NKG2C+ NK Cells for Immunotherapy of Glioblastoma Multiforme.

In glioblastoma, non-classical human leucocyte antigen E (HLA-E) and HLA-G are frequently overexpressed. HLA-E loaded with peptides derived from HLA class I and from HLA-G contributes to inhibition of natural killer (NK) cells with expression of the inhibitory receptor CD94/NKG2A. We investigated whether NK cells expressing the activating CD94/NKG2C receptor counterpart were able to exert anti-glioma effects. NKG2C+ subsets were preferentially expanded by a feeder cell line engineered to express an artificial disulfide-stabilized trimeric HLA-E ligand (HLA-E*spG). NK cells expanded by a feeder cell line, which facilitates outgrowth of conventional NKG2A+, and fresh NK cells, were included for comparison. Expansion via the HLA-E*spG feeder cells selectively increased the fraction of NKG2C+ NK cells, which displayed a higher frequency of KIR2DL2/L3/S2 and CD16 when compared to expanded NKG2A+ NK cells. NKG2C+ NK cells exhibited increased cytotoxicity against K562 and KIR:HLA-matched and -mismatched primary glioblastoma multiforme (GBM) cells when compared to NKG2A+ NK cells and corresponding fresh NK cells. Cytotoxic responses of NKG2C+ NK cells were even more pronounced when utilizing target cells engineered with HLA-E*spG. These findings support the notion that NKG2C+ NK cells have potential therapeutic value for treating gliomas.

Artificial feeder cells expressing ligands for killer cell immunoglobulin-like receptors and CD94/NKG2A for expansion of functional primary natural killer cells with tolerance to self.

BACKGROUND AIMS: Natural killer (NK) cells are promising cells for immunotherapy of cancer, and there are ongoing efforts to improve their ex vivo expansion to clinically relevant numbers. This study focused on the development of a C1-, C2-, Bw4 killer cell immunoglobulin-like receptor (KIR) ligand and NKG2A ligand-containing feeder cell line for autonomous expansion of functional NK cells. METHODS: PC3PSCA-derived feeder cells expressing IL-2, 4-1BBL and membrane-bound IL-15-mutDAP12 (mIL-15d) fusion protein in combinations or alone were generated and used for expansion. Expanded NK cells were analyzed with respect to subpopulations, expression of NK cell receptors and immune checkpoint molecules as well as their cytotoxicity against K562 cells, cetuximab-marked tumor cells and autologous B cells. RESULTS: Only combinatorial expression of IL-2 plus 4-1BBL or IL-2, 4-1BBL plus mIL-15d in feeder cells efficiently expanded NK cells and supported selective outgrowth of NK cells from peripheral blood mononuclear cell samples. Best expansion of NK cells was achieved using PC3PSCA-IL-2-4-1BBL-mIL-15d feeder cells. Such expanded NK cells exhibited upregulation of natural cytotoxicity receptors, DNAM-1 and NKG2C and induced expression of high affinity IL-2 receptor, which were paralleled by attenuated KIR and increased expression of NKG2A and ILT2. In addition, elevated TIM-3 levels were noted and PD-1 and T cell immunoreceptor with Ig and ITIM domain (TIGIT) levels remained low. Expanded NK cells were highly cytolytic when encountering K562 cells and cetuximab-marked target cells but remained unresponsive to autologous B cells and target cells with protective levels of human leukocyte antigen. CONCLUSIONS: Collectively, the results demonstrate the feasibility of PC3PSCA-IL-2-4-1BBL-mIL-15d feeder cells for robust expansion of NK cells, which remain tolerant to self and could be used in the future for adoptive cell therapy of cancer.

Immunogenetics in stem cell donor registry work: The DKMS example (Part 2).

DKMS is a leading stem cell donor registry with more than 9 million donors. Donor registry activities share many touch points with topics from immunogenetics or population genetics. In this two-part review article, we deal with these aspects of donor registry work by using the example of DKMS. In the second part of the review, we focus on donor typing of non-HLA genes, the impact of donor age, gender and CMV serostatus on donation probabilities, the identification of novel HLA, KIR and MIC alleles by high-throughput donor typing, the activities of the Collaborative Biobank and pharmacogenetics in the donor registry context.

Immunogenetics in stem cell donor registry work: The DKMS example (Part 1).

Currently, stem cell donor registries include more than 35 million potential donors worldwide to provide HLA-matched stem cell products for patients in need of an unrelated donor transplant. DKMS is a leading stem cell donor registry with more than 9 million donors from Germany, Poland, the United States, the United Kingdom, India and Chile. DKMS donors have donated hematopoietic stem cells more than 80,000 times. Many aspects of donor registry work are closely related to topics from immunogenetics or population genetics. In this two-part review article, we describe, analyse and discuss these areas of donor registry work by using the example of DKMS. Part 1 of the review gives a general overview on DKMS and includes typical donor registry activities with special focus on the HLA system: high-throughput HLA typing of potential stem cell donors, HLA haplotype frequencies and resulting matching probabilities, and donor file optimization with regard to HLA diversity.

DAP12-based activating chimeric antigen receptor for NK cell tumor immunotherapy.

NK cells are emerging as new effectors for immunotherapy of cancer. In particular, the genetic engraftment of chimeric Ag receptors (CARs) in NK cells is a promising strategy to redirect NK cells to otherwise NK cell-resistant tumor cells. On the basis of DNAX-activation protein 12 (DAP12), a signaling adaptor molecule involved in signal transduction of activating NK cell receptors, we generated a new type of CAR targeting the prostate stem cell Ag (PSCA). We demonstrate in this article that this CAR, designated anti-PSCA-DAP12, consisting of DAP12 fused to the anti-PSCA single-chain Ab fragment scFv(AM1) confers improved cytotoxicity to the NK cell line YTS against PSCA-positive tumor cells when compared with a CAR containing the CD3ζ signaling chain. Further analyses revealed phosphorylation of the DAP12-associated ZAP-70 kinase and IFN-γ release of CAR-engineered cells after contact with PSCA-positive target cells. YTS cells modified with DAP12 alone or with a CAR bearing a phosphorylation-defective ITAM were not activated. Notably, infused YTS cells armed with anti-PSCA-DAP12 caused delayed tumor xenograft growth and resulted in complete tumor eradication in a significant fraction of treated mice. The feasibility of the DAP12-based CAR was further tested in human primary NK cells and confers specific cytotoxicity against KIR/HLA-matched PSCA-positive tumor cells, which was further enhanced by KIR-HLA mismatches. We conclude that NK cells engineered with DAP12-based CARs are a promising tool for adoptive tumor immunotherapy.

Clonal evolution including partial loss of human leukocyte antigen genes favoring extramedullary acute myeloid leukemia relapse after matched related allogeneic hematopoietic stem cell transplantation.

BACKGROUND: Relapse of acute myeloid leukemia (AML) after allogeneic hematopoietic stem cell transplantation (HSCT) leaves few therapeutic options, and mechanisms of immune escape of recurring leukemic cells remain poorly understood. Recently, acquired loss of mismatched human leukocyte antigen (HLA) was demonstrated in patients with AML undergoing haploidentical allogeneic HSCT and was suggested not to occur in HLA-matched HSCT. We hypothesized that this mechanism applies to extramedullary AML relapse which occurs frequently after allogeneic HSCT and might also not be restricted to haploidentical HSCT. METHODS: DNA from extramedullary AML relapse after HSCT was compared with bone marrow at diagnosis with array comparative genomic hybridization to investigate relapse-specific genomic aberrations in relapsing AML after allogeneic HSCT. Formalin-fixed, paraffin-embedded tissues from the same points of time were assessed for HLA, major histocompatibility complex class I chain-related gene A, and TAP2 immunohistochemistry staining to assess cell surface expression of deleted loci encoded on chromosome 6p. RESULTS: Array comparative genomic hybridization revealed a partial loss of chromosome 6p in extramedullary myeloid sarcoma relapse of AML after sustained complete remission was achieved through matched related allogeneic HSCT. Among others, a deleted region 6p21.32-p21.33, which included several HLA class I genes, was detected. CONCLUSIONS: These results suggest that the loss of HLA class I haplotype also occurs in AML relapse after HLA-matched related HSCT. Partial loss of several HLA class I genes and subsequent reduced presentation of minor histocompatibility antigens and reduced ligation of activating natural killer-cell receptors may explain the loss of graft-versus-leukemia response and extramedullary AML relapse in tissue with reduced immunologic surveillance.

MHC class II genotype- and MHC class I and II phenotype-related parameters in sporadic colorectal cancer.

The underlying etiological cause of non-hereditary colorectal cancer has yet to be determined. The adenoma-carcinoma sequence is widely accepted, however, a sole trigger has not been specified. Therefore, we sought to further define genotypic and phenotypic parameters that could be involved in promoting a possible infection, inflammation and hyper-proliferation, followed by the adenoma-carcinoma sequence. Expression of phenotype-related parameters for MHC class I (HLA-A N-20 and ß2 microglobulin) and class II (HLA-DRα and HLA-DR) as well as CD45 and carcinoembryonic antigen (CEA) were investigated immunohistochemically in a series of 93 colorectal cancers. Additionally, in 49 of the tumours the MHC class II genotype was analysed. MHC class II genotype analyses revealed a tendency towards DRB1*08 and DQB1*04. A significant association among the MHC class I markers or the MHC class II markers was found. No difference in marker expression could be detected between tumour and stromal tissue, however a significant inverse expression existed for markers of the functionally different class I or II systems. With the exception of CEA, there was no correlation between expression of any marker and tumour grade. Only 2% of tumours expressed no markers for MHC class I and II. Further studies on MHC class I and II genotype and phenotype relation in colorectal cancer may help to identify trigger mechanisms for tumourigenesis, involved markers and possible mechanisms of subsequent immune escape.

Matched unrelated or matched sibling donors result in comparable survival after allogeneic stem-cell transplantation in elderly patients with acute myeloid leukemia: a report from the cooperative German Transplant Study Group.

PURPOSE: In patients with acute myeloid leukemia (AML), differential indications for matched sibling and unrelated hematopoietic stem-cell transplantation (HCT) are considered, and arbitrary age limits for HCT exist. We sought to determine whether donor type is a prognostic factor in elderly patients in the era of high-resolution DNA-based HLA typing. PATIENTS AND METHODS: We performed univariate and multivariate analyses of event-free survival (EFS) and overall survival (OS) in patients older than 50 years with standard- or high-risk AML who had received an allogeneic HCT between 1995 and 2005. Available DNA from donors and recipients of unrelated HCT was retyped so that the HLA-A, -B, -C, and -DRB1 alleles could be characterized in detail. Unrelated donors (UDs) were classified as matched (8/8), possibly matched (matched, but incomplete information), partially matched (one mismatch), or poorly matched (two or more mismatches) according to the final typing results. RESULTS: Data from 368 patients with a median age of 57 years (range, 50 to 73 years) were included. Multivariate Cox regression analysis revealed that patients' disease status at HCT (P < .001) and the cytogenetic risk (P < .001) highly significantly predicted EFS and OS. Compared with patients with matched sibling donors, the adjusted relative risk of EFS was 0.7 (95% CI, 0.4 to 1.1) for patients with matched UDs and 1.0 (95% CI, 0.7 to 1.6) for patients with partially matched UDs. CONCLUSION: Donor type is not a major prognostic factor for HCT in elderly patients with standard- or high-risk AML.

Altered phenotype of natural killer cell subsets after haploidentical stem cell transplantation.

OBJECTIVE: Haplotype-mismatched CD34(+) selected allogeneic stem cell transplantation (HASCT) has been described as a therapeutic option for patients with acute myeloid leukemia. The success of this regimen is based mainly on natural killer (NK) cell-mediated antileukemia effects. MATERIALS AND METHODS: We prospectively investigated NK-cell (CD56(+)/CD3(-)) reconstitution, including expression of antileukemia effector molecules in patients undergoing HASCT. RESULTS: Although absolute NK-cell numbers rapidly increased, their phenotype notably differed compared to healthy controls. In fact, the "effector" CD56(dim) subset was significantly reduced, as was the NKG2D expression on "regulatory" CD56(bright) cells. Perforin was completely absent on NK cells in one-third of patients. The expression of Fas-ligand (Fas-L) on NK cells as well as soluble Fas-L and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) plasma levels were also significantly lower after HASCT. In contrast, expression of TRAIL on CD56(dim) cells and interleukin-15 plasma levels were upregulated. Because the death rate due to relapse or infectious complications was high in the initial phase of the trial, subsequent patients received an adoptive infusion of donor NK cells followed by interleukin-2 in vivo in order to augment NK-cell function. This led to a distinct upregulation of perforin and Fas-L on the CD56(dim) subset accompanied by increased NK-cell cytotoxicity in vitro. CONCLUSION: The phenotype of reconstituting NK cells after HASCT is significantly altered. Whether the clinical outcomes of patients undergoing this regimen can be improved by a cytokine-based modulation of NK-cell activity needs to be determined.

Prevalence of HLA-DRB1 genotype and altered Fas/Fas ligand expression in adrenocortical carcinoma.

A distinctive feature of malignant adrenocortical neoplasms is decreased major histocompatibility complex (MHC) class II molecule expression. However, it is unknown whether there exists a restriction to certain MHC genotypes and whether this involves alterations of the Fas/Fas ligand system and thereby affects tissue homeostasis. Therefore, MHC class II phenotype and genotype and expression patterns of the Fas/Fas ligand system were investigated in 24 adrenocortical tumors (n(Adenomas) = 14, n(Carcinomas) = 10) and an adrenal cancer cell line (NCI-H295). No MHC class II antigen expression was detected in carcinomas. The DRB1*01 genotype was found in 54.5% of patients with carcinoma (P = 0.046). No prevalence of any genotype could be detected in patients with adenomas, which exhibited varying levels of antigen expression. Fas receptor expression was 75.0% in adenomas compared with 20.0% in carcinomas (P = 0.0196), whereas ligand expression was 37.7% in adenomas and reached almost 100% in the carcinomas investigated in this study (P = 0.0033). In summary, the DRB1*01 genotype may be correlated to a higher risk for malignancy. Additional studies on MHC class II genotype and phenotype and the altered Fas/Fas ligand system in adrenal neoplasms may help to identify mechanisms of immune escape and suggest new diagnostic approaches.

Analysis of HLA class I and II alleles in sporadic inclusion-body myositis.

Sporadic inclusion body myositis (s-IBM) is characterised by progressive weakness of proximal and distal limb muscles. Most patients are aged over 50 years at disease onset. Muscle biopsy reveals an inflammatory myopathy and cytoplasmic amyloid deposits. The mononuclear infiltrate is dominated by CD8+ T-cells. Several investigators have described associations between s-IBM and certain HLA antigens and alleles. However, to date neither HLA class I nor II alleles have been analysed in a large series of patients. We typed various HLA class I and II alleles in 47 patients suffering from s-IBM using sequence specific-primer pairs (SSPPCR). The results were compared with published German controls. Additional Bonferroni adjustment was performed over all allele groups corresponding to serologically defined antigens within one HLA class I or II locus. After Bonferroni adjustment, we found a significant increase in frequency of the following HLA alleles for s-IBM patients when compared with normal controls: A*03 (p = 0.0002), B*08 (p = 0.002), DRB1*03 (p = 0.0000002), and DQB1*05 (p = 0.02). HLA typing may be helpful to distinguish between subgroups of s-IBM patients. Moreover, HLA analysis may aid in identifying patients who might profit from future therapeutic strategies.

Prostate stem cell antigen: Identification of immunogenic peptides and assessment of reactive CD8+ T cells in prostate cancer patients.

Identification of TAAs recognized by CD8(+) CTLs paved the way for new concepts in cancer therapy. In view of the heterogeneity of tumors and their diverse escape mechanisms, CTL-based cancer therapy largely depends on an appropriate number of TAAs. In prostate cancer, the number of antigens defined as suitable targets of CTLs remains rather limited. PSCA is widely distributed in prostate cancer. In this report, we define immunogenic peptides of PSCA which are recognized by circulating CD8(+) T cells from prostate cancer patients and able to activate CTLs in vitro. Screening the amino acid sequence of PSCA for peptides containing a binding motif for HLA-A*0201 resulted in 8 candidate peptides. Specificity and affinity of peptide binding were verified in a competition assay. Frequencies of CD8(+) T lymphocytes reactive against selected epitopes were determined in the blood of prostate cancer patients using the ELISPOT assay. Increased frequencies were revealed for CD8(+) T cells recognizing the peptides ALQPGTALL and AILALLPAL. CTLs from prostate cancer patients were raised against these 2 peptides in vitro when presented by autologous DCs. They specifically recognized peptide-pulsed T2 target cells and prostate cancer cells that were HLA-A*0201- and PSCA-positive, indicating that these peptides were naturally generated by tumor cells. These data suggest that PSCA is a promising target for the immunotherapy of prostate cancer.