Elham Khatamzas

Use of overlapping peptide mixtures as antigens for cytokine flow cytometry

Intracellular cytokine staining and flow cytometry can be used to measure T-cell responses to defined antigens. Although CD8+ T-cell responses to soluble proteins are inefficiently detected by this approach, peptides can be used as antigens. Using overlapping peptides spanning an entire protein sequence, CD8+ T-cell responses can be detected to multiple epitopes, regardless of HLA type. In this study, overlapping peptide mixes of various lengths were compared and 15 amino acid peptides with 11 amino acid overlaps were found to stimulate both CD4+ and CD8+ T-cell responses. Such peptide mixes stimulated CD4+ T-cell responses equivalent to those observed with whole recombinant protein, while simultaneously stimulating CD8+ T-cell responses much higher than those observed with whole protein. Although 8-12 amino acid peptides produced the highest level of CD8+ T-cell responses, 15 amino acid peptides were still very effective. Peptides that were 20 amino acids in length, however, did not stimulate strong CD8+ T-cell responses at the same peptide dose. The cytokine responses to individual epitopes added up approximately to the response to the entire mix, demonstrating that large mixes can detect responses in a quantitative fashion. Unlike whole protein antigens, peptide mixes were effective at stimulating responses in both cryopreserved PBMC and blood stored for 24 h at room temperature. Thus, overlapping 15 amino acid peptide mixes may facilitate the analysis of antigen-specific CD4+ and CD8+ T-cell responses by cytokine flow cytometry, using clinical specimens that include shipped blood or cryopreserved PBMC.

Cytomegalovirus (CMV) phosphoprotein 65 makes a large contribution to shaping the T cell repertoire in CMV-exposed individuals

Antigen-specific, cytokine flow cytometry was used to analyze the prevalence and frequency of CD4 and CD8 memory T cells specific for the abundantly expressed cytomegalovirus (CMV) phosphoprotein 65 (pp65) in healthy CMV IgG-seropositive individuals. Stimulation of peripheral blood mononuclear cells with peptide pools and individual peptides derived from the pp65 amino acid sequence in 40 donors revealed that 63% of donors had a detectable CD4 T cell response and that 83% of donors had a detectable CD8 T cell response against this protein. The overall frequencies of T cells directed against pp65 were analyzed for 20 donors by stimulation with peptide pools covering the complete pp65 protein and were as high as 2 in 1000 and 9 in 1000 (median) peripheral blood CD4 and CD8 T cells, respectively. In addition, a comparison between CD4 responses to a CMV lysate containing various CMV proteins and pp65-specific responses in 9 donors indicated that pp65 was a dominant target of the CMV-specific CD4 T cell response in some, but not all, donors. Several new T cell epitopes were identified.

Distribution of human CMV‐specific memory T cells among the CD8pos. subsets defined by CD57, CD27, and CD45 isoforms

Chronic antigenic stimulation has been associated with peripheral blood expansions of CD8pos. Tu2004cells characterized by CD57 expression, loss of CD27 expression, and reversal of the CD45RObrightu2009/RAdim phenotype usually associated with immunological memory towards a CD45ROdimu2009/RAbright phenotype. However, the relationship and functional significance of these subset(s) has remained controversial. Here, this issue was addressed using a novel flow cytometric technique that allows simultaneous detection of human cytomegalovirus (HCMV)‐specific CD8pos. memory Tu2004cells by rapid (<u20096u2004h) HCMV peptide‐specific induction of cytokine synthesis, and their phenotypic characterization, including CD57, CD27 and CD45RA/RO. The vast majority of resting CD8pos. Tu2004cells capable of rapid induction of IFN‐γ and TNF‐α synthesis in response to HCMV peptides were found in a subset characterized by intermediate to high expression of CD57, down‐regulation/loss of CD27, and varying degrees of reversal of the classical “memory” CD45RObrightu2009/RAdim phenotype. This subpopulation likely includes the fully differentiated memory cells responsible for the long‐term immune defense against HCMV reactivation.

Measurement of Anti-Human Cytomegalovirus T Cell Reactivity in Transplant Recipients and Its Potential Clinical Use: A Mini-Review

By allowing direct determination of the frequencies of antigen-specific memory T cells in peripheral blood, novel techniques based on flow cytometry provide new diagnostic opportunities in various clinical settings, including organ transplantation. While the importance of the T cell compartment for the anti-human cytomegalovirus (HCMV) immune response is undisputed, efficient monitoring of this response was previously impossible because the conventional methods for measuring CD4+ and CD8+ T cell responses are too time-consuming and cost-intensive. We analyzed how the rapid induction of anti-HCMV CD4+ and CD8+ memory T cells by HCMV viral lysate or HCMV-derived peptides, respectively, followed by a flow-cytometric detection step, may be used to monitor HCMV-specific CD4+ and CD8+ memory T cells in solid-organ recipients. We also discuss a number of preconditions for integrating such testing into the clinical routine.