Soluble MHC-Peptide Complexes Induce Rapid Death of CD8+ CTL

Abstract

Soluble MHC-peptide (pMHC) complexes, commonly referred to as tetramers, are widely used to enumerate and to isolate Ag-specific CD8(+) CTL. It has been noted that such complexes, as well as microsphere- or cell-associated pMHC molecules compromise the functional integrity of CTL, e.g., by inducing apoptosis of CTL, which limits their usefulness for T cell sorting or cloning. By testing well-defined soluble pMHC complexes containing linkers of different length and valence, we find that complexes comprising short linkers (i.e., short pMHC-pMHC distances), but not those containing long linkers, induce rapid death of CTL. This cell death relies on CTL activation, the coreceptor CD8 and cytoskeleton integrity, but is not dependent on death receptors (i.e., Fas, TNFR1, and TRAILR2) or caspases. Within minutes of CTL exposure to pMHC complexes, reactive oxygen species emerged and mitochondrial membrane depolarized, which is reminiscent of caspase-independent T cell death. The morphological changes induced during this rapid CTL death are characteristic of programmed necrosis and not apoptosis. Thus, soluble pMHC complexes containing long linkers are recommended to prevent T cell death, whereas those containing short linkers can be used to eliminate Ag-specific CTL.

On the significance of CD8αα expression for T cell memory

“…Even if one discounts any CD8aa ligation-induced signalling or inhibition of signalling, the mere fact that some CD8ab + primary effector T cells express CD8aa is likely to aid their survival during the contraction phase, because CD8aa, or blocking of CD8ab, inhibits activation-induced cell death [11]. The finding that the expression of CD8aa is reversible and is absent on long term CD8 + memory cells allays the concern that selection of CD8aa + memory cells may compromise their sensitivity for recall responses.…”

Reversible Major Histocompatibility Complex I-Peptide Multimers Containing Ni2+-Nitrilotriacetic Acid Peptides and Histidine Tags Improve Analysis and Sorting of CD8+ T Cells

“…8D). Although the PE fluorescence reached background within 20 -30 s, the Cy5 fluorescence reached background only after 300 -350 s. The Cy5 fluorescence decrease exhibited half-maximal dissociation in the range of 98 -118 s. Importantly, the monomer dissociation reached background levels, which is in contrast to dissociation experiments with BSP multimers, in which complete dissociation is typically not reached because of multimer rebinding and/or internalization (8,9). The experiments gave well reproducible results when performed in the cold; at elevated temperatures the monomer dissociation were too rapid to be reliably measured (data not shown).…”

“…Because we have shown that the induced dissociation of DTB multimers from CTL is sufficiently rapid to prevent intracellular Ca 2ϩ mobilization (4), the same is expected for NTA-His tag multimers. Intracellular Ca 2ϩ mobilization is required for most CTL functions, including multimer-induced CD8ϩ T cell death (8,26,33).…”

“…A major shortcoming of these tetramers is that they avidly bind to and cross-link cell surface TCR and CD8, thereby inducing strong T cell activation, which frequently provokes T cell death (4,5,8,9). Thus, isolation of antigen-specific CD8ϩ T cells by tetramers harbors the risk of substantial T cell loss.…”

Studies on the CNS Histopathology of EAE and Its Correlation with Clinical and Immunological Parameters

“…For example, when a cell surface marker-specific antibody is injected to study the role of that molecule in EAE, the antibody might have a clinical effect on the disease, but it could be due to a multitude of mechanisms. The antibody could deplete the marker positive cells via the activation of complement, antibody-dependent cell-mediated cytotoxicity (ADCC) or apoptosis (Cebecauer et al, 2005), which in turn may be associated with a varying degree of inflammation causing unaccounted effects. Alternatively (or in addition) antibodies can inactivate or activate the marker positive cells, with variable bystander cell involvement.…”