Monoclonal antibodies derived from B cells in subjects with cystic fibrosis reduce Pseudomonas aeruginosa burden in mice.

Pseudomonas aeruginosa (PA) is an opportunistic, frequently multidrug-resistant pathogen that can cause severe infections in hospitalized patients. Antibodies against the PA virulence factor, PcrV, protect from death and disease in a variety of animal models. However, clinical trials of PcrV-binding antibody-based products have thus far failed to demonstrate benefit. Prior candidates were derivations of antibodies identified using protein-immunized animal systems and required extensive engineering to optimize binding and/or reduce immunogenicity. Of note, PA infections are common in people with cystic fibrosis (pwCF), who are generally believed to mount normal adaptive immune responses. Here we utilized a tetramer reagent to detect and isolate PcrV-specific B cells in pwCF and, via single-cell sorting and paired-chain sequencing, identified the B cell receptor (BCR) variable region sequences that confer PcrV-specificity. We derived multiple high affinity anti-PcrV monoclonal antibodies (mAbs) from PcrV-specific B cells across 3 donors, including mAbs that exhibit potent anti-PA activity in a murine pneumonia model. This robust strategy for mAb discovery expands what is known about PA-specific B cells in pwCF and yields novel mAbs with potential for future clinical use.

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.

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.

B cells are sufficient to prime the dominant CD4+ Tfh response to Plasmodium infection.

CD4+ T follicular helper (Tfh) cells dominate the acute response to a blood-stage Plasmodium infection and provide signals to direct B cell differentiation and protective antibody expression. We studied antigen-specific CD4+ Tfh cells responding to Plasmodium infection in order to understand the generation and maintenance of the Tfh response. We discovered that a dominant, phenotypically stable, CXCR5+ Tfh population emerges within the first 4 d of infection and results in a CXCR5+ CCR7+ Tfh/central memory T cell response that persists well after parasite clearance. We also found that CD4+ T cell priming by B cells was both necessary and sufficient to generate this Tfh-dominant response, whereas priming by conventional dendritic cells was dispensable. This study provides important insights into the development of CD4+ Tfh cells during Plasmodium infection and highlights the heterogeneity of antigen-presenting cells involved in CD4+ T cell priming.

Targeting Antigens to CD180 but Not CD40 Programs Immature and Mature B Cell Subsets to Become Efficient APCs.

Targeting Ags to the CD180 receptor activates both B cells and dendritic cells (DCs) to become potent APCs. After inoculating mice with Ag conjugated to an anti-CD180 Ab, B cell receptors were rapidly internalized. Remarkably, all B cell subsets, including even transitional 1 B cells, were programed to process, present Ag, and stimulate Ag-specific CD4+ T cells. Within 24-48 hours, Ag-specific B cells were detectable at T-B borders in the spleen; there, they proliferated in a T cell-dependent manner and induced the maturation of T follicular helper (TFH) cells. Remarkably, immature B cells were sufficient for the maturation of TFH cells after CD180 targeting: TFH cells were induced in BAFFR-/- mice (with only transitional 1 B cells) and not in µMT mice (lacking all B cells) following CD180 targeting. Unlike CD180 targeting, CD40 targeting only induced DCs but not B cells to become APCs and thus failed to efficiently induce TFH cell maturation, resulting in slower and lower-affinity IgG Ab responses. CD180 targeting induces a unique program in Ag-specific B cells and to our knowledge, is a novel strategy to induce Ag presentation in both DCs and B cells, especially immature B cells and thus has the potential to produce a broad range of Ab specificities. This study highlights the ability of immature B cells to present Ag to and induce the maturation of cognate TFH cells, providing insights toward vaccination of mature B cell-deficient individuals and implications in treating autoimmune disorders.

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.

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.

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.

Humoral immune responses to infection: common mechanisms and unique strategies to combat pathogen immune evasion tactics.

Humoral immune responses are crucial for protection against invading pathogens and are the underlying mechanism of protection for most successful vaccines. Our understanding of how humoral immunity develops is largely based on animal models utilizing experimental immunization systems. While these studies have made enormous progress for the field and have defined many of the fundamental principles of B cell differentiation and function, we are only now beginning to appreciate the complexities of humoral immune responses induced by infection. Co-evolution of the adaptive immune system and the pathogenic world has created a diverse array of B cell responses to infections, with both shared and unique strategies. In this review, we consider the common mechanisms that regulate the development of humoral immune responses during infection and highlight recent findings demonstrating the evolution of unique strategies used by either host or pathogen for survival.

Memory B cell heterogeneity: Remembrance of things past.

B cells that persist for long periods of time after antigen encounter exist as either antibody-producing plasma cells (long-lived plasma cells, LLPCs) that reside primarily in the bone marrow or rapidly responsive memory B cells (MBCs) that reside in the spleen and circulation. Although LLPCs are thought to be non-responsive to a secondary infection, MBCs respond to subsequent infection through the production of antibody-secreting cells, formation of new germinal centers (GCs), and repopulation of the memory pool. Dogma suggests that MBCs express class-switched, somatically hypermutated BCRs after undergoing a GC reaction. Yet this narrow view of MBCs has been challenged over the years and it is now well recognized that diverse MBC subsets exist in both rodents and humans. Here, we review current thoughts on the phenotypic and functional characteristics of MBCs, focusing on a population of somatically hypermutated, high affinity IgM+ MBCs that are rapidly responsive to a secondary malaria 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.

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.

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.

Clonotypic composition of the CD4+ T cell response to a vectored retroviral antigen is determined by its speed.

The mechanisms whereby different vaccines may expand distinct Ag-specific T cell clonotypes or induce disparate degrees of protection are incompletely understood. We found that several delivery modes of a model retroviral Ag, including natural infection, preferentially expanded initially rare high-avidity CD4(+) T cell clonotypes, known to mediate protection. In contrast, the same Ag vectored by human adenovirus serotype 5 induced clonotypic expansion irrespective of avidity, eliciting a predominantly low-avidity response. Nonselective clonotypic expansion was caused by relatively weak adenovirus serotype 5-vectored Ag presentation and was reproduced by replication-attenuated retroviral vaccines. Mechanistically, the potency of Ag presentation determined the speed and, consequently, completion of the CD4(+) T cell response. Whereas faster completion retained the initial advantage of high-avidity clonotypes, slower completion permitted uninhibited accumulation of low-avidity clonotypes. These results highlighted the importance of Ag presentation patterns in determining the clonotypic composition of vaccine-induced T cell responses and ultimately the efficacy of vaccination.

Removing T-cell epitopes with computational protein design.

Immune responses can make protein therapeutics ineffective or even dangerous. We describe a general computational protein design method for reducing immunogenicity by eliminating known and predicted T-cell epitopes and maximizing the content of human peptide sequences without disrupting protein structure and function. We show that the method recapitulates previous experimental results on immunogenicity reduction, and we use it to disrupt T-cell epitopes in GFP and Pseudomonas exotoxin A without disrupting function.

Opposing signals from the Bcl6 transcription factor and the interleukin-2 receptor generate T helper 1 central and effector memory cells.

Listeria monocytogenes infection generates T helper 1 (Th1) effector memory cells and CC chemokine receptor 7 (CCR7)(+) cells resembling central memory cells. We tracked endogenous L. monocytogenes-specific CD4(+) T cells to determine how these memory cells are formed. Two effector cell populations were already present several days after infection. One highly expressed the T-bet transcription factor and produced Th1 memory cells in an interleukin-2 (IL-2) receptor-dependent fashion. The other resided in the T cell areas, expressed CCR7 and CXC chemokine receptor 5 (CXCR5), and like follicular helper cells depended on the Bcl6 transcription factor and inducible costimulator ligand on B cells. The CCR7(+)CXCR5(+) effector cells produced similar memory cells that generated diverse effector cell populations in a secondary response. Thus, Th1 effector memory and follicular helper-like central memory cells are produced from early effector cell populations that diverge in response to signals from the IL-2 receptor, Bcl6, and B cells.

Robust antigen specific th17 T cell response to group A Streptococcus is dependent on IL-6 and intranasal route of infection.

Group A streptococcus (GAS, Streptococcus pyogenes) is the cause of a variety of clinical conditions, ranging from pharyngitis to autoimmune disease. Peptide-major histocompatibility complex class II (pMHCII) tetramers have recently emerged as a highly sensitive means to quantify pMHCII-specific CD4+ helper T cells and evaluate their contribution to both protective immunity and autoimmune complications induced by specific bacterial pathogens. In lieu of identifying an immunodominant peptide expressed by GAS, a surrogate peptide (2W) was fused to the highly expressed M1 protein on the surface of GAS to allow in-depth analysis of the CD4+ helper T cell response in C57BL/6 mice that express the I-A(b) MHCII molecule. Following intranasal inoculation with GAS-2W, antigen-experienced 2W:I-A(b)-specific CD4+ T cells were identified in the nasal-associated lymphoid tissue (NALT) that produced IL-17A or IL-17A and IFN-γ if infection was recurrent. The dominant Th17 response was also dependent on the intranasal route of inoculation; intravenous or subcutaneous inoculations produced primarily IFN-γ+ 2W:I-A(b+) CD4+ T cells. The acquisition of IL-17A production by 2W:I-A(b)-specific T cells and the capacity of mice to survive infection depended on the innate cytokine IL-6. IL-6-deficient mice that survived infection became long-term carriers despite the presence of abundant IFN-γ-producing 2W:I-A(b)-specific CD4+ T cells. Our results suggest that an imbalance between IL-17- and IFN-γ-producing CD4+ T cells could contribute to GAS carriage in humans.

Negative selection and peptide chemistry determine the size of naive foreign peptide-MHC class II-specific CD4+ T cell populations.

Naive CD4(+) T cell populations that express TCRs specific for different foreign peptide-MHC class II complex (pMHCII) ligands can vary in size over several orders of magnitude. This variation may explain why immune responses to some peptides are stronger than others. In this study, we used a sensitive pMHCII-tetramer-based cell enrichment method to study the derivation of two naive foreign pMHCII-specific naive CD4(+) T cell populations that differed in size by 8-fold in normal mice. Analysis of mice in which thymic negative selection was impaired revealed that the smaller population underwent more clonal deletion than the larger population. In addition, large naive cell populations tended to recognize peptides with tryptophan residues as TCR contacts. Thus, the foreign pMHCII that tend to be recognized by large naive populations induce minimal clonal deletion and contain certain amino acids with the capacity to interact favorably with TCRs.

Different routes of bacterial infection induce long-lived TH1 memory cells and short-lived TH17 cells.

We used a sensitive method based on tetramers of peptide and major histocompatibility complex II (pMHCII) to determine whether CD4(+) memory T cells resemble the T helper type 1 (T(H)1) and interleukin 17 (IL-17)-producing T helper (T(H)17) subsets described in vitro. Intravenous or intranasal infection with Listeria monocytogenes induced pMHCII-specific CD4(+) naive T cells to proliferate and produce effector cells, about 10% of which resembled T(H)1 or T(H)17 cells, respectively. T(H)1 cells were also present among the memory cells that survived 3 months after infection, whereas T(H)17 cells disappeared. The short lifespan of T(H)17 cells was associated with small amounts of the antiapoptotic protein Bcl-2, the IL-15 receptor and the receptor CD27, and little homeostatic proliferation. These results suggest that T(H)1 cells induced by intravenous infection are more efficient at entering the memory pool than are T(H)17 cells induced by intranasal infection.