Rakieb Andargachew

DNA-based nanoparticle tension sensors reveal that T-cell receptors transmit defined pN forces to their antigens for enhanced fidelity.

Significance T cells protect the body against pathogens and cancer by recognizing specific foreign peptides on the cell surface. Because antigen recognition occurs at the junction between a migrating T cell and an antigen-presenting cell (APC), it is likely that cellular forces are generated and transmitted through T-cell receptor (TCR)-ligand bonds. Here we develop a DNA-based nanoparticle tension sensor producing the first molecular maps of TCR-ligand forces during T cell activation. We find that TCR forces are orchestrated in space and time, requiring the participation of CD8 coreceptor and adhesion molecules. Loss or damping of TCR forces results in weakened antigen discrimination, showing that T cells harness mechanics to optimize the specificity of response to ligand. 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.

Neutrophil-derived JAML Inhibits Repair of Intestinal Epithelial Injury During Acute Inflammation

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 binds 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 are reversed with an anti-JAML monoclonal antibody that inhibits JAML–CAR binding. JAML released from transmigrating neutrophils across inflamed epithelia may thus 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 neutrophils would compromise intestinal barrier and inhibit mucosal healing. Thus, targeting JAML–CAR interactions may improve mucosal healing responses under conditions of dysregulated neutrophil recruitment.

The role of Cis dimerization of signal regulatory protein α (SIRPα) in binding to CD47

Interaction of SIRPα with its ligand, CD47, regulates leukocyte functions, including transmigration, phagocytosis, oxidative burst, and cytokine secretion. Recent progress has provided significant insights into the structural details of the distal IgV domain (D1) of SIRPα. However, the structural roles of proximal IgC domains (D2 and D3) have been largely unstudied. The high degree of conservation of D2 and D3 among members of the SIRP family as well as the propensity of known IgC domains to assemble in cis has led others to hypothesize that SIRPα forms higher order structures on the cell surface. Here we report that SIRPα forms noncovalently linked cis homodimers. Treatment of SIRPα-expressing cells with a membrane-impermeable cross-linker resulted in the formation of SDS-stable SIRPα dimers and oligomers. Biochemical analyses of soluble recombinant extracellular regions of SIRPα, including domain truncation mutants, revealed that each of the three extracellular immunoglobulin loops of SIRPα formed dimers in solution. Co-immunoprecipitation experiments using cells transfected with different affinity-tagged SIRPα molecules revealed that SIRPα forms cis dimers. Interestingly, in cells treated with tunicamycin, SIRPα dimerization but not CD47 binding was inhibited, suggesting that a SIRPα dimer is probably bivalent. Last, we demonstrate robust dimerization of SIRPa in adherent, stimulated human neutrophils. Collectively, these data are consistent with SIRPα being expressed on the cell surface as a functional cis-linked dimer.

Low-affinity CD4+T cells are major responders in the primary immune response

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.

Low-Affinity Memory CD8+ T Cells Mediate Robust Heterologous Immunity

Heterologous immunity is recognized as a significant barrier to transplant tolerance. Whereas it has been established that pathogen-elicited memory T cells can have high or low affinity for cross-reactive allogeneic peptide–MHC, the role of TCR affinity during heterologous immunity has not been explored. We established a model with which to investigate the impact of TCR-priming affinity on memory T cell populations following a graft rechallenge. In contrast to high-affinity priming, low-affinity priming elicited fully differentiated memory T cells with a CD45RBhi status. High CD45RB status enabled robust secondary responses in vivo, as demonstrated by faster graft rejection kinetics and greater proliferative responses. CD45RB blockade prolonged graft survival in low affinity–primed mice, but not in high affinity–primed mice. Mechanistically, low affinity–primed memory CD8+ T cells produced more IL-2 and significantly upregulated IL-2Rα expression during rechallenge. We found that CD45RBhi status was also a stable marker of priming affinity within polyclonal CD8+ T cell populations. Following high-affinity rechallenge, low affinity–primed CD45RBhi cells became CD45RBlo, demonstrating that CD45RB status acts as an affinity-based differentiation switch on CD8+ T cells. Thus, these data establish a novel mechanism by which CD45 isoforms tune low affinity–primed memory CD8+ T cells to become potent secondary effectors following heterologous rechallenge. These findings have direct implications for allogeneic heterologous immunity by demonstrating that despite a lower precursor frequency, low-affinity priming is sufficient to generate memory cells that mediate potent secondary responses against a cross-reactive graft challenge.

Surfactant Protein D (Sp-D) Binds to Membrane-proximal Domain (D3) of Signal Regulatory Protein α (SIRPα), a Site Distant from Binding Domain of CD47, while Also Binding to Analogous Region on Signal Regulatory Protein β (SIRPβ)

Background: Binding of SIRPα to its ligands CD47 and surfactant protein D (Sp-D) regulates many myeloid cell functions. Results: Sp-D binds to N-glycosylated sites in the membrane-proximal domain of SIRPα and SIRPβ, another related SIRP. Conclusion: Sp-D binds to a site on SIRPα distant from that of CD47. Significance: Multiple ligand binding sites on SIRPα may afford differential regulation of receptor function. Signal regulatory protein α (SIRPα), a highly glycosylated type-1 transmembrane protein, is composed of three immunoglobulin-like extracellular loops as well as a cytoplasmic tail containing three classical tyrosine-based inhibitory motifs. Previous reports indicate that SIRPα binds to humoral pattern recognition molecules in the collectin family, namely surfactant proteins D and A (Sp-D and Sp-A, respectively), which are heavily expressed in the lung and constitute one of the first lines of innate immune defense against pathogens. However, little is known about molecular details of the structural interaction of Sp-D with SIRPs. In the present work, we examined the molecular basis of Sp-D binding to SIRPα using domain-deleted mutant proteins. We report that Sp-D binds to the membrane-proximal Ig domain (D3) of SIRPα in a calcium- and carbohydrate-dependent manner. Mutation of predicted N-glycosylation sites on SIRPα indicates that Sp-D binding is dependent on interactions with specific N-glycosylated residues on the membrane-proximal D3 domain of SIRPα. Given the remarkable sequence similarity of SIRPα to SIRPβ and the lack of known ligands for the latter, we examined Sp-D binding to SIRPβ. Here, we report specific binding of Sp-D to the membrane-proximal D3 domain of SIRPβ. Further studies confirmed that Sp-D binds to SIRPα expressed on human neutrophils and differentiated neutrophil-like cells. Because the other known ligand of SIRPα, CD47, binds to the membrane-distal domain D1, these findings indicate that multiple, distinct, functional ligand binding sites are present on SIRPα that may afford differential regulation of receptor function.

Stepwise B-cell-dependent expansion of T helper clonotypes diversifies the T-cell response

Antigen receptor diversity underpins adaptive immunity by providing the ground for clonal selection of lymphocytes with the appropriate antigen reactivity. Current models attribute T cell clonal selection during the immune response to T-cell receptor (TCR) affinity for either foreign or self peptides. Here, we report that clonal selection of CD4+ T cells is also extrinsically regulated by B cells. In response to viral infection, the antigen-specific TCR repertoire is progressively diversified by staggered clonotypic expansion, according to functional avidity, which correlates with self-reactivity. Clonal expansion of lower-avidity T-cell clonotypes depends on availability of MHC II-expressing B cells, in turn influenced by B-cell activation. B cells clonotypically diversify the CD4+ T-cell response also to vaccination or tumour challenge, revealing a common effect.

Viral Escape Mutant Epitope Maintains TCR Affinity for Antigen yet Curtails CD8 T Cell Responses

T cells have the remarkable ability to recognize antigen with great specificity and in turn mount an appropriate and robust immune response. Critical to this process is the initial T cell antigen recognition and subsequent signal transduction events. This antigen recognition can be modulated at the site of TCR interaction with peptide:major histocompatibility (pMHC) or peptide interaction with the MHC molecule. Both events could have a range of effects on T cell fate. Though responses to antigens that bind sub-optimally to TCR, known as altered peptide ligands (APL), have been studied extensively, the impact of disrupting antigen binding to MHC has been highlighted to a lesser extent and is usually considered to result in complete loss of epitope recognition. Here we present a model of viral evasion from CD8 T cell immuno-surveillance by a lymphocytic choriomeningitis virus (LCMV) escape mutant with an epitope for which TCR affinity for pMHC remains high but where the antigenic peptide binds sub optimally to MHC. Despite high TCR affinity for variant epitope, levels of interferon regulatory factor-4 (IRF4) are not sustained in response to the variant indicating differences in perceived TCR signal strength. The CD8+ T cell response to the variant epitope is characterized by early proliferation and up-regulation of activation markers. Interestingly, this response is not maintained and is characterized by a lack in IL-2 and IFNγ production, increased apoptosis and an abrogated glycolytic response. We show that disrupting the stability of peptide in MHC can effectively disrupt TCR signal strength despite unchanged affinity for TCR and can significantly impact the CD8+ T cell response to a viral escape mutant.

CD4 T Cell Affinity Diversity Is Equally Maintained during Acute and Chronic Infection

TCR affinity for peptide MHC dictates the functional efficiency of T cells and their propensity to differentiate into effectors and form memory. However, in the context of chronic infections, it is unclear what the overall profile of TCR affinity for Ag is and if it differs from acute infections. Using the comprehensive affinity analysis provided by the two-dimensional micropipette adhesion frequency assay and the common indirect affinity evaluation methods of MHC class II tetramer and functional avidity, we tracked IAb GP61–80–specific cells in the mouse model of acute (Armstrong) and chronic (clone 13) lymphocytic choriomeningitis virus infection. In each response, we show CD4 T cell population affinity peaks at the effector phase and declines with memory. Of interest, the range and average relative two-dimensional affinity was equivalent between acute and chronic infection, indicating chronic Ag exposure did not skew TCR affinity. In contrast, functional and tetramer avidity measurements revealed divergent results and lacked a consistent correlation with TCR affinity. Our findings highlight that the immune system maintains a diverse range in TCR affinity even under the pressures of chronic Ag stimulation.

Differential IL-2 expression defines developmental fates of follicular versus nonfollicular helper T cells

(IL-)2 be or not to be? Immunological T follicular helper (TFH) cells are a subpopulation of CD4+ T cells that support B cell antibody production and the establishment of B cell memory. By contrast, non-TFH cells orchestrate enhanced innate immune cell functions at sites of pathogen encounter. The factors underlying differentiation into a TFH or non-TFH cell remain poorly understood, though there is evidence to suggest that the T cell growth factor interleukin-2 (IL-2) may play a role. Using IL-2 reporter mice, DiToro et al. show that naïve CD4+ T cells that produce IL-2 are fated to become TFH cells, whereas nonproducers, which receive IL-2, become non-TFH cells. The CD4+ T cell–fate decision was linked to T cell receptor strength—only those naïve CD4+ T cells that received the highest T cell receptor signals were able to produce IL-2. Science, this issue p. eaao2933 Expression of the cytokine IL-2 is linked with cell fate choice in immunological T cells. INTRODUCTION The adaptive immune system has evolved to mount different types of responses that are matched to the type of invading pathogen. For CD4+ T cells, this is predicated on the multipotentiality of clonally restricted naïve T cells, which differentiate into distinct subsets of effector T cells contingent on recognition of cognate antigen and cytokine cues from cells of the innate immune system. There are two broad divisions of effector CD4+ T cells: T follicular helper (TFH) cells, which are programmed to interact with B cells within lymphoid tissues to support production of high-affinity, class-switched antibodies, and non-TFH effector cells, including T helper 1 (TH1), TH2, and TH17 cells, which are programmed to egress from lymphoid tissues to orchestrate heightened innate immune cell function at sites of pathogen entry. The mechanisms controlling bifurcation into TFH versus non-TFH effector cell pathways are incompletely understood. RATIONALE An impediment to understanding mechanisms controlling TFH–non-TFH cell divergence is an absence of early markers to define cells destined for these alternative fates. Unlike effector CD4+ T cells, which produce a diversity of cytokines that define their phenotype and function, naïve CD4+ T cells are largely limited to the rapid production of interleukin-2 (IL-2) when activated by antigen. IL-2 is only produced by a subset of activated naïve T cells, suggesting a possible relationship between IL-2 production and effector cell fate determination. To explore this, we developed two IL-2 reporter mice strains with complementary features that enabled the tracking and deletion of T cells on the basis of differential IL-2 expression. This allowed us to determine whether naïve T cells that do, or do not, produce IL-2 are biased in their developmental programming and, if so, how. RESULTS RNA sequencing of naïve T cells sorted on the basis of IL-2 reporter expression identified cosegregation of transcripts encoding IL-2 and Bcl6—the signature transcription factor of TFH cells. Conversely, IL-2–negative (IL-2–) cells preferentially expressed the gene Prdm1, which encodes the transcriptional repressor Blimp1. Blimp1, in turn, antagonizes Bcl6 and the TFH developmental program. This suggested that IL-2 producers give rise to TFH cells, whereas IL-2 nonproducers give rise to non-TFH effector cells. Moreover, the fact that IL-2 receptor signaling induces expression of Prdm1 via Stat5 suggested that IL-2 producers resisted IL-2 signaling and activated IL-2 signaling in nonproducers in trans. Indeed, in vivo studies established that IL-2 signaling was mostly paracrine and that depletion of IL-2–producing cells selectively impaired TFH cell development. Finally, IL-2 expression was limited to a subset of naïve T cells that received the strongest T cell receptor (TCR) signals, establishing a link between TCR signal strength, IL-2 production, and TFH versus non-TFH differentiation. CONCLUSION This study provides new insights into the mechanisms that control early bifurcation of CD4+ T cells into TFH and non-TFH effectors. Naïve T cells that receive differing strengths of TCR signals stratify into those that exceed a threshold predisposing them to IL-2 production and early TFH commitment and those that do not express IL-2 yet receive IL-2 signaling, which reinforces non-TFH effector commitment. IL-2–producing CD4+ T cells become TFH cells, whereas IL-2 nonproducers become non-TFH cells. (Left) Strong TCR signaling via an antigen presenting cell induces Il2 and Bcl6 gene expression (red pathway); weaker signaling induces expression of non-TFH genes (blue pathway), including Prdm1 and S1pr1, which encodes the S1P receptor S1PR1. Bcl6+ cells (red) secrete IL-2 in trans to T regulatory (Treg) cells (yellow) and recently activated IL-2– cells (blue), up-regulating IL-2 receptor IL2rα and reinforcing Prdm1. (Top right) Bcl6+ cells engage cognate B cells (green) and migrate to germinal centers (GCs); Bcl6+ TFH cells mature into GC-TFH cells. (Bottom right) Prdm1+ cells migrate to efferent lymphatics and mature into non-TFH effectors in nonlymphoid tissues. MHCII, major histocompatibility complex class II; ICOS, inducible costimulator; CXCR5, a receptor for chemokine CXCL13. In response to infection, naïve CD4+ T cells differentiate into two subpopulations: T follicular helper (TFH) cells, which support B cell antibody production, and non-TFH 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 TFH cells, delivered IL-2 to nonproducers destined to become non-TFH 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.