Development of an epitope panel for consistent identification of antigen-specific T-cells in humans.

We aimed to establish a panel of MHC-peptide multimers suitable as a positive control in the detection of HLA A*0201 restricted antigen specific T cells (ASTC) by flow cytometry. MHC Dextramers were loaded with HLA A*0201 binding peptides from viral antigens and melanoma targets identified from a literature search and in silico prediction. Peripheral blood mononuclear cells (PBMC) from healthy donors were analysed with the MHC Dextramers using flow cytometry. The best performing epitopes were tested on PBMC from patients undergoing testing for Mycobacterium tuberculosis infection to assess the coverage of this epitope panel. Of 21 candidate epitopes, ASTC could be detected against 12 (57·1%) in at least one of 18 healthy blood donors. Reactivity to two or more epitopes was seen in 17 of the 18 donors (94·4%). We selected the six best-performing epitopes and demonstrated a positive response in 42 (97·7%) of 43 patient samples (healthy, latent and active M. tuberculosis infection). The selected panel of six antigenic epitopes sufficed as a positive control in the detection of ASTC in HLA A*0201. Performance was robust in different stages of latent and active M. tuberculosis infection, indicating reliability also during infection.

Neutralizing antibody and CD8+ T cell responses following BA.4/5 bivalent COVID-19 booster vaccination in adults with and without prior exposure to SARS-CoV-2.

As severe acute respiratory coronavirus 2 (SARS-CoV-2) variants continue to emerge, it is important to characterize immune responses against variants which can inform on protection efficacies following booster vaccination. In this study, neutralizing breadth and antigen-specific CD8+ T cell responses were analyzed in both infection-naïve and infection-experienced individuals following administration of a booster bivalent Wuhan-Hu-1+BA.4/5 Comirnaty® mRNA vaccine. Significantly higher neutralizing titers were found after this vaccination compared to the pre-third booster vaccination time point. Further, neutralizing breadth to omicron variants, including BA.1, BA.2, BA.5, BQ.1 and XBB.1, was found to be boosted following bivalent vaccination. SARS-CoV-2-specific CD8+ T cells were identified, but with no evidence that frequencies were increased following booster vaccinations. Spike protein-specific CD8+ T cells were the only responses detected after vaccination and non-spike-specific CD8+ T cells were only detected after infection. Both spike-specific and non-spike-specific CD8+ T cells were found at much lower frequencies than CD8+ T cells specific to cytomegalovirus (CMV), Epstein-Barr virus (EBV) and influenza (Flu). Taken together, these results show that the bivalent Wuhan-Hu-1+BA.4/5 Comirnaty® mRNA vaccine boosted the breadth of neutralization to newer SARS-CoV-2 variants and that vaccination is able to induce spike protein-specific CD8+ T cell responses, which are maintained longitudinally.

Significance of calcium binding, tyrosine phosphorylation, and lysine trimethylation for the essential function of calmodulin in vertebrate cells analyzed in a novel gene replacement system.

Calmodulin (CaM) was shown to be essential for survival of lower eukaryotes by gene deletion experiments. So far, no CaM gene deletion was reported in higher eukaryotes. In vertebrates, CaM is expressed from several genes, which encode an identical protein, making it difficult to generate a model system to study the effect of CaM gene deletion. Here, we present a novel genetic system based on the chicken DT40 cell line, in which the two functional CaM genes were deleted and one allele replaced with a CaM transgene that can be artificially regulated. We show that CaM is essential for survival of vertebrate cells as they die in the absence of CaM expression. Reversal of CaM repression or ectopic expression of HA-tagged CaM rescued the cells. Cells exclusively expressing HA-CaM with impaired individual calcium binding domains as well as HA-CaM lacking the ability to be phosphorylated at residues Tyr(99)/Tyr(138) or trimethylated at Lys(115) survived and grew well. CaM mutated at both Ca(2+) binding sites 3 and 4 as well as at both sites 1 and 2, but to a lesser degree, showed decreased ability to support cell growth. Cells expressing CaM with all calcium binding sites impaired died with kinetics similar to that of cells expressing no CaM. This system offers a unique opportunity to analyze CaM structure-function relationships in vivo without the use of pharmacological inhibitors and to analyze the function of wild type and mutated CaM in modulating the activity of different target systems without interference of endogenous CaM.

Ras-inducible immortalized fibroblasts: focus formation without cell cycle deregulation.

The Ras oncogene transforms cultured murine fibroblasts into malignant, focus-forming cells, whose lack of contact inhibition is evidenced by high saturation densities. In order to investigate the reversibility of Ras transformation, as well as the kinetics of Ras-induced changes, cell lines that conditionally express oncogenic Ras were constructed. Both focus formation and increased saturation density were inducible and fully reversible. In exponentially growing cells, oncogenic Ras-expression had no effect on proliferation rates, Erk phosphorylation, or the level of cyclin D1, and Ras-induction did not confer serum-independent growth. As expected, growth to high density in uninduced cells led to quiescence with a low level of cyclin D1 and no active Erk; in this setting, Ras induction prevented full downregulation of cyclin D1 and inactivation of Erk. Our results show that Ras expression to a level sufficient for transformation leads to relatively subtle effects on known downstream targets, and that the focus formation and increased saturation density growth induced by Ras is not a result of growth factor independence.

Dextramer reagents are effective tools for quantifying CMV antigen-specific T cells from peripheral blood samples.

The enumeration of antigen-specific T cells is increasingly relevant in clinical and research settings. This information is useful for evaluating immune responses to treatment, monitoring the efficacy of anticancer vaccines, and for detecting self-reactive T cells in autoimmune disorders. Quantifying antigen-specific T cells can be accomplished via IFNγ ELISpot assay, the measurement of intracellular cytokine production by flow cytometry, or by lymphocyte proliferation assays in response to antigen. While robust, these technologies are labor-intensive and can take several days to obtain results. New technology has led to more powerful tools for quickly and accurately measuring antigen-specific T cells by flow cytometry via fluorescently-labeled TCR-specific multimers. In this study, we evaluated the use of an assay based on Dextramer reagents for enumerating cytomegalovirus (CMV) antigen-specific T cells (CASTs). Assay performance characteristics were assessed by establishing Dextramers' sensitivity (median=0.4; range=0.1-1.4 CASTs µl(-1) ), determining their specificity (100%), evaluating assay robustness with different leukocyte sources and assay reproducibility via interlaboratory and interinstrument investigations. Furthermore, the levels of CASTs in 95 peripheral blood samples from 62 unique blood and marrow transplants recipients correlated well between Dextramers and Tetramers (R(2) =0.9042).