The microstructure in the placenta is influenced by the functional diversity of HLA-G allelic variants.

The main expression sites of HLA-G are human extravillous trophoblast cells. The interaction of HLA-G with uterine NK cells promotes their maturation and differentiation into decidual NK (dNK) cells. dNK cells secrete chemokines, cytokines, and proangiogenic factors in favor of a vascular remodeling and an immune suppressive microenvironment of the decidua. HLA-G is the most polymorphic member of the oligomorphic non-classical HLA molecule family; yet, the impact of polymorphic differences is not comprehensively understood. sHLA-G levels in embryo culture medium correlate with successful pregnancy; however, it remains questionable if HLA-G allelic diversity impacts on the outcome of dNK cell development. We utilized synthetic sHLA-G*01:01, 01:03, and 01:04 molecules and transduced K652/mHLA-G*01:01, 01:03, and 01:04 cells to study the biological interaction between HLA-G alleles and primary NK cells of human term placenta. Despite its low frequency, HLA-G*01:04 and not the most prevalent allele HLA-G*01:01 appear to be strong catalysts of dNK cell proliferation. Concluding, this study illustrates novel insights into the impact and binding efficiency of the three most common variants of HLA-G on primary placental NK cells.

Granulocyte colony-stimulating factor impairs CD8(+) T cell functionality by interfering with central activation elements.

Besides mobilizing stem cells into the periphery, granulocyte colony-stimulating factor (G-CSF) has been shown to influence various types of innate and adaptive immune cells. For example, it impairs the effector function of cytotoxic T lymphocytes (CTLs). It is assumed that this effect is mediated indirectly by monocytes, regulatory T cells and immunomodulatory cytokines influenced by G-CSF. In this study, isolated G-CSF-treated CD8(+) T cells were stimulated antigen-dependently with peptide-major histocompatibility complex (pMHC)-coupled artificial antigen-presenting cells (aAPCs) or stimulated antigen-independently with anti-CD3/CD28 stimulator beads. By measuring the changes in interferon (IFN)-γ and granzyme B expression at the mRNA and protein level, we showed for the first time that G-CSF has a direct effect on CD8(+) CTLs, which was confirmed based on the reduced production of IFN-γ and granzyme B by the cytotoxic T cell line TALL-104 after G-CSF treatment. By investigating further elements affected by G-CSF in CTLs from stem cell donors and untreated controls, we found a decreased phosphorylation of extracellular-regulated kinase (ERK)1/2, lymphocyte-specific protein tyrosine kinase (Lck) and CD3ζ after G-CSF treatment. Additionally, miRNA-155 and activation marker expression levels were reduced. In summary, our results show that G-CSF directly influences the effector function of cytotoxic CD8(+) T cells and affects various elements of T cell activation.

Lentivirus-induced 'Smart' dendritic cells: Pharmacodynamics and GMP-compliant production for immunotherapy against TRP2-positive melanoma.

Monocyte-derived conventional dendritic cells (ConvDCs) loaded with melanoma antigens showed modest responses in clinical trials. Efficacy studies were hampered by difficulties in ConvDC manufacturing and low potency. Overcoming these issues, we demonstrated higher potency of lentiviral vector (LV)-programmed DCs. Monocytes were directly induced to self-differentiate into DCs (SmartDC-TRP2) upon transduction with a tricistronic LV encoding for cytokines (granulocyte macrophage colony stimulating factor (GM-CSF) and interleukin-4 (IL-4)) and a melanoma antigen (tyrosinase-related protein 2 (TRP2)). Here, SmartDC-TRP2 generated with monocytes from five advanced melanoma patients were tested in autologous DC:T cell stimulation assays, validating the activation of functional TRP2-specific cytotoxic T lymphocytes (CTLs) for all patients. We described methods compliant to good manufacturing practices (GMP) to produce LV and SmartDC-TRP2. Feasibility of monocyte transduction in a bag system and cryopreservation following a 24-h standard operating procedure were achieved. After thawing, 50% of the initial monocyte input was recovered and SmartDC-TRP2 self-differentiated in vitro, showing uniform expression of DC markers, detectable LV copies and a polyclonal LV integration pattern not biased to oncogenic loci. GMP-grade SmartDC-TRP2 expanded TRP2-specific autologous CTLs in vitro. These results demonstrated a simpler GMP-compliant method of manufacturing an effective individualized DC vaccine. Such DC vaccine, when in combination with checkpoint inhibition therapies, might provide higher specificity against melanoma.

Modulation of heme oxygenase-1 by metalloporphyrins increases anti-viral T cell responses.

Heme oxygenase (HO)-1, the inducible isoform of HO, has immunomodulatory functions and is considered a target for therapeutic interventions. In the present study, we investigated whether modulation of HO-1 might have regulatory effects on in-vitro T cell activation. The study examined whether: (i) HO-1 induction by cobalt-protoporphyrin (CoPP) or inhibition by tin-mesoporphyrin (SnMP) can affect expansion and function of virus-specific T cells, (ii) HO-1 modulation might have a functional effect on other cell populations mediating effects on proliferating T cells [e.g. dendritic cells (DCs), regulatory T cells (T(regs)) and natural killer cells] and (iii) HO-1-modulated anti-viral T cells might be suitable for adoptive immunotherapy. Inhibition of HO-1 via SnMP in cytomegalovirus (CMV)pp65-peptide-pulsed peripheral blood mononuclear cells (PBMCs) led to increased anti-viral T cell activation and the generation of a higher proportion of effector memory T cells (CD45RA(-) CD62L(-)) with increased capability to secrete interferon (IFN)-γ and granzyme B. T(reg) depletion and SnMP exposure increased the number of anti-viral T cells 15-fold. To test the possibility that HO-1 modulation might be clinically applicable in conformity with good manufacturing practice (GMP), SnMP was tested in isolated anti-viral T cells using the cytokine secretion assay. Compared to control, SnMP treatment resulted in higher cell counts and purity without negative impact on quality and effector function [CD107a, IFN-γ and tumour necrosis factor (TNF)-α levels were stable]. These results suggest an important role of HO-1 in the modulation of adaptive immune responses. HO-1 inhibition resulted in markedly more effective generation of functionally active T cells suitable for adoptive T cell therapy.

A future with less HLA: potential clinical applications of HLA-universal cells.

The high variability of the human leukocyte antigen (HLA) remains a major obstacle to the application of allogeneic products in cell-based therapies. We have developed a strategy to decrease the immunogenicity of cell and tissues to improve their survival after allogeneic transplantation in the absence of immunosuppression. Using RNA interference technology, the expression of HLA class I and II was stably downregulated. HLA-silenced cells demonstrated to prevent a de novo and escape a pre-formed alloimmune response in vitro and in vivo. Also, they demonstrated to be capable of engraft and survive after allogeneic transplantation independently of the donor's and recipient's genetic background. The generation of HLA-universal cells has may open new horizons in the field of regenerative medicine. Some of the potential clinical applications of HLA universal cells will be discussed in this review.

Determination of unacceptable HLA antigen mismatches in kidney transplant recipients: recommendations of the German Society for Immunogenetics.

One of the major tasks of histocompatibility and immunogenetics laboratories is the pretransplant determination of unacceptable antigen mismatches (UAM) in kidney transplant recipients. In this procedure, human leucocyte antigen (HLA) specificities are defined against which the patient has circulating alloantibodies that are expected to harm the transplanted organ. Using the information on UAM and the potential donor's complete HLA typing, prediction of the crossmatch result, the so called 'virtual crossmatch', is possible. Currently, the laboratories are using different algorithms for the determination of UAM, and depending on the algorithm, more or fewer organ offers are excluded for patients with a similar antibody profile. In order to bring homogeneity into the allocation of organs to immunized patients in Germany, the German Society for Immunogenetics established, on the basis of current knowledge, recommendations for the determination of UAM. The UAM recommendations, which are thought to serve as a common tool for responsible physicians at different transplant centers, contain technical issues that need to be considered and are individualized for sensitized patients with a high or intermediate risk of antibody-mediated rejection. The present review contains these recommendations and puts them into perspective to current international practice.

Allogeneic and xenogeneic anti-tumor effect of callithrix jacchus natural killer cells is dependent on NKp30 and B7-H6 interaction.

Natural Killer (NK) cells mount a fast and efficient immune response against tumor cells and are currently a major focus in the development of anti-cancer cell-based therapies. Due to major differences between the murine and human NK cell receptor system, a non-human primate model would be helpful to evaluate the efficiency of NK-cell based therapies prior to clinical applications. In humans, B7-H6 has been shown to facilitate the elimination of lymphoma cells through the interaction with its receptor NKp30. The common marmoset (Callithrix jacchus) is a new world monkey readily used in biomedical research due to its easy management and proximity to humans. In this study, we demonstrated the expression of B7-H6 antigen in marmoset B-lymphoblastoid cell lines. In addition, a method was established to isolate B- or NK-cells from peripheral blood of marmosets with purities of up to 97%We detected the expression of B7-H6 in lymphoma cells and for the first time in leukemic blasts of human acute myeloid leukemia (AML). Marmoset NK cells were shown to lyse marmoset B lymphoblastoid cell line (B-LCL) cells by up to 28.4% and human B-LCL cells by up to 20%. This effect was abrogated when the NK cells were pre-treated with an anti-NKp30 specific antibody. Also, marmoset NK cells were able to lyse primary leukemic AML cells and lymphoma cells by up to 8.3 and 20.3%respectively. Stimulation of marmoset NK cells with recombinant B7-H6 induced phosphorylation of ERK1/2 and proliferation rates. Furthermore, the secretion of IL-1ß, IL-8, IFN-γ and TNF-α was significantly increased upon B7-H6 stimulation. In conclusion, we demonstrated that non-human primate NK cells have similar mechanisms for the lysis of tumor cells as human NK cells. Thus, this animal model constitutes a very promising tool for the development and evaluation of novel NK-cell based therapies.

The new HLA-C variant HLA-C*05:26 is likely to be structurally identical to the C*05:01P alleles.

The novel allele HLA-C*05:26 differs from HLA-C*05:01 by the non-synonymous amino acid exchange Gly16Ser.

The new HLA-B*58:21 allele is predicted to be functionally similar to the B*58:01P group of alleles.

The new human leukocyte antigen (HLA)-B*58:21 allele differs from B*58:01:01 by an amino acid exchange at codon 90.

Non-expression of HLA-C*07:55N is caused by a premature stop codon in exon 3.

Sequencing of HLA-C*07:55N assessed the presence of a premature TAG stop codon at residue 113.

The novel allele HLA-B*35:167 differs from HLA-B*35:03:01 by the amino acid exchange Val152Glu.

HLA-B*35:167 allele differs from HLA-B*35:03:01 and HLA-B*35:70 by an amino acid exchange at position 152.

HLA-B*08:01:08- joining the fold of silent alpha-1 proline mutations in HLA-B.

The sequence of HLA-B*08:01:08 differs from other HLA-B*08:01 alleles by at least two synonymous nucleotide exchanges.

The new allele HLA-B*35:87 was generated by recombination between HLA-B*35:01 and B*08:01 in intron 2.

The new allele HLA-B*35:87 was generated by recombination between HLA-B*35:01 and B*08:01 in intron 2.

Pocketcheck: updating the HLA class I peptide specificity roadmap.

The structural determination of peptide:HLA (human leucocyte antigen) class I complexes by X-ray crystallography has provided valuable information for understanding how peptides bind to individual HLA class I molecules and how this may influence the immune response. We compared 101 crystal structures of 9-mer peptide:HLA class I complexes available in the protein data bank (PDB) by performing a contact analysis using the Contact Map Analysis webserver http://ligin.weizmann.ac.il/cma. An InterSystems Caché 'post-relational' database containing residue position, amino acid (AA) and buried surface that contact a particular peptide position was then created allowing data comparison for all the structures (Pocketcheck). The analysis illustrates that the HLA class I residues 24, 45, 63 and 67 show high contact frequencies to both the p1 and/or p2 position of bound peptides, indicating that they might influence the nature of a peptide anchor. To determine the influence of these residues we utilized soluble HLA technology and mass spectrometry to analyze peptides derived from HLA-B*44:06 since it differs from the previously described allele B*44:02 by seven AA exchanges located in the alpha 1 domain (residues 24, 32, 41, 45, 63, 67 and 80). HLA-B*44:06 features an anchor motif of P or A at p2 and Y or W at the C-terminal. Additionally B*44:06-derived peptides feature an auxiliary anchor motif at p1, comprising D or E. Our results illustrate that structural analysis can provide valuable information to understand allogenicity and provides a further step towards intelligent HLA mismatching.

The new HLA allele, HLA-A*03:57, differs from HLA-A*03:01 by two amino acids at positions 76 and 77 in the α2 domain affecting the pocket F of the peptide-binding groove.

HLA-A*03:57 has two amino acid exchanges in exon 2 at adjacent positions of the peptide-binding groove.

Closing the gap: discrimination of the expression profile of HLA questionable alleles by a cytokine-induced secretion approach using HLA-A*32:11Q.

Matching of human leukocyte antigen (HLA) alleles between donors and recipients plays a major role in hematopoietic stem cell transplantation (HSCT). Null or questionably expressed HLA allelic variants are a major issue in HLA matching, because the aberrant expression of such alleles can have a major impact on the outcome of HSCT and/or its complications such as graft-versus-host disease. The goal of this study was to investigate the potential of a recently developed cytokine-induced secretion assay to differentiate the expression levels of HLA-A*32:11Q (questionable) into a null (N) or low (L) expression variant. An amino acid mutation at position 164 of HLA-A*32:11Q disrupts the disulfide bridge in the α2 domain. HLA-A*32:11Q is not detectable by standard microlymphocytotoxicity assay. To this end, we cloned soluble HLA-A*32:11Q and a reference allele (HLA-A*32:01) into expression vectors and transfected/transduced HEK293 and K562 cells. Allele-expressing K562 cells were simultaneously transfected/transduced with a ß2-microglobulin (B2M)-encoding vector to ensure the intact HLA structure with B2M. After treatment with proinflammatory cytokines, secreted soluble HLA molecules were determined by enzyme-linked immunosorbent assay in the supernatant and intracellular accumulation of the recombinant proteins by flow cytometry. HLA-A*32:11Q was nearly undetectable in untreated transfectants. Cytokine treatment increased the secretion of HLA-A*32:11Q to detectable levels and resulted in intracellular accumulation of the allele. There was no difference in mRNA transcription between the A*32 alleles. On the basis of these results, we recommend reclassification of HLA-A*32:11Q as a low expression (L) variant.

Modulatory role of calreticulin as chaperokine for dendritic cell-based immunotherapy.

Heat shock proteins (HSPs) play a regulatory role for maturation of antigen-presenting cells (APCs) such as dendritic cells (DCs) and macrophages. Whereas HSP70 has been shown to enhance the maturation of human DCs via a nuclear factor kappa-B (NF-κB)-dependent pathway, the regulatory role of calreticulin (CRT), which is a HSP with similar functions to HSP70, is not well studied. To investigate the role of CRT as adjuvant in cell activation and co-stimulatory responses we determined the effects of CRT on human APC maturation in comparison to that of HSP70. To facilitate eukaryotic endotoxin-free CRT protein expression, three different methods were compared. We demonstrate that CRT induces the maturation of human DCs and increases the production of proinflammatory cytokines via the NF-κB pathway. CRT-mediated maturation was qualitatively similar to that induced by HSP70. Interestingly, priming of monocytes with HSPs showed an even more prominent effect on maturation than exposure of immature DCs to these compounds. A higher expression of CD86, CD83 and CCR7 on mature DCs were found in response to CRT. Our data provide novel insights into the role of extracellular HSPs as chaperokines in the processes of APC generation and may thus be useful to improve adoptive immunotherapy.

The composition of the F pocket in HLA-A*74 generates C-terminal promiscuity among its bound peptides.

In this study we sequenced the bound peptides from three alleles belonging to the HLA-A*74 group (HLA-A*74:04, A*74:06 and A*74:07) that are distinguished by four polymorphic residues within the peptide-binding region. Our data illustrates that A*74:04 exhibits preference for L, M or I at P2 and L, S or P at PΩ, while for A*74:07 the P2 anchor prefers L, P or I and the PΩ anchor S, P, L. In contrast A*74:06 features a P2 anchor motif of S or L, while a PΩ anchor could not be defined; however, a preference for polar residues S, T, Q or the charged residue R at the PΩ position could be detected.

Regulation of HLA class II expression prevents allogeneic T-cell responses.

Human leukocyte antigen (HLA) class II molecules are polymorphic heterodimers that present peptides to CD4+ T-cells. The HLA-DM molecule contributes to assemble HLA class II-peptide complexes. We investigated the effect of silencing either HLA-DR or HLA-DM expression in the allogeneic T-cell responses. The delivery of HLA-DR- or HLA-DM-specific short hairpin RNAs (shRNAs) in a monocytic cell line caused a decrease by up to 85% and 75% at the respective mRNA level. Allogeneic T-cells stimulated with HLA-DM-silenced monocytes decreased to 30% granzyme B mRNA and interferon gamma (IFN-γ) production in comparison with T-cells stimulated with monocytes expressing a non-specific shRNA. By contrast, HLA-DR-silenced monocytes did not induce proliferation, up-regulation of granzyme B mRNA levels or high IFN-γ secretion by allogeneic T-cells vs HLA-DR expressing cells. Direct targeting of HLA-DR expression prevented more efficiently an allogeneic T-cell response in comparison with the knockdown of the expression of HLA-DM molecules. Silencing the expression of HLA-DR molecules might contribute to the development of new allogeneic cell-based therapeutic approaches.

The amino acid variation of HLA-A*02:182 in a highly conserved region is predicted to be functionally similar to HLA-A*02:01:01:01 allele.

We here describe the identification of the novel human leukocyte antigen allele HLA-A*02:182 which has been detected in a potential bone marrow donor. The new allele differs from the sequence of HLA-A*02:01:01:01 only by a non-synonymous nucleotide exchange of Guanin (G) → Cytosin (C) at position 199 in exon 3 replacing amino acid (AA) Arginine (Arg, R) by Threonine (Thr, T) in codon 157. Since the HLA-A*02:01:01:01 allele differs from A*02:182 only at AA position 157, it is assumed that the protein structures of these alleles are highly similar. A mismatch between HLA-A*02:01:01:01 and HLA-A*02:182 is predicted to have a very low allogeneic potential in hematopoietic stem cell transplantation.