Outcome of TCR/pMHC interactions is presently unpredictable

This week journal Cell published a very important paper from KC Garcia's lab at Stanford. The focus of the study was to understand and determine, if possible, all the parameters that makes a given peptide/MHC and its similar looking variants stimulatory versus non-stimulatory to a given TCR molecule.

First, they compared two TCR molecules specific for class II HLA-DR4-HIV_{(Gag164–183)}. While both TCR expressing clones, TCR6 and TCR11, could bind pHLA tetramer, only TCR6 "proliferated, secreted cytokines, and downregulated TCR in response to stimulation with" HLA-DR4-HIV_{(Gag164–183)}. Both TCR clones could respond directly or after transducing them into TCR αβ-deficient cell line to non-specific stimulation such as anti-CD3 (OKT3).

Similar observation were made using TCRs specific for class I HLA-B35-HIV_{(Pol448–456)}. Here too, both TCR clones designated as TCR589 and TCR55 could bind specific pMHC tetramer but only TCR589 showed dose-dependent secretion of IL-2 in response to peptide-pulsed APCs.

Next, the authors determined TCR/pMHC affinity by surface plasmon resonance. For TCR55 (non-stimulatory) and TCR589 (stimulatory) clones it was 17_μM and 4_μM K_D, respectively. Surprisingly, for TCR6 (stimulatory) and TCR11 (non-stimulatory) clones affinity was 10_μM and 1_μM K_D, respectively.

Analysis of TCR55 (non-stimulatory) and TCR589 (stimulatory) TCR/pMHC interaction on lipid bilayers by total internal reflection fluorescence (TIRF) microscopy showed similar mean dwell times of 10.1 ± 2.0 s and 8.7 ± 2.1 s, respectively. Structural analysis of the crystal structures of TCR589 and TCR55 in complex with HLA-B35-HIV_{(Pol448–456)} also showed "no substantial differences in the conformations of the constant regions that would indicate differential allostery correlating with signaling."

Functional analysis of TCR proximal signaling molecules showed that stimulatory TCR589 but not TCR55 could up-regulate CD69, induce calcium release and recruit ZAP70 in response to HLA-B35-HIV_{(Pol448–456)}.

Next, they generated peptide library similar to HIV_{(Pol448–456)} with potencies "ranging from full agonism to partial agonism to no detectable signaling". Importantly, "the peptides that elicited these disparate signaling outcomes were remarkably similar in sequence."

One common feature of agonist peptides such as pep20 and SQL that could signal TCR55 was that such interaction excluded CD45 with varying degrees of efficiency compared to non-stimulatory peptide ligands in "a cell-free system using giant unilamellar vesicles (GUVs) to eliminate effects of signaling in live cells".

In addition, another common feature of stimulatory peptide ligands were so called catch bond formation between TCR and stimulatory pMHC "under conditions of force using a Biomembrane Force Probe (BFP) bond lifetime assay". In contrast, non-stimulatory ligands formed slip bond formations.

In summary, this study indicates that "catch bond formation and CD45 exclusion as principal emergent properties differentiating stimulatory from non-stimulatory TCR-pMHC interactions" but "the same pMHC can be an agonist or non-agonist for different TCRs, although it is difficult to assign causality to either the TCR or the peptide-MHC. The formation of a catch or slip bond is a collective property of the entire TCR-pMHC interface."

My opinion: one drawback of this study is that not many labs can verify its conclusions. I have not even heard of some of the techniques they used here. I could only trust that the authors tried to interpret their data as unbiased as it is rationally possible. Another weakness of the study is a failure to identify parameters that control CD45 exclusion or catch bond formation when TCR interacts with pMHC. Basically, with the exception of few very high affinity ligands (< 2_μM), we cannot predict in silico whether a given peptide will be stimulatory or non-stimulatory without actually doing an experiment on it.   

posted by David Usharauli

pMHC tetramer-coated nanoparticles treat autoimmune conditions in mice

This week Nature published "figure-dense" paper showing that nanoparticles coated with auto-antigen+MHC complexes (pMHC) could treat spontaneous or experimentally-induced autoimmune diseases in mice. In fact, this study contains so many experimental results it should have been published in two part, in my view. I will try to condense its basic findings in few paragraphs and provide its interpretation below.

Non-obese diabetic (NOD) mice spontaneously develop [autoimmune] diabetes. Part of T cells that infiltrate and damage β cells in NOD mice express T-cell receptor (TCR) resembling diabetogenic BDC2.5-specific TCR that can be recognized by pMHC–2.5mi/I^Ag7 tetramers. When the authors injected nanoparticles (dextran-coated or pegylated iron oxide NPs) coated with 2.5mi/I^Ag7 tetramers (pMHC–NP) it induced expansion of cognate CD4+ T cells in blood and spleens of NOD mice. These cells had a memory-like (CD44^hiCD62L^low) FOXP3− T_R1-like phenotype.

These "activated/experienced" CD4+ T_R1 cells from donor mice treated with pMHC–2.5mi/I^Ag7-NPs suppressed diabetes development in T-cell-reconstituted NOD-scid hosts upon adoptive transfer. This effect of T_R1 cells was augmented by treating hosts with pMHC–NPs.

Moreover, 90–100% of the already diabetic mice that received nanoparticles coated with β cell antigens, 2.5mi/I^Ag7, IGRP_4–22/I^Ag7 or IGRP_128–145/I^Ag7, reverted to stable normoglycaemia.

Of note, treatment withdrawal resulted in loss of the normoglycaemic state in 25–60% of mice, in association with the loss of the tetramer+CD4+ T-cell pools. In other words, these data suggest that almost half of treated diabetic mice maintained normoglycaemic state after 5 weeks of treatment.

Beyond diabetes, the authors showed that nanoparticles coated with myelin oligodendrocyte glycoprotein, pMOG_38–49/IA^b–NP, dampended progression of experimental autoimmune encephalomyelitis (EAE, a model of multiple sclerosis), when given on day 14 after immunization and even restored motor function in paralytic mice when given on day 21.

Similar therapeutic effects were seen in 3rd autoimmune disease model, collagen-induced arthritis (CIA). HLA-DR4-IE-transgenic mice receiving nanoparticles displaying mouse collagen (mCII)_259–273/DR4-IE showed reduced joint inflammation. In all these models, pMHC–NPs effect was antigen-specific.

Indeed, the effects of pMHC–NP therapy were not associated with impaired systemic immunity because pMHC–NP-treated mice showed unimpaired anti-viral immunity and mounted antibodies against an exogenous antigen as efficiently as control mice.

Interestingly, pMHC–NPs could not expand tetramer+ T cells in non-diabetic control mice which harbor mostly naive T cells. Further experiments confirmed that therapeutic effect of pMHC–NP required presence of "experienced" T cells. For example, whereas pMHC–NP therapy afforded 100% diabetes protection to T-cell-reconstituted NOD-scid hosts bearing memory BDC2.5 T cells, such therapy was inconsequential in hosts receiving naive BDC2.5 T cells.

The role of "experienced", but not naive T cells, in therapeutic effect of pMHC–NP was also supported by observation that whereas diabetic NOD G6pc2−/− mice (which lack IGRP antigen and corresponding memory/experienced T cells, but most likely harbor IGRP_4–22 specific [aka, nonself-specific] naive T cells) responded to 2.5mi/I^Ag7–NPs like wild-type NOD mice, they did not respond to IGRP_4–22/I^Ag7–NPs.

At molecular level, the authors showed blockade of IL-10, TGF-β and IL-21R (but not IFNγ) abrogated the anti-diabetogenic properties of 2.5mi/I^Ag7–NPs or IGRP_4–22/I^Ag7–NPs in diabetic NOD mice.

However, to make things more complicated, the authors also showed that the development of the T_R1 precursors and/ or T_R1-like cells that expand in response to pMHC–NP therapy required IFNγ in addition to IL-10 in pre-diabetic NOD mice.

Finally, the authors found that at the cellular level, pMHC–NP treated NOD mice harbored increased numbers of so called regulatory B cells, B_REG cells (but only in pancreatic lymph nodes, PLNs). These B_REG cells were producing IL-10 in response to lipopolysaccharide (LPS). In vivo, these B cells suppressed diabetes development in T-cell-reconstituted NOD-scid hosts as compared to PLN B cells from control mice (and worked together with T_R1 cells). The role of B_REG cells was supported by observation that treatment of newly diabetic NOD mice with a B-cell depleting anti-CD20 mAb abrogated the anti-diabetogenic activity of 2.5mi/I^Ag7–NPs (see above).

In summary, this study suggests that when injected into mice these pMHC–nanoparticles expand antigen-specific and antigen-experienced T cells with regulatory properties. These T_R1 work with B_REG cells to block the chain of events that perpetuates autoimmune cycle. 

David Usharauli