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Surveillance of IL-2 inducing CD4+ T cell epitopes in acute HIV-1 infection for the emergence of escape mutants. R. Brad Jones 1 , Feng Yun Yue 2 , Colin Kovacs 3 , Ruqaya Mohamed 2 , Kelly Macdonald 1,2 , Mario Ostrowski 1,2. 1 Department of Immunology, University of Toronto

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slide1

Surveillance of IL-2 inducing CD4+ T cell epitopes in

acute HIV-1 infection for the emergence of escape mutants

R. Brad Jones1, Feng Yun Yue2, Colin Kovacs3,

Ruqaya Mohamed2, Kelly Macdonald1,2,

Mario Ostrowski1,2

1 Department of Immunology, University of Toronto

2 Clinical Sciences Division, University of Toronto

3 Canadian Immunodeficiency Collaborative

slide2

Introduction

  • CD4+ T cell responses are critical in control of other chronic viral
  • infections including: gamma herpesvirus (Cardin, 1996),
  • LCMV and vaccinia (Leist, 1989)
  • Strong HIV-specific CD4+ T cell proliferation is maintained only
  • in long-term nonprogressors (LTNP), (Pontesilli, 1999)
  • Vigorous HIV-1-specific IL-2 producing CD4+ T cell responses
  • are associated with control of viremia (Rosenberg, 1997)
  • Is this cause or effect? High level HIV-1 viremia suppresses viral
  • antigen specific CD4+ T cell proliferation (McNeil, 2001)
  • Prospective study suggests that IL-2 producing CD4+ T cell
  • response to gag does not have prognostic value for rate
  • of progression to AIDS (Miedema, 2006)
slide3

Delineating Cause and Effect

  • HIV-1 is capable of rapidly acquiring mutations which confer
  • escape from selective pressure
  • We see this with gp120 mutations which escape antibody
  • responses, drug-resistance mutations, and certain CD8+
  • T cell responses
  • If CD4+ T cells are capable of exerting immunological
  • pressure on HIV-1, we should see the emergence of CD4
  • epitope escape mutations
slide4

Subject: OM214

  • Acute seroconverter - symptomatic: fever, rash

HAART

slide5

Methods

Cloning:

  • Sample obtained from leukopheresis
  • After CD8+ depletion, cells were stimulated overnight with p55
  • Enrichment for HIV-p55 specific CD4+ T cells achieved with IL-2
  • secretion assay (Miltenyi) and MACS
  • Plated at limiting dilution with irradiated feeder cells
  • p55 specificity was screened by ELISPOT and confirmed by FACS

Determining Eptiope Specificity of Clones:

  • Gross specificity determined by overlapping gag peptide pool
  • ELISPOT and confirmed by FACS
  • Minimal epitope determined using truncated peptides
slide6

Results

Gag p17:

Gag p24:

“HIVWASRELER”

“FRDYVDRFYK”

Gag p24:

“REPRGSDIAGT”

slide7

HLA Restriction

  • ELISPOTs repeated with core peptides in presence of either anti-DQ,
  • anti-DR, or isotype controls

Peptide + clone +

B cell line

Clone +

BCL

Clone +

BCL + anti-DR

Clone +

BCL + anti-DQ

  • Two clones from OM214 ‘MREPRGSDIAGT’ and ‘FRDYVDRFYK’ are DQ
  • restricted
  • Specifically ‘FRDYVDRFYK’ is restricted by DQB1*05011/DQA1*010101
slide8

Epitope Responses in ex vivo PBMCs

Month 2:

Control

“FRDYVDRFYK”

Autologous p55

“REPRGSDIAGT”

“HIVWASRELER”

0.025

0.021

0

0.053

0.015

IL-2

CD69

Month 12:

Control

“FRDYVDRFYK”

Autologous p55

“REPRGSDIAGT”

“HIVWASRELER”

0.005

0

0

0

0

IL-2

CD69

slide9

Sequencing

  • Performed on circulating plasma viruses
  • Limiting dilution methodology with direct sequencing
  • from PCR product
  • Sequences screened for hypermutation
  • Phylogenetic trees constructed to ensure that patient’s
  • sequences cluster together
slide10

Types ofMutations Observed

Mutations in Core Epitope

Extended Epitope/Processing Mutations

slide13

Epitope Mutation

Clone A2:

FRDYVDRFYK

FRDYVDQFYK

55.9

0

PBMCs:

Control

FRDYVDQFYK

FRDYVDRFYK

0.006

0.025

0

slide14

Flanking Mutations

  • Clone and express autologous p55 with and without
  • flanking mutations
  • Test ability to stimulate clones

Flanking mutations(FM)

wt

2ug/ml

FM p55

2ug/ml

SUMO-CAT

2.5ug/ml

wt p55

10ug/ml

p55

1ug/ml

p55

10ug/ml

p55

1ug/ml

p55

Med

Med

SEB

SEB

Clone A2

‘FRDYVDRFYKT’

Clone B2

‘REPRGSDIAGT’

  • Flanking mutations do not confer escape to A2 or B2
slide15

Conclusions

  • Rapid progression occurred in OM214 despite early
  • induction of an IL-2 producing CD4+ T cell response -
  • including a potent MHR-directed response
  • Loss of IL-2 secreting CD4+ T-cell response accompanied
  • disease progression
  • An escape mutation in an IL-2 inducing CD4+ T cell
  • epitope within the MHR was confirmed
  • This mutation arose within 6 months of infection and was
  • maintained at a frequency of 10%, for at least 1 year
  • IL-2 producing CD4+ T cell responses are capable of
  • exerting immune pressure on HIV-1, resulting in escape
  • mutations
  • Generalized loss of IL-2 responses with time suggests that
  • immune dysfunction due to viremia is an important
  • mechanism for viral escape from immune pressure
slide16

Acknowledgments

Elizabeth Yue

Mario Ostrowski

Maple Leaf Clinic:

Colin Kovacs

Roberta Halpenny

Macdonald Lab:

Ruqaya Mohamed

David Willer

Kaul Lab

Sample Donors

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