims to investigate the role of IL6, TNFα, IL10, IL2 and IL12 gene polymorphisms in the outcome of AHSCT in a South East Asian population. A total of 67 patients and 59 donors who underwent HLA-identical matched sibling AHSCT were available for analysis. There was no significant association between the different cytokine genotypes of patients with the incidence and severity of acute GVHD. Patients with IL2 166∗T allele and patients who received donor stem cells who had IL2 166∗G allele appeared to have reduced incidence of cGVHD. Patients who received donor stem cells with IL12 1188∗C allele are found to be associated with better disease free survival. These results suggest a possible role of IL2 and IL12 gene polymorphisms in the outcome of AHSCT in a South East Asian population.

Clinical Analysis of Cytomegalovirus Infection after Different Patterns of Hematopoietic Stem Cell Transplantation

To investigate the clinical difference of cytomegalovirus (CMV) infection between HLA-matched allogeneic hematopoietic stem cell transplantation (allo-HSCT) and haploidentical hematopoietic stem cell transplantation (hi-HSCT). The clinical data of 83 patients who had undergone allo-HSCT were retrospectively analyzed. Out of them 50 patients underwent hi-HSCT and 33 patients received grafts from HLA-matched donors. The sera of all recipients and donors were CMV-negative before transplantation. All patients accepted myeloablative regimen without total body irradiation. PCR was performed to detect CMV in the peripheral blood twice a week after neutrophil recovery. CMV-DNA>500 copies/ml was defined as CMV viremia. 68 patients (81.9%) were diagnosed as CMV viremia before 100 days after transplantation. The incidence of CMV infection in hi-HSCT group was 90% and significantly higher than that in HLA-matched HSCT group (69.7%) (P < 0.05). All the patients responded well to anti-CMV therapy; however, 63 cases experienced CMV reactivation. The occurrence rate of CMV reactivation in hi-HSCT group (95.6%) was comparable to that in HLA-matched HSCT group (87.0%) (P > 0.05). Univariate analysis showed that the transplantation pattern, the recovery time of peripheral neutrophils and the occurrence of acute graft-versus-host disease (aGVHD) significantly related to the episode of CMV viremia, while the sex and age of the recipients, and the recovery time of platelets did not associate with the incidence. Further analysis found that the recovery time of neutropils and platelets in HLA-matched HSCT group were greatly shorter than those in hi-HSCT group (P < 0.05). The incidence of aGVHD was comparable between two groups however, incidence of severe aGVHD was significantly higher in hi-HSCT (P < 0.05). The hi-HSCT is more susceptible to CMV infection, which may be related to the higher incidence of severe aGVHD and the relative delay of hematopoietic reconstruction as compared with HLA-matched HSCT.

Donor CD4 T Cell Diversity Determines Virus Reactivation in Patients After HLA-Matched Allogeneic Stem Cell Transplantation

Delayed reconstitution of the T cell compartment in recipients of allogeneic stem cell grafts is associated with an increase of reactivation of latent viruses. Thereby, the transplanted T cell repertoire appears to be one of the factors that affect T cell reconstitution. Therefore, we studied the T cell receptor beta (TCRβ) gene rearrangements of flow cytometry–sorted CD4+ and CD8+ T cells from the peripheral blood of 23 allogeneic donors before G-CSF administration and on the day of apheresis. For this purpose, TCRβ rearrangements were amplified by multiplex PCR followed by high-throughput amplicon sequencing. Overall, CD4+ T cells displayed a significantly higher TCRβ diversity compared to CD8+ T cells irrespective of G-CSF administration. In line, no significant impact of G-CSF treatment on the TCR Vβ repertoire usage was found. However, correlation of the donor T cell repertoire with clinical outcomes of the recipient revealed that a higher CD4+ TCRβ diversity after G-CSF treatment is associated with lower reactivation of cytomegalovirus and Epstein–Barr virus. By contrast, no protecting correlation was observed for CD8+ T cells. In essence, our deep TCRβ analysis identifies the importance of the CD4+ T cell compartment for the control of latent viruses after allogeneic stem cell transplantation.

Possible role of minor h antigens in the persistence of donor chimerism after stem cell transplantation; relevance for sustained leukemia remission.

Persistent complete donor chimerism is an important clinical indicator for remissions of hematological malignancies after HLA-matched allogeneic stem cell transplantation (SCT). However, the mechanisms mediating the persistence of complete donor chimerism are poorly understood. The frequent coincidence of complete donor chimerism with graft-versus-leukemia effects and graft-versus-host disease suggests that immune responses against minor histocompatibility antigens (mHags) are playing an important role in suppressing the host hematopoiesis after allogeneic SCT. Here, we investigated a possible relationship between donor immune responses against the hematopoiesis-restricted mHag HA-1 and the long-term kinetics of host hematopoietic chimerism in a cohort of 10 patients after allogeneic HLA-matched, HA-1 mismatched SCT. Functional HA-1 specific CTLs (HA-1 CTLs) were detectable in 6/10 patients lysing host-type hematopoietic cells in vitro. Presence of HA-1 CTLs in the peripheral blood coincided with low host hematopoiesis levels quantified by highly sensitive mHag specific PCR. Additionally, co-incubation of host type CD34+ cells with HA-1 CTLs isolated after allogeneic SCT prevented progenitor and cobblestone area forming cell growth in vitro and human hematopoietic engraftment in immunodeficient mice. Conversely, absence or loss of HA-1 CTLs mostly coincided with high host hematopoiesis levels and/or relapse. In summary, in this first study, presence of HA-1 CTLs paralleled low host hematopoiesis levels. This coincidence might be supported by the capacity of HA-1 CTLs isolated after allogeneic SCT to specifically eliminate host type hematopoietic stem/progenitor cells. Additional studies involving multiple mismatched mHags in more patients are required to confirm this novel characteristic of mHag CTLs as factor for the persistence of complete donor chimerism and leukemia remission after allogeneic SCT.

HLA-matched allogeneic stem cell transplantation improves outcome of higher risk myelodysplastic syndrome A prospective study on behalf of SFGM-TC and GFM.

Allogeneic hematopoietic stem cell transplantation (HSCT) is considered the only a curative treatment in patients with higher risk myelodysplastic syndrome (MDS), although demethylating agents (DMA) have been reported to improve survival. The advantage of HSCT over other treatment comes from retrospective studies and the aim of the current study was to prospectively test this hypothesis, analyzing in particular patients from the pre-transplant period to avoid the selection bias of performing transplantation. This study was conducted to compare overall survival in MDS patients candidates to transplantation according to donor availability. The majority of patients (76%) received a treatment with DMA after registration, 69% had a human leukocyte antigen (HLA)-identical donor, 70% of whom were transplanted. Baseline patient and disease characteristics were similar according to donor availability. Four-year overall survival was significantly better in patients with an HLA matched donor (37%) compared to patients without donor (15%). There was also evidence that this overall survival advantage was because of transplantation. Mortality risk was decreased after transplantation but it became significant only after the second year post transplant, because of early transplant-related mortality. Our results appear to justify, in higher risk MDS, a transplantation approach in all potential candidates who have an HLA identical donor.

Pediatric donor cell leukemia after allogeneic hematopoietic stem cell transplantation in AML patient from related donor

Here we present a male patient with acute myeloid leukemia (AML) initially diagnosed as M5 and with karyotype 46,XY. After induction therapy, he underwent a HLA-matched allogeneic hematopoietic stem cell transplantation, and six years later he relapsed as AML M1 with an abnormal karyotype //47,XX,+10[2]/47,XX,+11[3]/48,XX,+10,+11[2]/46,XX[13]. Based on this, we tested the possibility of donor cell origin by FISH and molecular STR analysis. We found no evidence of Y chromosome presence by FISH and STR analysis consistent with the success of the allogeneic hematopoietic stem cell transplantation from the female donor. FISH studies confirmed trisomies and no evidence of MLL translocation either p53 or ATM deletion. Additionally 28 fusion common leukemia transcripts were evaluated by multiplex reverse transcriptase-polymerase chain reaction assay and were not rearranged. STR analysis showed a complete donor chimerism. Thus, donor cell leukemia (DCL) was concluded, being essential the use of cytological and molecular approaches. Pediatric DCL is uncommon, our patient seems to be the sixth case and additionally it presented a late donor cell leukemia appearance. Different extrinsic and intrinsic mechanisms have been considered to explain this uncommon finding as well as the implications to the patient.

Impact de la composition du greffon sur le devenir des patients après une allogreffe de cellules souches hématopoïétiques : corrélation entre proportion des lymphocytes T CD4+ du greffon exprimant le CCR7 et la survenue d’une GVH aiguë

In a previous prospective study on 62 patients who underwent an HLA-matched allogeneic stem cell transplantation, we have observed that proportion of donor-derived CCR7+/CD4+ T cells in the graft provided a predictive indicator of acute GVHD without interfering on chronic GVHD and relapse rate. Here we present our results on a confirmatory cohort of 137 consecutive patients. Indeed patients who received more than 76% of CCR7+/CD4+ T cells in the graft developed more often acute GVHD be it of low or high grade than those who did not. Determination of the CCR7+/CCR7neg ratio of CD4+ T cells in the graft provides a predictive indicator of acute GVHD and could help to define strategies of partial selective T cell depleted transplantation.

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

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. 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. 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. 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.

Collateral Damage of Non-Hematopoietic Tissue by Hematopoiesis-Specific T Cells Results in GvHD During An Ongoing Profound Gvl Reaction,

Abstract 4031Following HLA-matched allogeneic stem cell transplantation (SCT) donor T cells may recognize polymorphic antigens specifically expressed on cells of the recipient, designated as minor histocompatibility antigens (MiHA). It has been hypothesized that hematopoiesis-restricted MiHA may give rise to a specific graft versus leukemia (GvL) effect without graft versus host disease (GvHD), whereas ubiquitously expressed MiHA can be targets for both GvL and GvHD. We and others have observed that during an overt GvL response, associated with the occurrence of T cells specific for hematopoiesis-restricted MiHA, may coincide with limited GvHD. Therefore, we investigated whether allo-reactive T cells recognizing hematopoiesis-restricted MiHA on leukemic cells may induce collateral damage to surrounding non-hematopoietic tissues. To test this, we stimulated MiHA-specific cytotoxic T lymphocytes (CTLs) in vitro with MiHA positive hematopoietic targets and analyzed whether this resulted in lysis of surrounding MiHA negative primary human fibroblasts seeded in the same well. Using chromium release assays, quantitative FACS analysis and fluorescence microscopy, we demonstrated that MiHA-specific CTLs (CD8 n=3, CD4 n=2) and allo-reactive CTLs upon proper activation by MiHA-positive hematopoietic stimulator cells (EBV-LCL/ DCs), induced 80% (60–100%) bystander cytotoxicity to the surrounding fibroblasts at a 5/1 T cell/fibroblast-ratio within 24 hrs. T cell receptor (TCR)-specific T cell activation by the hematopoietic cells was pivotal for the induction of this collateral damage since no bystander kill was observed when MiHA-specific CTLs were exposed to MiHA negative stimulators. Next, we investigated the influence of the strength of T cell activation by stimulating the CTLs with MiHA-negative stimulator cells loaded with increasing concentrations of the relevant peptide resulting in increasing T cell activation measured by IFNg ELISA and expression of the T cell activation marker CD137. We demonstrated that this increase in the strength of T cell activation is correlated with an increased severity of collateral damage. Moreover, we showed also that T cell activation in a cell free system using PHA (0,5ug/ml) or antiCD3/CD28 beads resulted in induction of collateral damage of the surrounding non-hematopoietic cells illustrating that the MiHA-negative fibroblasts are not attacked due to cross-presentation of HLA/peptide complexes from apoptotic hematopoietic cells. Next, we further investigated the prerequisites for collateral damage induction to occur. Using a transwell system, we demonstrated that direct T cell-fibroblast interaction was required. Furthermore, ICAM-1 expression on the non-hematopoietic targets, induced by pro-inflammatory cytokines which were secreted by the activated CTLs, was demonstrated to be crucial for strong T cell-fibroblast interaction. To investigate the contribution of the different T cell effector mechanisms, we retrovirally overexpressed the anti-apoptotic proteins c-FLIP, Bcl-2 or PI-9 in the fibroblasts, thereby inhibiting the death receptor-, mitochondrial-, or granzyme-B (GzB)-mediated pathways, respectively. High expression of c-FLIP or Bcl-2 did not inhibit induction of collateral damage. In contrast, increased PI-9 expression in the fibroblasts partially inhibited induction of collateral damage. Moreover, blockade of Ca2+-dependent secretion of perforin by EGTA, could also inhibit collateral damage suggesting that the main killing pathway of collateral damage is via release of GzB- and perforin-containing granules by the T cells upon TCR-triggering. Misdirection of the release of cytotoxic granules towards the MiHA negative non-hematopoietic targets may induce collateral damage. In conclusion, these data suggest that hematopoiesis-restricted T cells actively participating in an overt GvL response can induce GvHD by collateral damage to MiHA-negative non-hematopoietic targets by misdirection of their cytotoxic execution machinery when they are in close proximity of the non-hematopoietic tissue. Disclosures:No relevant conflicts of interest to declare.