Lecture 6. Burr BIO 4353/6345 HIV/AIDS. Tetramer staining of T cells (CTL s) Andrew McMichael seminar: Background

Transcription

1 Lecture 6 Burr BIO 4353/6345 HIV/AIDS Andrew McMichael seminar: Background Tetramer staining of T cells (CTL s) 1. Vβ 19: There are 52 T cell receptor (TCR) Vβ gene segments in germ line DNA (See following page 208 in notes, Parham, fig 3.3, ), and there are commercially available DNA probes for all of them. In any person, any particular T cell clone will have a 1:52 chance of expressing a receptor that contains a particular Vβ-encoded variable region. Thus, if one sees, as McMichael did, that in patient 1, shortly after HIV-infection, 30% of CTL s express Vβ-19, that means that these Vβ-19 cells probably represent a single clone of CTLs that have proliferated massively, and are directed against one particular viral peptide. This is referred to as an immunodominant response. (CTL = cytotoxic lymphocyte; also known as killer T-cells; CD8-expressing T cells) 2. For each patient in their study, McMichael s group determined the allelic form of each of his or her 6 MHC-I genes (2 each of HLA-A, HLA-B, and HLA-C). For almost all HLA alleles, certainly the most common ones, the anchor residues are known. Since the amino acid sequences of all the HIV proteins are known, the McMichael group could determine which HIV peptides might be bound by a particular patient s set of MHC-I molecules. 3. Knowing the HLA-A, HLA-B, and HLA-C haplotypes of the patient, they could then create a set of engineered molecules (HLA + peptide) that could specifically stain clones of the patient s T-cells (ie, be recognized by the T-cell receptors of that patient); of particular interest, of course, are the T cell clones that would arise after infection, directed against one or all of the viral peptides which that particular patient s MHC-I molecules would be able to present. To recapitulate: each of these specific molecular T-cell stains consists of a molecule of fluorescently-tagged streptavidin, binding four identical avidin-tagged molecules of MHC-I molecules. (The MHC-I molecules being used would in each case correspond to one of the patient s 6 MHC-I isoforms). The four MHC-I molecules would each contain a bound peptide, corresponding to one of the viral peptides which that particular MHC-I isoform is known to bind (and present to T cells). A particular fluorescence-tagged MHC-I tetramer/peptide complex is then used to stain specific a specific clone of T-cells present in the blood of a patient; ie, those T-cells whose receptors (TCRs) are specifically directed against that particular peptide/mhc-i complex. This is referred to as tetramer staining. Stained T-cell populations are then resolved by fluorescence-activated cell sorting (FACS). (One has to have at least a tetramer of fluorescently-tagged MHC-I/peptide complexes, binding to 4 TCR molecules on a T cell, to get sufficiently tight binding of the fluorescent stain to the cells so that FACS experiments can be performed.) 207

2

3

4

5

6 Burr BIO 4353/6345 HIV/AIDS Andrew McMichael Seminar: Highlights Refer to Xeroxed lecture notes (numbered as pp M1-M14; first page says Lecture Notes for McMichael Seminar). Page M5, fig 7: a cytotoxic lymphocyte (CTL) (Killer T-cell) response (not antibodies) controls the early burst of virus production (and CTLs continue to control the virus thereafter, until HIV mutants finally arise that can no longer be controlled). Pages M6, M7; figs 9-12: a single immunodominant clone of CTLs, directed against a single viral peptide, is responsible for controlling HIV levels in many cases (in some cases not just one clone, but two or three clones) Page M8, fig 15: CTL are most effective if they can kill an infected cell within the first 20 hours after infection, before progeny virus are released from the infected cell. (On average, virus begins to be released from a cell approximately one day after infection.) Page M9, figs 17, 18: Patient 007: the immunodominant CTL clone in this patient that is controlling the virus recognizes a peptide derived from the p24 (capsid) protein; this peptide is presented on infected cells by the patient s HLA B27 (MHC-I) molecules. On this peptide there is an arginine (R) at position 2 that is an anchor residue for the binding of this peptide to the B27 molecule (see lecture notes, p194, to review the concept of an anchor residue ). P24 peptide amino acid sequence: K R W I I (L/M) G L M K Arginine anchor residue Lysine, another amino acid with a positively charged side chain like arginine, cannot substitute for arg in terms of the ability of the peptide to bind to the B27 MHC molecule. (But in many proteins, a mutational substitution of lys for arg has little effect on the folding and therefore function of a protein.) Why did a mutant virus, with an R to L substitution, not immediately arise in the infected patient 007? K K W I I (L/M) G L M K Page M9, fig 18: In 1989 (12 years after infection) this mutant did finally arise; because it could not be controlled by the patient s immunodominant CTL clone (the clone that recognized the K R W I I (L/M) G L M K peptide presented by the patients B27 molecule) that mutant virus then became the main virus expressed in the patient. This is called an escape mutant and because the immunodominant clone of CTL can no longer kill HIV-infected cells, the appearance of such mutants results in a transient increase in the concentration of HIV in the blood of the patient (and of course, a decline in T Helper cell counts, as the increased amount of virus kills more T Helper cells) (see fig 23, p. M11). Furthermore, the next best clone of CTL that subsequently arises can t respond as well to the next best immunodominant peptide in this mutant strain of virus, so the steady state level of virus will also be higher than it was before. As time goes on, steady state levels of virus will continue to increase in the patient, as each succeeding escape mutant arises, and CTL are less and less able to defend against these mutants. Page M11, fig 25: Why did it take 12 years for an escape mutant to arise in patient 007? Refer to lecture notes, pages 15 & 16 ( Andrew McMichael seminar take-home message ). M 14(b) 212

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21 Burr BIO 4353/6345 HIV/AIDS Andrew McMichael Seminar: Highlights Refer to Xeroxed lecture notes (numbered as pp M1-M14; first page says Lecture Notes for McMichael Seminar). Page M5, fig 7: a cytotoxic lymphocyte (CTL) (Killer T-cell) response (not antibodies) controls the early burst of virus production (and CTLs continue to control the virus thereafter, until HIV mutants finally arise that can no longer be controlled). Pages M6, M7; figs 9-12: a single immunodominant clone of CTLs, directed against a single viral peptide, is responsible for controlling HIV levels in many cases (in some cases not just one clone, but two or three clones) Page M8, fig 15: CTL are most effective if they can kill an infected cell within the first 20 hours after infection, before progeny virus are released from the infected cell. (On average, virus begins to be released from a cell approximately one day after infection.) Page M9, figs 17, 18: Patient 007: the immunodominant CTL clone in this patient that is controlling the virus recognizes a peptide derived from the p24 (capsid) protein; this peptide is presented on infected cells by the patient s HLA B27 (MHC-I) molecules. On this peptide there is an arginine (R) at position 2 that is an anchor residue for the binding of this peptide to the B27 molecule (see lecture notes, p194, to review the concept of an anchor residue ). P24 peptide amino acid sequence: K R W I I (L/M) G L M K Arginine anchor residue Lysine, another amino acid with a positively charged side chain like arginine, cannot substitute for arg in terms of the ability of the peptide to bind to the B27 MHC molecule. (But in many proteins, a mutational substitution of lys for arg has little effect on the folding and therefore function of a protein.) Why did a mutant virus, with an R to L substitution, not immediately arise in the infected patient 007? K K W I I (L/M) G L M K Page M9, fig 18: In 1989 (12 years after infection) this mutant did finally arise; because it could not be controlled by the patient s immunodominant CTL clone (the clone that recognized the K R W I I (L/M) G L M K peptide presented by the patients B27 molecule) that mutant virus then became the main virus expressed in the patient. This is called an escape mutant and because the immunodominant clone of CTL can no longer kill HIV-infected cells, the appearance of such mutants results in a transient increase in the concentration of HIV in the blood of the patient (and of course, a decline in T Helper cell counts, as the increased amount of virus kills more T Helper cells) (see fig 23, p. M11). Furthermore, the next best clone of CTL that subsequently arises can t respond as well to the next best immunodominant peptide in this mutant strain of virus, so the steady state level of virus will also be higher than it was before. As time goes on, steady state levels of virus will continue to increase in the patient, as each succeeding escape mutant arises, and CTL are less and less able to defend against these mutants. Page M11, fig 25: Why did it take 12 years for an escape mutant to arise in patient 007? Refer to lecture notes, pages 15 & 16 ( Andrew McMichael seminar take-home message ). M 14(b) 212

22

23

24