A novel category of antigens enabling CTL immunity to tumor escape variants: Cinderella antigens

Deficiencies in MHC class I antigen presentation are a common feature of tumors and allows escape from cytotoxic T lymphocyte (CTL)-mediated killing. It is crucial to take this capacity of tumors into account for the development of T-cell-based immunotherapy, as it may strongly impair their effectiveness. A variety of escape mechanisms has been described thus far, but progress in counteracting them is poor. Here we review a novel strategy to target malignancies with defects in the antigenic processing machinery (APM). The concept is based on a unique category of CD8+ T-cell epitopes that is associated with impaired peptide processing, which we named TEIPP. We characterized this alternative peptide repertoire emerging in MHC-I on tumors lacking classical antigen processing due to defects in the peptide transporter TAP (transporter associated with peptide processing). These TEIPPs exemplify interesting parallels with the folktale figure Cinderella: they are oppressed and neglected by a stepmother (like functional TAP prevents TEIPP presentation), until the suppression is released and Cinderella/TEIPP achieves unexpected recognition. TEIPP-specific CTLs and their cognate peptide-epitopes provide a new strategy to counteract immune evasion by APM defects and bear potential to targeting escape variants observed in a wide range of cancers

Promiscuous Binding of Invariant Chain-Derived CLIP Peptide to Distinct HLA-I Molecules Revealed in Leukemic Cells

Antigen presentation by HLA class I (HLA-I) and HLA class II (HLA-II) complexes is achieved by proteins that are specific for their respective processing pathway. The invariant chain (Ii)-derived peptide CLIP is required for HLA-II-mediated antigen presentation by stabilizing HLA-II molecules before antigen loading through transient and promiscuous binding to different HLA-II peptide grooves. Here, we demonstrate alternative binding of CLIP to surface HLA-I molecules on leukemic cells. In HLA-II-negative AML cells, we found plasma membrane display of the CLIP peptide. Silencing Ii in AML cells resulted in reduced HLA-I cell surface display, which indicated a direct role of CLIP in the HLA-I antigen presentation pathway. In HLA-I-specific peptide eluates from B-LCLs, five Ii-derived peptides were identified, of which two were from the CLIP region. In vitro peptide binding assays strikingly revealed that the eluted CLIP peptide RMATPLLMQALPM efficiently bound to four distinct HLA-I supertypes (-A2, -B7, -A3, -B40). Furthermore, shorter length variants of this CLIP peptide also bound to these four supertypes, although in silico algorithms only predicted binding to HLA-A2 or -B7. Immunization of HLA-A2 transgenic mice with these peptides did not induce CTL responses. Together these data show a remarkable promiscuity of CLIP for binding to a wide variety of HLA-I molecules. The found participation of CLIP in the HLA-I antigen presentation pathway could reflect an aberrant mechanism in leukemic cells, but might also lead to elucidation of novel processing pathways or immune escape mechanisms

Intrastriatal Administration of AAV5-miHTT in Non-Human Primates and Rats Is Well Tolerated and Results in miHTT Transgene Expression in Key Areas of Huntington Disease Pathology

Huntington disease (HD) is a fatal, neurodegenerative genetic disorder with aggregation of mutant Huntingtin protein (mutHTT) in the brain as a key pathological mechanism. There are currently no disease modifying therapies for HD; however, HTT-lowering therapies hold promise. Recombinant adeno-associated virus serotype 5 expressing a microRNA that targets HTT mRNA (AAV5-miHTT) is in development for the treatment of HD with promising results in rodent and minipig HD models. To support a clinical trial, toxicity studies were performed in non-human primates (NHP, Macaca fascicularis) and Sprague-Dawley rats to evaluate the safety of AAV5-miHTT, the neurosurgical administration procedure, vector delivery and expression of the miHTT transgene during a 6-month observation period. For accurate delivery of AAV5-miHTT to the striatum, real-time magnetic resonance imaging (MRI) with convection-enhanced delivery (CED) was used in NHP. Catheters were successfully implanted in 24 NHP, without neurological symptoms, and resulted in tracer signal in the target areas. Widespread vector DNA and miHTT transgene distribution in the brain was found, particularly in areas associated with HD pathology. Intrastriatal administration of AAV5-miHTT was well tolerated with no clinically relevant changes in either species. These studies demonstrate the excellent safety profile of AAV5-miHTT, the reproducibility and tolerability of intrastriatal administration, and the delivery of AAV5-miHTT to the brain, which support the transition of AAV5-miHTT into clinical studies

HLA-I binding affinity of eluted peptides derived from the invariant chain.

*<p>See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034649#pone-0034649-g002" target="_blank">Figure 2</a> for amino acid position in the invariant chain protein.</p>†<p>IC50 is the concentration used to obtain half maximal competition and represents the mean value of two independent experiments.</p>‡<p>Binding affinity is classified according to the following IC50 cut-off values: high affinity, ≤5 µM; intermediate (int) affinity, 5–15 µM; low affinity, 15–100 µM; no binding, >100 µM <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034649#pone.0034649-Kessler1" target="_blank">[21]</a>.</p

Surface display of the CLIP epitope on HLA-II-negative leukemic cells and influence of the invariant chain on HLA-I expression.

<p>(A) Surface expression of CLIP, HLA-DR, total HLA-II (‘HLA-DRPQ’) and invariant chain (‘CD74’) of myeloid cells from an acute promyelocytic leukemia (APL) patient, as determined by flow cytometry. Myeloid cells were defined as CD45^dim/SSC^low/int and expression thresholds were set according to unstained myeloid cells. (B) Quantitative analysis on frequencies of myeloid cells from APL patients that express CLIP (n = 9), HLA-DR (n = 9), total HLA-II (n = 6) and CD74 (n = 6). Frequencies indicate percentage tumor cells that reach threshold expression based on unstained leukemic cells. (C) The effect of invariant chain Ii down-modulation in KG-1 (CLIP^-) and THP-1 (CLIP⁺) leukemic cells on HLA-I expression at the cell surface. Intracellular staining (ICS) of Ii (PIN1.1) and surface staining of HLA-I (W6/32) were compared between Ii siRNA-transduced and non-transduced cells.</p

Promiscuous binding of CLIP-peptide to distinct HLA-I alleles, representing four supertypes.

<p>The CLIP peptide RMATPLLMQALPM (peptide 3) was tested for binding affinity in a competition-based cellular peptide binding assay. The four tested HLA-I alleles (HLA-A0201, -A0301, -B0702 and -B4002) harbor a completely distinct binding pocket and bind different peptide ligands. CLIP peptide shows intermediate to high binding affinity to all of these. Separate positive control peptides efficiently bind to their respective HLA allele: GILGFVFTL (A0201 peptide), QVPLRPMTYK (A0301 peptide), SPSVDKARAEL (B0702 peptide) and GEFGGFGSV (B4002 peptide) with IC50 values of 3.7, 0.2, 0.7 and 0.2, respectively <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034649#pone.0034649-Kessler1" target="_blank">[21]</a>. The peptide concentration started at 100 µg/ml for HLA-A0201, -A0301 and -B4002 and 25 µg/ml for HLA-B0702, followed by a serial dilution of a factor two. Exact IC50 values of the CLIP peptides are depicted in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034649#pone-0034649-t002" target="_blank">Table 2</a>.</p

Invariant chain-derived peptides identified in isolated HLA-I molecules of B-LCLs.

<p>Peptide elutions of purified HLA-I molecules from EBV-transformed B-LCLs resulted in the identification of five peptides originating from the invariant chain. HLA-I purification and subsequent mass spectrometry analysis are described in <i>Materials and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034649#s2" target="_blank">Methods</a></i>. Of note, peptide 3 and 4 are located in the CLIP region, known for universal binding to HLA-II molecules.</p

HLA-I binding affinity of peptide length variants located in the CLIP region of the invariant chain.

*<p>Peptide found with HLA-I elutions.</p>†<p>Amino acid (AA) position in the invariant chain protein.</p>‡<p>Predicted HLA-I allele to which the peptide binds. Peptide binding prediction was done with netMHC (<a href="http://www.cbs.dtu.dk/services/NetMHC" target="_blank">http://www.cbs.dtu.dk/services/NetMHC</a>). Binding predictions can be made for peptide lengths between 8 and 11 for all alleles with a novel approximation algorithm using artificial neural networks trained on 9-mer peptides <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034649#pone.0034649-Lundegaard1" target="_blank">[26]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034649#pone.0034649-Lundegaard2" target="_blank">[27]</a>. Only peptides are shown with a predicted binding affinity of 500 nM or stronger.</p>§<p>IC50 is the concentration used to obtain half maximal competition and represents the mean value of two independent experiments.</p>¶<p>Binding affinity is classified according to the following IC50 cut-off values: high affinity, ≤5 µM; intermediate (int) affinity, 5–15 µM; low affinity, 15–100 µM; no binding, >100 µM <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034649#pone.0034649-Kessler1" target="_blank">[21]</a>.</p

Specificity and Dynamics of Effector and Memory CD8 T Cell Responses in Human Tick-Borne Encephalitis Virus Infection

<div><p>Tick-borne encephalitis virus (TBEV) is transferred to humans by ticks. The virus causes tick-borne encephalitis (TBE) with symptoms such as meningitis and meningoencephalitis. About one third of the patients suffer from long-lasting sequelae after clearance of the infection. Studies of the immune response during TBEV-infection are essential to the understanding of host responses to TBEV-infection and for the development of therapeutics. Here, we studied in detail the primary CD8 T cell response to TBEV in patients with acute TBE. Peripheral blood CD8 T cells mounted a considerable response to TBEV-infection as assessed by Ki67 and CD38 co-expression. These activated cells showed a CD45RA-CCR7-CD127- phenotype at day 7 after hospitalization, phenotypically defining them as effector cells. An immunodominant HLA-A2-restricted TBEV epitope was identified and utilized to study the characteristics and temporal dynamics of the antigen-specific response. The functional profile of TBEV-specific CD8 T cells was dominated by variants of mono-functional cells as the effector response matured. Antigen-specific CD8 T cells predominantly displayed a distinct Eomes+Ki67+T-bet+ effector phenotype at the peak of the response, which transitioned to an Eomes-Ki67-T-bet+ phenotype as the infection resolved and memory was established. These transcription factors thus characterize and discriminate stages of the antigen-specific T cell response during acute TBEV-infection. Altogether, CD8 T cells responded strongly to acute TBEV infection and passed through an effector phase, prior to gradual differentiation into memory cells with distinct transcription factor expression-patterns throughout the different phases.</p></div