Ryan J. Martinez

Lower Affinity T Cells are Critical Components and Active Participants of the Immune Response

Kinetic and biophysical parameters of T cell receptor (TCR) and peptide:MHC (pMHC) interaction define intrinsic factors required for T cell activation and differentiation. Although receptor ligand kinetics are somewhat cumbersome to assess experimentally, TCR:pMHC affinity has been shown to predict peripheral T cell functionality and potential for forming memory. Multimeric forms of pMHC monomers have often been used to provide an indirect readout of higher affinity T cells due to their availability and ease of use while allowing simultaneous definition of other functional and phenotypic characteristics. However, multimeric pMHC reagents have introduced a bias that underestimates the lower affinity components contained in the highly diverse TCR repertoires of all polyclonal T cell responses. Advances in the identification of lower affinity cells have led to the examination of these cells and their contribution to the immune response. In this review, we discuss the identification of high- vs. low-affinity T cells as well as their attributed signaling and functional differences. Lastly, mechanisms are discussed that maintain a diverse range of low- and high-affinity T cells.

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.

Targeted loss of SHP1 in murine thymocytes dampens TCR signaling late in selection

SHP1 is a tyrosine phosphatase critical to proximal regulation of TCR signaling. Here, analysis of CD4‐Cre SHP1fl/fl conditional knockout thymocytes using CD53, TCRβ, CD69, CD4, and CD8α expression demonstrates the importance of SHP1 in the survival of post selection (CD53+), single‐positive thymocytes. Using Ca2+ flux to assess the intensity of TCR signaling demonstrated that SHP1 dampens the signal strength of these same mature, postselection thymocytes. Consistent with its dampening effect, TCR signal strength was also probed functionally using peptides that can mediate selection of the OT‐I TCR, to reveal increased negative selection mediated by lower‐affinity ligand in the absence of SHP1. Our data show that SHP1 is required for the survival of mature thymocytes and the generation of the functional T‐cell repertoire, as its absence leads to a reduction in the numbers of CD4+ and CD8+ naïve T cells in the peripheral lymphoid compartments.

Conserved Region C Functions To Regulate PD-1 Expression and Subsequent CD8 T Cell Memory

Expression of programmed death 1 (PD-1) on CD8 T cells promotes T cell exhaustion during chronic Ag exposure. During acute infections, PD-1 is transiently expressed and has the potential to modulate CD8 T cell memory formation. Conserved region C (CR-C), a promoter proximal cis-regulatory element that is critical to PD-1 expression in vitro, responds to NFATc1, FoxO1, and/or NF-κB signaling pathways. Here, a CR-C knockout mouse was established to determine its role on PD-1 expression and the corresponding effects on T cell function in vivo. Deletion of CR-C decreased PD-1 expression on CD4 T cells and Ag-specific CD8 T cells during acute and chronic lymphocytic choriomeningitis virus challenges, but did not affect the ability to clear an infection. Following acute lymphocytic choriomeningitis virus infection, memory CD8 T cells in the CR-C knockout mouse were formed in greater numbers, were more functional, and were more effective at responding to a melanoma tumor than wild-type memory cells. These data implicate a critical role for CR-C in governing PD-1 expression, and a subsequent role in guiding CD8 T cell differentiation. The data suggest the possibility that titrating PD-1 expression during CD8 T cell activation could have important ramifications in vaccine development and clinical care.

B cell activation and plasma cell differentiation are inhibited by de novo DNA methylation

B cells provide humoral immunity by differentiating into antibody-secreting plasma cells, a process that requires cellular division and is linked to DNA hypomethylation. Conversely, little is known about how de novo deposition of DNA methylation affects B cell fate and function. Here we show that genetic deletion of the de novo DNA methyltransferases Dnmt3a and Dnmt3b (Dnmt3-deficient) in mouse B cells results in normal B cell development and maturation, but increased cell activation and expansion of the germinal center B cell and plasma cell populations upon immunization. Gene expression is mostly unaltered in naive and germinal center B cells, but dysregulated in Dnmt3-deficient plasma cells. Differences in gene expression are proximal to Dnmt3-dependent DNA methylation and chromatin changes, both of which coincide with E2A and PU.1-IRF composite-binding motifs. Thus, de novo DNA methylation limits B cell activation, represses the plasma cell chromatin state, and regulates plasma cell differentiation.DNA methylation is known to contribute to B cell differentiation, but de novo methylation has not been studied in this context. Here the authors use a conditional Dnmt3a/b knockout mouse to map the function of de novo DNA methylation in B cell differentiation and the development of humoral immunity.

Identification of T cell clones without the need for sequencing

The brainbow recombination fluorescent protein system has been used for a multitude of applications in fate and lineage tracking. Here, we use a mouse with a ubiquitously expressed brainbow construct, termed the Confetti mouse, to perform T lymphocyte cell lineage tracking. We demonstrate that antigen-specific T lymphocyte clonotypes can be identified and phenotyped using flow cytometry instead of performing expensive and time-consuming methods of single cell sequencing.

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.