Peptide-MHC Class I Tetramers Can Fail To Detect Relevant Functional T Cell Clonotypes and Underestimate Antigen-Reactive T Cell Populations.

Peptide-MHC (pMHC) multimers, usually used as streptavidin-based tetramers, have transformed the study of Ag-specific T cells by allowing direct detection, phenotyping, and enumeration within polyclonal T cell populations. These reagents are now a standard part of the immunology toolkit and have been used in many thousands of published studies. Unfortunately, the TCR-affinity threshold required for staining with standard pMHC multimer protocols is higher than that required for efficient T cell activation. This discrepancy makes it possible for pMHC multimer staining to miss fully functional T cells, especially where low-affinity TCRs predominate, such as in MHC class II-restricted responses or those directed against self-antigens. Several recent, somewhat alarming, reports indicate that pMHC staining might fail to detect the majority of functional T cells and have prompted suggestions that T cell immunology has become biased toward the type of cells amenable to detection with multimeric pMHC. We use several viral- and tumor-specific pMHC reagents to compare populations of human T cells stained by standard pMHC protocols and optimized protocols that we have developed. Our results confirm that optimized protocols recover greater populations of T cells that include fully functional T cell clonotypes that cannot be stained by regular pMHC-staining protocols. These results highlight the importance of using optimized procedures that include the use of protein kinase inhibitor and Ab cross-linking during staining to maximize the recovery of Ag-specific T cells and serve to further highlight that many previous quantifications of T cell responses with pMHC reagents are likely to have considerably underestimated the size of the relevant populations

Survival of metastatic melanoma patients after dendritic cell vaccination correlates with expression of leukocyte phosphatidylethanolamine-binding protein 1/Raf kinase inhibitory protein

Immunotherapy for metastatic melanoma offers great promise but, to date, only a subset of patients have responded. There is an urgent need to identify ways of allocating patients to the most beneficial therapy, to increase survival and decrease therapy-associated morbidity and costs. Blood-based biomarkers are of particular interest because of their straightforward implementation in routine clinical care. We sought to identify markers for dendritic cell (DC) vaccine-based immunotherapy against metastatic melanoma through gene expression analysis of peripheral blood mononuclear cells. A large-scale microarray analysis of 74 samples from two treatment centers, taken directly after the first round of DC vaccination, was performed. We found that phosphatidylethanolamine binding protein 1 (_PEBP1_)/ Raf Kinase inhibitory protein (RKIP) expression can be used to identify a significant proportion of patients who performed poorly after DC vaccination. This result was validated by q-PCR analysis on blood samples from a second cohort of 95 patients treated with DC vaccination in four different centers. We conclude that low _PEBP1_ expression correlates with poor overall survival after DC vaccination. Intriguingly, this was only the case for expression of _PEBP1_ after, but not prior to, DC vaccination. Moreover, the change in _PEBP1_ expression upon vaccination correlated well with survival. Further analyses revealed that _PEBP1_ expression positively correlated with genes involved in T cell responses but inversely correlated with genes associated with myeloid cells and aberrant inflammation including _STAT3, NOTCH1,_ and _MAPK1_. Concordantly, _PEBP1_ inversely correlated with the myeloid/ lymphoid-ratio and was suppressed in patients suffering from chronic inflammatory disease

Tumour-reactive T cell subsets in the microenvironment of ovarian cancer.

Solid malignancies are frequently infiltrated with T cells. The success of adoptive cell transfer (ACT) with expanded tumour-infiltrating lymphocytes (TILs) in melanoma warrants its testing in other cancer types. In this preclinical study, we investigated whether clinical-grade TILs could be manufactured from ovarian cancer (OC) tumour specimens. Thirty-four tumour specimens were obtained from 33 individual patients with OC. TILs were analysed for phenotype, antigen specificity and functionality. Minimally expanded TILs (Young TILs) were successfully established from all patients. Young TILs contained a high frequency of CD3 <sup>+</sup> cells with a variable CD4/CD8 ratio. TILs could be expanded to clinical numbers. Importantly, recognition of autologous tumour cells was demonstrated in TILs in >50% of the patients. We confirmed with mass spectrometry the presentation of multiple tumour antigens, including peptides derived from the cancer-testis antigen GAGE, which could be recognised by antigen-specific TILs. Antigen-specific TILs could be isolated and further expanded in vitro. These findings support the hypothesis that patients with OC can benefit from ACT with TILs and led to the initiation of a pilot clinical trial at our institution . clinicaltrials.gov: NCT02482090