In Vivo CD4+ T Cell Differentiation and Function: Revisiting the Th1/Th2 Paradigm.

The discovery of CD4+ T cell subset-defining master transcription factors and framing of the Th1/Th2 paradigm ignited the CD4+ T cell field. Advances in in vivo experimental systems, however, have revealed that more complex lineage-defining transcriptional networks direct CD4+ T cell differentiation in the lymphoid organs and tissues. This review focuses on the layers of fate decisions that inform CD4+ T cell differentiation in vivo. Cytokine production by antigen-presenting cells and other innate cells influences the CD4+ T cell effector program [e.g., T helper type 1 (Th1), Th2, Th17]. Signals downstream of the T cell receptor influence whether individual clones bearing hallmarks of this effector program become T follicular helper cells, supporting development of B cells expressing specific antibody isotypes, or T effector cells, which activate microbicidal innate cells in tissues. These bifurcated, parallel axes allow CD4+ T cells to augment their particular effector program and prevent disease.

Principles and therapeutic applications of adaptive immunity.

Adaptive immunity provides protection against infectious and malignant diseases. These effects are mediated by lymphocytes that sense and respond with targeted precision to perturbations induced by pathogens and tissue damage. Here, we review key principles underlying adaptive immunity orchestrated by distinct T cell and B cell populations and their extensions to disease therapies. We discuss the intracellular and intercellular processes shaping antigen specificity and recognition in immune activation and lymphocyte functions in mediating effector and memory responses. We also describe how lymphocytes balance protective immunity against autoimmunity and immunopathology, including during immune tolerance, response to chronic antigen stimulation, and adaptation to non-lymphoid tissues in coordinating tissue immunity and homeostasis. Finally, we discuss extracellular signals and cell-intrinsic programs underpinning adaptive immunity and conclude by summarizing key advances in vaccination and engineering adaptive immune responses for therapeutic interventions. A deeper understanding of these principles holds promise for uncovering new means to improve human health.

Imprinted SARS-CoV-2-specific memory lymphocytes define hybrid immunity.

Immune memory is tailored by cues that lymphocytes perceive during priming. The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic created a situation in which nascent memory could be tracked through additional antigen exposures. Both SARS-CoV-2 infection and vaccination induce multifaceted, functional immune memory, but together, they engender improved protection from disease, termed hybrid immunity. We therefore investigated how vaccine-induced memory is shaped by previous infection. We found that following vaccination, previously infected individuals generated more SARS-CoV-2 RBD-specific memory B cells and variant-neutralizing antibodies and a distinct population of IFN-γ and IL-10-expressing memory SARS-CoV-2 spike-specific CD4+ T cells than previously naive individuals. Although additional vaccination could increase humoral memory in previously naive individuals, it did not recapitulate the distinct CD4+ T cell cytokine profile observed in previously infected subjects. Thus, imprinted features of SARS-CoV-2-specific memory lymphocytes define hybrid immunity.

Functional SARS-CoV-2-Specific Immune Memory Persists after Mild COVID-19.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus is causing a global pandemic, and cases continue to rise. Most infected individuals experience mildly symptomatic coronavirus disease 2019 (COVID-19), but it is unknown whether this can induce persistent immune memory that could contribute to immunity. We performed a longitudinal assessment of individuals recovered from mild COVID-19 to determine whether they develop and sustain multifaceted SARS-CoV-2-specific immunological memory. Recovered individuals developed SARS-CoV-2-specific immunoglobulin (IgG) antibodies, neutralizing plasma, and memory B and memory T cells that persisted for at least 3 months. Our data further reveal that SARS-CoV-2-specific IgG memory B cells increased over time. Additionally, SARS-CoV-2-specific memory lymphocytes exhibited characteristics associated with potent antiviral function: memory T cells secreted cytokines and expanded upon antigen re-encounter, whereas memory B cells expressed receptors capable of neutralizing virus when expressed as monoclonal antibodies. Therefore, mild COVID-19 elicits memory lymphocytes that persist and display functional hallmarks of antiviral immunity.

Elicitation of Potent Neutralizing Antibody Responses by Designed Protein Nanoparticle Vaccines for SARS-CoV-2.

A safe, effective, and scalable vaccine is needed to halt the ongoing SARS-CoV-2 pandemic. We describe the structure-based design of self-assembling protein nanoparticle immunogens that elicit potent and protective antibody responses against SARS-CoV-2 in mice. The nanoparticle vaccines display 60 SARS-CoV-2 spike receptor-binding domains (RBDs) in a highly immunogenic array and induce neutralizing antibody titers 10-fold higher than the prefusion-stabilized spike despite a 5-fold lower dose. Antibodies elicited by the RBD nanoparticles target multiple distinct epitopes, suggesting they may not be easily susceptible to escape mutations, and exhibit a lower binding:neutralizing ratio than convalescent human sera, which may minimize the risk of vaccine-associated enhanced respiratory disease. The high yield and stability of the assembled nanoparticles suggest that manufacture of the nanoparticle vaccines will be highly scalable. These results highlight the utility of robust antigen display platforms and have launched cGMP manufacturing efforts to advance the SARS-CoV-2-RBD nanoparticle vaccine into the clinic.

Universal Principled Review: A Community-Driven Method to Improve Peer Review.

Despite being a staple of our science, the process of pre-publication peer review has few agreed-upon standards defining its goals or ideal execution. As a community of reviewers and authors, we assembled an evaluation format and associated specific standards for the process as we think it should be practiced. We propose that we apply, debate, and ultimately extend these to improve the transparency of our criticism and the speed with which quality data and ideas become public.

Interleukin-4 downregulates transcription factor BCL6 to promote memory B cell selection in germinal centers.

Germinal center (GC)-derived memory B cells (MBCs) are critical for humoral immunity as they differentiate into protective antibody-secreting cells during re-infection. GC formation and cellular interactions within the GC have been studied in detail, yet the exact signals that allow for the selection and exit of MBCs are not understood. Here, we showed that IL-4 cytokine signaling in GC B cells directly downregulated the transcription factor BCL6 via negative autoregulation to release cells from the GC program and to promote MBC formation. This selection event required additional survival cues and could therefore result in either GC exit or death. We demonstrate that both increasing IL-4 bioavailability or limiting IL-4 signaling disrupted MBC selection stringency. In this way, IL-4 control of BCL6 expression serves as a tunable switch within the GC to tightly regulate MBC selection and affinity maturation.

The Emergence and Functional Fitness of Memory CD4+ T Cells Require the Transcription Factor Thpok.

Memory CD4+ T cells mediate long-term immunity, and their generation is a key objective of vaccination strategies. However, the transcriptional circuitry controlling the emergence of memory cells from early CD4+ antigen-responders remains poorly understood. Here, using single-cell RNA-seq to study the transcriptome of virus-specific CD4+ T cells, we identified a gene signature that distinguishes potential memory precursors from effector cells. We found that both that signature and the emergence of memory CD4+ T cells required the transcription factor Thpok. We further demonstrated that Thpok cell-intrinsically protected memory cells from a dysfunctional, effector-like transcriptional program, similar to but distinct from the exhaustion pattern of cells responding to chronic infection. Mechanistically, Thpok- bound genes encoding the transcription factors Blimp1 and Runx3 and acted by antagonizing their expression. Thus, a Thpok-dependent circuitry promotes both memory CD4+ T cells' differentiation and functional fitness, two previously unconnected critical attributes of adaptive immunity.

A Thpok-Directed Transcriptional Circuitry Promotes Bcl6 and Maf Expression to Orchestrate T Follicular Helper Differentiation.

The generation of high-affinity neutralizing antibodies, the objective of most vaccine strategies, occurs in B cells within germinal centers (GCs) and requires rate-limiting "help" from follicular helper CD4+ T (Tfh) cells. Although Tfh differentiation is an attribute of MHC II-restricted CD4+ T cells, the transcription factors driving Tfh differentiation, notably Bcl6, are not restricted to CD4+ T cells. Here, we identified a requirement for the CD4+-specific transcription factor Thpok during Tfh cell differentiation, GC formation, and antibody maturation. Thpok promoted Bcl6 expression and bound to a Thpok-responsive region in the first intron of Bcl6. Thpok also promoted the expression of Bcl6-independent genes, including the transcription factor Maf, which cooperated with Bcl6 to mediate the effect of Thpok on Tfh cell differentiation. Our findings identify a transcriptional program that links the CD4+ lineage with Tfh differentiation, a limiting factor for efficient B cell responses, and suggest avenues to optimize vaccine generation.

NKTeeing Up B Cell Responses to Viral Infection.

Activated B cells mature in germinal centers (GCs), but GC initiation during infection is poorly understood. Gaya et al. (2018) show that NKT cells, activated by CD169+ macrophages, produce an early wave of interleukin-4 (IL-4) that promotes GC formation during viral infection.

Somatically Hypermutated Plasmodium-Specific IgM(+) Memory B Cells Are Rapid, Plastic, Early Responders upon Malaria Rechallenge.

Humoral immunity consists of pre-existing antibodies expressed by long-lived plasma cells and rapidly reactive memory B cells (MBC). Recent studies of MBC development and function after protein immunization have uncovered significant MBC heterogeneity. To clarify functional roles for distinct MBC subsets during malaria infection, we generated tetramers that identify Plasmodium-specific MBCs in both humans and mice. Long-lived murine Plasmodium-specific MBCs consisted of three populations: somatically hypermutated immunoglobulin M(+) (IgM(+)) and IgG(+) MBC subsets and an unmutated IgD(+) MBC population. Rechallenge experiments revealed that high affinity, somatically hypermutated Plasmodium-specific IgM(+) MBCs proliferated and gave rise to antibody-secreting cells that dominated the early secondary response to parasite rechallenge. IgM(+) MBCs also gave rise to T cell-dependent IgM(+) and IgG(+)B220(+)CD138(+) plasmablasts or T cell-independent B220(-)CD138(+) IgM(+) plasma cells. Thus, even in competition with IgG(+) MBCs, IgM(+) MBCs are rapid, plastic, early responders to a secondary Plasmodium rechallenge and should be targeted by vaccine strategies.

Interleukin-2-Dependent Allergen-Specific Tissue-Resident Memory Cells Drive Asthma.

Exposure to inhaled allergens generates T helper 2 (Th2) CD4(+) T cells that contribute to episodes of inflammation associated with asthma. Little is known about allergen-specific Th2 memory cells and their contribution to airway inflammation. We generated reagents to understand how endogenous CD4(+) T cells specific for a house dust mite (HDM) allergen form and function. After allergen exposure, HDM-specific memory cells persisted as central memory cells in the lymphoid organs and tissue-resident memory cells in the lung. Experimental blockade of lymphocyte migration demonstrated that lung-resident cells were sufficient to induce airway hyper-responsiveness, which depended upon CD4(+) T cells. Investigation into the differentiation of pathogenic Trm cells revealed that interleukin-2 (IL-2) signaling was required for residency and directed a program of tissue homing migrational cues. These studies thus identify IL-2-dependent resident Th2 memory cells as drivers of lung allergic responses.

Cytotoxic T Cells Targeting Spike Glycoprotein Are Associated with Hybrid Immunity to SARS-CoV-2.

mRNA vaccination of individuals with prior SARS-CoV-2 infection provides superior protection against breakthrough infections with variants of concern compared with vaccination in the absence of prior infection. However, the immune mechanisms by which this hybrid immunity is generated and maintained are unknown. Whereas genetic variation in spike glycoprotein effectively subverts neutralizing Abs, spike-specific T cells are generally maintained against SARS-CoV-2 variants. Thus, we comprehensively profiled human T cell responses against the S1 and S2 domains of spike glycoprotein in a cohort of SARS-CoV-2-naive (n = 13) or -convalescent (n = 17) individuals who received two-dose mRNA vaccine series and were matched by age, sex, and vaccine type. Using flow cytometry, we observed that the overall functional breadth of CD4 T cells and polyfunctional Th1 responses was similar between the two groups. However, polyfunctional cytotoxic CD4 T cell responses against both S1 and S2 domains trended higher among convalescent subjects. Multimodal single-cell RNA sequencing revealed diverse functional programs in spike-specific CD4 and CD8 T cells in both groups. However, convalescent individuals displayed enhanced cytotoxic and antiviral CD8 T cell responses to both S1 and S2 in the absence of cytokine production. Taken together, our data suggest that cytotoxic CD4 and CD8 T cells targeting spike glycoprotein may partially account for hybrid immunity and protection against breakthrough infections with SARS-CoV-2.

De novo design of potent and selective mimics of IL-2 and IL-15.

We describe a de novo computational approach for designing proteins that recapitulate the binding sites of natural cytokines, but are otherwise unrelated in topology or amino acid sequence. We use this strategy to design mimics of the central immune cytokine interleukin-2 (IL-2) that bind to the IL-2 receptor ßγc heterodimer (IL-2Rßγc) but have no binding site for IL-2Rα (also called CD25) or IL-15Rα (also known as CD215). The designs are hyper-stable, bind human and mouse IL-2Rßγc with higher affinity than the natural cytokines, and elicit downstream cell signalling independently of IL-2Rα and IL-15Rα. Crystal structures of the optimized design neoleukin-2/15 (Neo-2/15), both alone and in complex with IL-2Rßγc, are very similar to the designed model. Neo-2/15 has superior therapeutic activity to IL-2 in mouse models of melanoma and colon cancer, with reduced toxicity and undetectable immunogenicity. Our strategy for building hyper-stable de novo mimetics could be applied generally to signalling proteins, enabling the creation of superior therapeutic candidates.

Affinity alone does not drive long-lived plasma cell differentiation.

Long-lived plasma cells are important for preventing infection by maintaining baseline antibody titers. However, the cues leading to plasma cell differentiation remain unclear. In this article, we discuss recent work assessing the role of affinity on plasma cell differentiation.

Antibodies to Interleukin-2 Elicit Selective T Cell Subset Potentiation through Distinct Conformational Mechanisms.

Interleukin-2 (IL-2) is a pleiotropic cytokine that regulates immune cell homeostasis and has been used to treat a range of disorders including cancer and autoimmune disease. IL-2 signals via interleukin-2 receptor-ß (IL-2Rß):IL-2Rγ heterodimers on cells expressing high (regulatory T cells, Treg) or low (effector cells) amounts of IL-2Rα (CD25). When complexed with IL-2, certain anti-cytokine antibodies preferentially stimulate expansion of Treg (JES6-1) or effector (S4B6) cells, offering a strategy for targeted disease therapy. We found that JES6-1 sterically blocked the IL-2:IL-2Rß and IL-2:IL-2Rγ interactions, but also allosterically lowered the IL-2:IL-2Rα affinity through a "triggered exchange" mechanism favoring IL-2Rα(hi) Treg cells, creating a positive feedback loop for IL-2Rα(hi) cell activation. Conversely, S4B6 sterically blocked the IL-2:IL-2Rα interaction, while also conformationally stabilizing the IL-2:IL-2Rß interaction, thus stimulating all IL-2-responsive immune cells, particularly IL-2Rß(hi) effector cells. These insights provide a molecular blueprint for engineering selectively potentiating therapeutic antibodies.

ICOS coreceptor signaling inactivates the transcription factor FOXO1 to promote Tfh cell differentiation.

T follicular helper (Tfh) cells are essential in the induction of high-affinity, class-switched antibodies. The differentiation of Tfh cells is a multi-step process that depends upon the co-receptor ICOS and the activation of phosphoinositide-3 kinase leading to the expression of key Tfh cell genes. We report that ICOS signaling inactivates the transcription factor FOXO1, and a Foxo1 genetic deletion allowed for generation of Tfh cells with reduced dependence on ICOS ligand. Conversely, enforced nuclear localization of FOXO1 inhibited Tfh cell development even though ICOS was overexpressed. FOXO1 regulated Tfh cell differentiation through a broad program of gene expression exemplified by its negative regulation of Bcl6. Final differentiation to germinal center Tfh cells (GC-Tfh) was instead FOXO1 dependent as the Foxo1(-/-) GC-Tfh cell population was substantially reduced. We propose that ICOS signaling transiently inactivates FOXO1 to initiate a Tfh cell contingency that is completed in a FOXO1-dependent manner.