T cells are triggered when the T-cell receptor (TCR) encounters its antigenic ligand, the peptide-major histocompatibility complex (pMHC), on the surface of antigen presenting cells (APCs). Because T cells are highly migratory and antigen recognition occurs at an intermembrane junction where the T cell physically contacts the APC, there are long-standing questions of whether T cells transmit defined forces to their TCR complex and whether chemomechanical coupling influences immune function. Here we develop DNA-based gold nanoparticle tension sensors to provide, to our knowledge, the first pN tension maps of individual TCR-pMHC complexes during T-cell activation. We show that naïve T cells harness cytoskeletal coupling to transmit 12–19 pN of force to their TCRs within seconds of ligand binding and preceding initial calcium signaling. CD8 coreceptor binding and lymphocyte-specific kinase signaling are required for antigen-mediated cell spreading and force generation. Lymphocyte function-associated antigen 1 (LFA-1) mediated adhesion modulates TCR-pMHC tension by intensifying its magnitude to values >19 pN and spatially reorganizes the location of TCR forces to the kinapse, the zone located at the trailing edge of migrating T cells, thus demonstrating chemomechanical crosstalk between TCR and LFA-1 receptor signaling. Finally, T cells display a dampened and poorly specific response to antigen agonists when TCR forces are chemically abolished or physically “filtered” to a level below ∼12 pN using mechanically labile DNA tethers. Therefore, we conclude that T cells tune TCR mechanics with pN resolution to create a checkpoint of agonist quality necessary for specific immune response.

In response to infection, naïve CD4 T cells differentiate into two subpopulations: T follicular helper (T) cells, which support B cell antibody production, and non-T cells, which enhance innate immune cell functions. Interleukin-2 (IL-2), the major cytokine produced by naïve T cells, plays an important role in the developmental divergence of these populations. However, the relationship between IL-2 production and fate determination remains unclear. Using reporter mice, we found that differential production of IL-2 by naïve CD4 T cells defined precursors fated for different immune functions. IL-2 producers, which were fated to become T cells, delivered IL-2 to nonproducers destined to become non-T cells. Because IL-2 production was limited to cells receiving the strongest T cell receptor (TCR) signals, a direct link between TCR-signal strength, IL-2 production, and T cell fate determination has been established.

A robust primary immune response has been correlated with the precursor number of antigen-specific T cells, as identified using peptide MHCII tetramers. However, these tetramers identify only the highest-affinity T cells. Here we show the entire CD4+ T-cell repertoire, inclusive of low-affinity T cells missed by tetramers, using a T-cell receptor (TCR) signalling reporter and micropipette assay to quantify naive precursors and expanded populations. In vivo limiting dilution assays reveal hundreds more precursor T cells than previously thought, with higher-affinity tetramer-positive T cells, comprising only 5–30% of the total antigen-specific naive repertoire. Lower-affinity T cells maintain their predominance as the primary immune response progresses, with no enhancement of survival of T cells with high-affinity TCRs. These findings demonstrate that affinity for antigen does not control CD4+ T-cell entry into the primary immune response, as a diverse range in affinity is maintained from precursor through peak of T-cell expansion.

Neutrophil transepithelial migration (TEM) during acute inflammation is associated with mucosal injury. Using models of acute mucosal injury in-vitro and in-vivo, we describe a new mechanism by which neutrophils infiltrating the intestinal mucosa disrupt epithelial homeostasis. We report that junctional adhesion molecule-like protein (JAML) is cleaved from neutrophil surface by zinc-metalloproteases during TEM. Neutrophil-derived soluble JAML bound to the epithelial tight junction protein coxsackie-adenovirus receptor (CAR) resulting in compromised barrier and inhibition of wound repair, through decreased epithelial proliferation. The deleterious effects of JAML on barrier and wound repair were reversed with an anti-JAML mAb that inhibits JAML-CAR binding. Thus, JAML released from transmigrating neutrophils across inflamed epithelia can promote recruitment of leukocytes and aid in clearance of invading microorganisms. However, sustained release of JAML under pathologic conditions associated with persistence of large numbers of infiltrated neutrophil would compromise intestinal barrier and inhibit mucosal healing. Targeting JAML-CAR interactions may thus improve mucosal healing responses under conditions of dysregulated neutrophil recruitment.

The LCMV GP33 CD8 epitope has long been one of the most widely used antigens in viral immunology. Of note, almost all of the in vitro analyses of CD8 T cell responses to this epitope make use of an altered peptide ligand (APL) in which the cysteine from the original 9-mer peptide (KAVYNFATC) is substituted by a methionine at position 41 (KAVYNFATM). In addition, it is possible that the antigen processed during natural LCMV infection is an 11-mer peptide (KAVYNFATCGI) rather than the widely used 9-mer. Although previous affinity measurements using purified proteins for these antigen variants revealed minimal differences, we applied highly sensitive two dimensional (2D) biophysical based techniques to further dissect TCR interaction with these closely related GP33 variants. The kinetic analyses of affinity provided by the 2D micropipette adhesion frequency assay (2D-MP) and bond lifetime under force analyzed using a biomembrane force probe (BFP) revealed significant differences between 41M, 41C and the 11-mer 41CGI antigen. We found a hierarchy in 2D affinity as 41M peptide displayed augmented TCR 2D affinity compared to 41C and 41CGI. These differences were also maintained in the presence of CD8 coreceptor and when analysis of total TCR:pMHC and CD8:pMHC bonds were considered. Moreover, the three ligands displayed dramatic differences in the bond lifetimes generated under force, in particular the 41CGI variant with the lowest 2D affinity demonstrated a 15-fold synergistic contribution of the CD8 coreceptor to overall bond lifetime. Our analyses emphasize the sensitivity of single cell and single bond 2D kinetic measurements in distinguishing between related agonist peptides.