Elisabetta Zino

Effect of T-cell-epitope matching at HLA-DPB1 in recipients of unrelated-donor haemopoietic-cell transplantation: a retrospective study

BACKGROUND The risks after unrelated-donor haemopoietic-cell transplantation with matched HLA-A, HLA-B, HLA-C, HLA-DRB1, HLA-DQB1 alleles between donor and recipient (10/10 matched) can be decreased by selection of unrelated donors who also match for HLA-DPB1; however, such donors are difficult to find. Classification of HLA-DPB1 mismatches based on T-cell-epitope groups could identify mismatches that might be tolerated (permissive) and those that would increase risks (non-permissive) after transplantation. We did a retrospective study to compare outcomes between permissive and non-permissive HLA-DPB1 mismatches in unrelated-donor haemopoietic-cell transplantation. METHODS HLA and clinical data for unrelated-donor [corrected] transplantations submitted to the International Histocompatibility Working Group in haemopoietic-cell transplantation were analysed retrospectively. HLA-DPB1 T-cell-epitope groups were assigned according to a functional algorithm based on alloreactive T-cell crossreactivity patterns. Recipients and unrelated donors matching status were classified as HLA-DPB1 match, non-permissive HLA-DPB1 mismatch (those with mismatched T-cell-epitope groups), or permissive HLA-DPB1 mismatch (those with matched T-cell-epitope groups). The clinical outcomes assessed were overall mortality, non-relapse mortality, relapse, and severe (grade 3-4) acute graft-versus-host disease (aGvHD). FINDINGS Of 8539 transplantations, 5428 (64%) were matched for ten of ten HLA alleles (HLA 10/10 matched) and 3111 (36%) for nine of ten alleles (HLA 9/10 matched). Of the group overall, 1719 (20%) were HLA-DPB1 matches, 2670 (31%) non-permissive HLA-DPB1 mismatches, and 4150 (49%) permissive HLA-DPB1 mismatches. In HLA 10/10-matched transplantations, non-permissive mismatches were associated with a significantly increased risk of overall mortality (hazard ratio [HR] 1·15, 95% CI 1·05-1·25; p=0·002), non-relapse mortality (1·28, 1·14-1·42; p<0·0001), and severe aGvHD (odds ratio [OR] 1·31, 95% CI 1·11-1·54; p=0·001), but not relapse (HR 0·89, 95% CI 0·77-1·02; p=0·10), compared with permissive mismatches. There were significant differences between permissive HLA-DPB1 mismatches and HLA-DPB1 matches in terms of non-relapse mortality (0·86, 0·75-0·98; p=0·03) and relapse (1·34, 1·17-1·54; p<0·0001), but not for overall mortality (0·96, 0·87-1·06; p=0·40) or aGvHD (OR 0·84, 95% CI 0·69-1·03; p=0·09). In the HLA 9/10 matched population, non-permissive HLA-DPB1 mismatches also increased the risk of overall mortality (HR 1·10, 95% CI 1·00-1·22; p=0·06), non-relapse mortality (1·19, 1·05-1·36; p=0·007), and severe aGvHD (OR 1·37, 95% CI 1·13-1·66; p=0·002) compared with permissive mismatches, but the risk of relapse was the same in both groups (HR 0·93, 95% CI 0·78-1·11; p=0·44). Outcomes for HLA 10/10-matched transplantations with non-permissive HLA-DPB1 mismatches did not differ substantially from those for HLA 9/10-matched transplantations with permissive HLA-DPB1 mismatches or HLA-DPB1 matches. INTERPRETATION T-cell-epitope matching defines permissive and non-permissive HLA-DPB1 mismatches. Avoidance of an unrelated donor with a non-permissive T-cell-epitope mismatch at HLA-DPB1 might provide a practical clinical strategy for lowering the risks of mortality after unrelated-donor haemopoietic-cell transplantation. FUNDING National Institutes of Health; Associazione Italiana per la Ricerca sul Cancro; Telethon Foundation; Italian Ministry of Health; Cariplo Foundation; National Cancer Institute; National Heart, Lung and Blood Institute; National Institute of Allergy and Infectious Diseases; Office of Naval Research; IRGHET Paris; Swedish Cancer Society; Childrens Cancer Foundation; Swedish Research Council; Cancer Society in Stockholm; Karolinska Institutet; and Leukemia and Lymphoma Society.

Temporal, quantitative, and functional characteristics of single-KIR-positive alloreactive natural killer cell recovery account for impaired graft-versus-leukemia activity after haploidentical hematopoietic stem cell transplantation

In this study, we have characterized reconstitution of the natural killer (NK) cell repertoire after haploidentical CD34(+) selected hematopoietic stem cell transplantation (HSCT) for high-risk hematologic malignancies. Analysis focused on alloreactive single-KIR(+) NK cells, which reportedly are potent antileukemic effectors. One month after HSCT, CD56(bright)/CD56(dim) NK-cell subsets showed inverted ratio and phenotypic features. CD25 and CD117 down-regulation on CD56(bright), and NKG2A and CD62L up-regulation on CD56(dim), suggest sequential CD56(bright)-to-CD56(dim) NK-cell maturation in vivo. Consistently, the functional potential of these maturation intermediates against leukemic blasts was impaired. Mature receptor repertoire reconstitution took at least 3 months. Importantly, at this time point, supposedly alloreactive, single-KIR(+) NK cells were not yet fully functional. Frequency of these cells was highly variable, independently from predicted NK alloreactivity, and below 1% of NK cells in 3 of 6 alloreactive patients studied. In line with these observations, no clinical benefit of predicted NK alloreactivity was observed in the total cohort of 56 patients. Our findings unravel the kinetics, and limits, of NK-cell differentiation from purified haploidentical hematopoietic stem cells in vivo, and suggest that NK-cell antileukemic potential could be best exploited by infusion of mature single-KIR(+) NK cells selected from an alloreactive donor.

Nonpermissive HLA-DPB1 disparity is a significant independent risk factor for mortality after unrelated hematopoietic stem cell transplantation

The importance of donor-recipient human leukocyte antigen (HLA)-DPB1 matching for the clinical outcome of unrelated hematopoietic stem cell transplantation (HSCT) is controversial. We have previously described an algorithm for nonpermissive HLA-DPB1 disparities involving HLA-DPB1*0901,*1001,*1701,*0301,*1401,*4501, based on T-cell alloreactivity patterns. By revisiting the immunogenicity of HLA-DPB1*02, a modified algorithm was developed and retrospectively tested in 621 unrelated HSCTs facilitated through the Italian Registry for oncohematologic adult patients. The modified algorithm proved to be markedly more predictive of outcome than the original one, with significantly higher Kaplan-Meier probabilities of 2-year survival in permissive compared with nonpermissive transplantations (55% vs 39%, P = .005). This was the result of increased adjusted hazards of nonrelapse mortality (hazard ratio [HR] = 1.74; confidence interval [CI], 1.19-2.53; P = .004) but not of relapse (HR = 1.02; CI, 0.73-1.42; P = .92). The increase in the hazards of overall mortality by nonpermissive HLA-DPB1 disparity was similar in 10 of 10 (HR = 2.12; CI, 1.23-3.64; P = .006) and 9 of 10 allele-matched transplantations (HR = 2.21; CI, 1.28-3.80; P = .004), both in early-stage and in advanced-stage disease. These data call for revisiting current HLA matching strategies for unrelated HSCT, suggesting that searches should be directed up-front toward identification of HLA-DPB1 permissive, 10 of 10 or 9 of 10 matched donors.

Molecular and functional characterization of allogantigen specific anergic T cells suitable for cell therapy

Background CD4+ regulatory T cells are a specialized subset of T cells that actively control immune responses. Several experimental protocols have been used to expand natural regulatory T cells and to generate adaptive type 1 regulatory T cells for regulatory T-cell-based therapies. Design and Methods The ability of exogenous recombinant human interleukin-10 to induce alloantigen-specific anergy in T cells was investigated and compared to that of interleukin-10 derived from tolerogenic dendritic cells, in mixed lymphocyte cultures. A detailed characterization of the effector functions of the resulting anergized T cells is reported. Results Interleukin-10, whether exogenous or derived from tolerogenic dendritic cells, induces a population of alloantigen-specific T cells (interleukin-10-anergized T cells) containing type 1 regulatory T cells, which are anergic and actively suppress alloantigen-specific effector T cells present within the mixed population. Interleukin-10-induced anergy is transforming growth factor-β independent, and is associated with a decreased frequency of alloantigen-specific cytotoxic T lymphocyte precursors, but interleukin-10-anergized T cells are still responsive to third-party, bacterial, and viral antigens. Tolerogenic dendritic cells are more powerful than exogenous interleukin-10 in generating type 1 regulatory T-cell precursors, and are also effective in the context of HLA-matched donors. Conclusions Based on these studies, we have developed an efficient and reproducible in vitro method to generate antigen-specific type 1 regulatory T-cell precursors starting from total peripheral blood cells with minimal cell manipulation and suitable for generating type 1 regulatory T cells for regulatory T-cell-based therapies.

Incidence, risk factors and clinical outcome of leukemia relapses with loss of the mismatched HLA after partially incompatible hematopoietic stem cell transplantation

Genomic loss of the mismatched human leukocyte antigen (HLA) is a recently described mechanism of leukemia immune escape and relapse after allogeneic hematopoietic stem cell transplantation (HSCT). Here we first evaluated its incidence, risk factors and outcome in 233 consecutive transplants from partially HLA-mismatched related and unrelated donors (MMRD and MMUD, respectively). We documented 84 relapses, 23 of which with HLA loss. All the HLA loss relapses occurred after MMRD HSCT, and 20/23 in patients with acute myeloid leukemia. Upon MMRD HSCT, HLA loss variants accounted for 33% of the relapses (23/69), occurring later than their ‘classical’ counterparts (median: 307 vs 88 days, P<0.0001). Active disease at HSCT increased the risk of HLA loss (hazard ratio (HR): 10.16; confidence interval (CI): 2.65–38.92; P=0.001), whereas older patient ages had a protective role (HR: 0.16; CI: 0.05–0.46; P=0.001). A weaker association with HLA loss was observed for graft T-cell dose and occurrence of chronic graft-versus-host disease. Outcome after ‘classical’ and HLA loss relapses was similarly poor, and second transplantation from a different donor appeared to provide a slight advantage for survival. In conclusion, HLA loss is a frequent mechanism of evasion from T-cell alloreactivity and relapse in patients with myeloid malignancies transplanted from MMRDs, warranting routine screening in this transplantation setting.

Significantly higher frequencies of alloreactive CD4+ T cells responding to nonpermissive than to permissive HLA-DPB1 T-cell epitope disparities

To the editor: Increasing evidence suggests that donor-recipient disparities for human leukocyte antigen (HLA)–DPB1 can be of clinical importance in unrelated hematopoietic stem cell transplantation (HSCT).[1][1] Two overlapping algorithms for functional T-cell epitope (TCE) matching involving 3

T cell neoepitope discovery in colorectal cancer by high throughput profiling of somatic mutations in expressed genes

Objective Patient-specific (unique) tumour antigens, encoded by somatically mutated cancer genes, generate neoepitopes that are implicated in the induction of tumour-controlling T cell responses. Recent advancements in massive DNA sequencing combined with robust T cell epitope predictions have allowed their systematic identification in several malignancies. Design We undertook the identification of unique neoepitopes in colorectal cancers (CRCs) by using high-throughput sequencing of cDNAs expressed by standard cancer cell cultures, and by related cancer stem/initiating cells (CSCs) cultures, coupled with a reverse immunology approach not requiring human leukocyte antigen (HLA) allele-specific epitope predictions. Results Several unique mutated antigens of CRC, shared by standard cancer and related CSC cultures, were identified by this strategy. CD8+ and CD4+ T cells, either autologous to the patient or derived from HLA-matched healthy donors, were readily expanded in vitro by peptides spanning different cancer mutations and specifically recognised differentiated cancer cells and CSC cultures, expressing the mutations. Neoepitope-specific CD8+ T cell frequency was also increased in a patient, compared with healthy donors, supporting the occurrence of clonal expansion in vivo. Conclusions These results provide a proof-of-concept approach for the identification of unique neoepitopes that are immunogenic in patients with CRC and can also target T cells against the most aggressive CSC component.

Platelet transfusion refractoriness in highly immunized beta thalassemia children undergoing stem cell transplantation.

Marktel S, Napolitano S, Zino E, Cappelli B, Chiesa R, Poli F, Crocchiolo R, Ronchi P, Rossini S, Ciceri F, Roncarolo MG, Fleischhauer K. Platelet transfusion refractoriness in highly immunized beta thalassemia children undergoing stem cell transplantation.
Pediatr Transplantation 2010: 14:393–401.

Sequencing of a new HLA-A*32 subtype (A*3202)

A healthy Caucasian donor (donor MP) enrolled in the Italian National Bone Marrow Registry (IBMDR) was typed as HLA A3/A32, B15/B38, Cw4/by standard serology. Molecular typing of theHLA-A locus was routinely performed using a previously described polymerase chain reaction (PCR) sequence-specific oligonucleotide probe (SSOP) approach (Oh et al. 1992; Benazzi et al. 1995). This analysis revealed an unexpected hybridization pattern forA*32 involving probes derived from the region around codons 151 and 156 of the L2 domain. cDNA was prepared by reverse transcription of mRNA extracted from peripheral blood lymphocytes of donor MP. The full-lengthA*32 coding region was PCR-amplified by use of a primer pair suitable for amplification of A*32 but not of A*03 genes. Primers were a generous gift of S. Y. Yang. The 1.1 kilobase PCR products from two independent PCR were subcloned into pBluescript and sequenced on both strands by the Sanger dideoxy chain termination method (Sanger et al. 1977). The new A*32 allele, now referred to asA*3202, was shown to differ fromA*3201by three nucleotide substitutions in exon 3. Base pair (bp) 182 in exon 3 was changed from G in A*3201 to A in A*3202 and bps 196/197 were changed from TT in A*3201to CA in A*3202 (Fig. 1A). This changed the predicted amino acid sequence from Arg ( A*3201) to His (A*3202) at codon 151 and from Leu ( A*3201) to Gln (A*3202) at codon 156 of the L2 domain (Fig. 1B). The combination 151His/156Gln is also found in someA*24 subtypes, inA*3202, and in A*0212 (Arnett et al. 1995).A*3202 may have arisen by gene conversion of A*3201 with one of these alleles. In order to assess the frequency of A*3202 in Caucasians from Northern Italy, sixty-eight additional samples from unrelated, healthy individuals enrolled in the IBMDR and typed as HLA-A32 by standard serology were screened for the presence of A*3202by PCR-SSOP typing. A*3202was found in two additional samples. Therefore, the frequency of A*3202 in A*32+ Caucasians from Northern Italy amounts to 3/69 = 4.34%. TheA*32 allele has been reported to have a frequency of 5.6% in Italian Caucasians (Imanishi et al. 1991). Therefore, the estimated overall frequency ofA*3202 in Italians is 0.24%. Interestingly, two of the three individuals carrying the A*3202gene came from the region of Valtellina, while only twelve of the sixty-nineA*32+ donors tested came from this region. This raises the possibility that A*3202 might occur with increased frequency in the population of Valtellina which, as judged by the highly restricted number of family names, is relatively inbred, although the exact extent of any inbreeding is unknown. The nucleotide sequence data reported in this paper have been submitted to the EMBL nucleotide sequence database and have been assigned the accession number X97120. The name A*3202 was officially assigned by the WHO Nomenclature Committee in June 1996. This follows the agreed policy that, subject to the conditions stated in the most recent Nomenclature Report (Bodmer et al. 1995), names will be assigned to new sequences as they are identified. Lists of such new names will be published in the following WHO Nomenclature Report

Effects of transmembrane region variability on cell surface expression and allorecognition of HLA-DP3

The functional relevance of polymorphisms outside the peptide binding groove of HLA molecules is poorly understood. Here we have addressed this issue by studying HLA-DP3, a common antigen relevant for functional matching algorithms of unrelated hematopoietic stem cell transplantation (HSCT) encoded by two transmembrane (TM) region variants, DPB1(*)03:01 and DPB1(*)104:01. The two HLA-DP3 variants were found at a overall allelic frequency of 10.4% in 201 volunteer stem cell donors, at a ratio of 4.2:1. No significant differences were observed in cell surface expression levels of the two variants on B lymphoblastoid cell lines (BLCL), primary B cells or monocytes. Three different alloreactive T cell lines or clones showed similar levels of activation marker CD107a and/or CD137 upregulation in response to HLA-DP3 encoded by DPB1(*)03:01 and DPB1(*)104:01, either endogenously on BLCL or after lentiveral-vector mediated transfer into the same cellular background. These data provide, for the first time, direct evidence for a limited functional role of a TM region polymorphism on expression and allorecognition of HLA-DP3 and are compatible with the notion that the two variants can be considered as a single functional entity for unrelated stem cell donor selection.