Most of what we know about adaptive immunity has come from inbred mouse studies, using methods that are often difficult or impossible to confirm in humans. In addition, vaccine responses in mice are ...often poorly predictive of responses to those same vaccines in humans. Here we use human tonsils, readily available lymphoid organs, to develop a functional organotypic system that recapitulates key germinal center features in vitro, including the production of antigen-specific antibodies, somatic hypermutation and affinity maturation, plasmablast differentiation and class-switch recombination. We use this system to define the essential cellular components necessary to produce an influenza vaccine response. We also show that it can be used to evaluate humoral immune responses to two priming antigens, rabies vaccine and an adenovirus-based severe acute respiratory syndrome coronavirus 2 vaccine, and to assess the effects of different adjuvants. This system should prove useful for studying critical mechanisms underlying adaptive immunity in much greater depth than previously possible and to rapidly test vaccine candidates and adjuvants in an entirely human system.
The Ab response to HIV is of great interest, particularly in the context of a protective vaccine and broadly neutralizing Abs, but research is typically geared toward elite controllers because of ...their ability to successfully control the virus. In this study, we studied the evolution of the Ab repertoire over the first year of HIV infection in people classified as rapid progressors (RP) compared with typical progressors. HIV RPs are an important yet understudied group of HIV patients classified by a rapid decline in CD4 counts and accelerated development of AIDS. We found that the global IgG somatic hypermutation load negatively correlated with disease progression, possibly because of exaggerated isotype switching of unmutated sequences in patients with low CD4 counts. We measured Ab sequence evolution over time using longitudinal samples taken during the early stages of infection and 1 year postinfection. Within clonal lineages spanning both timepoints, visit 2-derived sequences harbored considerably more mutations than their visit 1 relatives. Despite extensive ongoing somatic hypermutation, the initially strong signs of Ag selection pressure observed in visit 1-derived sequences decayed by visit 2. These data suggest that excessive immune activation in RPs leads to a hyperactive B cell response that fails to confer protection.
Accurately measuring antibody repertoire sequence composition in a small amount of blood is challenging yet important for understanding repertoire responses to infection and vaccination. We develop ...molecular identifier clustering-based immune repertoire sequencing (MIDCIRS) and use it to study age-related antibody repertoire development and diversification before and during acute malaria in infants (< 12 months old) and toddlers (12-47 months old) with 4-8 ml of blood. Here, we show this accurate and high-coverage repertoire-sequencing method can use as few as 1000 naive B cells. Unexpectedly, we discover high levels of somatic hypermutation in infants as young as 3 months old. Antibody clonal lineage analysis reveals that somatic hypermutation levels are increased in both infants and toddlers upon infection, and memory B cells isolated from individuals who previously experienced malaria continue to induce somatic hypermutations upon malaria rechallenge. These results highlight the potential of antibody repertoire diversification in infants and toddlers.Somatic hypermutation of antibodies can occur in infants but are difficult to track. Here the authors present a new method called MIDCIRS for deep quantitative repertoire sequencing with few cells, and show infants as young as 3 months can expand antibody lineage complexity in response to malaria infection.
Unique molecular identifiers (MIDs) have been demonstrated to effectively improve immune repertoire sequencing (IR-seq) accuracy, especially to identify somatic hypermutations in antibody repertoire ...sequencing. However, evaluating the sensitivity to detect rare T cells and the degree of clonal expansion in IR-seq has been difficult due to the lack of knowledge of T cell receptor (TCR) RNA molecule copy number and a generalized approach to estimate T cell clone size from TCR RNA molecule quantification. This limited the application of TCR repertoire sequencing (TCR-seq) in clinical settings, such as detecting minimal residual disease in lymphoid malignancies after treatment, evaluating effectiveness of vaccination and assessing degree of infection. Here, we describe using an MID Clustering-based IR-Seq (MIDCIRS) method to quantitatively study TCR RNA molecule copy number and clonality in T cells. First, we demonstrated the necessity of performing MID sub-clustering to eliminate erroneous sequences. Further, we showed that MIDCIRS enables a sensitive detection of a single cell in as many as one million naïve T cells and an accurate estimation of the degree of T cell clonal expression. The demonstrated accuracy, sensitivity, and wide dynamic range of MIDCIRS TCR-seq provide foundations for future applications in both basic research and clinical settings.
CXCR5 is a key marker of follicular helper T (TFH) cells. Using primary lymph nodes (LNs) from HIV-infected patients, we identified a population of CXCR5− CD4+ T cells with TFH-cell-like features. ...This CXCR5− subset becomes expanded in severe HIV infection and is characterized by the upregulation of activation markers and high PD-1 and ICOS surface expression. Integrated analyses on the phenotypic heterogeneity, functional capacity, T cell receptor (TCR) repertoire, transcriptional profile, and epigenetic state of CXCR5−PD-1+ICOS+ T cells revealed a shared clonal relationship with TFH cells. CXCR5−PD-1+ICOS+ T cells retained a poised state for CXCR5 expression and exhibited a migratory transcriptional program. TCR sequence overlap revealed a contribution of LN-derived CXCR5−PD-1+ICOS+ T cells to circulating CXCR5− CD4+ T cells with B cell help function. These data link LN pathology to circulating T cells and expand the current understanding on the diversity of T cells that regulate B cell responses during chronic inflammation.
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•High-dimensional profiling of CD4+ T cells in HIV-infected lymph nodes•A subset of CXCR5− CD4+ T cells in lymph nodes are clonally related to TFH cells•CXCR5−PD-1+ICOS+ CD4+ T cells exhibit TFH-like functional features•TFH-like CXCR5− T cells contribute to circulating T cells with B cell help function
Follicular helper T (TFH) cells are critical for antibody production. Del Alcazar et al. showed that TFH cells can lose their characteristic chemokine receptor, giving rise to migratory populations of CXCR5− T cells that retain B cell help function and are poised for CXCR5 expression.
Follicular-helper T cells (T
) are an essential arm of the adaptive immune system. Although T
were first discovered through their ability to contribute to antibody affinity maturation through ...co-stimulatory interactions with B cells, new light has been shed on their ability to remain a complex and functionally plastic cell type. Due to a lack sample availability, however, many studies have been limited to characterizing T
in mice or non-canonical tissue types, such as peripheral blood. Such constraints have resulted in a limited, and sometimes contradictory, understanding of this fundamental cell type. One subset of T
receiving attention in chronic infection are CXCR3-expressing T
cells (CXCR3
T
) due to their abnormal accumulation in secondary lymphoid tissues. Their function and clonal relationship with other T
subsets in lymphoid tissues during infection, however, remains largely unclear. We thus systematically investigated this and other subsets of T
within untreated HIV-infected human lymph nodes using Mass CyTOF and a combination of RNA and TCR repertoire sequencing. We show an inflation of the CXCR3
T
compartment during HIV infection that correlates with a lower HIV burden. Deeper analysis into this population revealed a functional shift of CXCR3
T
away from germinal center T
(GC-T
), including the altered expression of several important transcription factors and cytokines. CXCR3
T
also upregulated cell migration transcriptional programs and were clonally related to peripheral T
populations. In combination, these data suggest that CXCR3
T
have a greater tendency to enter circulation than their CXCR3
counterparts, potentially functioning through distinct modalities that may lead to enhanced defense.
T cells recognize and kill a myriad of pathogen-infected or cancer cells using a diverse set of T cell receptors (TCRs). The affinity of TCR to cognate antigen is of high interest in adoptive T cell ...transfer immunotherapy and antigen-specific T cell repertoire immune profiling because it is widely known to correlate with downstream T cell responses. We introduce the in situ TCR affinity and sequence test (iTAST) for simultaneous measurement of TCR affinity and sequence from single primary CD8(+) T cells in human blood. We demonstrate that the repertoire of primary antigen-specific T cells from pathogen-inexperienced individuals has a surprisingly broad affinity range of 1000-fold composed of diverse TCR sequences. Within this range, samples from older individuals contained a reduced frequency of high-affinity T cells compared to young individuals, demonstrating an age-related effect of T cell attrition that could cause holes in the repertoire. iTAST should enable the rapid selection of high-affinity TCRs ex vivo for adoptive immunotherapy and measurement of T cell response for immune monitoring applications.
Advancements in high-throughput sequencing and molecular identifier-based error correction have opened the door to antibody repertoire sequencing with single mutation precision, increasing both the ...breadth and depth of immune response characterization. However, improvements in sequencing technology cannot resolve one key aspect of antibody repertoire sequencing accuracy: the possibility of undocumented novel germline alleles. Somatic hypermutation (SHM) calling requires a reference germline sequence, and the antibody variable region gene alleles collected by the IMGT database, although large in number, are not comprehensive. Mismatches, resulted from single nucleotide polymorphisms or other genetic variation, between the true germline sequence and the closest IMGT allele can inflate SHM counts, leading to inaccurate antibody repertoire analysis. Here, we developed a streamlined approach to novel allele prediction and validation using bulk PBMC antibody repertoire sequencing data and targeted genomic DNA amplification and sequencing using PBMCs from only 4 ml of blood to quickly and effectively improve the fidelity of antibody repertoire analysis. This approach establishes a framework for comprehensively annotating novel alleles using a small amount of blood sample, which is extremely useful in studying young children's immune systems.
Plasmodium falciparum malaria is a deadly infectious disease in which Abs play a critical role in naturally acquired immunity. However, the specificity and nature of Abs elicited in response to ...malaria are only partially understood. Autoreactivity and polyreactivity are common features of Ab responses in several infections and were suggested to contribute to effective pathogen-specific Ab responses. In this article, we report on the regulation of B cells expressing the inherently autoreactive VH4-34 H chain (identified by the 9G4 mAb) and 9G4
plasma IgG in adults and children living in a P. falciparum malaria-endemic area in West Africa. The frequency of 9G4
peripheral blood CD19
B cells was similar in United States adults and African adults and children; however, more 9G4
B cells appeared in classical and atypical memory B cell compartments in African children and adults compared with United States adults. The levels of 9G4
IgG increased following acute febrile malaria but did not increase with age as humoral immunity is acquired or correlate with protection from acute disease. This was the case, even though a portion of 9G4
B cells acquired phenotypes of atypical and classical memory B cells and 9G4
IgG contained equivalent numbers of somatic hypermutations compared with all other VHs, a characteristic of secondary Ab repertoire diversification in response to Ag stimulation. Determining the origin and function of 9G4
B cells and 9G4
IgG in malaria may contribute to a better understanding of the varied roles of autoreactivity in infectious diseases.
Follicular helper CD4
T cells (T
) play an integral role in promoting B cell differentiation and affinity maturation. Whereas T
cell frequencies are increased in lymph nodes (LNs) from individuals ...infected with HIV, humoral immunity remains impaired during chronic HIV infection. Whether HIV inhibits T
responses in LNs remains unclear. Advances in this area have been limited by the difficulty of accessing human lymphoid tissues. Here, we combined high-dimensional mass cytometry with T cell receptor repertoire sequencing to interrogate the composition of T
cells in primary human LNs. We found evidence for intact antigen-driven clonal expansion of T
cells and selective utilization of specific complementarity-determining region 3 (CDR3) motifs during chronic HIV infection, but the resulting T
cells acquired an activation-related T
cell signature characterized by interleukin-21 (IL-21) dominance. These IL-21
T
cells contained an oligoclonal HIV-reactive population that preferentially accumulated in patients with severe HIV infection and was associated with aberrant B cell distribution in the same LN. These data indicate that T
cells remain capable of responding to HIV antigens during chronic HIV infection but become functionally skewed and oligoclonally restricted under persistent antigen stimulation.
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