Analysis, Isolation, and Activation of Antigen-Specific CD4(+) and CD8(+) T Cells by Soluble MHC-Peptide Complexes

Abstract

T cells constitute the core of adaptive cellular immunity and protect higher organisms against pathogen infections and cancer. Monitoring of disease progression as well as prophylactic or therapeutic vaccines and immunotherapies call for conclusive detection, analysis, and sorting of antigen-specific T cells. This is possible by means of soluble recombinant ligands for T cells, i.e., MHC class I-peptide (pMHC I) complexes for CD8(+) T cells and MHC class II-peptide (pMHC II) complexes for CD4(+) T cells and flow cytometry. Here we review major developments in the development of pMHC staining reagents and their diverse applications and discuss perspectives of their use for basic and clinical investigations.

Keywords: MHC; T cell receptor; T cells; coreceptor; flow cytometry; tetramers.

Figure 1

Evolution of soluble pMHC complexes and their applications for T cell analysis and sorting. (A) Chronological listing of milestones in the use of pMHC complexes for the detection and analysis of antigen-specific CD8⁺ and CD4⁺ T cells. (B) Cartoons of the most frequently used pMHC oligomers; the red oval represents PE.

Figure 2

Role of valence and spacer distances of soluble pMHC complexes on binding and activation of CD8⁺ T cells. (A) Cartoons of mono, di, tetra, and octamer pMHC complexes, in which x and y indicate maximal spacer distances in Å, X a pMHC monomer and SA streptavidin. The spacer distances for the pMHC dimers varies from 18 to 90 Å. (B) Relative binding on CD8⁺ T cells of monomer (black), short dimer (blue), and long dimer (dotted blue). (C) Same as (B) with short tetramer (green), long tetramer (dotted green), short octamer (red), and long octamer (dotted red). Short distances are<20 Å and long distances are>80 Å. (D) Relative activation (Ca²⁺ mobilization) of CD8⁺ T cells after binding of the represented pMHC complexes. (E) Specific lysis of target cells (GP33 peptide sensitized P815/Db cells) was assessed following incubation with LCMV d8 CD8⁺ CTL in the presence of Db/GP33 monomer (black bars) or Db/GP33 long octamer (red bars). (F) Specific lysis of GP33 peptide sensitized P815/Db cells was assessed in LCMV infected mice that were or were not (LCMV) injected previously with octamer. Presented data were derived from Ref. (–38).

Figure 3

Sorting of antigen-specific T cells with reversible pMHC multimers. (A) Cartoon illustrating the principle of sorting of “untouched” T cells. After cell staining and sorting, reversible, but not conventional multimers can be removed and the T cells cultured at 37°C in the absence of potentially harmful pMHC complexes. (B) Representation of the three types of reversible bonds used for the preparation of pMHC oligomers and agent used to provoke dissociation. (C) Flu matrix_58–66 peptide stimulated PBMC were stained at 4°C with A2/Flu multimers containing conventional irreversible (red), NTA₄-His (green), streptactin-streptag (orange), or desthiobiotin–streptavidin (blue) scaffolds, washed and cell-associated fluorescence measured after different periods of incubation at 20°C. (D) Alternatively cells labeled with BSP (red) or NTAmer were FACS sorted and cloned by limiting dilution. Hundred percent refers to the number of clones obtained from BSP multimer sorted cells. (E) Randomly chosen clones were incubated with BSP multimer at 37°C, stained with AnnexinV, and analyzed by flow cytometry. Presented data were derived from (35).

Figure 4

Peptide-MHC oligomers built on switchable Ni²⁺-NTA-His-tag chelate complexes. (A,B) Cartoons illustrating conventional BSP multimers pMHC (B) and reversible monomers directly coupled on PE via the NTA strategy (A), where PE designates phycoerythrin. (C) The structure of a phycoerythrin molecule is shown in which lysine side chains reacted with a maleimido-N-hydroxysuccinimide ester and the resulting maleimido-PE will subsequently reacted with thio-NTA derivative. (D) Room temperature isotherms were shown for HLA-A2-Flu matrix_58–66 BSP multimers (red circles) or PE-NTAmer (green squares) as assessed on Flu peptide stimulated PBMC. (E) Cartoon illustrating the two-step dissociation of PE labeled pMHC NTAmer from T cells. Imidazole induced rapid decay of cell-associated NTAmers and disappearance of PE fluorescence, followed by slower dissociation of Cy5 labeled blue monomers from the cells. (F) Cloned Flu matrix-specific CD8⁺ T cells were stained in the cold with conventional A2/Flu_58–66 PE multimers, washed and cell-associated PE fluorescence measured by flow cytometry after the indicated periods of time. (G) Alternatively the cells were labeled with PE-NTAmers containing Cy5 conjugated A2/Flu_58–66 monomers and the monomer dissociation from the cells was assessed by flow cytometry as illustrated in (E). The inserted numbers indicate the dissociation half-lives T_1/2. Presented data were derived from (33) and (35).

Figure 5

Molecularly defined pMHC class II multimers detect low avidity CD4⁺ T cells. (A,C,F) Cartoons illustrating the use of photocleavable (red asterisk) peptide tags to discriminate pMHC II complexes containing the peptide of interest (green ball) or irrelevant peptides (blue ball) (A), to affinity purify correctly loaded complexes (C) and to remove the peptide tag, either His₆ or desthiobiotin that is linked to the peptide via a β-nitrophenylglycine residue, which can be cleft by UV irradiation. (B) “Empty” HLA-DR4 was incubated for 6, 24, and 48 h at 37°C at pH 6, 32°C for 4 h with His₆-HA_306–318 (black bars) or His₆-NY-ESO-1_119–143 (white bars). Peptide occupancy was calculated as (amount of His₆-peptide bound to the DR4)/(amount of total DR4). After gel-filtration the peptide occupancy was determined by ELISA using affinity-purified complexes as reference (100%). (D) Staining of DR52b/NY-ESO-1_119–143-specific (top) or an irrelevant (SSX2-specific) CD4⁺ T cell clone (bottom) were stained with 5.6 nM of conventional (left) and “immunopure” DR52b/NY-ESO-1_119–143 multimers (right) at 37°C for 1 h and analyzed by flow cytometry. (E) Ex vivo CD4⁺ T cells from PBMC of a DR52b⁺ healthy donors (HD) or of DR52b melanoma patients either pre- or post-NY-ESO-1 vaccination were stained as in E plus anti-CD45RA antibody and analyzed by flow cytometry. (G,H) The 37°C binding isotherms of “immunopure” DR4/HA_306–318 (blue) and conventional multimers (red) on the DR4/HA_306–318-specific clones 9 (G) or 8 (H). The immunopure multimers contained [dark blue or not (light blue)] the N-terminal His₆-tag that was used for affinity purification. Presented data were derived from (31).

Figure 6

Degree of peptide loading determines the avidity detection threshold on pMHC II multimer detection. (A,B) Cognate (DR4/HA_306–318) and irrelevant (DR4/CLIP) pMHC II monomers were mixed in different ratios (x-axis; fractions of one) and reacted with streptavidin–PE to make tetramers. For 0, 20, 50, 80, and 100% of cognate monomers the compositions of tetramers containing X = 0, 1, 2, 3, or 4 cognate monomers were calculated according to the binominal distribution and expressed in %, indicated by the inserted numbers (A), with 100% being the sum of each row; alternatively it was plotted on the y-axis as fraction of 1 against the fraction of cognate/non-cognate complexes (x-axis) for the indicated four avidity levels T cell binding (red: all four complexes bind; green: three; blue: two; and purple: one). (C) Seven DR4/HA_306–318-specific CD4⁺ T cell clones were incubated with 18.5 nM DR4 tetramers with DR4 tetramers containing the indicated factions of HA_306–318 and CLIP peptides (x-axis; fraction of one) for 1 h at 37°C and cell-associated fluorescence assessed by flow cytometry and expressed in % of maximal binding (y-axis). (D–F) From these binding isotherms those observed on the clones 11, 17, and 18 match the high avidity binding curve (in red) (D), those recorded on the clones 19 and 20, match the green curve (E) and the one measured on the clone 12 the purple curve (F). (G,H) The cartoon representation of high (G) and low avidity (H) CD4⁺ T cells as defined by their ability to bind mixed tetramers.