Direct enumeration of Borrelia-reactive CD4 T cells ex vivo by using MHC class II tetramers

Direct enumeration of Borrelia-reactive CD4 T cells ex vivo by using MHC class II tetramers

^‡Tufts University School of Medicine, Department of Pathology, Boston, MA 02111; ^§Laboratory of Molecular Immunology, Center for Neurologic Diseases, Brigham and Women's Hospital and Harvard Medical School, 77 Avenue Louis Pasteur, Boston, MA 02115; ^¶Howard Hughes Medical Institute, Department of Immunology, National Jewish Medical and Research Center, Denver, CO 80206; and ^‖Tufts University School of Medicine, Division of Rheumatology/Immunology, New England Medical Center, Boston, MA 02111

Contributed by John Kappler

Abstract

We characterized antigen-specific CD4⁺ T cells in six patients with treatment-resistant Lyme arthritis, using an HLA-DRB1*0401 major histocompatibility complex (MHC) class II tetramer covalently loaded with OspA_164–175, an immunodominant epitope of Borrelia burgdorferi. Direct analysis of OspA-tetramer binding CD4⁺ cells in patients expressing the HLA-DRB1*0401 allele revealed frequencies of between <0.005 and 0.1% in peripheral blood (n = 6), and between <0.005 and 3.1% in synovial fluid (n = 3). OspA-tetramer⁺CD4⁺ cells were directly cloned at 1 cell per well and expanded by mitogen and IL-2 on allogeneic feeder cells. As measured by [³H]thymidine incorporation, 95% of 168 T cell clones from synovial fluid binding the OspA-tetramer were antigen-reactive. Clones generated from peripheral blood revealed a different pattern of responsiveness when compared with clones generated from synovial fluid, as measured by proliferation, IFN-γ, and IL-13 secretion. These clones, selected on the basis of their peptide binding, also responded to whole protein, but with a different cytokine profile. Our studies demonstrate that MHC class II tetramers can be used in humans to directly identify, isolate, and characterize antigen-reactive T cells from an inflammatory compartment.

Footnotes

↵ † A.L.M. and C.T. contributed equally to this work.
↵ ‡‡ To whom reprint requests should be addressed at: Howard Hughes Medical Institute, National Jewish Medical and Research Center, 1400 Jackson Street, Denver, CO 80206. E-mail: kapplerj{at}njc.org.
Article published online before print: Proc. Natl. Acad. Sci. USA, 10.1073/pnas.190335897.
Article and publication date are at www.pnas.org/cgi/doi/10.1073/pnas.190335897
Abbreviations:

Bb, Borrelia burgdorferi,

EBV, Epstein–Barr virus;

MCC,

mouse cytochrome c;

OspA,

outer surface protein A;

MFI,

mean fluorescence intensity;

PBMC,

peripheral blood mononuclear cell;

r,

recombinant;

SF,

synovial fluid;

THy,

T cell hybridomas;

TCR,

T cell receptor;

APC,

antigen-presenting cells;

LFA,

lymphocyte function-associated antigen