Antigen affinity and antigen dose exert distinct influences on CD4 T-cell differentiation Simone Kecka, Mathias Schmalerb, Stefan Gantera, Lena Wyssa, Susanne Oberlec, Eric S. Husebyd, Dietmar Zehnc, and Carolyn G. Kinga,1 aLaboratory of Transplantation Immunology, and bLaboratory of Immunoregulation, Department of Biomedicine, University Hospital Basel, University of Basel, CH-4031 Basel, Switzerland; cSwiss Vaccine Research Institute, and Division of Immunology and Allergy, Department of Medicine, Lausanne University Hospital (CHUV), 1011 Lausanne, Switzerland; and dDepartment of Pathology, University of Massachusetts Medical School, Worcester, MA 01655 Edited by Marc K. Jenkins, University of Minnesota, Minneapolis, MN, and accepted by the Editorial Board September 5, 2014 (received for review February 25, 2014) Cumulative T-cell receptor signal strength and ensuing T-cell re- (13–15). However, another study examining high and low avidity sponses are affected by both antigen affinity and antigen dose. Here CD4 T-cell responses during viral infection found significant we examined the distinct contributions of these parameters to CD4 differences in Th1 but not TFH generation (16). Sustained TCR– T-cell differentiation during infection. We found that high antigen pMHC interactions have also been shown to promote memory affinity positively correlates with T helper (Th)1 differentiation at T-cell differentiation, which is associated with increased TCR avidity both high and low doses of antigen. In contrast, follicular helper T (17, 18). These studies, however, have focused on the development cell (TFH) effectors are generated after priming with high, intermedi- of the Th1 memory compartment, which is phenotypically and ate, and low affinity ligand. Unexpectedly, memory T cells generated functionally distinct from the TFH memory compartment (19, 20). after priming with very low affinity antigen remain impaired in their Thus, although strong TCR signals resulting from high antigen af- ability to generate secondary Th1 effectors, despite being recalled finity or high antigen dose can clearly affect the extent and quality of with high affinity antigen. These data challenge the view that only T-cell differentiation, whether or not T cells can discriminate these strongly stimulated CD4 T cells are capable of differentiating into the signals, and how this contributes to T-cell differentiation during in- T and memory T-cell compartments and reveal that differential fection, has not been determined. FH To address this question, we infected mice with varying con- strength of stimulation during primary T-cell activation imprints INFLAMMATION unique and long lasting T-cell differentiation programs. centrations of Listeria expressing either high or low affinity IMMUNOLOGY AND antigens for the TCR. By normalizing the degree of proliferation induced by high and low affinity antigens we were able to discern follicular helper | infection | lymphocytes distinct influences of antigen affinity and antigen dose on Th cell differentiation. We observed a strong positive correlation be- ollowing infection, T-cell receptor (TCR) interactions with tween antigen affinity and Th1 differentiation that occurs early Fforeign peptide/MHC (pMHC) drive the rapid clonal ex- and is dose independent. Importantly, high antigen dose does not pansion and differentiation of T cells into distinct effector sub- compensate for the low efficiency of Th1 differentiation induced by sets specialized against different classes of microbes. An early low affinity antigen. In contrast, early TFH effector generation was bifurcation in CD4 T-cell responses results in the generation of observed after priming with high, intermediate, and low affinity T helper (Th)1 effectors, which regulate innate cell microbicidal antigen, but was not maintained at later time points under con- function and follicular helper T (TFH) cells, which migrate to B-cell ditions of low antigen dose. In addition, we found that T cells ac- follicles to regulate germinal center (GC) responses and antimi- tivated by either high or low affinity antigen are equally capable of crobial antibody production (1). After pathogen is cleared, T cells memory T-cell differentiation. Surprisingly, memory T cells gen- undergo a contraction phase during which the majority of effectors erated by either low antigen affinity or low antigen dose maintained die by apoptosis, leaving behind a population of long-lived memory their biased effector lineages following recall activation with high cells to provide protection upon subsequent reinfection. The de- affinity antigen. These data indicate that differential strength of cision to differentiate into Th1 and TFH lineagesappearstooccur very early in the immune response (2, 3). Initial T-cell priming by Significance dendritic cells (DCs) is sufficient to induce fate-committed Th1 and TFH cells as early as 3 d after infection, whereas maintenance and T-cell receptor recognition of antigen is an essential first step in further expansion of the TFH compartment depends on T-cell the initiation of a T-cell response. This report demonstrates that interactions with B cells (2). Similarly, memory T-cell differentiation CD4 T cells responding during an infection can discriminate be- occurs very early after infection and is critically dependent on B-cell tween antigen affinity and antigen dose, resulting in distinct interactions for optimal priming (4, 5). Importantly, CD4 T-cell types of effector and memory cell generation. Moreover, mem- differentiation is coupled to division, and unlike CD8 T-cell dif- ory T cells “remember” the strength of primary T-cell activation ferentiation, requires constant antigen recognition (6, 7). and maintain a biased recall response upon secondary infection. Although the strength of TCR–pMHC interactions has been These data reveal that antigen affinity exerts an important in- shown to directly modulate T-cell expansion and clonal domi- fluence on T-cell differentiation that is not compensated for by nance within the Th cell compartment (8, 9), how this influences high antigen dose. Understanding the rules of CD4 T-cell differ- CD4 T-cell fate is not well understood. Cumulative TCR sig- entiation is integral to effective vaccine design. naling can be influenced by both antigen affinity and antigen dose (10). In terms of proliferation, higher antigen dose can Author contributions: S.K. and C.G.K. designed research; S.K., M.S., S.G., L.W., and C.G.K. compensate for lower antigen affinity to some extent, but several performed research; S.O., E.S.H., and D.Z. contributed new reagents/analytic tools; S.K., reports have shown independent effects on T-cell responses both M.S., S.G., and C.G.K. analyzed data; and C.G.K. wrote the paper. in vitro and in vivo (10–12). These data indicate that antigen The authors declare no conflict of interest. affinity and antigen dose may promote qualitatively distinct TCR This article is a PNAS Direct Submission. M.K.J. is a guest editor invited by the signals. Recently, modulation of the overall TCR signal by Editorial Board. varying either TCR affinity or antigen dose was shown to in- 1To whom correspondence should be addressed. Email: [email protected]. fluence the pattern of effector T-cell differentiation, with higher This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. affinity ligands or higher antigen dose promoting TFH generation 1073/pnas.1403271111/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1403271111 PNAS Early Edition | 1of6 Downloaded by guest on September 28, 2021 stimulation during primary T-cell activation can imprint unique and generate consistent levels of inflammation, mice infected with long lasting T-cell differentiation programs. low dose Lm.3K were also infected with wild-type bacteria not expressing 3K. Results Establishing the TCR Ligand Affinity Hierarchy. Several models have Antigen Affinity Influences the Pattern of Effector T-cell Differentiation. been proposed to explain the sensitivity of TCR recognition of Infection results in the generation of two distinct effector pop- pMHC. The receptor occupancy model uses the affinity of the ulations. Th1 effector cells express high levels of the transcription γ TCR for pMHC (KD) to predict ligand affinity, whereas the kinetic factor T-bet, produce IFN , and are important for inducing mac- proofreading model suggests that ligand affinity is dependent on rophage microbicidal function (1). TFH cells express low levels of the half-life (t1/2)ofTCR–pMHC interactions (10, 21–23). A recent the surface marker Ly6c (20) and high levels of the chemokine variation of this model suggests that serial rebinding of pMHC receptor CXCR5, which directs T-cell migration to the B-cell results in an aggregate half-life of TCR binding (ta), that can allow areas of lymphoid structures where they provide signals to enhance antigens with fast on-rates to activate T cells more strongly than B-cell antibody secretion (1). TFH cells expressing high levels of their t1/2 would predict (24). To study the effect of TCR ligand PD-1 and the transcription factor Bcl6 further migrate into B-cell affinity on CD4 T-cell activation, we used B3K508 TCR transgenic germinal centers where they drive B-cell affinity maturation (31), b T cells, which are specific for an H-2A bound peptide FEAQ- whereas TFH cells that express low levels of PD-1 and intermediate KAKANKAKAVD (3K). The KD, t1/2,andta values for 3K and levels of Bcl6 are suggested to be precursors to central memory a panel of single amino acid substitutions 3K variants have been cells (3, 31). To understand how ligand affinity affects CD4 effector previously established (Fig. S1A) (24, 25). Although we use the T-cell differentiation, we examined the phenotype of B3K508 T cells term “affinity” to indicate the strength of TCR–pMHC inter- responding to infection with high affinity Lm.3K or low affinity actions, the strength and hierarchy of the ligands used in our study Lm.P2A. At day 6 after infection with high dose Lm.3K, B3K508 is independently reflected by all three biophysical parameters.