Use of overlapping peptide mixtures as antigens for cytokine flow cytometry

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Abstract

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.

Introduction

Cytokine flow cytometry (CFC) assays can measure T-cell immune responses to a variety of antigens after short-term activation of whole blood or PBMC (Waldrop et al., 1997, Komanduri et al., 1998, Suni et al., 1998, Weynants et al., 1998, He et al., 1999, Kern et al., 1999a, Kern et al., 1999b, Pitcher et al., 1999, Asanuma et al., 2000, Karanikas et al., 2000, Kern et al., 2000, Maino et al., 2000, Maecker et al., 2001; reviewed in Maecker et al., 2000, Maecker et al., 2002). Cells are incubated with antigen, often in the presence of costimulatory antibodies to molecules such as CD28 and CD49d. Addition of a secretion inhibitor such as brefeldin A allows for intracellular accumulation of cytokines in activated cells. After fixation and permeabilization, the cells are stained for intracellular cytokine(s) and analyzed by flow cytometry Suni et al., 1998, Nomura et al., 2000.

When whole proteins or viral lysates are used as antigens in CFC assays, host antigen presenting cells (APC) preferentially process the antigen(s) via the exogenous pathway of antigen presentation, leading primarily to presentation of peptides on MHC class II molecules and stimulation of CD4+ T-cells. However, CD8+ T-cell responses require antigen to be processed via the endogenous pathway of antigen presentation, leading to presentation of peptides on MHC class I molecules Braciale et al., 1987, Monaco, 1995. While some “cross-priming” of CD8+ T-cell responses can occur in the presence of exogenous antigen, this process is generally inefficient Carbone and Bevan, 1990, Rock et al., 1990, Rock et al., 1993, Harding and Song, 1994, Kovacsovics-Bankowski and Rock, 1995, Martinez-Kinader et al., 1995, Norbury et al., 1995, Reis e Sousa and Germain, 1995, Harding, 1996, Rock, 1996, Song and Harding, 1996, Brossart and Bevan, 1997, Day et al., 1997, Oh et al., 1997, Shen et al., 1997. Thus, CD8+ T-cell responses to complex antigens have been difficult to assess using CFC, requiring strategies such as infection of APC with recombinant vaccinia virus vectors (Gea-Banacloche et al., 2000).

To circumvent the requirement for endogenous antigen processing to stimulate CD8+ T-cells, peptides representing immunodominant epitopes can be used as antigens Kern et al., 1998, He et al., 2001. Such peptides, if they are of the proper length and sequence to bind to host MHC class I molecules, can efficiently stimulate CD8+ T-cell responses in CFC assays. Presumably, this occurs through direct binding of the peptides to cell-surface MHC molecules, bypassing the need for internalization and processing. However, use of such peptides necessitates both knowledge of the immunodominant epitopes of the antigen in question and knowledge of the host MHC type. In addition, responses to single immunodominant epitopes may be very low in some donors and are often not reflective of responses to the entire antigen (Betts et al., 2000 and our unpublished observations). Thus, use of many peptides would be required to assess the full repertoire of CD8+ T-cell responses to an entire protein, virus, or bacterial antigen.

Recently, mixtures of overlapping peptides have been shown capable of efficiently stimulating CD8+ T-cell responses Betts et al., 2000, Kern et al., 2000. Using a set of overlapping peptides spanning the CMV glycoprotein pp65, Kern et al. showed that CD8+ T-cell responses could be efficiently detected by CFC. The peptides used were 15 amino acids in length, overlapping by 11 amino acids each. One hundred thirty-eight peptides were required to span the entire pp65 sequence. These 138 peptides, when combined into a single mixture, stimulated a vigorous CD8+ T-cell response, and some evidence was shown that the quantitative responses to individual peptides were preserved in the presence of all 138 peptides as a mixture.

In the present study, such mixtures of 15 amino acid peptides were used to stimulate both CD4+ and CD8+ T-cell responses. The CD4+ T-cell responses were approximately equal to those detected when whole soluble protein was used as an antigen, while the CD8+ responses were significantly higher than the often negligible responses detected with soluble protein stimulation. Furthermore, the CD8+ T-cell responses to a mixture of 15 amino acid peptides were similar to those obtained with a mix of 8–12 amino acid peptides, selected to represent known CD8-restricted epitopes of the same protein. The CD8+ response to a mixture of 20 amino acid peptides was significantly lower than that obtained with either the 8–12-mer or 15-mer mixes, for the equivalent dose of peptide. Also, the responses to individual immunodominant peptides within the 15-mer mix approximately added up to the response seen with the entire mix, demonstrating that large mixtures of peptides represent a viable approach for detection of both CD4+ and CD8+ T-cell responses by CFC.

Immunomonitoring of clinical samples often requires the use of blood that has been shipped overnight from a remote site or the use of cryopreserved PBMC. In part, because APC function can deteriorate significantly under these conditions (our unpublished data), it has previously been difficult to detect CFC responses in such samples. However, because T-cell responses to peptide antigens are presumably less dependent upon APC function than responses to whole antigens, we reasoned that it might be possible to stimulate day-old or cryopreserved PBMC with peptides. In this study, CFC assays using a 15-mer peptide mix detected T-cell responses in stored or cryopreserved specimens with nearly equal efficiency to those detected in fresh samples from the same donor. Thus, 15-mer peptide mixes may be very useful as antigens for CFC monitoring of clinical trials.

Section snippets

Peptides

Individual peptides consisting of 15 amino acid residues, overlapping by 11 amino acids each (synthesized by Ashok Khatri, Massachusetts General Hospital, Boston, MA), were designed to span the sequences of CMV pp65 or HIV p55 gag (SF2 strain). This required 138 peptides for pp65 and 127 peptides for p55 gag (including four alternate peptides, numbered 28A, 29A, 30A and 31A, for p55 gag, to account for potential AA to DT mutations at amino acids 121–122 present in the MN strain of HIV).

Cytokine response to 15-mer peptide mix

A mixture of overlapping 15 amino acid peptides spanning the sequence of the CMV glycoprotein pp65 was used to stimulate both CD4+ and CD8+ T-cell responses in 6-h CFC assays. Fig. 1A shows a titration of this peptide mix on a typical CMV seropositive donor. Note that the CD4+ and CD8+ T-cell responses plateau at a titer of approximately 1:400, which represents a final concentration of 1.75 μg/ml of each peptide in the mix. This is similar to concentrations typically used for stimulation of CFC

Discussion

In this study, peptide mixes comprised of overlapping peptides of various lengths were compared for their ability to stimulate CD4+ and CD8+ T-cell responses by CFC. The results indicate that 15 amino acid peptides with 11 amino acid overlaps may be particularly useful for the detection of both CD4+ and CD8+ responses. While 20-mer peptides were able to generate similar CD4+ responses at a given peptide dose, they were much less efficient at generating CD8+ responses. While suitable for

Acknowledgements

We thank the HIV+ and CMV+ subjects for their participation in this study and Joyce Ruitenberg for her technical assistance. This research was supported by grants from the NIH UCSF Center for AIDS Research (P30 A127763); NIH National Clinical Research Centers (MO1-RR-00083); the NIH National Institute of Allergy and Infectious Diseases (R01-AI47062, to JMM, and AI47606 to LJP); and Center for AIDS Research (5P 30 AI36214). JMM is an Elizabeth Glaser Scientist of the Elizabeth Glaser Pediatric

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