Evidence That HLA-B*40:02 and HLA-Α*31:01 Are Strongly Involved in the Presentation of Autoantigens to CTLs Responsible for the Development of Acquired Aplastic Anemia

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 2948-2948
Author(s):  
Yoshitaka Zaimoku ◽  
Hiroyuki Maruyama ◽  
Kana Maruyama ◽  
Takamasa Katagiri ◽  
An T. T. Dao ◽  
...  
Keyword(s):  
T Cells ◽  
Aplastic Anemia ◽  
Nk Cells ◽  
Conflicts Of Interest ◽  
Neutral Loss ◽  
The Other ◽  
Class I ◽  
Allelic Loss ◽  
Hla Alleles ◽  
Allele Specific

Abstract The presence of leukocytes lacking one haplotype of the human leukocyte antigen (HLA) gene as a result of copy-neutral loss of heterozygosity of chromosome 6p (6pLOH) is compelling evidence that cytotoxic T cells (CTLs) have a key role in the development of acquired aplastic anemia (AA). Pathogenic auto-antigens are presumed to be presented by particular HLAs, such as HLA-B*40:02, HLA-A*31:01, HLA-A*02:01 and HLA-A*02:06, all of which are frequently lost due to 6pLOH (Katarigi, Blood 2011). However, this presumption may be incorrect, because the frequency of lacking haplotypes that contain KIR-ligands (KIR-Ls), such as HLA-A*24:02 and HLA-B*52:01, the most frequent alleles of HLA-A and HLA-B, may be underestimated as a result of the killing of 6pLOH(+) leukocytes by NK cells. Indeed, all four frequently missing HLA alleles are non-KIR-Ls. To address these issues, we conducted a mass screening of 6pLOH(+) leukocytes in newly diagnosed AA patients using an assay that allows us to detect 6pLOH(+) cells within one day, and reanalyzed haplotypes that are likely to be lost. The HLA-B and –C allele-specific duplex real time PCR (2qPCR) that we established compared the copy number of heterozygous HLA alleles in a single reaction mixture using two allele-specific TaqMan probes labeled with two different fluorochromes (VIC and FAM) and one primer pair complementary to consensus sequences, which allowed us to detect as small as 5% 6pLOH(+) leukocytes in the total leukocytes. The HLA haplotypes of 6pLOH(+) patients were determined based on the HLA-A antigen expression as demonstrated by flow cytometry and the HLA-B and -C allele data from the 2qPCR, in combination with the haplotype database of the Japanese population available in the HLA laboratory website. A total of 498 patients with AA were subjected to this analysis and 60 6pLOH(+) patients, including 39 6pLOH(+) patients that had been identified by our previous study were used in this analysis. The allelic loss frequencies of HLA-B*40:02 (25%, 33/132) and HLA-A*31:01 (19%, 14/75) were markedly higher than those of the other HLA-B (2.9%, 24/814, P = 7x10-16) and HLA-A (4.9%, 36/731, P = 0.002) alleles, while the frequencies of HLA-A*02:01 (11%, 11/99) and HLA-A*02:06 (11%, 11/99) were similar to those of the other HLA-A alleles. In 41 6pLOH(+) patients possessing either HLA-B*40:02 or HLA-A*31:01, these alleles were all contained in the missing haplotype. On the other hand, four of the 15 patients with HLA-A*02:01(+) haplotypes and two of the 13 patients with HLA-A*02:06(+) haplotypes had these alleles in the retained haplotype. The 6pLOH(+) patients could be divided into three groups according to the status of KIR-Ls (HLA-Bw4 and HLA-C1/C2) of their haplotype; A, the lack of the KIR-L does not occur when either of the two haplotypes is lost; B, the lack of KIR-L occurs when one specific haplotypes is lost; C, the lack of KIR-L occurs when either haplotype is lost. These groups comprised 42%, 53% and 5% of the total. The proportion of group C was much lower than that expected in the general population (16%) and only five (16%) of the 31 group B patients lacked KIR-Ls, thus suggesting that NK cells had an effect on the appearance of 6pLOH(+) leukocytes in AA patients. However, the very high frequency of HLA-loss in the HLA-B*40:02 and HLA-A*31:01 alleles could not be explained by the absence of KIR-Ls in the missing haplotype. Of particular note, the lack of KIR-Ls occurred in eight patients as a result of 6pLOH; six of the eight lost a haplotype containing HLA-B*40:02 and one lost an A*31:01-containing haplotype, suggesting that CTLs specific for autoantigens presented by these class I alleles more dominantly inhibit HSCs than NK cells. Together, these results indicate that HLA-B*40:02 and HLA-A*31:01 have particularly important roles in the presentation of autoantigens to T cells in AA. Studies of T-cell responses to autoantigens restricted by these class I alleles are thus warranted. Figure 1 Figure 1. Figure 2 Figure 2. Disclosures No relevant conflicts of interest to declare.

Immunologically-Selected Hematopoiesis Caused by HLA Allelic Loss In Patients with Aplastic Anemia

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 198-198
Author(s):  
Takamasa Katagiri ◽  
Aiko Matsubara ◽  
Koichi Kashiwase ◽  
Motohiro Kato ◽  
Yasuo Morishima ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
Aplastic Anemia ◽  
Myeloid Leukemia ◽  
Genetic Alterations ◽  
Class I ◽  
Allelic Loss ◽  
Allogeneic Bone ◽  
Hematopoietic Stem ◽  
Hla Alleles

Abstract Abstract 198 Background: Idiopathic aplastic anemia (AA) is a syndrome characterized by pancytopenia and bone marrow (BM) hypoplasia, which are caused by the auto-immune destruction of hematopoietic stem cells (HSCs). However, little is known about the nature of HSCs that survive the occurrence of autoimmune insults and maintain hematopoiesis both during and after aplastic diathesis. While the severity of the autoimmunity is the major determinant of BM failure in patients with AA, some intrinsic genetic changes in HSCs could also be involved in the disease process, since the clonality of the residual, persistent hematopoiesis under the aplastic state has been well recognized. Objectives/Methods: To characterize the nature of the HSCs that support hematopoiesis in AA, peripheral blood (PB) specimens obtained from 317 patients with AA were subjected to the genome-wide analysis of genetic lesions using Affymetrix® 500K SNP arrays. For the normal controls, 1746 PB specimens from the Japan Marrow Donation Program (JMDP) were also analyzed. All specimens were genotyped for HLA-A, -B, -C, -DRB1, -DQB1 and -DPB1 alleles. In the eligible cases, PB leukocytes and CD34+ BM cells were also examined to determine their expression of HLA-A antigens using allele-specific monoclonal antibodies by flow cytometry (FCM). To identity the common HLA types associated with AA, a total of 6,629 registries from JMDP who had received allogeneic bone marrow transplantation between 1992 and 2008 were employed, where the HLA frequencies in AA (N=406) were compared with those among other hematopoietic disorders, in acute myeloid leukemia (N=1,822), acute lymphocytic leukemia (N=1,406), chronic myeloid leukemia (N=1,014), myelodysplastic syndromes (N=824), non-Hodgkin's lymphoma (N=565), and other neoplastic disorders (N=392). Results: A number of genetic alterations were detected in our AA case series, among which the most conspicuous was acquired uniparental disomy (UPD), or the copy number-neutral loss of heterozygosity, involving the 6p arms (6pUPD). The 6pUPD was identified in 38 patients (12%) but not in any JMDP donor specimens, and it commonly affected the HLA locus, which was expected to result in the loss of one HLA haplotype. In fact, loss of HLA-A expression from the missing haplotype was confirmed by FCM in all 13 6pUPD-positive cases thus far tested, whereas the HLA-A expression from both haplotypes was preserved in the 58 samples without 6pUPD. The loss of HLA-A expression in the 6pUPD-positive cases was found in multiple lineages of leukocytes, including granulocytes, monocytes, B cells, BM CD34+ cells, and to a lesser extent in T cells. Of particular interest was the fact that the missing HLA haplotypes that were predicted from the SNP array data were extremely biased to particular class I HLA alleles, including HLA-A*02:01, HLA-A*02:06, A*31:01, B*40:02, and B*40:06. Moreover, when the frequencies of these alleles were compared among the 6,629 JMDP registries, they were shown to be strongly associated with AA in comparison to other non-significant HLA alleles, where the odds ratios for these alleles with regard to non-significant alleles as for the risk of the development of AA were 2.00 (95%CI; 1.52 – 2.62) for A*02:01, 2.37 (95%CI; 1.80 – 3.12) for A*02:06, 1.46 (95%CI; 1.06 – 2.02) for A*31:01, 2.07 (1.56 - 2.77) for B*40:02, and 2.67 (1.95 - 3.66) for B*40:06. Conclusions: AA patients frequently show permissive hematopoiesis with 6pUPD, which is thought to develop due to the occurrence of auto-immune insult based on the Darwinian principle of “survival of the fittest” (Figure). The tight association of AA with particular class I antigens that are invariably missing in permissive hematopoiesis with 6pUPD strongly supports the hypothesis that the auto-immunity responsible for AA is primarily mediated by cytotoxic T cells which target a relatively limited species of auto-antigens presented on HSCs through these relevant HLAs. Our findings therefore also provide a solid basis for isolating the target auto-antigens responsible for the development of AA in the future studies. Disclosures: No relevant conflicts of interest to declare.

Immunobiology and Pathophysiology of Hodgkin Lymphomas

Hematology ◽  
2005 ◽  
Vol 2005 (1) ◽  
pp. 231-238 ◽  
Author(s):  
Sibrand Poppema
Keyword(s):  
T Cells ◽  
Immune Responses ◽  
Nk Cells ◽  
Intracellular Signaling ◽  
Transforming Growth Factor ◽  
Hla Class I ◽  
The Other ◽  
Class I ◽  
Immunoglobulin Gene ◽  
Cellular Interactions

Abstract Classical Hodgkin lymphoma (HL) is characterized by the presence of Reed-Sternberg (RS) cells, which are transformed post-germinal center B cells destined for apoptosis since they have not undergone successful immunoglobulin gene rearrangement. Several mechanisms, including latent infection by Epstein-Barr virus (EBV), allow these cells to survive. It is remarkable that many of the signaling pathways that promote survival are shared between the EBV-induced proteins, such as EBNA1, LMP1, and LMP2, and other molecules that are upregulated in RS cells. A key role is played by the presence of constitutive nuclear factor (NF)-κB, which is induced by LMP1, as well as by CD30, CD40, tumor necrosis factor (TNF)-α, and Notch1 interactions, and results in the upregulation of at least 45 genes including chemokines, cytokines, receptors, apoptotic regulators, intracellular signaling molecules, and transcription factors. The other characteristic of classical HL is the presence of an extensive inflammatory infiltrate. Key features of this infiltrate are that it comprises Th2 and T regulatory cells and generally lacks Th1 cells, CD8 cytotoxic T cells, and natural killer (NK) cells. The RS cells appear to induce this infiltrate by the secretion of Th2 type chemokines such as TARC and MDC. The RS cells also produce cytokines that inhibit Th1 responses, as interleukin (IL)-10 and transforming growth factor (TGF)-β express CD95 ligand, which induces apoptosis of activated Th1 and CD8 T cells. Other important mechanisms that allow the RS cells to escape an effective anti-EBV immune response include the downregulation of HLA class I in EBV-negative cases or the presence of a polymorphism in HLA class I in EBV-positive cases that allow escape from CD8-mediated cytotoxicity. On the other hand, expression of HLA-G allows the escape from NK cells that would normally recognize the HLA class I-negative RS cells. Overall, the cellular infiltrate in HL appears to play a decisive role in allowing the RS cells to survive by providing an environment that suppresses cytotoxic immune responses and providing cellular interactions and cytokines that support the growth and survival of RS cells. Future therapeutic strategies could focus directly on the NF-κB activation, on various receptors to ligand interactions, on the chemokine and cytokine network, or on the induction of effective anti-EBV latent protein immune responses.

Inhibitory MHC class I receptors on NK cells and T cells

1996 ◽  
Vol 17 (2) ◽  
pp. 86-91 ◽  
Author(s):  
Lewis L. Lanier ◽  
Joseph H. Phillips
Keyword(s):  
T Cells ◽  
Nk Cells ◽  
Mhc Class I ◽  
Class I

scholarly journals Recognition of virus-infected cells by natural killer cell clones is controlled by polymorphic target cell elements.

1993 ◽  
Vol 178 (3) ◽  
pp. 961-969 ◽  
Author(s):  
M S Malnati ◽  
P Lusso ◽  
E Ciccone ◽  
A Moretta ◽  
L Moretta ◽  
...  
Keyword(s):  
T Cells ◽  
Nk Cells ◽  
Natural Killer ◽  
Target Cell ◽  
Nk Cell ◽  
Class I ◽  
Target Cells ◽  
Cell Recognition ◽  
Infected Cells ◽  
Nk Cell Recognition

Natural killer (NK) cells provide a first line of defense against viral infections. The mechanisms by which NK cells recognize and eliminate infected cells are still largely unknown. To test whether target cell elements contribute to NK cell recognition of virus-infected cells, human NK cells were cloned from two unrelated donors and assayed for their ability to kill normal autologous or allogeneic cells before and after infection by human herpesvirus 6 (HHV-6), a T-lymphotropic herpesvirus. Of 132 NK clones isolated from donor 1, all displayed strong cytolytic activity against the NK-sensitive cell line K562, none killed uninfected autologous T cells, and 65 (49%) killed autologous T cells infected with HHV-6. A panel of representative NK clones from donors 1 and 2 was tested on targets obtained from four donors. A wide heterogeneity was observed in the specificity of lysis of infected target cells among the NK clones. Some clones killed none, some killed only one, and others killed more than one of the different HHV-6-infected target cells. Killing of infected targets was not due to complete absence of class I molecules because class I surface levels were only partially affected by HHV-6 infection. Thus, target cell recognition is not controlled by the effector NK cell alone, but also by polymorphic elements on the target cell that restrict NK cell recognition. Furthermore, NK clones from different donors display a variable range of specificities in their recognition of infected target cells.

scholarly journals Heterogeneous phenotypes of expression of the NKB1 natural killer cell class I receptor among individuals of different human histocompatibility leukocyte antigens types appear genetically regulated, but not linked to major histocompatibililty complex haplotype.

1996 ◽  
Vol 183 (4) ◽  
pp. 1817-1827 ◽  
Author(s):  
J E Gumperz ◽  
N M Valiante ◽  
P Parham ◽  
L L Lanier ◽  
D Tyan
Keyword(s):  
T Cells ◽  
Cell Surface ◽  
Nk Cells ◽  
Natural Killer ◽  
Killer Cell ◽  
Peripheral Blood Lymphocytes ◽  
Class I ◽  
Cell Surface Expression ◽  
Target Cells ◽  
Hla Type

Natural killer (NK) cells that express the NKB1 receptor are inhibited from killing target cells that possess human histocompatibility leukocyte antigen (HLA) B molecules bearing the Bw4 serological epitope. To investigate whether NKB1 expression is affected by HLA type, peripheral blood lymphocytes of 203 HLA-typed donors were examined. Most donors had a single population of NKB1+ cells, but some had two populations expressing different cell surface levels of NKB1, and others had no detectable NKB1+ cells. Among the donors expressing NKB1, both the relative abundance of NKB1+ NK cells and their level of cell surface expression varied substantially. The percentage of NKB1+ NK cells ranged from 0 to >75% (mean 14.7%), and the mean fluorescence of the positive population varied over three orders of magnitude. For each donor, the small percentage of T cells expressing NKB1 (usually <2%), had a pattern of expression mirroring that of the NK cells. NKB1 expression by NK and T cells remained stable over the 2-yr period that five donors were tested. Patterns of NKB1 expression were not associated with Bw4 or Bw6 serotype of the donor or with the presence of any individual HLA-A or -B antigens. Cells expressing NKB1 are often found in donors who do not possess an appropriate class I ligand, and can be absent in those who express Bw4+ HLA-B antigens. Family studies further suggested that the phenotype of NKB1 expression is inherited but not HLA linked. Whereas identical twins show matching patterns of NKB1 expression, HLA-identical siblings can differ in NKB1 expression, and conversely, HLA-disparate siblings can be similar. Thus NKB1 expression phenotypes are tightly regulated and extremely heterogeneous, but not correlated with HLA type.

Anti-Leukemia Activity of Alloreactive NK Cells in Haploidentical HSCT in Pediatric Patients: Re-Defining the Role of Activating and Inhibitory KIR

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 3002-3002 ◽  
Author(s):  
Daniela Pende ◽  
Stefania Marcenaro ◽  
Michela Falco ◽  
Stefania Martini ◽  
Maria Ester Bernardo ◽  
...  
Keyword(s):  
T Cell ◽  
Nk Cells ◽  
Nk Cell ◽  
Cell Subset ◽  
Hla Class I ◽  
Class I ◽  
Leukemia Cells ◽  
Hla Alleles ◽  
Time Points ◽  
Selection Of

Abstract T-cell depleted hematopoietic stem cell transplantation from haploidentical donors (haplo-HSCT) has been reported to benefit from the graft-versus-leukemia effect mediated by natural killer (NK) cells when donor displays NK alloreactivity versus the recipient. NK alloreactivity is mediated by NK receptors, namely Killer Ig-like receptors (KIR) which are specific for allotypic determinants that are shared by different HLA-class I alleles (referred to as KIR ligands). It is known that KIR2DL1 recognizes HLA-C alleles characterized by Lys at position 80 (C2 group), KIR2DL2/3 recognize HLA-C alleles characterized by Asn at position 80 (C1 group), KIR3DL1 recognizes HLA-B alleles sharing the Bw4 supertypic specificity (Bw4 group) and KIR3DL2 recognizes HLA-A3 and –A11 alleles. KIR2D/3DL are inhibitory receptors that, upon engagement with the cognate ligand, inhibit lysis. Activating KIRs, highly homologous in the extracellular domain to the inhibitory counterparts, are KIR2DS1, KIR2DS2 and KIR3DS1, but only KIR2DS1 has been shown to specifically recognize C2 group of alleles expressed on B-EBV cells. We analyzed 21 children with leukemia receiving haplo-HSCT from a relative after a myeloablative conditioning regimen; in all pairs, the expression of a given KIR ligand (HLA class I allele) of the donor was missing in the patient (i.e. KIR ligand-mismatched haplo-HSCT). T-cell depletion was performed through positive selection of CD34+ cells; no pharmacological immune suppression was employed after HSCT. KIR genotype of all donors was evaluated to detect the presence of the various inhibitory and activating KIR genes. Phenotypic analyses were performed on NK cells derived from the donor and the patient at different time points after HSCT. Thanks to the availability of new mAbs able to discriminate between the inhibitory and the activating forms of a certain KIR, we could identify the alloreactive NK cell subset at the population level. These alloreactive NK cells express the KIR specific for the KIR ligand-mismatch (permissive inhibitory KIR) and the activating KIR (if present), while they do not express all inhibitory KIR specific for the patient HLA alleles and NKG2A. Thus, in most instances, we could precisely identify the size of the alloreactive NK cell subset in the donor and in the reconstituted repertoire of the recipient. Functional assays were performed to assess alloreactivity, using appropriate B-EBV cell lines and, if available, patient’s leukemia blasts. In some cases, also NK cell clones were extensively studied, for phenotype and receptor involvement in killing activity. We found that, in most transplanted patients, variable proportions of donor-derived alloreactive NK cells displaying anti-leukemia activity were generated and maintained even at late time-points after transplantation. Donor-derived KIR2DL1+ NK cells isolated from the recipient displayed the expected capability of selectively killing C1/C1 target cells, including patient leukemia blasts. Differently, KIR2DL2/3+ NK cells displayed poor alloreactivity against leukemia cells carrying HLA alleles belonging to the C2 specificity. Unexpectedly, this was due to recognition of C2 by KIR2DL2/3, as revealed by receptor blocking experiments and by binding assays of soluble KIR to HLA-C transfectants. Remarkably, however, C2/C2 leukemia blasts were killed by KIR2DL2/3+ (or by NKG2A+) NK cells that co-expressed KIR2DS1. This could be explained by the ability of KIR2DS1 to directly recognize C2 on leukemia cells. A role for the KIR2DS2 activating receptor in leukemia cell lysis could not be established. Taken together, these findings provide new information on NK alloreactivity in haplo-HSCT that may greatly impact on the selection of the optimal donor.

HLA and Aplastic Anemia: associations In Large Brazilian Cohorts

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1237-1237
Author(s):  
Marco Aurelio Salvino ◽  
Larissa A Medeiros ◽  
Alessandro Moura ◽  
Marianna Batista ◽  
Marilda Souza Goncalves ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Risk Factor ◽  
Aplastic Anemia ◽  
Protective Factor ◽  
Conflicts Of Interest ◽  
Hla Antigens ◽  
Hla Typing ◽  
Control Group ◽  
Hla Alleles ◽  
Northeast Region

Abstract Introduction Aplastic anemia (AA) is perceived as an immune mediated disease where T-lymphocytes recognize and destroy bone marrow elements leading to varying degrees of failure of hematopoiesis. Many autoimmune diseases have been linked to certain HLA alleles and such a relationship has been also been reported in AA. Expansion of CD8+ oligoclones has been reported in AA and likely contributes to pathogenesis. However, the interaction of CD4+ and CD8+ T cells and their targets mediated by human leukocyte antigen (HLA) class I and II peptides remain elusive. Thus, it has been speculated that polymorphic loci of these genes could be implicated in the susceptibility to the disease. Various alleles and haplotypes of HLA molecules have been implicated in the predisposition of AA development. The influence of HLA has been studied in North America, European and Asian countries. Data from Latin America, where there is a large mixture of Hispanic, European, and African descendants, is still lacking. This study focuses on the association between HLA alleles in AA patients in different regions of Brazil with particular ethnic groups. Patients and methods From 2000 to 2013, all patients with a diagnosis of acquired AA in the Brazilian state of Bahia (BA) followed at the Federal University of Bahia Hospital/ Foundation Hemoba who tested the HLA typing were included, totaling 215 patients. In this northeast region there is a predominance of African descendant (25% white, 75% brown/black). The genes in the analysis included HLA A, B, DR and DQ. SPSS was used to statistical calculations. Qui-square test/Fisher test were using the p-value correction of Bonferroni (p significant <0,0016) for comparison of genetic varieties.The HLA of patients with acquired AA Bahia (n = 215) were compared to the control group (3680 healthy non-related bone marrow volunteers donors from Bahia). To address regional differences within the country we also analyzed the HLA of AA patients (n = 344) from the State of Paraná located in the southern portion of the country and is characterized by a diverse ethnic mixture (71.3% white and 27% black/brown). Thus, we compared the findings of HLA associated with acquired AA in the two states, Bahia (northeast region) and Parana( south region). Of those statistically associated with AA (p<0,0016), we considered HLA clinically relevant only those present in at least 10% of cases and/or controls. Results From the 559 AA patients analysed, 45,1% were women, and 54.9% were men.The mean age was 23.4 (± 12.3). Among the HLA antigens with OR of risk or protection only HLA DR15 and B15, were significant, respectively (in both populations: Bahia and Parana). Identified as a risk factor for development of AA, HLA DR15 was found in 41.6% of patients (Bahia) versus 24% in controls (OR: 2.23 - CI: 1.68 to 2.9) (p <0.0001). As protective factor for AA development the HLA B15 was found in only 6% of patients (Bahia) versus 21.3% of controls (OR: 0.213, CI 0.12 to 0.370) (p <0.0001). A stratified analysis was conducted to assess the presence of interaction between the antigens DR15 and B15 in AA patients. When analyzing synergistically, the effects of HLA DR15 and B15, we observed that, in the AA group, the positivity of DR15+ of 41,6% falls significantly to 14,8%, when concurrently with the presence of B15+. The AA risk factor of HLA DR15+ loses its statistical risk power in presence of B15+. The incidence of B15+ patients (6% in AA patients) falls to 4% in the presence of DR15 negativity (p=ns). Conclusion We observed in 2 large AA cohorts (totaling 559 patients) from very distinct ethnic regions of Brazil, that, in both, HLA DR15 positivity was associated with a higher risk of disease, while B15 positivity was associated with a lesser likelihood of developing AA. The synergic combination of these alleles appears to be further associated with AA development. New studies analyzing synergic effect between HLA antigens/alleles should be conducted in immuno-mediated diseases. Disclosures: No relevant conflicts of interest to declare.

Development of an Alpha/Beta T-Cell Depleted Allogeneic Stem Cell Transplantation Protocol Form Matched Related and Unrelated Donor Grafts in Patients with Poor Risk Leukemia

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 3885-3885 ◽  
Author(s):  
Moniek A DeWitte ◽  
Liane te Boome ◽  
Lotte van der Wagen ◽  
Jurgen H Kuball
Keyword(s):  
T Cells ◽  
Nk Cells ◽  
Conflicts Of Interest ◽  
Γδ T Cells ◽  
Great Promise ◽  
Tumor Control ◽  
Unrelated Donor ◽  
Post Transplantation ◽  
Poor Risk ◽  
Unrelated Donors

Abstract Introduction: The outcome of allo-SCT in patients with poor risk leukemia is still hampered by GVHD and relapse. The innate immune system has been reported to contribute to tumor control, with lower incidence of GVHD. Specific depletion of αβ T- cells – key players in the development of GVHD – will render NK cells and γδ T cells within the allograft. Recently reported results have shown the great promise of this approach in haploindentical transplantations. Within this study, we aim to extend αβT- cell depleted allo-SCT to patients with a MRD or MUD. Methods: Patients with either ‘poor-risk’ or ‘very poor-risk’ leukemia were included in this phase I study. Either HLA matched siblings (MRD) or fully matched HLA matched (10/10) unrelated donors (MUD) were eligible. abT-cell reduction was performed by negative selection with anti-abTCR antibodies in combination with magnetic microbeads, using the automated CliniMACS device (Miltenyi Biotec, Bergisch Gladbach, Germany). The maximal contamination with αβT-cells for all dose levels was 5x105/kg. Three conditioning regimens have been investigated (I): fludarabine 120 mg/m2 + cyclophosfamide 4800 mg/m2, (II): fludarabine 120 mg/m2 + busilvex AUC=90 and (III): ATG (Genzyme®) 4 mg/m2 + fludarabine 120 mg/m2 + busilvex AUC=90 followed by αβT- cell depleted grafts from matched related or unrelated donors. Within cohort II and III, no additional immune suppression was given after allo-SCT. Results: Products for 14 patients have been successfully processed and used for αβT-cell depleted allo-SCT between 2011 and 2013. A ~4 log depletion of αβT-cells has been observed in the product with a recovery of ~75% of CD34+ cells. In cohort I and cohort II, 60% and 25% primary graft failures were observed, whereas in cohort III primary engraftment (chimerism > 95%) was observed in all patients. The combination of ATG/fludarabine/busilvex was well tolerated with a hematological recovery of within 3 weeks. In all 14 patients immune reconstitution primarily consisted of innate cells (NK cells and γδ T cells) the first 6 months post transplantation. In addition, no increase in CMV or EBV reactivations has been observed so far under the profound “innate control”. Conclusion: ATG Busulfan Fludarabine is a low toxicity platform for abTCR-depleted transplantations, resulting in a swift reconstitution of innate cells (NK cells and γδ T cells) the first 6 months post transplantation. This transplantation strategy can serve as a tool for future immunological interventions such as a low dose DLI or genetically modified T cells. Disclosures No relevant conflicts of interest to declare.

Improved Immune Recovery after Transplantation of TCRαβ/CD19 Depleted Allografts from Haploidentical Donors in Pediatric Patients

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 852-852
Author(s):  
Peter Lang ◽  
Tobias Feuchtinger ◽  
Heiko-Manuel Teltschik ◽  
Wolfgang Schwinger ◽  
Patrick Schlegel ◽  
...  
Keyword(s):  
Stem Cells ◽  
T Cells ◽  
T Lymphocytes ◽  
Nk Cells ◽  
Pediatric Patients ◽  
Conflicts Of Interest ◽  
Γδ T Cells ◽  
Control Group ◽  
Unrelated Donor ◽  
Immune Recovery

Abstract Transplantation of haploidentical stem cells has become an accepted option for pediatric patients and adults with high risk malignancies who lack a matched related or unrelated donor. In recent years, the majority of pediatric transplant centers chose the CD34 positive selection of peripheral stem cells, which allowed minimizing GvHD by effective reduction of T cells in the graft. However, infectious complications caused by delayed immune recovery were a major reason for transplant related mortality (TRM). In order to improve the immune recovery, we have established a new T-cell depletion method which removes αβ+ T-lymphocytes via a biotinylated anti-TcRαβ antibody followed by an anti-biotin antibody conjugated to magnetic microbeads while retaining γδ+ T-lymphocytes, natural killer (NK) cells and other cells in the graft. In addition, CD19+ B-lymphocytes were concomitantly depleted for the prevention of post-transplant EBV-associated lymphoproliferative disease. Immune recovery was retrospectively analyzed in a cohort of 41 patients with acute leukemia, MDS and non-malignant diseases, who received αβ T and B cell depleted allografts from haploidentical family donors. Conditioning regimens consisted of fludarabine or clofarabine, thiotepa, melphalan and serotherapy with OKT3 or ATG-Fresenius®. Graft manipulation was carried out with anti TCRαβ and anti CD19 antibodies and immunomagnetic microbeads. γδ T cells and NK cells remained in the grafts. Primary engraftment occurred in 88%, acute graft versus host disease (aGvHD) grade II and III-IV occurred in 10% and 15%. Immune recovery data were available in 26 patients and comparable after OKT3 (n=7) or ATG-F® (n=19). Median time to reach > 100 CD3+/µl, > 200 CD19+ cells/µl and > 200 CD56+ cells/µl for the whole group was 13, 127 and 12.5 days. Compared to a historical control group of patients with CD34 positive selected grafts, significantly higher cell numbers were found for CD3+ at days +30 and +90 (267 vs. 27 and 397 vs. 163 cells/µl), for CD3+4+ at day +30 (58 vs. 11 cells/µl) and for CD56+ at day +14 (622 vs. 27 cells/µl). The clinical impact of this accelerated immune recovery will be evaluated in an ongoing prospective multi-center trial. Disclosures No relevant conflicts of interest to declare.

Induction of NK and T Cell Immune Responses Against Leukemia Cells By Bispecific NKG2D-CD16 and -CD3 Fusion Proteins

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 2558-2558
Author(s):  
Samuel Koerner ◽  
Lothar Kanz ◽  
Ludger Grosse-Hovest ◽  
Gundram Jung ◽  
Helmut R Salih
Keyword(s):  
T Cells ◽  
Nk Cells ◽  
Fusion Proteins ◽  
Conflicts Of Interest ◽  
Ex Vivo ◽  
Specific Binding ◽  
Leukemia Cells ◽  
Dose Titration ◽  
Immune Effector ◽  
Effector Cells

Abstract Monoclonal antibodies (mAbs) have by now become an established tool in therapy of many malignancies. The interaction of a mAb's Fc portion with Fcγ receptors (FcγR) on immune effector cells is important for its efficacy, but often insufficient to potently induce antitumor immunity e.g. due to FcγR polymorphisms. Moreover, Fc parts of mAbs may bind to FcγRs expressed on non-cytotoxic cells (e.g., platelets and B cells) and interact with FcγRs that do not trigger cytotoxicity (e.g. CD16b on granulocytes). These shortcomings can be overcome by novel antibody formats like bispecific antibodies allowing for improved activation of a specific pool of effector cells. Here we report on the development and preclinical characterization of two bispecific fusion proteins that target ligands of the immunoreceptor NKG2D (NKG2DL) which are widely expressed on malignant cells but generally absent on healthy tissue. Our fusion proteins consist of the extracellular domain of NKG2D as targeting moiety for tumor-expressed NKG2DL fused to Fab-fragments of either an agonistic CD3 or CD16 antibody. Specific binding of these NKG2D-CD3 and NKG2D-CD16 constructs was confirmed using NKG2DL-transfectants with regard to their target arm and either NK cells or T cells with regard to their different effector parts. Dose titration assays revealed an increased affinity of NKG2D-CD16 to the FcγR on NK cells as compared to our previously described (e.g., Steinbacher et al, 2014) Fc-optimized NKG2D-IgG1 fusion protein, which was mirrored by a potently increased ability to induce lysis of NKG2DL-tranfectants and NKG2DL-positive primary acute myeloid leukemia (AML) cells by allogeneic NK cells. The novel NKG2D-CD3 construct in turn was found to potently activate allogeneic and autologous CD4+ and CD8+ T cells. Next we comparatively analyzed the efficacy of T cells and NK cells to lyse autologous leukemia cells upon treatment with NKG2D-CD3 and NKG2D-CD16, respectively, by using PBMC of AML patients (blast counts 30-70%) directly ex vivo in long term cytotoxicity assays. NKG2D-CD16 potently induced AML cell lysis, and this was, in line with their proliferative and higher effector potential, by far exceeded upon stimulation of T cells with NKG2D-CD3. Taken together, we here introduce novel "antibody-like" bispecific constructs that take advantage of the highly tumor-restricted expression of NKG2DL and potently activate the reactivity of NK or T cells for immunotherapy of leukemia. Disclosures No relevant conflicts of interest to declare.

Sign in / Sign up

Export Citation Format

Share Document